Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.Gene therapy is a potential treatment option for genetic diseases, malignant diseases, and other acquired diseases. To this end, safe and efficient gene transfer into specific target cells is a central requirement, and a variety of nonviral and viral vector systems have been developed (6, 44). Recombinant viruses can be used for efficient gene transfer. Retroviruses, adeno-associated viruses, and lentiviruses are able to integrate foreign genes into host genomes and are suitable for gene therapeutics by virtue of their permanent expression of the therapeutic genes, whereas adenoviruses, herpesviruses, and baculoviruses can transiently express foreign genes (6, 12, 44). Pseudotype particles bearing other viral envelope proteins have been developed to improve transduction efficiency and the safety of viral vectors, including retrovirus (4, 7), lentivirus (25), vesicular stomatitis virus (VSV) (29), and baculovirus (17, 42). Pseudotype retroviruses and lentiviruses bearing the baculovirus envelope protein GP64 of Autographa californica nucleopolyhedrosis virus (AcNPV) have been shown to exhibit efficient gene transduction into a wide variety of cells with a lower cytotoxicity compared to those bearing the VSV envelope protein G (VSVG), which is commonly used for pseudotyping (18, 32, 35, 36).However, a drawback of gene transduction by viral vectors is that human sera inactivate the vectors (11, 40). Complement is a major element of the innate immune response and serves to link innate and adaptive immunity (8). Complement activation can occur via classical, lectin, and alternative pathways (2, 8). All pathways invoke several responses, such as virus opsonization, virolysis, anaphylatoxin, and chemotaxin production, as well as others (2, 8). VSV and baculovirus are inactivated by human sera via the classical pathway (1, 11). Because complement activation also induces potential damage to host cells, the complement system is tightly regulated by the complement regulatory proteins (CRPs), including CD55/decay-accelerating factor (DAF), CD46/membrane cofactor protein (MCP), and CD59 (2, 8, 15). DAF and CD46 inhibit activation of C3/C5-converting enzymes, which regulate the activation of classical and alternative pathways, whereas CD59 regulates the assembly of the membrane attack complex (2, 8, 15).Viral vectors can be manipulated to confer resistance to the complement inactivation. Human immunodeficiency virus (HIV) is known to develop resistance to human complement through the incorporation of DAF, CD46, and CD59 to the viral particles (22, 30, 31, 38). Moloney murine leukemia virus vectors produced in HT1080 cells are resistant to complement inactivation (5). Baculovirus and lentivirus vectors bearing DAF or the fusion protein between the functional domains of human DAF and the GP64 were resistant to complement inactivation (9, 13). It has been shown that lentivirus vectors pseudotyped with the GP64 are more resistant to inactivation in the sera of mice and rats (14, 32) and are capable of executing longer expression of the transgenes in nasal epithelia compared to those pseudotyped with the VSVG (35, 36). However, the precise mechanisms underlying the resistance to complement inactivation by pseudotyping of the GP64 is not known.To clarify the molecular mechanisms underlying the resistance of the viral vectors pseudotyped with the GP64 to the complement inactivation, we produced pseudotype and recombinant VSVs bearing the GP64. The recombinant VSVs carrying the gp64 gene generated in human cells but not in insect cells exhibited incorporation of human DAF on the viral particles and were resistant to the complement inactivation. Furthermore, production of the gp64 pseudotype VSV in the DAF knockdown human cells impaired serum resistance, whereas production of the gp64 recombinant VSV in the CHO cell lines stably expressing human DAF and the recombinant baculoviruses in the insect cells stably expressing human DAF or encoding the DAF gene in the genome conferred resistance to the complement inactivation. These results suggest that DAF incorporation into viral particles bearing baculovirus GP64 confers resistance to serum inactivation.     

Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus

Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% β-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.The entry mechanism of enveloped viruses includes two major steps: virus binding to host receptors and subsequent fusion of the viral membrane with the cell membrane. For many viruses the processes of binding and fusion are mediated by a machinery composed of several membrane proteins working in concert with sequential events triggered by conformational changes upon interaction with host (co)receptors. Examples are herpes simplex virus (HSV) (4) and vaccinia virus (23), which have an entry machinery composed of four and eight proteins, respectively. The entry of the occlusion-derived virus (ODV) form of baculoviruses into insect midgut epithelial cells upon oral infection of insect larvae may involve a similar strategy, but little is known about the role of ODV membrane proteins.Baculovirus ODVs are orally infectious, enveloped virus particles embedded in a protein crystal called an occlusion body (OB) that infect midgut epithelial cells (24). After ingestion of OBs by the host, the proteinaceous OB crystal dissolves quickly due to the alkaline conditions (pH 10 to 11) in the midgut, and the ODV particles are released (reviewed in reference 24). After passage through the peritrophic membrane, ODVs bind and fuse with the microvilli of columnar epithelial cells, resulting in the release of nucleocapsids into the cytosol and subsequent initiation of infection (10, 12, 24). A second type of virus particle, the budded virus (BV), is produced in these cells and infects other cells and tissues in the insect, causing a systemic infection (reviewed in reference 22). While the entry mechanisms of BVs have been studied at least to a certain extent (16, 31, 32), the entry mechanism of ODVs is still rather enigmatic due to its complexity and the lack of proper cell lines supporting ODV entry.ODVs contain more than 10 different envelope proteins (3). Five of these, denoted PIF1, PIF2, PIF3, PIF4, and P74, have been identified to be essential for per os infection of insect larvae (6, 7, 14, 18, 20). These PIF proteins function in the early stage of virus infection, and deletion of any of these pif genes leads to a block in infection prior to viral gene expression in midgut epithelial cells (7, 10, 18). Until now, three of these proteins, PIF1, PIF2, and P74, have been reported to function in virus binding (10, 18). Deletion of any of these three proteins leads to a loss of oral infectivity, while only a 3-fold reduction in binding is measured, and no significant reduction in fusion efficiency is observed (10, 18). This suggests that the three PIF proteins, apart from binding to midgut epithelial cells, may have other unknown functions for which they may have to work together. The functions of PIF3 and PIF4 are rather enigmatic although there has been speculation that PIF3 functions in nucleocapsid translocation along the microvilli as it seemed to be dispensable for ODV binding and fusion (18, 24).All five proteins are highly conserved in Baculoviridae and are encoded by so-called core genes (3, 6, 11, 29). Recent work further revealed that these proteins have homologues in other large invertebrate DNA viruses which replicate in the nucleus, such as salivary gland hypertrophy viruses (SGHVs) (9), nudiviruses (30) and white spot syndrome virus (WSSV) (Nimaviridae) (J. A. Jehle, personal communication). pif genes are also found in polydnaviruses of braconid wasps (2). This high conservation of pif genes in a diverse range of large, circular, double-stranded DNA viruses suggests that the PIF proteins are associated with a conserved and evolutionarily ancient entry mechanism of viruses into invertebrate hosts.The aim of the present study is to follow the ODV entry process by investigating whether the PIF proteins form a complex on the ODV membrane. Based on immunogold labeling, cross-linking, differential temperature SDS-PAGE, and coimmunoprecipitation (CoIP) analysis with a panel of recombinant viruses, we provide strong evidence that PIF1, PIF2, PIF3, and P74 form a complex on the ODV surface. This complex is likely to play an essential role in virus entry into midgut epithelial cells of susceptible insect larvae.     

Host Insect Inhibitor-of-Apoptosis SfIAP Functionally Replaces Baculovirus IAP but Is Differentially Regulated by Its N-Terminal Leader

The inhibitor-of-apoptosis (IAP) proteins encoded by baculoviruses bear a striking resemblance to the cellular IAP homologs of their invertebrate hosts. By virtue of the acquired selective advantage of blocking virus-induced apoptosis, baculoviruses may have captured cellular IAP genes that subsequently evolved for virus-specific objectives. To compare viral and host IAPs, we defined antiapoptotic properties of SfIAP, the principal cellular IAP of the lepidopteran host Spodoptera frugiperda. We report here that SfIAP prevented virus-induced apoptosis as well as viral Op-IAP3 (which is encoded by the Orgyia pseudotsugata nucleopolyhedrovirus) when overexpressed from the baculovirus genome. Like Op-IAP3, SfIAP blocked apoptosis at a step prior to caspase activation. Both of the baculovirus IAP repeats (BIRs) were required for SfIAP function. Moreover, deletion of the C-terminal RING motif generated a loss-of-function SfIAP that interacted and dominantly interfered with wild-type SfIAP. Like Op-IAP3, wild-type SfIAP formed intracellular homodimers, suggesting that oligomerization is a functional requirement for both cellular and viral IAPs. SfIAP possesses a ∼100-residue N-terminal leader domain, which is absent among all viral IAPs. Remarkably, deletion of the leader yielded a fully functional SfIAP with dramatically increased protein stability. Thus, the SfIAP leader contains an instability motif that may confer regulatory options for cellular IAPs that baculovirus IAPs have evolved to bypass for maximal stability and antiapoptotic potency. Our findings that SfIAP and viral IAPs have common motifs, share multiple biochemical properties including oligomerization, and act at the same step to block apoptosis support the hypothesis that baculoviral IAPs were derived by acquisition of host insect IAPs.Apoptosis is a prevalent host cell response to virus infection. Representing an important antivirus defense, apoptotic cell death can limit multiplication and virus dissemination in the host. Thus, the mechanisms by which a host organism detects a viral intruder and initiates the apoptotic response are critical to the outcome of the infection for both the host and virus. The cellular inhibitor-of-apoptosis (IAP) proteins are important candidates for sensing virus infection and determining cell fate by virtue of their central position in the apoptosis pathway (reviewed in references 35, 36, and 44). Affirming their importance in regulation of apoptosis, IAPs are encoded by multiple DNA viruses, including baculoviruses, entomopoxviruses, iridoviruses, and African swine fever virus (reviewed in 3). Nonetheless, the molecular mechanisms by which viral IAPs regulate virus-induced apoptosis and how they biochemically differ from cellular IAPs are poorly understood.The IAPs were first discovered in baculoviruses because of their capacity to prevent virus-induced apoptosis and thereby facilitate virus multiplication (4, 8). The baculovirus IAPs bear a striking resemblance to the cellular IAPs carried by the host insects that they infect. Cellular IAPs are a highly conserved family of survival factors that regulate developmental and stress-induced apoptosis, as well as inflammation, the cell cycle, and other signaling processes (35, 38, 44). Importantly, misregulation or overexpression of IAPs is associated with neoplasia and tumor chemoresistance (24, 49). The IAPs are defined by the presence of one or more ∼80-residue baculovirus IAP repeat (BIR) domains. The BIRs consist of a conserved Zn²⁺-coordinating arrangement of Cys and His residues (CCHC) that interact with diverse proteins, including the cysteinyl aspartate-specific proteases called caspases that execute apoptosis (reviewed in 16 and 37). The antiapoptotic activity of some, but not all, IAPs is derived from their ability to bind and neutralize caspases (reviewed in 35 and 44). The BIRs also interact with proapoptotic factors that contain IAP binding motifs (IBMs). IBM-containing factors have the capacity to bind and dissociate the IAP-caspase complex, thereby liberating active caspases to execute apoptosis (16, 35, 36, 48). Many IAPs, including viral IAPs, also possess a C-terminal RING domain, which is a Zn²⁺-coordinating motif with E3-ubiquitin ligase activity, which can contribute to antiapoptotic activity (48).The best-studied baculovirus IAP is Op-IAP3, which is encoded by Orgyia pseudotsugata nucleopolyhedrovirus. This small IAP (268 residues) contains two BIRs and a C-terminal RING (Fig. ​(Fig.1A).1A). Both BIRs are required for Op-IAP3 antiapoptotic activity (19, 50, 53). Truncation of the Op-IAP3 RING creates a loss-of-function dominant inhibitor (19). Op-IAP3''s capacity to form a complex with this RING-lacking (RINGless) dominant inhibitor and with itself suggests that oligomerization is necessary for IAP function. Upon overexpression, Op-IAP3 blocks apoptosis triggered by diverse signals in cells from certain insects and mammals, suggesting that it acts through a conserved mechanism (7, 11, 15, 33, 51, 54, 56). In the baculovirus host moth Spodoptera frugiperda (Lepidoptera: Noctuidae), Op-IAP3 prevents apoptosis by blocking the activation of effector caspases (25, 32, 40). However, in contrast to host insect IAPs, Op-IAP3 fails to inhibit active caspases (45, 51, 54). Thus, the host cell target(s) and the mechanism by which they are neutralized by this viral IAP remain unclear.Open in a separate windowFIG. 1.SfIAP structure and mutagenesis. (A) Viral and cellular IAPs. Viral Op-IAP3 (268 residues) and SfIAP (377 residues) each contain two BIR motifs (black boxes) and an E3 ligase RING domain (cross-hatched box). Each representing a potential start site, four methionines (M1 to M4) exist in the N-terminal leader of SfIAP. (B) SfIAP^M4 mutations. SfIAP^M4 (281 residues) begins with the M4 methionine. SfIAP^M4ΔR (227 residues) lacks the C-terminal RING. Amino acid substitutions of Zn-coordinating residues are indicated. An epitope tag (HA) was inserted at the N terminus. (C) Marker rescue assay. The antiapoptotic activity of wild-type or mutated forms of SfIAP^M4 was assayed by virus marker rescue in which replication of p35-deficient vΔp35/lacZ was restored in proportion to the antiapoptotic activity of the mutated Sfiap gene acquired by integration of the SfIAP-encoding plasmid (2). Virus yields were determined by plaque assay using apoptosis-sensitive SF21 cells. Antiapoptotic activity is reported as the ratio of nonapoptotic, lacZ-expressing plaques produced by transfection of the indicated Sfiap to those produced by wild-type Sfiap. Values shown are the averages ± standard deviations obtained from triplicate transfections.Among the cellular IAPs, SfIAP from Spodoptera frugiperda is most closely related to viral Op-IAP3. SfIAP (Fig. ​(Fig.1A)1A) is 42% identical to Op-IAP3, with a higher degree of amino acid identity localized to its two BIRs and C-terminal RING (20). As the principal IAP in Spodoptera, SfIAP suppresses a constitutive push toward apoptosis (34); ablation of SfIAP leads to immediate apoptosis of cultured Spodoptera cells. Upon overexpression, SfIAP also rescues the multiplication of apoptosis-inducing baculoviruses and can prevent apoptosis in certain mammalian cell lines (20, 26). In contrast to viral Op-IAP3, SfIAP can bind and inhibit caspases, including Spodoptera frugiperda caspase-1 (Sf-caspase-1) and human caspase-9 (20, 45). Thus, despite their structural similarities, there exist fundamental differences in the biochemical activities of these two IAPs. Importantly, SfIAP fails to prevent baculovirus-induced apoptosis when produced at endogenous levels in permissive Spodoptera cells. Thus, it is expected that SfIAP also possesses regulatory motifs that respond to cellular signals triggered upon virus infection.SfIAP provides an unprecedented opportunity to investigate the functional and evolutionary relationships between host and viral IAPs and to test the intriguing hypothesis that viral IAPs were acquired by host gene capture (21). We have investigated the biochemical properties of SfIAP as a means to define its molecular mechanisms and to test its relatedness to viral IAPs. We report here that SfIAP shares many biochemical and functional features with viral IAPs. Like Op-IAP3, overexpressed SfIAP prevented virus-induced apoptosis at a step upstream of caspase activation by a mechanism that required BIR1, BIR2, and the RING. SfIAP formed a complex with itself and with a RINGless dominant inhibitor, suggesting that oligomerization is also required for function of cellular IAPs. Unlike viral IAPs, SfIAP possesses an N-terminal leader, which modulates intracellular SfIAP levels and may respond to apoptotic signals to regulate cell survival. Our data are consistent with a model in which baculoviruses acquired a host cell IAP and modified it for virus-specific needs, thereby increasing virus fitness by preventing virus-induced apoptosis.     

Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

Molecules composed of β-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.Cryptococcus neoformans is the etiologic agent of cryptococcosis, a disease still characterized by high morbidity and mortality despite antifungal therapy (3). Pathogenic species belonging to the Cryptococcus genus also include Cryptococcus gattii, which causes disease mostly in immunocompetent individuals (24). A unique characteristic of Cryptococcus species is the presence of a polysaccharide capsule, which is essential for virulence (7-9, 19, 25, 33).C. neoformans has a complex cell surface. The thick fungal cell wall is composed of polysaccharides (29), pigments (11), lipids (35), and proteins (36). External to the cryptococcal cell wall, capsular polysaccharides form a capsule (19). Seemingly, the assembly of the surface envelope of C. neoformans requires the interaction of cell wall components with capsular elements. Some of the cryptococcal cell wall-capsule connectors have been identified, including the structural polysaccharide α-1,3-glucan and chitooligomers (29, 30, 32).Chitin-like molecules in fungi are polymerized by chitin synthases, which use cytoplasmic pools of UDP-GlcNAc (N-acetylglucosamine) to form β-1,4-linked oligosaccharides and large polymers. In C. neoformans, the final cellular site of chitin accumulation is the cell wall. The polysaccharide is also used for chitosan synthesis through enzymatic deacetylation (1). Eight putative cryptococcal chitin synthase genes and three regulator proteins have been identified (2). The chitin synthase Chs3 and regulator Csr2 may form a complex with chitin deacetylases for conversion of chitin to chitosan (1). Key early events in the synthesis of chitin/chitosan require the activity of glucosamine 6-phosphate synthase, which promotes the glutamine-dependent amination of fructose 6-phosphate to form glucosamine 6-phosphate, a substrate used for UDP-GlcNAc synthesis (23).In a previous study, we demonstrated that β-1,4-linked GlcNAc oligomers, which are specifically recognized by the wheat germ agglutinin (WGA), form bridge-like connections between the cell wall and the capsule of C. neoformans (32). In fact, other reports indicate that molecules composed of GlcNAc or its deacetylated derivative play key roles in C. neoformans structural biology. For example, mutations in the genes responsible for the expression of chitin synthase 3 or of the biosynthetic regulator Csr2p caused the loss of the ability to retain the virulence-related pigment melanin in the cell wall (1, 2). These cells were also defective in the synthesis of chitosan, which has also been demonstrated to regulate the retention of cell wall melanin (1). Treatment of C. neoformans acapsular mutants with chitinase affected the incorporation of capsular components into the cell wall (32). Considering that melanin and capsular components are crucial for virulence, these results strongly suggest that GlcNAc-derived molecules are key components of the C. neoformans cell surface. The expression of GlcNAc-containing molecules is likely to be modulated during infection since chitinase expression by host cells is induced during lung cryptococcosis (37).In this study, we used β-1,4-linked GlcNAc oligomers and an inhibitor of UDP-GlcNAc synthesis to evaluate the role played by GlcNAc-containing molecules in the surface architecture of C. neoformans. The results point to a direct relationship between the expression of GlcNAc-containing molecules and capsular assembly, indicating that chitin and chitooligomers are required for capsule organization in C. neoformans.     

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ΔBbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.Mycoinsecticides are considered promising biological control agents and alternatives or supplements to chemical pesticides (15). However, the dearth of physiological, genetic, and molecular knowledge of entomopathogenic fungi has retarded their widespread application.For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13, 14, 18, 20, 29, 49, 50, 51, 52, 53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle. M. anisopliae and B. bassiana produce hydrophobic spores which contact and adhere to the insect cuticle (12). Once attached, the conidium germinates and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via cuticle-degrading enzymes (11, 19, 46) as well as mechanical pressure (24, 53). Hyphae proliferate within the hemocoel, emerge from inside the insect, and subsequently conidiate on the cadaver (15). However, much remains to be elucidated regarding the mechanisms of insect fungal pathogenesis.To obtain detailed knowledge of the mechanisms of fungal pathogenesis, a pool of B. bassiana transfer DNA (T-DNA) insertional mutants had been generated through an Agrobacterium-mediated-transformation method (21). A mutant, designated T12, characterized by the presence of more conidia, was isolated, and its flanking sequence was obtained by T-DNA tagging. The flanking fragment contained an open reading frame (ORF), which corresponded to a gene termed JEN1, encoding a transporter of carboxylates (http://www.ncbi.nlm.nih.gov/Blast.cgi). Organic acid transportation is important for the metabolism of almost all cells of multicellular organisms and unicellular microorganisms (17, 25, 26). Transport across the plasma membrane is the first step in the metabolism of these substrates, which may affect many aspects of the organism, including regulation of energy metabolism (9, 34) and acid-base equilibrium status (10).JEN1p has been identified in several fungal species, e.g., Saccharomyces cerevisiae, Candida albicans, and Kluyveromyces lactis (9, 35, 45), which is a lactate/pyruvate symporter (1, 9, 34). The enzyme imports lactate or some short-chain monocarboxylates across the plasma membrane into cells. Then, the lactate is stereo-specifically oxidized to pyruvate. This reaction is performed by ferricytochrome c oxidoreductase in mitochondria (23, 33) and is tightly connected to the respiratory chain (34). JEN1 was induced by lactic, pyruvic, acetic, and propionic acids and repressed by glucose (2, 9, 35, 45). Nevertheless, for entomopathogenic fungi, the characterization of JEN1p has not been investigated, and its role in infection is still a mystery.For this paper, we studied the functions of a putative carboxylate transport gene, JEN1, in B. bassiana (BbJEN1). Our results demonstrated that BbJEN1 is involved in conidiation of B. bassiana and that the gene is a new factor influencing virulence in entomopathogenic fungi.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.Members of the family Baculoviridae are large rod-shaped enveloped viruses containing a circular double-stranded DNA genome that varies in size from 80 to 180 kb (3). Baculoviruses are unique viruses that only replicate in invertebrates. In general, isolates of each baculovirus species exhibit a narrow host range. For example, Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) is known to infect only the spruce budworm (Choristoneura fumiferana), but Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in hosts derived from several families of Lepidoptera (14). The restriction of baculovirus replication in nonpermissive hosts has been studied, and a number of genes, expressed at different points in the virus replication cycle, have been identified as playing some role in this restriction (40). Most of these identified genes are associated with viral DNA replication and late gene expression.Nine AcMNPV genes (ie-1, ie-2, p143, dnapol, lef-1, lef-2, lef-3, pe38, and p35) are required for directing transient replication of plasmids in transfected cells, suggesting that these genes are involved in baculovirus DNA replication (19, 27, 46). Only two of these genes, p143 and dnapol, have been shown to be essential for AcMNPV DNA replication in vivo (26, 41). Another gene, lef-11, although not essential for replication in transient assays, is also essential for DNA replication in vivo (24), indicating that questions concerning DNA replication need to be studied within the context of the whole virus genome.LEF-3 is a single-stranded DNA-binding protein (SSB) that self-localizes to the nucleus (15, 45). LEF-3 is also responsible for transporting P143, a predicted DNA unwinding (helicase) protein, into the nucleus, where it is required for viral DNA replication (26, 29, 45). LEF-3 may also regulate the activity of a viral alkaline nuclease (AN) during viral DNA replication (32). We have previously mapped the region carrying the nuclear localization signal of LEF-3 to residues 26 to 32 within the N-terminal 56-amino-acid domain (1, 7). By fusing this domain in frame with P143 and testing the construct in transient plasmid replication assays, we showed that additional functions of LEF-3 are required during replication, in addition to interacting with P143 to transport it into the nucleus. In fact, we have demonstrated that there is a close interaction between LEF-3 and P143 (as well as the immediate-early 1 [IE-1] protein) on viral DNA in the nucleus (17), suggesting that direct interaction of LEF-3 and P143 is required during viral DNA replication. The LEF-3 domain necessary for directing P143 to the nucleus is included within the N-terminal 125 amino acids (7). Two conserved cysteine residues in this region (C82 and C106) are not essential for this function, so it is unknown which specific amino acids are involved in the LEF-3-P143 interaction (1).In this study, a lef-3 knockout genome was constructed by exploiting a baculovirus shuttle vector (bacmid) system. Bacmids (a baculovirus genome carrying independent origins for replication in either bacteria or insect cells) were originally developed to prepare recombinant baculoviruses in Escherichia coli prior to transfection into insect cells (28). The system takes advantage of the site-specific transposition properties of the Tn7 transposon to simplify and enhance the process of generating recombinant bacmid DNA. In our case, we used the AcMNPV-derived bacmid as a template for deletion of the AcMNPV lef-3 gene and then examined the effect of this deletion on viral protein synthesis, budded virus (BV) production, and viral DNA replication. We also examined the ability of LEF-3 from another Alphabaculovirus species member, CfMNPV, to substitute for AcMNPV in a recombinant bacmid.     

Chitinases Are Essential for Sexual Development but Not Vegetative Growth in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic pathogen that mainly infects immunocompromised individuals. The fungal cell wall of C. neoformans is an excellent target for antifungal therapies since it is an essential organelle that provides cell structure and integrity. Importantly, it is needed for localization or attachment of known virulence factors, including melanin, phospholipase, and the polysaccharide capsule. The polysaccharide fraction of the cryptococcal cell wall is a complex structure composed of chitin, chitosan, and glucans. Chitin is an indispensable component of many fungal cell walls that contributes significantly to cell wall strength and integrity. Fungal cell walls are very dynamic, constantly changing during cell division and morphogenesis. Hydrolytic enzymes, such as chitinases, have been implicated in the maintenance of cell wall plasticity and separation of the mother and daughter cells at the bud neck during vegetative growth in yeast. In C. neoformans we identified four predicted endochitinases, CHI2, CHI21, CHI22, and CHI4, and a predicted exochitinase, hexosaminidase, HEX1. Enzymatic analysis indicated that Chi2, Chi22, and Hex1 actively degraded chitinoligomeric substrates. Chi2 and Hex1 activity was associated mostly with the cellular fraction, and Chi22 activity was more prominent in the supernatant. The enzymatic activity of Hex1 increased when grown in media containing only N-acetylglucosamine as a carbon source, suggesting that its activity may be inducible by chitin degradation products. Using a quadruple endochitinase deletion strain, we determined that the endochitinases do not affect the growth or morphology of C. neoformans during asexual reproduction. However, mating assays indicated that Chi2, Chi21, and Chi4 are each involved in sexual reproduction. In summary, the endochitinases were found to be dispensable for routine vegetative growth but not sexual reproduction.Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcosis in immunocompromised individuals. The incidence of cryptococcosis continues to rise in direct proportion to the spread of the human immunodeficiency virus (for review, see Casadevall and Perfect [7]). It is estimated that up to 13% of AIDS patients in the United States will develop life-threatening cryptococcal meningitis, and in some parts of Africa this estimate increases to 40% (7). Current antifungal therapies for treatment of cryptococcosis are inadequate. Amphotericin B, which is believed to interact with membrane sterols (ergosterol) to produce an aggregate that forms a transmembrane channel is effective, but toxic (50, 62). Fluconazole inhibits cytochrome P-450-dependent 14α-sterol demethylase, which leads to the depletion of ergosterol and the accumulation of sterol precursors and results in the formation of a plasma membrane with altered structure and function. It is fungistatic and has high relapse rates (18, 41, 42, 50, 62). Flucytosine can be toxic and resistance occurs frequently (9, 41, 42, 50, 62). The newest class of antifungals to emerge is the echinocandins that targets an essential fungal enzyme required for the synthesis of a β-(1,3)-glucan in the fungal cell wall (17, 34). In addition, the echinocandins have been shown to be safe and effective for treatment of specific fungal infections, including candidiasis and aspergillosis caused by Candida albicans and Aspergillus fumigatus, respectively (23, 59). However, even though C. neoformans possesses the target enzyme β-(1,3)-glucan synthase and in vitro assays have shown the enzyme''s activity to be inhibited by the echinocandin caspofungin (34), C. neoformans still exhibits resistance to this class of drugs (26).Because fungi are eukaryotes and share many biochemical processes with their host, antifungal drug design has been problematic. The cell wall is a prominent structure that differentiates fungi from mammalian host cells. For all fungi, this organelle is essential and provides structure as well as integrity; thus, the cell wall components or their biosynthetic pathways make attractive drug targets. In addition, the cell wall of C. neoformans is associated with a variety of known virulence factors that are important for host-pathogen interactions, and it contains polymers including chitin and chitosan that are necessary for the viability of C. neoformans. The first virulence factor that a host cell encounters is the polysaccharide capsule. The capsule attachment to the outer portion of the cell wall requires α-(1-3)-glucan (15, 46). Another cell wall associated virulence factor is the melanin pigment (61) that is produced by two laccase proteins, Lac1 and Lac2 (38, 44). Lac1 is responsible for generating the majority of melanin and is localized to the cell wall (38, 63, 69). Chitin and chitosan are essential components of the cell wall that have been shown to contribute to the overall strength and integrity of the cell wall (4, 5). The essentiality of the chitin component and the lack of it being present in host cells make chitin and its biosynthetic components attractive targets for drug design.Chitin is one of the most abundant polymers found in nature (1, 12). It is a linear polymer of β-(1,4)-linked N-acetylglucosamine (GlcNAc), and in fungi it is formed from cytoplasmic pools of UDP-GlcNAc. C. neoformans has eight predicted chitin synthases and three putative chitin synthase regulators for synthesis of chitin polymers. Mutational analysis indicate that two chitin synthases, Chs4 and Chs5, produce the majority of vegetative chitin, and one, Chs3, produces the majority of chitin that is converted to chitosan during vegetative growth (5). Chitosan, the deacetylated version of chitin, is produced by chitin deacetylases (EC 3.5.1.41) that remove acetyl groups from nascent chitin polymers. In C. neoformans the chitin produced by Chs3 and the chitin synthase regulator, Csr2, is deacetylated to chitosan by up to three chitin deacetylases (Cda1, Cda2, and Cda3) (4, 5). Strains of C. neoformans lacking either CHS3 or CSR2 have significantly reduced chitosan levels and are sensitive to a variety of cell wall inhibitors (5). Similarly, strains lacking all three chitin deacetylases are unable to convert chitin to chitosan and are sensitive to cell wall inhibitors (4). This indicates that chitosan is essential for the proper maintenance of cell wall integrity in C. neoformans and Chs3, Csr2, and the chitin deacetylases contribute to its formation (4, 5). Chitosan polymers of other fungi have been reported to possess various degrees of deacetylation (57). Chitin and chitosan are located throughout the lateral cell wall and bud neck regions of C. neoformans (4). During growth cellular chitin and chitosan need to be continuously remodeled, presumably through the enzymatic digestion of chitin and chitosan polymers by chitinases and or chitosanases.Chitinases (EC 3.2.1.14) are enzymes that hydrolyze the β-(1-4) linkages in polymers of chitin. Besides being in fungi, these enzymes occur in a wide variety of organisms, including viruses, bacteria, plants, and animals (1, 12). There are two major categories of chitinases: endochitinases and exochitinases. Generally, the endochitinases cleave chitin chains internally to generate low-molecular-mass multimers of GlcNAc. In contrast, the exochitinases are divided into two subcategories: chitobiosidases (EC 3.2.1.29) release diacetylchitobiose from the nonreducing end of chitin chains, and β-(1,4)-N-acetylhexosaminidases (EC 3.2.1.52) release GlcNAc from the nonreducing end of chitin oligosaccharides; both types are usually processive (12). Fungal chitosanases (EC 3.2.1.132) are less understood. They have been found in Aspergillus spp. and Gongronella sp. strain JG. Although these chitosanases have been shown to degrade chitosan, their in vitro physiological relevance has not been elucidated (8, 60).In other fungal systems chitinases are known to be involved in cell separation, hyphal growth and branching, development of reproductive structures, spore germination, and autolysis (1, 12). In the nonpathogenic model yeast Saccharomyces cerevisiae two chitinases, Cts1p and Cts2p, function independently in bud separation and spore formation, respectively (25, 27). Cts1p is the only chitinase expressed during vegetative growth, and strains lacking this enzyme display incomplete cell separation (27) that can lead to pseudohyphalike growth (25). The synthesis of the spore wall is adversely affected by the deletion of CTS2 and affects the ability of the yeast to form mature asci (19).C. neoformans reproduces predominantly by budding, but also has a defined sexual cycle that culminates in the production of basidiospores. Both the yeast and the spore forms are thought to be infectious particles (7). C. neoformans typically colonizes the lungs of a immunocompromised host, from where it can disseminate to the central nervous system (7). As such, reproduction by budding has been shown to occur within host macrophages and dendritic cells (3, 28). Because fungal chitinases in other systems such as S. cerevisiae and C. albicans have been shown to be necessary for the completion of cell division (11, 27), understanding the biosynthesis and activity of chitinases could determine whether interfering with chitinase activity would impair the ability of C. neoformans to reproduce.We hypothesized that the chitinases in C. neoformans would be involved in growth and, like the chitinases in S. cerevisiae and C. albicans, that they would degrade specific chitin during either bud separation, hyphal growth, or sporulation. In the present study we utilized a homology-based search to identify five potential chitinases in C. neoformans, the four endochitinases CHI2, CHI21, CHI22, and CHI4 and one exochitinase, HEX1. Using a panel of chitinase deletion strains we discovered that the chitinases are dispensable for “normal” vegetative growth but were necessary during development of the sexual phase of C. neoformans.     

Levels of the Secreted Vibrio cholerae Attachment Factor GbpA Are Modulated by Quorum-Sensing-Induced Proteolysis

Vibrio cholerae is the etiologic agent of cholera in humans. Intestinal colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of V. cholerae. HapR represses the expression of genes encoding regulators involved in V. cholerae virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, ΔhapA ΔprtV strains attach to chitin beads more efficiently than either the WT or a ΔhapA ΔprtV ΔgbpA strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues.Vibrio cholerae has adapted to lifestyles in dual environments, allowing survival in aquatic locations, as well as the ability to colonize the epithelium of the human small intestine. This intestinal colonization by V. cholerae is a prerequisite for the disease cholera in humans. Intestinal colonization proceeds in a stepwise manner, initiating with attachment to the epithelial cell layer by multiple attachment factors (26). This stable attachment localizes the bacterium in an environment conducive for activation of subsequent virulence factors, including the toxin-coregulated pilus, a type IVb pilus that mediates cell-cell interactions and microcolony formation (27). Cholera toxin (CT) is produced and extracellularly secreted by bacteria within the microcolonies and enters into intestinal epithelial cells. CT causes the disruption of fluid and electrolyte balance and results in the voluminous rice water diarrhea characteristically observed with cholera patients.The ability of V. cholerae to bind to surfaces is crucial for the initial stages of colonization of both the aquatic and intestinal environments. Previous studies observing V. cholerae in the aquatic setting identified the ability of the bacteria to attach to zooplankton and phytoplankton, binding to surface structures that include chitin as a major component (7, 10, 11, 19, 21, 42). Chitin, a polymer consisting primarily of a β-1,4 linkage of GlcNAc monomers, is the most abundant aquatic carbon source and, when presented on the surfaces of zooplankton, aquatic exoskeletons, algae, and plants, provides a substrate for V. cholerae surface binding (8, 19-22). V. cholerae is able to break down chitin into carbon to use as a nutrient source via degradation by secreted chitinases (12). We have described a protein, GbpA (GlcNAc binding protein A), which facilitates the binding of V. cholerae to chitin, specifically to the chitin monomer GlcNAc, a sugar residue that is also found on the surface of epithelial cells (3, 16, 26). GbpA mediates binding to chitin, GlcNAc, and exoskeletons of Daphnia magna, as well as participates in effective intestinal colonization within the infant mouse model of cholera (26). GbpA is a secreted protein that exits the cell via the type 2 secretion system by which it mediates attachment by a yet uncharacterized mechanism (26). Previous studies examining the role of GbpA in binding to surfaces have been conducted utilizing various wild-type (WT) strains of V. cholerae, specifically O395 (26) and N16961 (33). These strains both are of the O1 serogroup but are differentially classified as classical (43) and El Tor biotypes (18), respectively. The classical biotype was responsible for the first six pandemics of cholera, whereas El Tor is the cause of the current pandemic (39).Quorum sensing regulates multiple bacterial processes, including virulence, formation of biofilms, and bioluminescence (25, 35, 36). In contrast to many other bacterial quorum-sensing systems, virulence gene expression and biofilm formation in V. cholerae is expressed under conditions of low cell density and repressed at high cell density (17, 35, 48). HapR, a member of the TetR family of regulatory proteins, is a central regulator on which the three parallel inputs of the V. cholerae quorum-sensing system converge (30, 35). During low-cell-density conditions, characteristic of growth within the aquatic environment or stages of early intestinal colonization, the quorum-sensing system is not engaged. Under conditions of high cell density, bacterial numbers and secreted autoinducer molecules are increased to a level that triggers the V. cholerae quorum-sensing system.HapR regulates gene function in two ways, serving as both an activator and repressor. At high cell density, HapR functions in the capacity of a repressor of the toxin-coregulated pilus and CT virulence cascade (29, 31) as well as a repressor of vps gene expression (17), preventing biofilm formation. In addition to repressing gene expression, at high cell density HapR activates the expression of genes encoding extracellularly secreted proteases HapA and PrtV (14, 17, 23, 45-47). HapA, also referred to as hemagglutinin/protease (HA/P), was first reported as a mucinase by Burnet (6) and later characterized as a zinc- and calcium-dependent metalloprotease (4). Extracellularly secreted via the V. cholerae type 2 secretion pathway (40), HA/P has been demonstrated to cleave fibronectin, lactoferrin, and mucin (15), as well as to participate in the activation of the CT A subunit (5). Further studies have led to the suggestion that HA/P is a detachase, critical for the release of V. cholerae from the surface of intestinal cells (2, 14, 38). PrtV is a second protease encoded by a gene that is activated by HapR (47). It has been demonstrated to be essential for both V. cholerae killing of Caenorhabditis elegans, as well as protecting V. cholerae from predator grazing by various flagellates (32, 45).The data presented here indicate that HapA and PrtV participate in the targeted degradation of the attachment factor GbpA. We demonstrate that GbpA is present during the logarithmic phase of growth and conditions of low cell density but that it is not present in the supernatant of high-cell-density cultures of strains that express functional HapR. Further studies revealed that during stages of high cell density, proteases HapA and PrtV, encoded by HapR-activated genes, are responsible for GbpA degradation in the culture supernatant. These findings suggest that the attachment factor GbpA is potentially a ligand targeted for protease degradation during the epithelial detachment process. This process could aid in the release of V. cholerae back into the aquatic environment following late stages of intestinal colonization.     

Bacillus thuringiensis Bel Protein Enhances the Toxicity of Cry1Ac Protein to Helicoverpa armigera Larvae by Degrading Insect Intestinal Mucin

Bacillus thuringiensis has been used as a bioinsecticide to control agricultural insects. Bacillus cereus group genomes were found to have a Bacillus enhancin-like (bel) gene, encoding a peptide with 20 to 30% identity to viral enhancin protein, which can enhance viral infection by degradation of the peritrophic matrix (PM) of the insect midgut. In this study, the bel gene was found to have an activity similar to that of the viral enhancin gene. A bel knockout mutant was constructed by using a plasmid-free B. thuringiensis derivative, BMB171. The 50% lethal concentrations of this mutant plus the cry1Ac insecticidal protein gene were about 5.8-fold higher than those of the BMB171 strain. When purified Bel was mixed with the Cry1Ac protein and fed to Helicoverpa armigera larvae, 3 μg/ml Cry1Ac alone induced 34.2% mortality. Meanwhile, the mortality rate rose to 74.4% when the same amount of Cry1Ac was mixed with 0.8 μg/ml of Bel. Microscopic observation showed a significant disruption detected on the midgut PM of H. armigera larvae after they were fed Bel. In vitro degradation assays showed that Bel digested the intestinal mucin (IIM) of Trichoplusia ni and H. armigera larvae to various degrading products, similar to findings for viral enhancin. These results imply Bel toxicity enhancement depends on the destruction of midgut PM and IIM, similar to the case with viral enhancin. This discovery showed that Bel has the potential to enhance insecticidal activity of B. thuringiensis-based biopesticides and transgenic crops.Bacillus thuringiensis is a ubiquitous gram-positive, spore-forming soil bacterium and produces insecticidal crystal proteins during the sporulation phase of its growth cycle. Because these insecticidal crystal proteins have activity against certain insect species, B. thuringiensis has been extensively used as a biopesticide to control crop pests in commercial agriculture and forest management. It is also a key source of genes for transgenic expression and provides pest resistance in plants (2, 20, 30).The viral enhancin protein was originally described for granuloviruses (GVs) as a 126-kDa protein that showed an ability to enhance the infectivity of nucleopolyhedroviruses (NPVs) (36, 37, 39). It has also been found in several other GVs (13) and NPVs (19, 27). Considered a pathogenicity factor, it is not essential for growth of viruses in cell culture or infected insects but has the function of facilitating GV and NPV infection and decreasing larval survival time (14, 17, 19, 27).The widely accepted action mode of the viral enhancin protein, which has been identified as a metalloprotease (17), is that it can disrupt the protective peritrophic matrix (PM), allowing virion access to the underlying epithelial cells of the insect gut (17). The PM has a lattice structure formed by chitin and insect intestinal mucin (IIM), and the viral enhancin protein targets the IIM for degradation (33).Enhancin-like genes with 24 to 25% nucleotide identity to viral enhancin genes have been found in Yersinia pestis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus genome sequences (16, 25, 28). When B. cereus enhancin-like protein was expressed in recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded viruses and polyhedral inclusion bodies, it was found to be cytotoxic compared to viral enhancin protein. However, larval bioassays indicated that this enhancin-like protein did not enhance infection (8). Hajaij-Ellouze et al. (12) isolated a B. thuringiensis enhancin-like gene from a 407 crystal-minus strain and found that this enhancin-like protein has a typical metalloprotease zinc-binding domain (HEIAH) and belongs to the PlcR regulon. When the enhancin-like mutant was fed to Galleria mellonella larvae, no significant reduction in virulence was observed.In the present study, we report a B. thuringiensis enhancin-like gene (bel) encoding a protein (Bel) that has 20 to 30% identity to the viral enhancin protein and 95% identity to bacterial enhancin-like proteins. Therefore, Bel function may have a synergistic action similar to that of the virus enhancin protein. To understand the biochemical activity of this novel bacterial gene, bel was knocked out in the plasmid-free strain BMB171. We expected that this bel mutant would have no significant reduction in toxicity according to the reports of Galloway et al. (8) and Hajaij-Ellouze et al. (12). However, the bel mutant surprisingly resulted in dramatically reduced Cry1Ac toxicity to Helicoverpa armigera larvae. To further confirm this result, purified Bel was fed together with the Cry1Ac protein to H. armigera larvae. We found that Bel can function as a synergist of Cry1Ac toxicity against H. armigera. In vivo and in vitro observations showed that Bel can disrupt the insect midgut PM and degrade IIM of insect midgut PM. The target of Bel is the IIM of PM, similar to the results found with viral enhancin.     

OpnS,an Outer Membrane Porin of Xenorhabdus nematophila,Confers a Competitive Advantage for Growth in the Insect Host

The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.The bacterium Xenorhabdus nematophila forms a mutualistic association with the entomopathogenic nematode Steinernema carpocapsae (2). The nonfeeding infective juvenile form of the nematode (IJ) exists in the soil and carries the bacteria in a specialized receptacle region in the anterior intestine (4, 39). The IJ invades susceptible insect species and enters the hemocoel, where exposure to insect hemolymph stimulates the movement of bacteria down the intestine and out of the anus (36, 39). Together, the nematode and bacteria kill the insect host. X. nematophila not only helps to kill the insect but also promotes bioconversion of host macromolecules and tissues to provide nutrients for nematode reproduction and secretes diverse antimicrobial products to suppress competition for the nutrient resources of the insect cadaver (11, 13, 18, 19, 38). In turn, the nematode vectors X. nematophila to new insect hosts and protects it from the competitive environment of the soil. Colonization of the nematode receptacle is predominantly a monoculture process that is initiated by a single cell followed by bacterial proliferation (24, 39). The level of colonization varies from a few cells to several hundreds per nematode and is higher in nematodes reproducing in insects than on bacterial lawns, suggesting that the insect environment provides additional nutrients for bacterial growth (16, 39).Hydrophilic nutrients and antibiotics passively diffuse across the outer membrane of gram-negative bacteria through general porins and substrate-specific channels (17, 29). The most extensively studied general porins, OmpF and OmpC of Escherichia coli (30), are 16-stranded β-barrel proteins that are reciprocally regulated by changes in external osmolarity (12, 21, 41). Although the flow rate through OmpF is greater than OmpC (28), comparison of the resolved crystal structures does not reveal significant physiochemical differences between the two porins (3). The biological significance of the differential regulation of porins with distinct functional properties remains unclear. The major outer membrane protein of X. nematophila, OpnP, was shown to be produced at high levels in exponentially growing cells and is a homologue of OmpF and OmpC (14). OpnP production was not affected by changes in medium osmolarity, and the flow rate measured for the OpnP porin was more similar to the restrictive porin OmpC than to the more permissive OmpF porin (3). As cells transitioned to stationary phase, de novo synthesis of OpnP decreased, while the synthesis of the outer membrane protein, designated OpnS, increased (15, 22).Porin function and regulation have been studied in both pathogenic and symbiotic bacteria. In Vibrio cholerae two well-studied porins, OmpU and OmpT, that possess distinct functional properties have been shown to be differentially regulated (37). OmpU confers resistance to sodium deoxycholate (DC), a major component of bile, as well as polymixin B, detergents, and antimicrobial peptides, while the expression of OmpT alone sensitizes the cell to DC (26, 33). OmpU was thought to be expressed when V. cholerae colonizes the intestine, suggesting that it was required for host colonization (33); however, subsequent findings indicated that neither OmpU nor OmpT were essential for intestinal colonization (34). Recent findings indicated that OmpU may sense membrane perturbations and activate DegS which in turn modulates σ^E activity (25, 26). In the symbiotic bacterium Vibrio fischeri the deletion of ompU was shown to reduce the efficiency of colonization of the light organ of the Euprymna scolopes squid and increase sensitivity to bile, antimicrobial peptides, and detergent (1). Interestingly, the ompU strain did not display a competitive defect for colonization in the presence of the wild-type strain.In the present study the growth-phase-regulated outer membrane protein OpnS of X. nematophila was identified as a general porin that conferred a competitive advantage for growth in the insect host. OpnP and OpnS were the only general porins identified in the genome of X. nematophila. The reciprocal expression of OpnP and OpnS suggest that they serve distinct biological roles.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Partial Functional Rescue of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Infectivity by Replacement of F Protein with GP64 from Autographa californica Multicapsid Nucleopolyhedrovirus

Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64-null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64-rescued group II NPV of Helicoverpa armigera (vHaBacΔF-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBacΔF-gp64 compared to the HaF rescue control virus vHaBacΔF-HaF. Electron microscopy further showed that most vHaBacΔF-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBacΔF-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.The envelope fusion protein (EFP) of budded viruses (BV) (30) of baculoviruses is critical for virus entry (attachment and fusion) and egress (assembly and budding) (7, 13, 21). Two types of BV EFPs have been identified in the Baculoviridae family of viruses. The F proteins are similar in structure, but they are very diverse in their amino acid sequences (20 to 40% identity). They are widespread within the baculovirus family (group II NPVs of the alphabaculoviruses and in beta- and deltabaculoviruses) (23) and are thought to be carried by ancestral members (26). In contrast, the baculovirus GP64 homologs are all closely related EFPs (>74% sequence identity) and found only in group I NPVs of the alphabaculoviruses (23). It has been suggested that a gp64 gene was acquired relatively recently by an ancestral virus of the group II NPV, thereby giving these viruses a selective advantage and obviating the need of the envelope fusion function of the F protein (23). A nonfusogenic F homolog (F-like protein), however, is maintained in the genome of group I NPVs, functioning as a virulence factor (9, 17, 24, 32).GP64 and F proteins play similar roles during the baculovirus infection processes, such as virus-cell receptor attachment, membrane fusion, and efficient budding. However, there are striking differences between the receptor usage of GP64 and F proteins as well. These two types of proteins are very different in structure, mode of action, and receptor exploitation. The crystal structure reveals that GP64 belongs to class III viral fusion proteins, with its fusion loop located in the internal region of the protein, and proteolytic cleavage is not required for activation of fusion activity (10). F proteins by contrast share common features of class I viral fusion proteins (12). The proteolytic cleavage of the F precursor (F₀) by a furin-like protease generates an N-terminal F₂ fragment and a C-teminal F₁ fragment. This cleavage is essential for exposing the N-terminal fusion peptide of F₁ and for activating F fusogenicity (8, 36). Although the nature of the baculovirus host cell receptors is still enigmatic, it has been reported that Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV)) and Orgyia pseudotsugata MNPV (OpMNPV), both using GP64 as their EFPs, exploit the same insect cell receptor, while Lymantria dispar MNPV (LdMNPV) with an F protein as the EFP utilizes a cell receptor different from that used by AcMNPV (7, 37). Additionally, in the case of SeMNPV, using competition assays, it was confirmed that the baculovirus F protein and GP64 recognized distinct receptors to gain entry into cultured insect cells (34).Pseudotyping viral nucleocapsid with heterologous EFPs to form pseudotype virions is a valuable approach to studying the structure, function, and specificity of heterologous EFPs. It has been a successful strategy to expand or alter viral host range, i.e., in gene delivery (3). For example, vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus and AcMNPV gp64-pseudotyped HIV-1 exhibit high virus titers and wider tropism (5, 14, 38); the gp64-pseudotyped human respiratory syncytial virus (HRSV) lacking its own glycoproteins is of high and stable infectivity (22); furthermore, pseudotyped lentiviruses with modified fusion proteins of GP64 with targeting peptides (i.e., hepatitis B virus PreS1 peptide, involved in viral attachment) or with the decay accelerating factor (DAF) facilitate the targeting to specific cell types or confer stability against serum inactivation, respectively (6, 19). For the Baculoviridae, a series of pseudotyping studies have investigated the functional analogy between GP64 and F proteins. F proteins of group II NPVs (SeMNPV, LdMNPV, and Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus [HearNPV]) can substitute for GP64 in gp64-null AcMNPV viruses (15, 16). Recent studies indicated that many granulovirus (GV) F proteins, but not F protein from Plutella xylostella GV (PxGV), can rescue a gp64-null AcMNPV (16, 39). These results demonstrated that baculovirus F proteins are functional analogues to GP64. Since it was postulated that GP64 was captured by a baculovirus during evolution (24), one would expect the functional incorporation of GP64 into the BV of an F-null group II NPV. However, the reverse substitution of a group II NPV (SeMNPV) F protein by GP64 failed to produce infectious progeny viruses (35).In this paper, we show that AcMNPV gp64 could be inserted into an F-null HearNPV genome and produce infectious progeny virus upon transfection of insect cells. The infectivity of the pseudotyped virus, however, was greatly impaired, and large amounts of morphologically defective BV were produced. Bioassay experiments indicated that the infectivity of GP64-pseudotyped F-null HearNPV for insect larvae was not reduced, but that the time to death was significantly delayed. These results demonstrate that GP64 alone can only partially complement HearNPV F protein function.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Class III Chitin Synthase ChsB of Aspergillus nidulans Localizes at the Sites of Polarized Cell Wall Synthesis and Is Required for Conidial Development

Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.Chitin, a polymer of β-1,4-linked N-acetylglucosmine, is one of the major structural components of the fungal cell wall. Its metabolism, including synthesis, degradation, assembly, and cross-linking to other cell wall components, is thought to be very important for many fungi (5, 22, 24, 36, 45). Fungal chitin synthases have been classified into seven groups, classes I to VII, depending on the structures of their conserved regions (6). The genes encoding the synthases belonging to classes III, V, VI, and VII are only found in fungi with high chitin contents in their cell walls. We have identified six chitin synthase genes from Aspergillus nidulans and designated them chsA, chsB, chsC, chsD, csmA, and csmB; these gene products belong to classes II, III, I, IV, V, and VI, respectively (9, 13, 30, 31, 44, 52). The chsB deletion mutant grew very slowly and formed small colonies with highly branched hyphae, suggesting its important role in hyphal tip growth (3, 52). Repression of chsB expression in the deletion mutant of chsA, chsC, or chsD exaggerated the defects in the formation of aerial hyphae, the production of cell mass, or the growth under high-osmolarity conditions, respectively, compared to each single mutant. These results indicate that chsB functions at various stages of development (15, 16).The deletion of class III chitin synthase-encoding genes leads to severe defects in most of the filamentous fungi thus far investigated. However, their detailed functions are currently unknown. In Neurospora crassa, inactivation of the gene encoding Chs-1, a class III chitin synthase with 63% identity to A. nidulans ChsB, leads to slow growth, aberrant hyphal morphology, and a decrease in chitin synthase activity. The mutant of chs-1 became sensitive to Nikkomycin Z, a chitin synthase inhibitor (53). In Aspergillus fumigatus, two genes encoding class III chitin synthases, chsC and chsG, have been identified. Their gene products showed 66 and 89% identity, respectively, to A. nidulans ChsB. The chsG deletion mutant showed slow growth and defects in conidiation, and its hyphae were highly branched. chsC deletion did not cause any phenotypic change. The chsC chsG double deletion mutant showed almost the same phenotype as the chsG single deletion mutant (28). Class III chitin synthases have been reported to be involved in the virulence of some pathogens. Deletion of Bcchs3a in the phytopathogenic fungus Botrytis cinerea and double deletion of WdCHS3 and class I chitin synthase WdCHS2 in the human pathogen Wangiella dermatitidis both caused a reduction of virulence (40, 48). On the other hand, the deletion mutant of a class III chitin synthase-encoding gene, CgChsIII, of the maize pathogen Colletotrichum graminicola did not exhibit the significant phenotypic difference from the wild-type strain (50). Deletion of a gene, chs1, encoding a class III chitin synthase of the maize pathogenic dimorphic fungi Ustilago maydis caused minor defects in the growth of haploid yeastlike cells and conjugation tube formation (49). These results indicate that the functions of class III chitin synthases has evolutionally diverged.In the present study, we characterized the cytological defects of the A. nidulans chsB deletion mutant and investigated the localization of ChsB using FLAG- or enhanced green fluorescent protein (EGFP)-tagged ChsB. We reveal that the deletion mutant formed hyphae with aberrant cell wall structures and that ChsB tagged with EGFP primarily localized at polarized growth sites during germination, hyphal growth, septation, and conidiation. These findings suggest that ChsB functions at the polarized growth sites and forming septa during the hyphal growth and conidia development.     

Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV^CAR) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV^CAR to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV^CAR-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV^CARGFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV^CAR-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV^CAR to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV^CAR-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV^CAR in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV^CAR-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV^CAR-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV^CAR-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV^CAR-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.Adenoviruses (Ads) are extensively used today as gene transfer vectors for in vitro, ex vivo, and in vivo gene transfer protocols (reviewed in reference 65). Cell entry of human Ad type 5 (Ad5), the serotype most widely used as a gene vector, occurs most efficiently by the receptor-mediated endocytosis pathway (reviewed in references 64 and 65), via the coxsackievirus B-adenovirus receptor (CAR) (3, 77) and αvβ3/αvβ5 integrins (84, 85), although alternative receptors have been described (11, 12, 14, 27). Cell surface expression of CAR differs with different cell types, and this represents one of the major determinants of the efficiency of Ad5-mediated transduction (43). The ubiquitous nature of CAR is responsible for transduction of nontarget tissues by Ad vectors. Paradoxically, many target cells such as dermal fibroblasts, synoviocytes, mesenchymal stem cells (MSCs), peripheral blood mononuclear cells (PBMCs), and dendritic cells (DCs), express no or very low levels of CAR at their surface and are relatively resistant to Ad transduction (14, 15, 19). Much work has been done with different strategies to promote the entry of Ad5 into CAR-defective cells. These strategies include (i) the genetic modification of Ad capsid proteins to carry cell ligands (2, 15, 20, 28, 49, 50), (ii) pseudotyping Ad5 vectors with fibers from other serotypes (13, 57, 74, 86), (iii) using bispecific adapters or peptides (25, 40), (iv) chemical modification of Ad (9, 42), and (v) tethering on nanoparticles (7). The limitations to these strategies are that modifications of the Ad capsid are susceptible to negatively affecting the virus growth or viability, due to an alteration of virion assembly, stability, the viral uncoating process, and/or intracellular trafficking (13, 51).Other viruses which are gaining popularity as gene transfer vectors are the baculoviruses (BVs). Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) is an insect virus with a large double-stranded DNA genome packaged in a membrane-enveloped, rod-shaped protein capsid (70). Since the 1980s, the BV-insect cell expression system has been highly exploited for the production of recombinant proteins. In the mid-1990s, it was shown that recombinant BVs carrying reporter genes under cytomegalovirus (CMV) or retroviral Rous sarcoma virus promoter efficiently expressed reporter genes in mammalian cells (6, 22, 38, 41, 44, 69), as well as in avian cells (72) and fish cells (45). Since then, BVs have been reported to transduce numerous cells originating from species as various as humans, bovines, and fish (8, 32, 41, 73). As gene transfer vectors, BVs have been found to be rapidly inactivated by human serum complement (23), but exposing decay-accelerating factor (DAF) at the surface of BV by fusion with the baculoviral envelope glycoprotein can overcome this inactivation (33). BVs also have a good biosafety profile due to their incapacity to replicate in mammalian cells (31).Taking advantage of the ability of BVs to transduce a large repertoire of cells of invertebrate and vertebrate origins, including human primary cells, we investigated whether a recombinant AcMNPV could act as a carrier or macroadapter for Ad5 vectors to enter Ad5-refractory cells. To this aim, we pseudotyped AcMNPV virions with the high-affinity receptor for Ad5, the human CAR glycoprotein (BV^CAR), to enable the formation of complexes between vector particles of BV^CAR and Ad5-green fluorescent protein (Ad5GFP) mediated by Ad5 fiber and CAR interaction. We found that transduction of cell lines which were poorly permissive to Ad5, including human cancer cells and primary cells, was significantly improved using this strategy of piggybacking Ad5 vector on BV^CAR. More importantly, the increase in BV^CAR-Ad5-mediated transduction was obtained with a low range of Ad5 inputs, i.e., at multiplicities of infection (MOI) of less than 50 Ad5 vector particles per cell. We also found that the cell transduction enhancement observed with BV^CAR-Ad5 required the direct coupling of Ad5 to BV^CAR via fiber-CAR binding and that the cell attachment of the complex was mediated by the baculoviral envelope glycoprotein gp64. Kinetic analysis of virus-cell binding showed that the presence of BV^CAR in the complex was beneficial to Ad5 vector, not only in terms of tropism but also in terms of number of cell-bound virions and rate of cell attachment. In addition, the endocytic pathway of BV^CAR-Ad5 did not require Ad5 penton base RGD-integrin interaction. When administered in vivo to nude BALB/c mice, BV^CAR-Ad5 complex showed the same biodistribution as that of control Ad5 vector injected alone. In vitro, transduction of CAR-negative cells by BV^CAR-Ad5 was insensitive to coagulation factor X (FX), in contrast to Ad5 vector alone.Our novel strategy of gene delivery using the BV^CAR-Ad5 duo could be advantageously applied to various situations in vitro or ex vivo, e.g., for transducing Ad5-refractory cells when Ad5 capsid modifications cannot be envisaged, when oncolytic Ads need to be delivered to tumors via nonpermissive cell carriers belonging to the immune system, or when the simultaneous delivery of two transgenes by two separate vectors might be beneficial in terms of timing and/or level of cellular expression of the transgene products.