A Pseudomonas aeruginosa Type VI Secretion Phospholipase D Effector Targets Both Prokaryotic and Eukaryotic Cells

1. Download : Download high-res image (192KB)
2. Download : Download full-size image

     

Salmonella Type III Secretion Effector SlrP Is an E3 Ubiquitin Ligase for Mammalian Thioredoxin

Salmonella enterica encodes two virulence-related type III secretion systems in Salmonella pathogenicity islands 1 and 2, respectively. These systems mediate the translocation of protein effectors into the eukaryotic host cell, where they alter cell signaling and manipulate host cell functions. However, the precise role of most effectors remains unknown. Using a genetic screen, we identified the small, reduction/oxidation-regulatory protein thioredoxin as a mammalian binding partner of the Salmonella effector SlrP. The interaction was confirmed by affinity chromatography and coimmunoprecipitation. In vitro, SlrP was able to mediate ubiquitination of ubiquitin and thioredoxin. A Cys residue conserved in other effectors of the same family that also possess E3 ubiquitin ligase activity was essential for this catalytic function. Stable expression of SlrP in HeLa cells resulted in a significant decrease of thioredoxin activity and in an increase of cell death. The physiological significance of these results was strengthened by the finding that Salmonella was able to trigger cell death and inhibit thioredoxin activity in HeLa cells several hours post-infection. This study assigns a functional role to the Salmonella effector SlrP as a binding partner and an E3 ubiquitin ligase for mammalian thioredoxin.Protein secretion is a basic function in all groups of bacteria. Many secretion systems have been found in Gram-negative bacteria, from the relatively simple type I secretion systems to the complex type III or type IV machines or the recently described type VI systems (reviewed in Refs. 1 and 2). Many pathogenic or symbiotic Gram-negative bacteria rely on type III secretion systems (T3SS)² for their interaction with host organisms. The T3SS is a protein export pathway that spans the inner membrane, periplasmic space, outer membrane, and host cell membrane. These complex structures are related to flagella and consist of at least 20 different subunits that enable the bacteria to translocate substrates into the cytosol of the eukaryotic host cell (reviewed in Ref. 3). These systems have also been referred to as injectisomes or molecular needles (4).Proteins secreted and translocated into eukaryotic cells through T3SS are called “effectors.” These effectors have the ability to suppress host defense signaling. Effectors of plant pathogens may target salicylic acid- and abscisic acid-dependent defenses, host vesicle trafficking, or interfere with host RNA metabolism. Effectors from animal pathogens modify the cytoskeleton to facilitate bacterial entry, modulate Rho GTPases and NF-κB, inhibit the host inflammatory response, elicit death of immune cells, and disrupt both adaptative and innate immune responses (reviewed in Ref. 5).Salmonella enterica produces two distinct T3SS essential for virulence that are encoded by genes located in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), respectively. The SPI-1 T3SS secretes at least 16 proteins: AvrA, GogB, SipA, SipB, SipC, SipD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SptP, SspH1, SteA, and SteB (6–8). Six of them have been shown to regulate actin cytoskeleton dynamics (reviewed in Ref. 9). 19 SPI-2 T3SS effectors have been identified: GogB, PipB, PipB2, SifA, SifB, SopD2, SseF, SlrP, SseG, SseI/SrfH, SseJ, SseK1, SseK2, SseL, SspH1, SspH2, SteA, SteB, and SteC. However, the biochemical functions of most of them remain unknown (reviewed in Ref. 10).The conventional paradigm, supported by in vivo and in vitro studies, is that the SPI-1-encoded T3SS is required for the invasion of M cells and cultured epithelial cells (11, 12) as well as for the inflammatory response of the intestinal cells, and that the SPI-2-encoded T3SS is essential for replication and survival within macrophages and the progression of a systemic infection (13). Recent evidence suggests that the boundaries between SPI-1 and SPI-2 function are not sharply defined: some SPI-1 effectors are detected hours or days after infection and SPI-2-encoded genes may be expressed before penetration of the intestinal epithelium. In addition, as can be noticed comparing the lists of effectors above, some effectors, including SlrP, can be secreted by both T3SS.SlrP (for Salmonella leucine-rich repeat protein) was identified as a S. enterica serovar Typhimurium host range factor by signature-tagged mutagenesis (14). A mutant in this gene has no difference in virulence with the wild-type strain when infecting calves but is 6-fold attenuated for mouse virulence after oral infection. This gene is located in a 2.9-kb DNA region with features of horizontal acquisition and has similarity to yopM from Yersinia spp. and ipaH from Shigella flexneri. The predicted protein contains 10 copies of a leucine-rich repeat signature, a protein motif frequently involved in protein-protein interactions. Other members of the leucine-rich repeat family in Salmonella are the effectors SspH1 and SspH2, which share 39 and 38% amino acid identity with SlrP, respectively. Similarity in the amino-terminal region of these three proteins helped to define a translocation signal that was also found in four other T3SS effectors: SifA, SifB, SseI, and SseJ (15). Although SlrP can be delivered into mammalian cells by both T3SS, its expression seems to be induced by RtsA, one of the main regulators of SPI-1, independently of HilA or InvF (16).Although the function of SlrP was completely unknown, the presence of leucine-rich repeats in this protein suggested that it may bind eukaryotic proteins during infection. In addition, recent reports have shown an enzymatic activity, E3 ubiquitin ligase, for effectors of the same family (17, 18).In this work we demonstrate that SlrP interacts with mammalian thioredoxin-1 (Trx). We also show that SlrP is an E3 ubiquitin ligase that can use Trx as a substrate. Our results support a model in which interaction of SlrP with Trx leads to a decrease in thioredoxin activity and triggers host cell death.     

Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.     

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.     

A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.     

IcsA Is a Shigella flexneri Adhesin Regulated by the Type III Secretion System and Required for Pathogenesis

1. Download : Download high-res image (196KB)
2. Download : Download full-size image

     

The Shigella flexneri Type 3 Secretion System Is Required for Tyrosine Kinase-Dependent Protrusion Resolution,and Vacuole Escape during Bacterial Dissemination

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.     

Random Mutagenesis Identifies a C-Terminal Region of YopD Important for Yersinia Type III Secretion Function

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168–203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.     

Cag3 Is a Novel Essential Component of the Helicobacter pylori Cag Type IV Secretion System Outer Membrane Subcomplex

Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein.Helicobacter pylori infects 50% of the world population. Stomach infection with this bacterium is associated with the development of several gastric diseases, including chronic active gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. Factors influencing disease outcomes are not completely understood, but bacterial, host, and environmental factors have been identified that affect the dynamics of this bacterium-host interaction (30). A hallmark of H. pylori infection is the induction of mucosal inflammation, which is a risk factor for developing more severe pathology (27).Epidemiological studies have established that infection with strains harboring the cag pathogenicity island (PAI) leads to a higher risk for development of severe disease (27). The cag PAI size varies between 35 and 40 kb and encodes 27 putative proteins (1, 13). Several of the encoded proteins share sequence similarities with components of the prototypical type IV secretion (T4S) system VirB/D4 of Agrobacterium tumefaciens (15, 16). Based on research done in A. tumefaciens, the components of the molecular machinery have been divided into channel or core complex components (VirB6, VirB7, VirB8, VirB9, and VirB10), energetic components (VirB11, VirB4, and VirD4), and extracellular appendage components (VirB2 and VirB5). VirB6, VirB8, and VirB10 are components anchored at the inner membrane with domains spanning the periplasm, while VirB7 and VirB9 are located at the outer membrane. Energetic components are located at the inner membrane, and pilus components include the main subunit VirB2 and accessory components, such as VirB5, which functions as an adhesin (15, 16). The VirB/D4 T4S is thought to be energized by the inner membrane ATPases, and this energy is transduced to VirB10 and the outer membrane complex for protein translocation (11). The lipoprotein VirB7 is critical for the stability of HpVirB9 at the outer membrane (19).While the extent of homology of the H. pylori cag T4S components is often limited, sequence analysis has allowed the identification of the VirB11 (HP0525 and HpVirB11), VirB10 (HP0527 and HpVirB10), VirB9 (HP0528 and HpVirB9), and VirD4 (HP0524 and HpVirD4) homologues as summarized in Table S1 of the supplemental material (1, 13, 28). HpVirB9 and HpVirB10 homologies are not distributed along the entire length of the protein. For example, HpVirB10 is a very large protein with only a short domain similar to VirB10. HpVirB10 is also reported to localize on the external surface of the pilus (31), while VirB10 is tethered in the inner membrane. HP0529 (HpVirB6) and HP0530 (HpVirB8) have been assigned as homologs of VirB6 and VirB8, respectively (28). HP0523 (HpVirB1) has lytic transglycosylase activity, supporting its designation as a VirB1 homolog (38). HP0532 (HpVirB7) has a lipoprotein attachment site, suggesting a role as a VirB7 homolog (1, 28), and has been suggested to stabilize a Cag T4S outer membrane subcomplex containing CagM, HpVirB9, and HpVirB10 (28).The activity of the cag PAI-encoded T4S system is responsible for the translocation of the effector protein CagA and induction of proinflammatory chemokine and cytokine secretion, including the chemokine interleukin-8 (IL-8) (7). CagA T4S-mediated translocation into host cells is followed by tyrosine phosphorylation on specific tyrosine phosphorylation motifs (EPIYA motifs) at the C-terminal region of the protein and both phosphorylation-dependent and -independent interference with host cellular pathways. The induction of proinflammatory chemokine production is mediated by a still-uncharacterized Cag T4S-mediated delivery of peptidoglycan into host cells and subsequent activation of Nod receptors (37), and it has also been reported that CagA itself has proinflammatory properties (9). The molecular mechanisms responsible for Cag T4S system assembly and activity remain unclear.Null alleles of the genes with homology to T4S components (HpVirB11, HpVirB4, HpVirB6, HpVirB7, HpVirB8, HpVirB9, and HpVirB10) abolish both CagA translocation and IL-8 induction, with the exception of HpVirD4, which affects CagA translocation but not IL-8 induction (20). Other genes of the island also essential for Cag T4S function do not share sequence or structural homology with known T4S components. More detailed analysis of these Cag T4S essential genes allowed the recent assignment of several proteins as functional homologs of additional VirB components. HP0546 was suggested as a VirB2 homolog, the main subunit of other T4S system pili (3). Ultrastructural work suggested that HpVirB10 is also a major subunit of the Cag T4S system pilus (31, 35), but clear evidence that either HpVirB2 or HpVirB10 is the main pilus subunit is still lacking. CagL (HP0539) has been identified (29) as an adhesin (functionally similar to VirB5) whose binding to host cell receptors is required for activation of the secretion process, and CagF (HP0543) has been characterized as a CagA chaperone (17). CagD (HP0545) has been recently reported as a multifunctional Cag T4S component essential for CagA translocation and full IL-8 secretion induction (12).We have characterized the biochemical role of an additional essential H. pylori-specific gene, HP0522/cag3, in Cag T4S. A previous yeast two-hybrid screen that investigated interactions among cag PAI proteins suggested Cag3 could interact with HpVirB8, HpVirB7, CagM (HP0537), and CagG (HP0542) (10). To begin to understand the molecular basis of Cag3 function in T4S we investigated the subcellular localization of the Cag3 protein and the protein-protein interactions this protein establishes in H. pylori cells. We found evidence suggesting that Cag3 is an integral part of the Cag T4S outer membrane subcomplex required to maintain HpVirB7 levels.     

A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion

ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA (NpABC1) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense.     

A Conserved Domain in the Coronavirus Membrane Protein Tail Is Important for Virus Assembly

Coronavirus membrane (M) proteins play key roles in virus assembly, through M-M, M-spike (S), and M-nucleocapsid (N) protein interactions. The M carboxy-terminal endodomain contains a conserved domain (CD) following the third transmembrane (TM) domain. The importance of the CD (SWWSFNPETNNL) in mouse hepatitis virus was investigated with a panel of mutant proteins, using genetic analysis and transient-expression assays. A charge reversal for negatively charged E₁₂₁ was not tolerated. Lysine (K) and arginine (R) substitutions were replaced in recovered viruses by neutrally charged glutamine (Q) and leucine (L), respectively, after only one passage. E₁₂₁Q and E₁₂₁L M proteins were capable of forming virus-like particles (VLPs) when coexpressed with E, whereas E₁₂₁R and E₁₂₁K proteins were not. Alanine substitutions for the first four or the last four residues resulted in viruses with significantly crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. All recovered viruses with alanine substitutions in place of SWWS residues had second-site, partially compensating, changes in the first TM of M. Alanine substitution for proline had little impact on the virus. N protein coexpression with some M mutants increased VLP production. The results overall suggest that the CD is important for formation of the viral envelope by helping mediate fundamental M-M interactions and that the presence of the N protein may help stabilize M complexes during virus assembly.Coronaviruses are widespread, medically important respiratory and enteric pathogens of humans and a wide range of animals. New human coronaviruses (HCoV), including severe acute respiratory syndrome CoV (SARS-CoV), HCoV-NL63, and HCoV-HKU1, were recently identified (40, 47). The potential for emergence of other new viruses and the zoonotic nature of some coronaviruses strongly warrants understanding old and new viruses. Understanding vital interactions that take place during virus assembly and conserved domains (CDs) that mediate these interactions can provide insight toward identification of targets for development of antiviral therapeutics and vaccines.Coronaviruses are enveloped positive-stranded RNA viruses that belong to the Coronaviridae family in the Nidovirales order. The virion envelope contains at least three structural proteins, the membrane (M), spike (S), and envelope (E) proteins. The genomic RNA is encapsidated by the N phosphoprotein to form a helical nucleocapsid. The S glycoprotein is the viral attachment protein that facilitates infection through fusion of viral and cellular membranes and is the major target of neutralizing antibodies (13). The M glycoprotein is the most abundant component of the viral envelope and plays required, key roles in virus assembly (9, 20, 31, 33, 41). The E protein is a minor component of the viral envelope that plays an important, not clearly defined, role(s) during virus assembly and release (2, 5, 41).Coronavirus M proteins are divergent in their amino acid content, but all share the same overall basic structural characteristics. The proteins have three TM domains, flanked by a short amino-terminal glycosylated domain and a long carboxy-terminal tail located outside and inside the virion, respectively (14) (Fig. ​(Fig.11 A). M localizes in the Golgi region when expressed alone (20, 22). M molecules interact with each other and also with the spike and nucleocapsid during virus assembly (8-10, 23, 31, 33). M-M interactions constitute the overall scaffold for the viral envelope. The S protein and a small number of E molecules are interspersed in the M protein lattice in mature virions. Previous studies from a number of labs implicated multiple M domains and residues as being important for coronavirus assembly (6, 8, 9, 17, 43). Coronaviruses assemble and bud at intracellular membranes in the region of the endoplasmic reticulum (ER) Golgi intermediate compartment (ERGIC) (22, 39). Coexpression of only the M and the E proteins is sufficient for virus-like particle (VLP) assembly for most coronaviruses (2, 41).Open in a separate windowFIG. 1.M protein conserved domain and mutants. (A) A linear schematic of the M protein illustrating the relative positions of the three TM domains (black boxes) and the position of the CD in the tail. (B) Alignment of CDs from representative coronaviruses. Full-length amino acid sequences from transmissible gastroenteritis virus (TGEV), feline coronavirus (FeCoV), human coronavirus 229E, human coronavirus NL63, mouse hepatitis virus (MHV), bovine coronavirus (BCoV), human coronavirus OC43, porcine hemagglutinating encephalomyelitis virus (HEV), human coronavirus HKU1, SARS-CoV, infectious bronchitis virus (IBV), and turkey coronavirus (TCoV) were aligned by using CLUSTAL W (25). (C) Mutations introduced into the MHV CD, with + and − symbols used to indicate VLP production and virus recovery for each mutant.The long intravirion (cytoplasmic) tail of M consists of an amphipathic domain following the third TM and a short hydrophilic region at the carboxyl end of the tail (Fig. ​(Fig.1A).1A). The amphipathic domain appears to be closely associated with the membrane (34). At the amino terminus of the amphipathic domain, there is a highly conserved 12-amino-acid domain (SWWSFNPETNNL), consisting of residues 114 to 125 in the mouse hepatitis virus (MHV) A59 M protein (Fig. ​(Fig.1B)1B) (19). These residues are almost identically conserved across the entire Coronaviridae family. Because of the crucial role that M plays in virus assembly and the high conservation of this domain, we hypothesized that it is functionally important for virus assembly. To test this, a series of changes were introduced in the CD. The functional impact of the changes was studied in the context of the virus by genetic analysis and the ability of the mutant M proteins to participate in VLP assembly. The results show that the CD is functionally important for M protein to participate in virus assembly. The domain may help mediate important lateral interactions between M molecules. The results suggest that the N protein helps stabilize M complexes during virus assembly.     

A Conserved Tryptophan-Rich Motif in the Membrane-Proximal Region of the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Is Important for Env-Mediated Fusion and Virus Infectivity

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an α-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.     

Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts

Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+, Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 and pmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.     

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.     

Essential Role of the SycP Chaperone in Type III Secretion of the YspP Effector

The Ysa type III secretion (T3S) system enhances gastrointestinal infection by Yersinia enterocolitica bv. 1B. One effector protein targeted into host cells is YspP, a protein tyrosine phosphatase. It was determined in this study that the secretion of YspP requires a chaperone, SycP. Genetic analysis showed that deletion of sycP completely abolished the secretion of YspP without affecting the secretion of other Ysps by the Ysa T3S system. Analysis of the secretion and translocation signals of YspP defined the first 73 amino acids to form the minimal region of YspP necessary to promote secretion and translocation by the Ysa T3S system. Function of the YspP secretion/translocation signals was dependent on SycP. Curiously, when YspP was constitutively expressed in Y. enterocolitica bv. 1B, it was recognized and secreted by the Ysc T3S system and the flagellar T3S system. In these cases, the first 21 amino acids were sufficient to promote secretion, and while SycP did enhance secretion, it was not essential. However, neither the Ysc T3S system nor the flagellar T3S system translocated YspP into mammalian cells. This supports a model where SycP confers secretion/translocation specificities for YspP by the Ysa T3S system. A series of biochemical approaches further established that SycP specifically interacts with YspP and protected YspP degradation in the cell prior to secretion. Collectively, the evidence suggests that YspP secretion by the Ysa T3S system is a posttranslational event.Many gram-negative bacteria have evolved sophisticated delivery systems termed type III secretion (T3S) systems to transport effector proteins into the cytosols of eukaryotic host cells (10, 21, 22). The translocated effectors manipulate host cell activities in various ways, thereby permitting the establishment of a pathogenic or symbiotic interaction (20). T3S systems are ancestrally related to the flagellar T3S system, having in common a basal body spanning the inner and outer bacterial membranes responsible for the appropriate selection of polypeptides delivered into a hollow channel leading out of the bacterium. At the outer surface, flagellar polypeptides travel the length of the adjoining hook and filament, but in T3S systems, the secreted polypeptides pass through a special hollow needle that extends away from the bacterium to the targeted host cell (10, 21, 22). Heterologous multimeric proteins localized to the tip of the needle form the translocon, a porelike channel that is assembled in the eukaryotic plasma membrane, enabling the injection of bacterial effectors (24, 48, 51).Two terminologies are distinctly used to describe protein transport by T3S systems. While “secretion” is a transport event for proteins from the bacterial cytosol into the extracellular milieu, “translocation” is a transport event for proteins from the bacterial cytosol into the eukaryotic host''s cytosol. Generally, secretion but not translocation is mediated by the first 20 amino acids of effector proteins (41, 46, 47), albeit mRNA sequences at the N terminus of some proteins have been also considered to function as the secretion signals (3, 44). This secretion event is independent of the presence of cognate effector chaperones (46, 59). Despite no conservation of the amino acids among the secretion signals, amphipathic or disordered secondary structures of the peptides are thought to function as the secretion signals recognized by the T3S apparatuses (22, 34, 35). In contrast, translocation usually requires both the secretion (the first 20 amino acids) and the translocation (amino acids 20 to 100) signals (46, 47, 59). This translocation event is efficiently mediated by the presence of the cognate chaperones (9, 14, 30), and the chaperone-effector complexes have been proposed to function as the three-dimensional signals recognized by the T3S apparatuses (5, 33, 38, 49, 50).Many T3S effectors employ cognate chaperones in the bacterial cytoplasm (43, 57). The effector chaperones have been categorized into two subgroups, class 1A and class 1B, primarily based on the substrate properties (and the gene locations) (13, 43). Class 1A chaperones commonly bind to one effector, and most of them are encoded by genes located adjacent to the gene encoding the cognate effectors. In contrast, class 1B chaperones bind to multiple effectors and are encoded by genes located within operons that code for structural components of the T3S apparatus that are distant to the cognate effector genes. Evolutionally, this subgroup of chaperones is thought to be an archetype of effector chaperones. Although T3S effector chaperones lack primary sequence similarity even in same subgroup, overall the effector chaperones whose three-dimensional structures are solved share similar folds, consisting of three α-helices and five β-strands (5, 36, 38, 49, 54). Similarly, effector chaperones share the common biochemical characteristics of acidic properties (pI 4 to 5) and low molecular masses (12 to 15 kDa), with a tendency to form homodimers (43). These homodimers recognize the chaperone binding domains (CBD) of the cognate effectors, which are usually located in the amino-terminal 20 to 100 amino acids (translocation signal) of the effector (19, 30, 59). Despite the wealth of information about individual chaperones, a universally accepted model for the mechanisms by which they promote secretion is lacking. One study shows that the guidance of chaperone-effector complexes toward the T3S apparatus is provided by the affinity of their chaperones to the ATPase of the T3S apparatus, whereby the ATPase releases the chaperones from the complexes and then unfolds the cognate effector for secretion (2). Several additional functions of T3S effector chaperones have been reported, including the prevention of effector aggregation prior to delivery to the secretion system, limitation of premature interactions, and protection of effectors from protease degradation in bacterial cells (17, 43). When an organism has multiple T3S pathways, as is the case for some Yersinia spp., there is the opportunity to gain new insight into how a given chaperone might influence T3S system specificity for substrates. Without direct testing of the aforementioned mechanistic models, the role of a chaperone in T3S and how it affects the overall sequence of pathogenic events is, at best, a conjecture.Highly virulent strains of Yersinia enterocolitica bv. 1B have a total of three T3S systems. The first T3S system (Ysc) is encoded by the virulence plasmid, and it secretes six effectors termed Yops. Ysc T3S is important for systemic infection (11, 12, 42). This T3S system is common to all Yersinia species pathogenic to humans, including another enteropathogen, Yersinia pseudotuberculosis, and the plague pathogen Yersinia pestis. The second system (Ysa) is encoded by a cluster of genes mapping to the Ysa pathogenicity island (25, 53). The Ysa T3S system secretes a set of eight effectors termed Ysps and, interestingly, also secretes three Yops, YopE, YopN, and YopP/YopJ (39, 58, 61). This Ysa T3S system is restricted to clinical isolates of Y. enterocolitica bv. 1B and promotes the initial establishment of infection in gastrointestinal tissue (39, 55). The third T3S system is an integral part of the flagellum and secretes proteins termed Fops to the extracellular milieu (64).Previously, we identified the suite of Ysp proteins secreted by the Ysa T3S system (39). However, little is known about the detailed mechanism by which these proteins are secreted and translocated by this system. Among the Ysp proteins identified, YspP is a protein tyrosine phosphatase (PTPase) whose activity is required for full virulence (39). Here, we found a small open reading frame (ORF) immediately downstream of yspP and designated it sycP. The SycP protein was demonstrated to be a YspP-specific chaperone essential for both the secretion and the translocation of YspP by the Ysa T3S system. In addition, we also examined the secretion specificity requirements for YspP secretion by three different T3S systems as model cases. Interestingly, our data suggest that the mechanisms by which the secretion and translocation signals are recognized are different, depending on the type of T3S system examined.     

Epstein-Barr Virus BARF1 Protein Is Dispensable for B-Cell Transformation and Inhibits Alpha Interferon Secretion from Mononuclear Cells

The Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus.