Antibody-Mediated Immobilization of Cryptococcus neoformans Promotes Biofilm Formation

Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.Cryptococcus neoformans is a fungal pathogen that is ubiquitous in the environment and enters the body via the inhalation of airborne particles. The C. neoformans cell is surrounded by a layer of polysaccharide that consists predominantly of glucuronoxylomannan (GXM), which forms a protective capsule around the microbe. The capsule has been shown to be essential for virulence in murine models of infection (5-7) and, thus, is considered a key virulence factor. C. neoformans is the causative agent of cryptococcosis, a disease that primarily affects individuals with impaired immune systems, and is a significant problem in AIDS patients (21, 31). The most common manifestation of cryptococcosis is meningoencephalitis.Biofilms are communities of microbes that are attached to surfaces and held together by an extracellular matrix, often consisting predominantly of polysaccharides (8, 10). A great deal is known about bacterial biofilms (3, 9, 24, 30), but fungal biofilm formation is much less studied. Candida albicans is known to synthesize biofilms (11, 28, 29), as is C. neoformans. Biofilm-like structures consisting of innumerable cryptococcal cells encased in a polysaccharide matrix have been reported in human cases of cryptococcosis (32). Biofilm formation confers upon the microbe the capacity for drug resistance, and microbial cells in biofilms are less susceptible to host defense mechanisms (2, 4, 9, 12). In this regard, cells within C. neoformans biofilms are significantly less susceptible to caspofungin and amphotericin B than are planktonic cells (19). The cells within the biofilm are also resistant to the actions of fluconazole and voriconazole and various microbial oxidants and peptides (17, 19).Bacterial and fungal biofilms form readily on prosthetic materials, which poses a tremendous risk of chronic infection (10, 13, 15, 27). C. neoformans biofilms can form on a range of surfaces, including glass, polystyrene, and polyvinyl, and material devices, such as catheters (16). C. neoformans can form biofilms on the ventriculoatrial shunts used to decompress intracerebral pressure in patients with cryptococcal meningoencephalitis (32).The polysaccharide capsule of C. neoformans is essential for biofilm formation (18), and biofilm formation involves the shedding and accumulation of large amounts of GXM into the biofilm extracellular matrix (16). Previously, we reported that antibody to GXM in solution could inhibit biofilm formation through a process that presumably involves interference with polysaccharide shedding (18, 20). However, the effect of antibody-mediated immobilization of C. neoformans cells on cryptococcal biofilm formation has not been explored. In this paper, we report that the monoclonal antibody (MAb) 18B7, which is specific for the capsular polysaccharide GXM, can capture and immobilize C. neoformans to surfaces, a process that promotes biofilm formation. Interestingly, we identified planktonic variant C. neoformans cells that appeared to escape from the biofilm, but whose functions are not known. The results provide new insights on biofilm formation.     

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.     

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.     

Isolation and Characterization of Senescent Cryptococcus neoformans and Implications for Phenotypic Switching and Pathogenesis in Chronic Cryptococcosis

Although several virulence factors and associated genes have been identified, the mechanisms that allow Cryptococcus neoformans to adapt during chronic infection and to persist in immunocompromised hosts remain poorly understood. Characterization of senescent cells of C. neoformans demonstrated that these cells exhibit a significantly enlarged cell body and capsule but still cross the blood-brain barrier. C. neoformans cells with advanced generational age are also more resistant to phagocytosis and killing by antifungals, which could promote their selection during chronic disease in humans. Senescent cells of RC-2, a C. neoformans strain that undergoes phenotypic switching, manifest switching rates up to 11-fold higher than those of younger cells. Infection experiments with labeled cells suggest that senescent yeast cells can potentially accumulate in vivo. Mathematical modeling incorporating different switching rates demonstrates how increased switching rates promote the emergence of hypervirulent mucoid variants during chronic infection. Our findings introduce the intriguing concept that senescence in eukaryotic pathogens could be a mechanism of microevolution that may promote pathoadaptation and facilitate evasion of an evolving immune response.Cryptococcus neoformans is an encapsulated yeast that causes chronic meningoencephalitis predominantly in patients with advanced human immunodeficiency virus infection. Worldwide, this disease is estimated to cause more than 600,000 deaths per year (22). This high death rate may result in part from the fact that this chronic infection is notoriously difficult to eradicate, despite effective antifungal therapy. Even in successfully treated patients with access to antiretroviral therapy, the organism persists and can cause relapse both before and after the start of highly active antiretroviral treatment.In murine infection models, C. neoformans strain RC-2 can augment virulence by undergoing phenotypic switching from a smooth (SM) parent colony to a more virulent mucoid (MC) colony variant (6). Phenotypic switching occurs in both species and varieties of this fungus (6, 8, 11). Although differentially regulated genes associated with switching have been described (10), the precise molecular mechanism that controls phenotypic switching in C. neoformans is unknown. In vitro investigations have demonstrated that phenotypic switching occurs at a stable rate and MC switch variants spontaneously arise at a rate of 1 in about 20,000 plated SM colonies. Environmental signals that induce or alter this process have not been identified to date. Phenotypic switching occurs during chronic murine infection and alters the outcome (6), but it is noteworthy that MC switch variants consistently emerge late in the course of infection (after 6 weeks), although the fungal burden is highest on day 14 after intratracheal (i.t.) infection. This could reflect altering selection pressure by an evolving host response or, alternatively, a change in the microbe''s propensity to undergo phenotypic switching over time. In Candida albicans, the process of phenotypic switching is controlled by pathways that are involved in silencing and maintenance of genomic stability (14, 20, 34, 35). Furthermore, in the context of research on aging in Saccharomyces cerevisiae, it has become evident that senescent yeast cells (old cells with advanced generational age) exhibit less genomic stability (17, 18). C. neoformans is also a unicellular haploid yeast that replicates clonally in vivo. Consequently, we reasoned that it was conceivable that replicative aging of C. neoformans during chronic infection could alter genomic stability and its propensity to undergo phenotypic switching. We therefore investigated the effect of generational aging on phenotypic switching in C. neoformans and also compared it to C. albicans.     

A Putative P-Type ATPase,Apt1, Is Involved in Stress Tolerance and Virulence in Cryptococcus neoformans

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of Cryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the APT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of the APT1 gene resulted in phenotypes consistent with similar roles in C. neoformans. These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and the apt1 mutant strains at either 30°C or 37°C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, and apt1 mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response in C. neoformans through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised individuals (44). One million cases of cryptococcosis are estimated to occur each year, and approximately two-thirds of these are fatal (43). Key virulence traits for the fungus include growth at the mammalian host temperature, production of a polysaccharide capsule, deposition of laccase-synthesized melanin in the cell wall, secretion of enzymes, and resistance to host defenses, such as oxidative and nitrosative killing (44).The polysaccharide capsule is a key virulence factor and is both cell associated and released during infection (4). The two species of polysaccharide in the capsule, an abundant glucuronoxylomannan (GXM) and a minor galactoxylomannan (GalXM), cause a number of deleterious effects in mammalian hosts (4, 44). Extracellular vesicles (exosomes) containing capsule polysaccharide are present in culture supernatants, in lysates of macrophages containing C. neoformans, and in association with fungal cells during murine infection (41, 49, 50, 54). These so-called “virulence factor delivery bags” are thought to pass through the cell wall to deliver material outside the cell (50). Proteomic analysis of the vesicles identified 76 proteins, and many of these are associated with virulence, including urease, laccase, heat shock proteins, superoxide dismutase, thiol-specific antioxidants, and catalases (49).The mechanisms of trafficking of capsule polysaccharide and laccase are being actively pursued. For example, analysis of a mutant with a defect in the exocyst GTPase Sec4/Rab8 (designated Sav1) revealed the accumulation of intracellular vesicles containing capsule polysaccharide, thus providing support for intracellular synthesis and secretion via exocytosis (60). In addition, reduced expression of the exocyst protein Sec6 due to RNA interference (RNAi) resulted in partial attenuation of virulence as well as defects in melanin production and the export of urease and soluble capsule polysaccharide (42). The RNAi strains were also completely defective in the production of extracellular exosomes but retained wild-type (WT) levels of cell-associated capsule. Trafficking of the laccase required for melanin production and virulence has also been examined. Hu et al. (25) showed that C. neoformans lacking Vps34 (vacuolar protein sorting 34) had a marked reduction in melanin formation, suggesting that laccase-containing vesicles are derived from the endocytic pathway. Overall, the current evidence suggests that exocytic, endocytic, and specialized extracellular vesicles mediate the export of capsule and other virulence factors in C. neoformans (42, 49, 60).We demonstrated previously that vesicle trafficking functions in C. neoformans are regulated by the cAMP signal transduction pathway, which also controls the elaboration of both the capsule and melanin (28). We found that treatment of C. neoformans with inhibitors of Golgi apparatus-mediated transport (e.g., brefeldin A or monensin) or with lithium chloride results in inhibition of capsule expression (28). In addition, we found that cAMP-dependent protein kinase regulated the expression of a predicted phospatidylethanolamine binding protein, Ova1, which negatively influences capsule and melanin formation. These findings focused our attention on the roles of intracellular trafficking functions and phospholipids in virulence factor expression.In the context of phospholipid trafficking, some aminophospholipid translocases within the P-type ATPases are known to play roles in fungal virulence. For example, the aminophospholipid translocase MgApt2 is required for exocytosis during plant infection by the rice blast pathogen Magnaporthe grisea (18). P-type ATPases are a large family of multitransmembrane domain, ATP-dependent transporters, and three subfamilies are found in eukaryotes (29): (i) heavy metal ion ATPases (e.g., copper transporters), (ii) non-heavy-metal ion ATPases (e.g., Ca²⁺, H⁺, Na⁺, and K⁺ ATPases), and (iii) aminophospholipid translocases (APTs/flippases of the type IV or Drs2 family). APTs maintain the asymmetrical distribution of aminophospholipids in membranes by translocating phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) from one leaflet of the bilayer to the other. Phospholipid asymmetry is important in membrane fusion events (vesicle budding and docking) at the plasma membrane and in the trans-Golgi network (3). Thus, APTs are required for efficient Golgi function and play roles in both endocytosis and exocytosis. Some disorders in humans have been linked or attributed to genes from the APT subfamily, including familial intrahepatic cholestasis and Angelman syndrome (32, 55).Previously, we constructed a deletion of the APT1 gene, encoding a putative aminophospholipid translocase, as part of a study to examine disomy at chromosome 13 in C. neoformans (27). Our preliminary phenotypic analysis suggested a connection to nitrosative stress and prompted further investigation of virulence-related functions. In the present study, we show that Apt1 is functionally related to Drs2 in Saccharomyces cerevisiae and has roles in membrane trafficking and sensitivity to stress (oxidative and nitrosative) and drugs targeting ergosterol biosynthesis and secretion. Importantly, loss of Apt1 does not influence capsule and melanin formation, but the protein is required for intracellular growth in macrophages and for full virulence in mice.     

The Genome of the Amoeba Symbiont “Candidatus Amoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria

Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “Candidatus Amoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.Free-living amoebae, such as Acanthamoeba spp., are ubiquitous protozoa which can be found in such diverse habitats as soil, marine water, and freshwater and in many engineered environments (62, 100). They are important predators of prokaryotic and eukaryotic microorganisms, thereby having great influence on microbial community composition, soil mineralization, plant growth, and nutrient cycles (14, 100). Interestingly, many well-known pathogens of humans are able to infect, survive, and multiply within amoebae (39, 51). These protozoa can thus serve as reservoirs and vectors for the transmission of pathogenic bacteria to humans, as demonstrated for L. pneumophila and Mycobacterium avium (2, 115). It is also increasingly being recognized that protozoa are important for the adaptation of (pathogenic) bacteria to higher eukaryotic cells as a niche for growth (2, 24, 42, 78, 89).In addition to the many recognized transient associations between amoeba and pathogens, stable and obligate relationships between bacteria and amoebae also were described for members of the Alphaproteobacteria (11, 34, 48), the Betaproteobacteria (49), the Bacteroidetes (50), and the Chlamydiae (4, 12, 35, 52). These obligate amoeba symbionts show a worldwide distribution, since phylogenetically highly similar strains were found in amoeba isolates from geographically distant sources (51, 107). The phylogenetic diversity and the different lifestyles of these obligate intracellular bacteria—some are located directly in the host cell cytoplasm (11, 34, 48-50, 52), while others are enclosed in host-derived vacuoles (4, 35, 44)—suggest fundamentally different mechanisms of host cell interaction. However, with the exception of chlamydia-related amoeba symbionts (37, 46, 47), our knowledge of the biology of obligate intracellular symbionts of amoebae is still scarce.Comparative genomics has been extremely helpful for the analysis of intracellular bacteria. Numerous genome sequences from the Alpha- and Gammaproteobacteria and the Chlamydiae are available and have contributed significantly to our understanding of genome evolution, the biology of intracellular bacteria, and the interactions with their host cells (24, 26, 46, 79, 82). In this study, we determined and analyzed the complete genome sequence of “Candidatus Amoebophilus asiaticus” strain 5a2 in order to gain novel insights into its biology. “Ca. Amoebophilus asiaticus” is a Gram-negative, obligate intracellular amoeba symbiont belonging to the Bacteroidetes which has been discovered within an amoeba isolated from lake sediment (107). “Ca. Amoebophilus asiaticus” shows highest 16S rRNA similarity to “Candidatus Cardinium hertigii,” an obligate intracellular parasite of arthropods able to manipulate the reproduction of its hosts (131). According to 16S rRNA trees, both organisms are members of a monophyletic group within the phylogenetically diverse phylum Bacteroidetes, consisting only of symbionts and sequences which were directly retrieved from corals (113). Among members of the Bacteroidetes, the genome sequences of only three symbionts, which are only distantly related (75 to 80% 16S rRNA sequence similarity) to “Ca. Amoebophilus asiaticus,” have been determined to date: two strains of “Candidatus Sulcia muelleri,” a symbiont of sharpshooters, and “Azobacteroides pseudotrichonymphae,” a symbiont of an anaerobic termite gut ciliate (45, 72, 74, 127).The genome of “Ca. Amoebophilus asiaticus” is only moderately reduced in size compared to those of many other obligate intracellular bacteria (75, 123), but nevertheless, its biosynthetic capabilities are extremely limited. A large fraction of the genome consists of IS elements and an unparalleled high number of proteins with eukaryotic domains, such as ankyrin repeats, TPR/SEL1 repeats, leucine-rich repeats, and domains from the eukaryotic ubiquitin system, all of them most likely important for host cell interaction. Feature enrichment analysis across a nonredundant data set of all bacterial genomes showed that these domains are enriched in the genomes of bacteria (including several pathogens of humans) known to be able to infect amoebae, providing further evidence for an important role of amoebae in the evolution of mechanisms for host cell interaction in intracellular bacteria.     

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.     

Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-Infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.Cryptosporidium (Apicomplexa) is a genus of protozoan parasites with species and genotypes that infect humans, domesticated livestock, companion animals, and wildlife worldwide (5, 6, 14, 15, 20, 23). The majority of cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or C. hominis (8, 10, 19, 24), although rare cases due to species such as C. meleagridis, C. felis, or C. canis have been reported (8, 9, 11-13, 17, 18, 22). The specific identification and characterization of Cryptosporidium species are central to the control of this disease in humans and a wide range of animals.One of the most widely adopted techniques for the identification of microorganisms in complex microbial communities is fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes (2-4). This method relies on the hybridization of synthetic oligonucleotide probes to specific regions within the rRNA of the organism. While FISH has been applied for the detection of Cryptosporidium oocysts in water samples (21), no FISH probes that successfully differentiate C. hominis from C. parvum have been reported.We have reported previously on the design of a species-specific probe, Cpar677, that detects C. parvum (1). In this study, we report on the design and validation of a C. hominis species-specific probe, Chom253. Together, the two probes were used here for the development of a two-color, microscopy-based FISH assay for the simultaneous detection of C. parvum and C. hominis.     

Inhibiting the Initiation of Clostridium difficile Spore Germination using Analogs of Chenodeoxycholic Acid,a Bile Acid

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is pathogenic for both humans and animals (33, 44). Infections caused by C. difficile range from mild diarrhea to more life-threatening conditions, such as pseudomembranous colitis (33). In the classic case, prior antibiotic treatment that disrupts the normally protective colonic flora makes patients susceptible to C. difficile infection (CDI) (35, 53). Other antibiotics, such as vancomycin and metronidazole, are the most commonly used treatments for CDI (54). However, because these antibiotics also disrupt the colonic flora, 10 to 40% of patients whose symptoms have been ameliorated suffer from relapsing CDI (15, 24). The annual treatment-associated cost for CDI in the United States is estimated to be between $750 million and $3.2 billion (8, 9, 16, 31). Moreover, the number of fatal cases of CDI has been increasing rapidly (14, 39). Thus, there is an urgent need to find alternative therapies for CDI.C. difficile infection likely is initiated by infection with the spore form of C. difficile (12). C. difficile elicits disease through the actions of two secreted toxins, TcdA and TcdB (48). TcdB was recently shown to be critical for pathogenesis in an animal model of disease (18). Since the toxins are produced by vegetative cells, not by spores (17), germination and outgrowth are prerequisites for pathogenesis.Spore germination is triggered by the interaction of small molecules, called germinants, with receptors within the spore inner membrane. These germinants vary by bacterial species and can include ions, amino acids, sugars, nucleotides, surfactants, or combinations thereof (43). The recognition of germinants triggers irreversible germination, leading to Ca²⁺-dipicolinic acid release, the uptake of water, the degradation of the cortex, and, eventually, the outgrowth of the vegetative bacterium (43). The germination receptors that C. difficile uses to sense the environment have not been identified. Based on homology searches, C. difficile germination receptors must be very different from known germination receptors (42), but they appear to be proteinaceous (13).Taurocholate, a primary bile acid, has been used for approximately 30 years by researchers and clinical microbiologists to increase colony formation by C. difficile spores from patient and environmental samples (3, 49, 51, 52). This suggested that C. difficile spores interact with bile acids along the gastrointestinal (GI) tract and that spores use a host-derived signal to initiate germination.The liver synthesizes the two major primary bile acids, cholate and chenodeoxycholate (40). These compounds are modified by conjugation with either taurine (to give taurocholate or taurochenodeoxycholate) or glycine (producing glycocholate or glycochenodeoxycholate). Upon secretion into the digestive tract, bile aids in the absorption of fat and cholesterol; much of the secreted bile is actively absorbed and recycled back to the liver for reutilization (40). Though efficient, enterohepatic recirculation is not complete; bile enters the cecum of the large intestine at a concentration of approximately 2 mM (30).In the cecum, bile is modified by the normal, benign colonic flora. First, bile salt hydrolases found on the surfaces of many bacterial species remove the conjugated amino acid, producing the deconjugated primary bile acids cholate and chenodeoxycholate (40). These deconjugated primary bile acids are further metabolized by only a few species of intestinal bacteria, including Clostridium scindens. C. scindens actively transports unconjugated primary bile acids into the cytoplasm, where they are 7α-dehydroxylated, converting cholate to deoxycholate and chenodeoxycholate to lithocholate (21, 40). The disruption of the colonic flora by antibiotic treatment abolishes 7α-dehydroxylation activity (41).Building upon the work on Wilson and others (51, 52), we demonstrated that taurocholate and glycine, acting together, trigger the loss of the birefringence of C. difficile spores (45). All cholate derivatives (taurocholate, glycocholate, cholate, and deoxycholate) stimulate the germination of C. difficile spores (45). Recently it was shown that taurocholate binding is prerequisite to glycine binding (37). At physiologically relevant concentrations, chenodeoxycholate inhibits taurocholate-mediated germination (46) and also inhibits C. difficile vegetative growth, as does deoxycholate (45). In fact, C. difficile spores use the relative concentrations of the various bile acids as cues for germination within the host (10).Since chenodeoxycholate is absorbed by the colonic epithelium and metabolized to lithocholate by the colonic flora (25, 40), the use of chenodeoxycholate as a therapy against C. difficile disease might be hindered by its absorption and conversion to lithocholate.Here, we further characterize the interaction of C. difficile spores with various bile acids and demonstrate that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination. Further, we identify chemical analogs of chenodeoxycholate that are more potent inhibitors of germination and that resist 7α-dehydroxylation by the colonic flora, potentially increasing their stability and effectiveness as inhibitors of C. difficile spore germination in the colonic environment.     

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).     

Asp1, a Conserved 1/3 Inositol Polyphosphate Kinase,Regulates the Dimorphic Switch in Schizosaccharomyces pombe

The ability to undergo dramatic morphological changes in response to extrinsic cues is conserved in fungi. We have used the model yeast Schizosaccharomyces pombe to determine which intracellular signal regulates the dimorphic switch from the single-cell yeast form to the filamentous invasive growth form. The S. pombe Asp1 protein, a member of the conserved Vip1 1/3 inositol polyphosphate kinase family, is a key regulator of the morphological switch via the cAMP protein kinase A (PKA) pathway. Lack of a functional Asp1 kinase domain abolishes invasive growth which is monopolar, while an increase in Asp1-generated inositol pyrophosphates (PP) increases the cellular response. Remarkably, the Asp1 kinase activity encoded by the N-terminal part of the protein is regulated negatively by the C-terminal domain of Asp1, which has homology to acid histidine phosphatases. Thus, the fine tuning of the cellular response to environmental cues is modulated by the same protein. As the Saccharomyces cerevisiae Asp1 ortholog is also required for the dimorphic switch in this yeast, we propose that Vip1 family members have a general role in regulating fungal dimorphism.Eucaryotic cells are able to define and maintain a particular cellular organization and thus cellular morphology by executing programs modulated by internal and external signals. For example, signals generated within a cell are required for the selection of the growth zone after cytokinesis in the fission yeast Schizosaccharomyces pombe or the emergence of the bud in Saccharomyces cerevisiae (37, 44, 81). Cellular morphogenesis is also subject to regulation by a wide variety of external signals, such as growth factors, temperature, hormones, nutrient limitation, and cell-cell or cell-substrate contact (13, 34, 66, 75, 81). Both types of signals will lead to the selection of growth zones accompanied by the reorganization of the cytoskeleton.The ability to alter the growth form in response to environmental conditions is an important virulence-associated trait of pathogenic fungi which helps the pathogen to spread in and survive the host''s defense system (7, 32). Alteration of the growth form in response to extrinsic signals is not limited to pathogenic fungi but is also found in the model yeasts S. cerevisiae and S. pombe, in which it appears to represent a foraging response (1, 24).The regulation of polarized growth and the definition of growth zones have been studied extensively with the fission yeast S. pombe. In this cylindrically shaped organism, cell wall biosynthesis is restricted to one or both cell ends in a cell cycle-regulated manner and to the septum during cytokinesis (38). This mode of growth requires the actin cytoskeleton to direct growth and the microtubule cytoskeleton to define the growth sites (60). In interphase cells, microtubules are organized in antiparallel bundles that are aligned along the long axis of the cell and grow from their plus ends toward the cell tips. Upon contact with the cell end, microtubule growth will first pause and then undergo a catastrophic event and microtubule shrinkage (21). This dynamic behavior of the microtubule plus end is regulated by a disparate, conserved, microtubule plus end group of proteins, called the +TIPs. The +TIP complex containing the EB1 family member Mal3 is required for the delivery of the Tea1-Tea4 complex to the cell tip (6, 11, 27, 45, 77). The latter complex docks at the cell end and recruits proteins required for actin nucleation (46, 76). Thus, the intricate cross talk between the actin and the microtubule cytoskeleton at specific intracellular locations is necessary for cell cycle-dependent polarized growth of the fission yeast cell.The intense analysis of polarized growth control in single-celled S. pombe makes this yeast an attractive organism for the identification of key regulatory components of the dimorphic switch. S. pombe multicellular invasive growth has been observed for specific strains under specific conditions, such as nitrogen and ammonium limitation and the presence of excess iron (1, 19, 50, 61).Here, we have identified an evolutionarily conserved key regulator of the S. pombe dimorphic switch, the Asp1 protein. Asp1 belongs to the highly conserved family of Vip1 1/3 inositol polyphosphate kinases, which is one of two families that can generate inositol pyrophosphates (PP) (17, 23, 42, 54). The inositol polyphosphate kinase IP6K family, of which the S. cerevisiae Kcs1 protein is a member, is the “classical” family that can phosphorylate inositol hexakisphosphate (IP₆) (70, 71). These enzymes generate a specific PP-IP₅ (IP₇), which has the pyrophosphate at position 5 of the inositol ring (20, 54). The Vip1 family kinase activity was unmasked in an S. cerevisiae strain with KCS1 and DDP1 deleted (54, 83). The latter gene encodes a nudix hydrolase (14, 68). The mammalian and S. cerevisiae Vip1 proteins phosphorylate the 1/3 position of the inositol ring, generating 1/3 diphosphoinositol pentakisphosphate (42). Both enzyme families collaborate to generate IP₈ (17, 23, 42, 54, 57).Two modes of action have been described for the high-energy moiety containing inositol pyrophosphates. First, these molecules can phosphorylate proteins by a nonenzymatic transfer of a phosphate group to specific prephosphorylated serine residues (2, 8, 69). Second, inositol pyrophosphates can regulate protein function by reversible binding to the S. cerevisiae Pho80-Pho85-Pho81 complex (39, 40). This cyclin-cyclin-dependent kinase complex is inactivated by inositol pyrophosphates generated by Vip1 when cells are starved of inorganic phosphate (39, 41, 42).Regulation of phosphate metabolism in S. cerevisiae is one of the few roles specifically attributed to a Vip1 kinase. Further information about the cellular function of this family came from the identification of the S. pombe Vip1 family member Asp1 as a regulator of the actin nucleator Arp2/3 complex (22). The 106-kDa Asp1 cytoplasmic protein, which probably exists as a dimer in vivo, acts as a multicopy suppressor of arp3-c1 mutants (22). Loss of Asp1 results in abnormal cell morphology, defects in polarized growth, and aberrant cortical actin cytoskeleton organization (22).The Vip1 family proteins have a dual domain structure which consists of an N-terminal “rimK”/ATP-grasp superfamily domain found in certain inositol signaling kinases and a C-terminal part with homology to histidine acid phosphatases present in phytase enzymes (28, 53, 54). The N-terminal domain is required and sufficient for Vip1 family kinase activity, and an Asp1 variant with a mutation in a catalytic residue of the kinase domain is unable to suppress mutants of the Arp2/3 complex (17, 23, 54). To date, no function has been described for the C-terminal phosphatase domain, and this domain appears to be catalytically inactive (17, 23, 54).Here we describe a new and conserved role for Vip1 kinases in regulating the dimorphic switch in yeasts. Asp1 kinase activity is essential for cell-cell and cell-substrate adhesion and the ability of S. pombe cells to grow invasively. Interestingly, Asp1 kinase activity is counteracted by the putative phosphatase domain of this protein, a finding that allows us to describe for the first time a function for the C-terminal part of Vip1 proteins.     

Increase in Acid Tolerance of Campylobacter jejuni through Coincubation with Amoebae

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga. Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.Campylobacter jejuni is a major cause of human bacterial enteritis, with an incidence exceeding that of Salmonella spp. or Escherichia coli O157 (6, 28). Most infections are associated with consumption of contaminated food, primarily undercooked chicken meat, but unchlorinated water and unpasteurized milk can also be sources of Campylobacter infection (reviewed in reference 13). Apart from food-borne sources, additional risk factors include close contact with pets or farm animals and activities in recreational waters (reviewed in reference 13). C. jejuni is widely distributed in many animals and has also been reported to be isolated from surface waters (15) and, occasionally, even from groundwater (31). However, the bacterium has been shown to be relatively sensitive to environmental stress outside its hosts, including heating, disinfectants, oxygen exposure, osmotic stress, desiccation, and acidity (5, 9, 19, 35).Several hygiene practices have been implemented in broiler production facilities to reduce C. jejuni carriage in live birds. Such measures include hygiene barriers such as changing clothes before entering the broiler houses and disinfection of the interior of the building with acid between flock rotations (20). Such efforts may reduce the number of C. jejuni organisms, but the bacterium is still difficult to eradicate from contaminated farms, and subsequent outbreaks at the same farm are not rare (11). Contradictory to its fragility in different in vitro settings, C. jejuni seems to be well adapted to survive the acidic milieu of the human stomach during the passage to the lower intestinal tract, where infection is established. This is illustrated by the very low infectious dose for both broiler chickens (7) and humans (4) and indicates that the bacterium has developed strategies to avoid or withstand low pH in order to survive the transit. The gastric acid is the first line of defense against ingested pathogens. During fasting conditions in healthy humans, the luminal pH in the stomach is usually around 2.0, but it may range from 1.5 to 5.5 depending on food intake, such as a diet with a high pH, or the use of proton pump inhibitors (36). Laboratory studies have demonstrated that C. jejuni in solution survives a maximum of 30 min at pH levels below pH 2.5 and for up to 60 min at pH 3 (5, 23). When the bacterium is mixed with food, it seems to be protected, and it has been shown that C. jejuni inoculated onto ground beef survived at pH 2.5 for 2 h at 37°C (37).In the last few years, laboratory studies have identified a new potential epidemiological pathway for C. jejuni in which the bacterium colonizes unicellular eukaryotic organisms (protozoa) and thereby acquires protection from adverse environmental conditions (2, 17, 29). C. jejuni can colonize protozoa and survive longer in its protozoan host than as a free-living bacterium, and given the right temperature, the bacterium can also replicate intracellularly (1, 2). Protozoa, especially amoebae, serve as natural reservoirs or vehicles for the dissemination of several other pathogenic bacteria, including Legionella pneumophila (25), Vibrio cholerae (34), and Helicobacter pylori (38). Amoebae are abundant in virtually all natural water systems and can be found grazing on biofilms in water supply systems (14). In their trophozoite form, amoebae are naturally resistant to many environmental factors that are lethal to Campylobacter, and they can multiply at pHs ranging from 4 to 12 (16). Moreover, amoebae can enter a cyst form when challenged with unfavorable conditions. These cysts generally have a double cell wall that might explain their capability to survive chlorination, antimicrobials, and changes in pH and osmotic pressure. This resistance feature of amoebae makes them suitable hosts for other, less-resistant microorganisms (16, 32).In this study, we built on the advances gained in protozoa-Campylobacter research and investigated whether internalization of C. jejuni into Acanthamoeba affects bacterial tolerance to hydrochloric acid. Using an in vitro setup, we found that C. jejuni survived better in an acidic environment when it was coincubated with amoebae than when it was incubated as bacteria in solution. Furthermore, we show that bacterial motility and adhesion to and internalization into amoeba are trigged by moderately acidic conditions. The implications of these findings for the survival of C. jejuni in food production as well as in transit through the human stomach are discussed.