We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.Toxigenic strains of the Gram-negative bacterium Vibrio cholerae cause a life-threatening diarrheal disease that can kill its victims within hours of the onset of symptoms. The disease is endemic in the Indian subcontinent and continues to reemerge elsewhere in Asia, Africa, and the Americas with the incidence estimated to exceed 5 million cases each year (1-3). Although over 180 serogroups of V. cholerae exist, only the O1 and O139 serovars are known to cause epidemic cholera. The O1 serogroup can be divided further into two biotypes, classical and El Tor, based on various biochemical and phenotypic differences (4). Since 1817, seven cholera pandemics have occurred, of which the classical biotype is responsible for at least the fifth (1881-1896) and the sixth . The El Tor biotype is responsible for the seventh and current pandemic (1961-present). Toxigenic strains of O139 serovar appeared in India and Bangladesh in late 1992 as the first non-O1 serovar to cause epidemic cholera (5, 6). These strains are closely related and probably derived from toxigenic El Tor O1 strains (7). The factors that allowed El Tor biotype and O139 serotype to overtake the classical strains and establish pandemics remain a mystery.Work focusing on the pathogenesis of V. cholerae led to the identification of several key virulence factors, including the cholera toxin responsible for the diarrhea and the toxincoregulated pilus essential for colonizing the human small intestine (8, 9). Several groups tried to construct live, attenuated V. cholerae vaccines by deleting the genes for cholera toxin and several other putative accessory toxins (10-16). However, many of these strains remain reactogenic in human subjects, causing diarrhea or other significant symptoms (17).The virulence factors responsible for the residual reactogenicity remain to be identified.Bacterial genomics offers an opportunity to identify undiscov...
The Hgt4 protein of Candida albicans (orf19.5962) is orthologous to the Snf3 and Rgt2 glucose sensors of Saccharomyces cerevisiae that govern sugar acquisition by regulating the expression of genes encoding hexose transporters. We found that HGT4 is required for glucose induction of the expression of HGT12, HXT10, and HGT7, which encode apparent hexose transporters in C. albicans. An hgt4⌬ mutant is defective for growth on fermentable sugars, which is consistent with the idea that Hgt4 is a sensor of glucose and similar sugars. Hgt4 appears to be sensitive to glucose levels similar to those in human serum (ϳ5 mM). HGT4 expression is repressed by high levels of glucose, which is consistent with the idea that it encodes a high-affinity sugar sensor. Glucose sensing through Hgt4 affects the yeast-to-hyphal morphological switch of C. albicans cells: hgt4⌬ mutants are hypofilamented, and a constitutively signaling form of Hgt4 confers hyperfilamentation of cells. The hgt4⌬ mutant is less virulent than wild-type cells in a mouse model of disseminated candidiasis. These results suggest that Hgt4 is a high-affinity glucose sensor that contributes to the virulence of C. albicans.Candida albicans is responsible for most yeast infections in humans. In addition to causing ordinary vaginal and oral (thrush) infections, this fungus is a serious threat to immunocompromised patients, for whom disseminated, invasive candidiasis is common and often fatal (31, 56). The fungus is normally maintained as a harmless commensal on skin, on mucosa, and in the gut (23) but becomes an opportunistic pathogen upon entry into the bloodstream of immunocompromised (particularly neutropenic) individuals (25,45).
Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.
A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. One class, called type IV, are defined as having homology to the conjugal transfer systems of naturally occurring plasmids. It has been proposed that pathogens with type IV secretion systems have acquired and adapted the conjugal transfer systems of plasmids and now use them to export toxins. Several well-characterized intracellular pathogens, including Legionella pneumophila, Coxiella burnetii, Brucella abortus, and Rickettsia prowazekii, contain type IV systems which are known or suspected to be of critical importance in their ability to cause disease. Specifically, these systems are believed to be the key factors determining intracellular fate, and thus the ability to replicate and cause disease.
Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ⌬dotU ⌬icmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ⌬dotU ⌬icmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.
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