Genomic Analysis of Immune Response against Vibrio Cholerae Hemolysin in Caenorhabditis elegans

Vibrio cholerae cytolysin (VCC) is among the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, known as poreforming toxins (PFTs). V. cholerae infects and kills Caenorhabditis elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection. However, little is known about the host gene expression responses against VCC. To address this question we performed a microarray study in C. elegans exposed to V. cholerae strains with intact and deleted hlyA genes. Many of the VCC regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/asparagine [N]-rich)-domain containing genes, genes regulated by insulin/ IGF-1-mediated signaling (IIS) pathway, were previously reported as mediators of innate immune response against other bacteria in C. elegans. Protective function of the subset of the genes up-regulated by VCC was confirmed using RNAi. By means of a machine learning algorithm called FastMEDUSA, we identified several putative VCC induced immune regulatory transcriptional factors and transcription factor binding motifs. Our results suggest that VCC is a major virulence factor, which induces a wide variety of immune response- related genes during V. cholerae infection in C. elegans

Variation in hemolytic activity of Brachyspira hyodysenteriae strains from pigs

Brachyspira hyodysenteriae is the primary cause of swine dysentery, which is responsible for major economic losses to the pig industry worldwide. The hemolytic activity of 10 B. hyodysenteriae strains isolated from stools of pigs with mild to mucohemorrhagic diarrhea was compared and seven hemolysis associated genes were sequenced. Hemolysis induced by these strains varied from strong to near absent. One weakly hemolytic B. hyodysenteriae strain showed sequence changes in five hemolysis associated genes (tlyA, tlyB, hemolysin III, hemolysin activation protein and hemolysin III channel protein) resulting in amino acid substitutions. The occurrence of weakly hemolytic strains identifiable as B. hyodysenteriae should be taken into account in swine dysentery diagnostics. The presence of these strains may affect herd dysentery status, with great impact on a farms trading opportunities

Escherichia coli α-Hemolysin Counteracts the Anti-Virulence Innate Immune Response Triggered by the Rho GTPase Activating Toxin CNF1 during Bacteremia

International audienceThe detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1 beta signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1 beta antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1 beta and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the Phi-hemolysin toxin inhibits IL-1 beta secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia

Bacterial hemolysins as virulence factors

No abstract availabl

Uropathogenic E. coli Induce Different Immune Response in Testicular and Peritoneal Macrophages: Implications for Testicular Immune Privilege

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells

Secretion of alpha-hemolysin by<i> Escherichia coil</i> disrupts tight junctions in ulcerative colitis patients

OBJECTIVES: The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated. METHODS: E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coliisolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). ProbioticE. coliNissle and pathogenicE. coliLF82 were used as controls. RESULTS: E. colistrains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probioticE. coliNissle, pathogenicE. coliLF82, fourE. colifrom patients with inactive UC and threeE. colistrains from healthy controls did not disrupt tight junctions.E. colip19A WT as well ascnf1, and single loci ofhlymutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of bothhlyloci (P<0.001). CONCLUSIONS: UC-associated E. coliproducing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. colipathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD

El Tor biotype Vibrio cholerae activates the caspase-11-independent canonical Nlrp3 and Pyrin inflammasomes

Vibrio cholerae is a Gram-negative enteropathogen causing potentially life-threatening cholera disease outbreaks, for which the World Health Organization currently registers 2-4 million cases and similar to 100.000 cholera-associated deaths annually worldwide. Genomic Vibrio cholerae research revealed that the strains causing this ongoing cholera pandemic are members of the El Tor biotype, which fully replaced the Classical biotype that caused former cholera pandemics. While both of these biotypes express the characteristic Cholera Toxin (CT), the El Tor biotype additionally expresses the accessory toxins hemolysin (hlyA) and multifunctional auto-processing repeat-in-toxin (MARTX). Previous studies demonstrated that the Classical biotype of Vibrio cholerae triggers caspase-11-dependent non-canonical inflammasome activation in macrophages following CT-mediated cytosolic delivery of LPS. In contrast to the Classical biotype, we here show that El Tor Vibrio cholerae induces IL-1 beta maturation and secretion in a caspase-11- and CT-independent manner. Instead, we show that El Tor Vibrio cholerae engages the canonical Nlrp3 inflammasome for IL-1 beta secretion through its accessory hlyA toxin. We further reveal the capacity of this enteropathogen to engage the canonical Pyrin inflammasome as an accessory mechanism for IL-1 beta secretion in conditions when the pro-inflammatory hlyA-Nlrp3 axis is blocked. Thus, we show that the V. cholerae El Tor biotype does not trigger caspase-11 activation, but instead triggers parallel Nlrp3- and Pyrin-dependent pathways toward canonical inflammasome activation to induce IL-1 beta-mediated inflammatory responses. These findings further unravel the complex inflammasome activating mechanisms that can be triggered when macrophages face the full arsenal of El Tor Vibrio cholerae toxins, and as such increase our understanding of host-pathogen interactions in the context of the Vibrio cholerae biotype associated with the ongoing cholera pandemic

ppGpp, the general stress response alarmone, is required for the expression of the alpha-hemolysin toxin in the uropathogenic EScherichia coli isolate, J96.

ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection

Biochemistry of Hemolysin Toxin Activation by Fatty Acylation: Characterization of an Internal Protein Acyltransferase

Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA) is made toxic by post-translational fatty acylation of two internal lysine residues. HlyC, C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. ProHlyA and HlyC were separately subcloned, expressed, and purified, and acyl-ACPs with diverse radioactive acyl groups were synthesized. With these proteins, the conversion of proHlyA to HlyA by acyltransfer was assayed. Acyl-ACP was the obligate acyl donor. Acyltransfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was detected and shown to catalyze the reverse reaction. The reaction mechanism was examined by steady state kinetics, and the nature of inhibitions by reaction products was determined. The kinetic mechanism of the internal protein acylation was compatible with an uni uni iso uni uni ping pong with isomerization of the F form of the enzyme. Clues to the chemical mechanism for the acyltransferase were elucidated by both chemical modification studies and site directed mutagenesis of the enzyme. Chemical modification experiments ruled out any critical cysteines, serines, and lysine residues, but suggested a role for histidine(s) and tyrosine(s) in acyltransferase function. In order to examine the function of specific residues and possibly corroborate the chemical findings, site directed mutagenesis studies of the acyltransferase were employed. Seventeen residues that were conserved among 13 different RTX toxin acyltransferases were individually mutated, and the respective HlyCs expressed, and characterized. Residues that were critical for acyltransferase function included Gly 11, His 23, Tyr 70, and Gly 85. As with chemical modification data, mutagenesis ruled out any conserved, essential, cysteines or serines critical for HlyC acyltransferase activity

A new role for Zinc limitation in bacterial pathogenicity: modulation of α-hemolysin from uropathogenic Escherichia coli.

Metal limitation is a common situation during infection and can have profound effects on the pathogen's success. In this report, we examine the role of zinc limitation in the expression of a virulence factor in uropathogenic Escherichia coli. The pyelonephritis isolate J96 carries two hlyCABD operons that encode the RTX toxin α-hemolysin. While the coding regions of both operons are largely conserved, the upstream sequences, including the promoters, are unrelated. We show here that the two hlyCABD operons are differently regulated. The hly II operon is efficiently silenced in the presence of zinc and highly expressed when zinc is limited. In contrast, the hly I operon does not respond to zinc limitation. Genetic studies reveal that zinc-responsive regulation of the hly II operon is controlled by the Zur metalloregulatory protein. A Zur binding site was identified in the promoter sequence of the hly II operon, and we observe direct binding of Zur to this promoter region. Moreover, we find that Zur regulation of the hly II operon modulates the ability of E. coli J96 to induce a cytotoxic response in host cell lines in culture. Our report constitutes the first description of the involvement of the zinc-sensing protein Zur in directly modulating the expression of a virulence factor in bacteria