Role of NleH from attaching effacing bacteria in host pathogen interactions

Enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), and Citrobacter rodentium are diarrhoeal pathogens grouped together on the basis of their ability to intimately adhere to host intestinal epithelia and efface brush border microvilli to form the broader category of attaching effacing (A/E) pathogens. While EPEC and EHEC are human pathogens of global health concern, C. rodentium is a natural murine pathogen which serves as an excellent animal model for in vivo studies. A/E pathogens encode a filamentous type 3 secretion system (T3SS) which is used to deliver virulence factors called effectors directly into the host cell. Both in vivo and in vitro studies have demonstrated that the T3SS is integral to the A/E pathogen virulence strategy. Once translocated to the host cell, effector proteins modulate and disrupt a wide range of host cell signalling pathways and processes including the immune response, cytoskeletal dynamics, GTPase signalling pathways, phagocytosis and apoptosis. As a defence strategy, the host responds by activating an immune response and apoptosis. However, several studies have reported that epithelial cells infected with EPEC do not undergo apoptosis despite the presence of early markers of apoptosis. Recently the NleH effectors were identified in a secretomic and genomic analysis, however their function remains unknown. NleH effectors are present in all sequenced A/E pathogen strains and can be found in duplicate copies (nleH1 and nleH2). We verified that EPEC NleH1 and NleH2 are secreted and translocated in a T3SS dependent manner. In this study we demonstrate that NleH effectors have anti-apoptotic function. During infection of cultured cell lines, we found increased nuclear condensation, membrane blebbing, caspase-3 cleavage and cell death and detachment in an nleH1 and nleH2 double mutant in comparison to wild type EPEC. Furthermore, treatment with a global caspase inhibitor abolished cell loss due to cell death or detachment. Using ectopic expression, we showed that NleH1 alone is sufficient to inhibit caspase-3 cleavage in the presence of the general apoptosis inducers staurosporine and the ER stress apoptosis inducers tunicamycin and brefeldin A. Interestingly we found that NleH effectors are kinases, however their kinase activity is not involved in the inhibition of apoptosis. However, an intact C terminus is essential for NleH’s anti-apoptotic activity. To determine the pathway by which NleH effectors inhibit apoptosis a HeLa human cDNA library was used to screen for potential binding partners using a Y2H assay. The ER antiapoptotic protein Bax inhibitor 1 (BI-1) was identified as a putative binding partner and verified using a direct 2 hybrid assay. Knockdown of BI-1 resulted in loss of NleH’s cytoprotective function. As BI-1 plays a role in calcium homeostasis, the effect of NleH effectors on cytosolic Ca2+ levels was assessed. We found that NleH effectors reduced cytosolic Ca2+ levels in a BI-1 dependent manner. Using the C. rodentium animal model we verified the ability of NleH to inhibit apoptosis in vivo by demonstrating that NleH inhibits caspase-3 activation and nuclear condensation at the site of bacterial attachment. Furthermore, we found a reduction in cell exfoliation in the presence of NleH. Additionally we showed that the NleH effectors play a role in competitiveness and also induce a mild but significant increase in NF-KB activation and TNF-[alpha] expression. Together our results demonstrate that NleH effectors are multi-functional proteins that inhibit apoptosis both in vitro and in vivo, and are kinases with an unknown function. Furthermore, NleH effectors induce a local NF-KB and TNF-[alpha] upregulation which could be linked to its anti-apoptotic activity. NleH effectors may provide a competitive advantage by preventing shedding of infected cells to prolong infection

The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway

Role of NleH from attaching effacing bacteria in host pathogen interactions

Enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), and Citrobacter rodentium are diarrhoeal pathogens grouped together on the basis of their ability to intimately adhere to host intestinal epithelia and efface brush border microvilli to form the broader category of attaching effacing (A/E) pathogens. While EPEC and EHEC are human pathogens of global health concern, C. rodentium is a natural murine pathogen which serves as an excellent animal model for in vivo studies. A/E pathogens encode a filamentous type 3 secretion system (T3SS) which is used to deliver virulence factors called effectors directly into the host cell. Both in vivo and in vitro studies have demonstrated that the T3SS is integral to the A/E pathogen virulence strategy. Once translocated to the host cell, effector proteins modulate and disrupt a wide range of host cell signalling pathways and processes including the immune response, cytoskeletal dynamics, GTPase signalling pathways, phagocytosis and apoptosis. As a defence strategy, the host responds by activating an immune response and apoptosis. However, several studies have reported that epithelial cells infected with EPEC do not undergo apoptosis despite the presence of early markers of apoptosis. Recently the NleH effectors were identified in a secretomic and genomic analysis, however their function remains unknown. NleH effectors are present in all sequenced A/E pathogen strains and can be found in duplicate copies (nleH1 and nleH2). We verified that EPEC NleH1 and NleH2 are secreted and translocated in a T3SS dependent manner. In this study we demonstrate that NleH effectors have anti-apoptotic function. During infection of cultured cell lines, we found increased nuclear condensation, membrane blebbing, caspase-3 cleavage and cell death and detachment in an nleH1 and nleH2 double mutant in comparison to wild type EPEC. Furthermore, treatment with a global caspase inhibitor abolished cell loss due to cell death or detachment. Using ectopic expression, we showed that NleH1 alone is sufficient to inhibit caspase-3 cleavage in the presence of the general apoptosis inducers staurosporine and the ER stress apoptosis inducers tunicamycin and brefeldin A. Interestingly we found that NleH effectors are kinases, however their kinase activity is not involved in the inhibition of apoptosis. However, an intact C terminus is essential for NleH’s anti-apoptotic activity. To determine the pathway by which NleH effectors inhibit apoptosis a HeLa human cDNA library was used to screen for potential binding partners using a Y2H assay. The ER antiapoptotic protein Bax inhibitor 1 (BI-1) was identified as a putative binding partner and verified using a direct 2 hybrid assay. Knockdown of BI-1 resulted in loss of NleH’s cytoprotective function. As BI-1 plays a role in calcium homeostasis, the effect of NleH effectors on cytosolic Ca2+ levels was assessed. We found that NleH effectors reduced cytosolic Ca2+ levels in a BI-1 dependent manner. Using the C. rodentium animal model we verified the ability of NleH to inhibit apoptosis in vivo by demonstrating that NleH inhibits caspase-3 activation and nuclear condensation at the site of bacterial attachment. Furthermore, we found a reduction in cell exfoliation in the presence of NleH. Additionally we showed that the NleH effectors play a role in competitiveness and also induce a mild but significant increase in NF-KB activation and TNF-[alpha] expression. Together our results demonstrate that NleH effectors are multi-functional proteins that inhibit apoptosis both in vitro and in vivo, and are kinases with an unknown function. Furthermore, NleH effectors induce a local NF-KB and TNF-[alpha] upregulation which could be linked to its anti-apoptotic activity. NleH effectors may provide a competitive advantage by preventing shedding of infected cells to prolong infection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection

The human pathogens enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli and the related mouse pathogen Citrobacter rodentium subvert a variety of host cell signaling pathways via their plethora of type III secreted effectors, including triggering of an early apoptotic response. EPEC-infected cells do not develop late apoptotic symptoms, however. In this study we demonstrate that the NleH family effectors, homologs of the Shigella effector kinase OspG, blocks apoptosis. During EPEC infection, NleH effectors inhibit elevation of cytosolic Ca(2+) concentrations, nuclear condensation, caspase-3 activation, and membrane blebbing and promote cell survival. NleH1 alone is sufficient to prevent procaspase-3 cleavage induced by the proapoptotic compounds staurosporine, brefeldin A, and tunicamycin. Using C. rodentium, we found that NleH inhibits procaspase-3 cleavage at the bacterial attachment sites in vivo. A yeast two-hybrid screen identified the endoplasmic reticulum six-transmembrane protein Bax inhibitor-1 (BI-1) as an NleH-interacting partner. We mapped the NleH-binding site to the N-terminal 40 amino acids of BI-1. Knockdown of BI-1 resulted in the loss of NleH’s antiapoptotic activity. These results indicate that NleH effectors are inhibitors of apoptosis that may act through BI-1 to carry out their cytoprotective function

Role of NleH, a Type III Secreted Effector from Attaching and Effacing Pathogens, in Colonization of the Bovine, Ovine, and Murine Gut▿

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC ΔnleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain