Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection.

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

Iron regulatory proteins 1 and 2 have opposing roles in regulating inflammation in bacterial orchitis.

Acute bacterial orchitis (AO) is a prevalent cause of intrascrotal inflammation, often resulting in sub- or infertility. A frequent cause eliciting AO is uropathogenic Escherichia coli (UPEC), a gram negative pathovar, characterized by the expression of various iron acquisition systems to survive in a low-iron environment. On the host side, iron is tightly regulated by iron regulatory proteins 1 and 2 (IRP1 and -2) and these factors are reported to play a role in testicular and immune cell function; however, their precise role remains unclear. Here, we showed in a mouse model of UPEC-induced orchitis that the absence of IRP1 results in less testicular damage and a reduced immune response. Compared with infected wild-type (WT) mice, testes of UPEC-infected Irp1-/- mice showed impaired ERK signaling. Conversely, IRP2 deletion led to a stronger inflammatory response. Notably, differences in immune cell infiltrations were observed among the different genotypes. In contrast with WT and Irp2-/- mice, no increase in monocytes and neutrophils was detected in testes of Irp1-/- mice upon UPEC infection. Interestingly, in Irp1-/- UPEC-infected testes, we observed an increase in a subpopulation of macrophages (F4/80+CD206+) associated with antiinflammatory and wound-healing activities compared with WT. These findings suggest that IRP1 deletion may protect against UPEC-induced inflammation by modulating ERK signaling and dampening the immune response.

Helicobacter pylori CagA Induces Cortactin Y-470 Phosphorylation-Dependent Gastric Epithelial Cell Scattering via Abl, Vav2 and Rac1 Activation.

The pathogen Helicobacter pylori is the first reported bacterial type-1 carcinogen playing a role in the development of human malignancies, including gastric adenocarcinoma. Cancer cell motility is an important process in this scenario, however, the molecular mechanisms are still not fully understood. Here, we demonstrate that H. pylori subverts the actin-binding protein cortactin through its type-IV secretion system and injected oncoprotein CagA, e.g., by inducing tyrosine phosphorylation of cortactin at Y-470, which triggers gastric epithelial cell scattering and motility. During infection of AGS cells, cortactin was discovered to undergo tyrosine dephosphorylation at residues Y-421 and Y-486, which is mediated through inactivation of Src kinase. However, H. pylori also profoundly activates tyrosine kinase Abl, which simultaneously phosphorylates cortactin at Y-470. Phosphorylated cortactin interacts with the SH2-domain of Vav2, a guanine nucleotide exchange factor for the Rho-family of GTPases. The cortactin/Vav2 complex then stimulates a previously unrecognized activation cascade including the small GTPase Rac1, to effect actin rearrangements and cell scattering. We hypothesize that injected CagA targets cortactin to locally open the gastric epithelium in order to get access to certain nutrients. This may disturb the cellular barrier functions, likely contributing to the induction of cell motility, which is important in gastric cancer development.

Campylobacter Virulence Factors and Molecular Host-Pathogen Interactions.

Campylobacter jejuni and Campylobacter coli can be frequently isolated from poultry and poultry-derived products, and in combination these two species cause a large portion of human bacterial gastroenteritis cases. While birds are typically colonized by these Campylobacter species without clinical symptoms, in humans they cause (foodborne) infections at high frequencies, estimated to cost billions of dollars worldwide every year. The clinical outcome of Campylobacter infections comprises malaise, diarrhea, abdominal pain and fever. Symptoms may continue for up to two weeks and are generally self-limiting, though occasionally the disease can be more severe or result in post-infection sequelae. The virulence properties of these pathogens have been best-characterized for C. jejuni, and their actions are reviewed here. Various virulence-associated bacterial determinants include the flagellum, numerous flagellar secreted factors, protein adhesins, cytolethal distending toxin (CDT), lipooligosaccharide (LOS), serine protease HtrA and others. These factors are involved in several pathogenicity-linked properties that can be divided into bacterial chemotaxis, motility, attachment, invasion, survival, cellular transmigration and spread to deeper tissue. All of these steps require intimate interactions between bacteria and host cells (including immune cells), enabled by the collection of bacterial and host factors that have already been identified. The assortment of pathogenicity-associated factors now recognized for C. jejuni, their function and the proposed host cell factors that are involved in crucial steps leading to disease are discussed in detail.

Campylobacter jejuni Serine Protease HtrA Cleaves the Tight Junction Component Claudin-8.

Campylobacter jejuni express the high temperature requirement protein A (HtrA), a secreted serine protease, which is implicated in virulence properties of the pathogen. Previous studies have shown that C. jejuni HtrA can cleave the epithelial transmembrane proteins occludin and E-cadherin in the tight and adherens junctions, respectively. In the present report, we studied the interaction of HtrA with another human tight junction protein, claudin-8. Confocal immunofluorescence experiments have shown that C. jejuni infection of the intestinal polarized epithelial cells in vitro leads to a relocation of claudin-8. Wild-type C. jejuni induced the downregulation of claudin-8 signals in the tight junctions and an accumulation of claudin-8 agglomerates in the cytoplasm, which were not seen during infection with isogenic ΔhtrA knockout deletion or protease-inactive S197A point mutants. Western blotting of protein samples from infected vs. uninfected cells revealed that an 18-kDa carboxy-terminal fragment is cleaved-off from the 26-kDa full-length claudin-8 protein, but not during infection with the isogenic ΔhtrA mutant. These results were confirmed by in vitro cleavage assays using the purified recombinant C. jejuni HtrA and human claudin-8 proteins. Recombinant HtrA cleaved purified claudin-8 in vitro giving rise to the same 18-kDa sized carboxy-terminal cleavage product. Mapping studies revealed that HtrA cleavage occurs in the first extracellular loop of claudin-8. Three-dimensional modeling of the claudin-8 structure identified an exposed HtrA cleavage site between the amino acids alanine 58 and asparagine 59, which is in well agreement with the mapping studies. Taken together, HtrA operates as a secreted virulence factor targeting multiple proteins both in the tight and adherens junctions. This strategy may help the bacteria to open the cell-to-cell junctions, and to transmigrate across the intestinal epithelium by a paracellular mechanism and establish an acute infection.

Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions.

BACKGROUND: Serine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins. METHODS: The in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of ß-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy. RESULTS: We demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of ß-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage. CONCLUSION: Here we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen.

Campylobacter jejuni enters gut epithelial cells and impairs intestinal barrier function through cleavage of occludin by serine protease HtrA.

Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81-176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells.

Expression of CEACAM1 or CEACAM5 in AZ-521 cells restores the type IV secretion deficiency for translocation of CagA by Helicobacter pylori.

Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin-associated genes pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c-Src and c-Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ-521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA-dependent fashion. In contrast, we discovered that AZ-521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin-ß1 or carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ-521 cells readily express integrin-ß1 , but overexpression of integrin-ß1 constructs did not restore the T4SS defect. We further show that AZ-521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ-521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ-521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori-infected AGS cells. Our results suggest the existence of an integrin-ß1 - and CEACAM1- or CEACAM5-dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.

Helicobacter pylori Employs a Unique Basolateral Type IV Secretion Mechanism for CagA Delivery.

The Helicobacter pylori (Hp) type IV secretion system (T4SS) forms needle-like pili, whose binding to the integrin-ß1 receptor results in injection of the CagA oncoprotein. However, the apical surface of epithelial cells is exposed to Hp, whereas integrins are basolateral receptors. Hence, the mechanism of CagA delivery into polarized gastric epithelial cells remains enigmatic. Here, we demonstrate that T4SS pilus formation during infection of polarized cells occurs predominantly at basolateral membranes, and not at apical sites. Hp accomplishes this by secreting another bacterial protein, the serine protease HtrA, which opens cell-to-cell junctions through cleaving epithelial junctional proteins including occludin, claudin-8, and E-cadherin. Using a genetic system expressing a peptide inhibitor, we demonstrate that HtrA activity is necessary for paracellular transmigration of Hp across polarized cell monolayers to reach basolateral membranes and inject CagA. The contribution of this unique signaling cascade to Hp pathogenesis is discussed.

Overexpression of serine protease HtrA enhances disruption of adherens junctions, paracellular transmigration and type IV secretion of CagA by Helicobacter pylori.

BACKGROUND: The serine protease HtrA is an important factor for regulating stress responses and protein quality control in bacteria. In recent studies, we have demonstrated that the gastric pathogen Helicobacter pylori can secrete HtrA into the extracellular environment, where it cleaves-off the ectodomain of the tumor suppressor and adherens junction protein E-cadherin on gastric epithelial cells. RESULTS: E-cadherin cleavage opens cell-to-cell junctions, allowing paracellular transmigration of the bacteria across polarized monolayers of MKN-28 and Caco-2 epithelial cells. However, rapid research progress on HtrA function is mainly hampered by the lack of ΔhtrA knockout mutants, suggesting that htrA may represent an essential gene in H. pylori. To circumvent this major handicap and to investigate the role of HtrA further, we overexpressed HtrA by introducing a second functional htrA gene copy in the chromosome and studied various virulence properties of the bacteria. The resulting data demonstrate that overexpression of HtrA in H. pylori gives rise to elevated rates of HtrA secretion, cleavage of E-cadherin, bacterial transmigration and delivery of the type IV secretion system (T4SS) effector protein CagA into polarized epithelial cells, but did not affect IL-8 chemokine production or the secretion of vacuolating cytotoxin VacA and γ-glutamyl-transpeptidase GGT. CONCLUSIONS: These data provide for the first time genetic evidence in H. pylori that HtrA is a novel major virulence factor controlling multiple pathogenic activities of this important microbe.

Exploiting the Gastric Epithelial Barrier: Helicobacter pylori's Attack on Tight and Adherens Junctions.

Highly organized intercellular tight and adherens junctions are crucial structural components for establishing and maintenance of epithelial barrier functions, which control the microbiota and protect against intruding pathogens in humans. Alterations in these complexes represent key events in the development and progression of multiple infectious diseases as well as various cancers. The gastric pathogen Helicobacter pylori exerts an amazing set of strategies to manipulate these epithelial cell-to-cell junctions, which are implicated in changing cell polarity, migration and invasive growth as well as pro-inflammatory and proliferative responses. This chapter focuses on the H. pylori pathogenicity factors VacA, CagA, HtrA and urease, and how they can induce host cell signaling involved in altering cell-to-cell permeability. We propose a stepwise model for how H. pylori targets components of tight and adherens junctions in order to disrupt the gastric epithelial cell layer, giving fresh insights into the pathogenesis of this important bacterium.

Urmylation and tRNA thiolation functions of ubiquitin-like Uba4·Urm1 systems are conserved from yeast to man.

The ubiquitin-like protein Urm1 from budding yeast and its E1-like activator Uba4 have dual roles in protein urmylation and tRNA thiolation pathways. To study whether these are conserved among eukaryotes, we used gene shuffles to replace the yeast proteins by their human counterparts, hURM1 and hUBA4/MOCS3. As judged from biochemical and genetical assays, hURM1 and hUBA4 are functional in yeast, albeit at reduced efficiencies. They mediate urmylation of the peroxiredoxin Ahp1, a known urmylation target in yeast, and support tRNA thiolation. Similar to hUBA4, yeast Uba4 itself is modified by Urm1 and hURM1 suggesting target overlap between eukaryal urmylation pathways. In sum, our study shows that dual-function ubiquitin-like Urm1·Uba4 systems are conserved and exchangeable between human and yeast cells.