Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cgamma but not Grb2-associated binder 1 or growth factor receptor-bound protein 2. The H. pylori-induced motogenic response is suppressed and blocked by the inhibition of PLCgamma and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori-infected epithelial cells suggests that CagA could be involved in tumor progression.

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden. C. jejuni can cross the intestinal epithelial barrier as visualized in biopsies derived from human patients and animal models, however, the underlying molecular mechanisms and associated immunopathology are still not well understood. We have recently shown that the secreted serine protease HtrA (high temperature requirement A) plays a key role in C. jejuni cellular invasion and transmigration across polarized epithelial cells in vitro. In the present in vivo study we investigated the role of HtrA during C. jejuni infection of mice. We used the gnotobiotic IL-10(-/-) mouse model to study campylobacteriosis following peroral infection with the C. jejuni wild-type (WT) strain NCTC11168 and the isogenic, non-polar NCTC11168ΔhtrA deletion mutant. Six days post infection (p.i.) with either strain mice harbored comparable intestinal C. jejuni loads, whereas ulcerative enterocolitis was less pronounced in mice infected with the ΔhtrA mutant strain. Moreover, ΔhtrA mutant infected mice displayed lower apoptotic cell numbers in the large intestinal mucosa, less colonic accumulation of neutrophils, macrophages and monocytes, lower large intestinal nitric oxide, IFN-γ, and IL-6 as well as lower TNF-α and IL-6 serum concentrations as compared to WT strain infected mice at day 6 p.i. Notably, immunopathological responses were not restricted to the intestinal tract given that liver and kidneys exhibited mild histopathological changes 6 days p.i. with either C. jejuni strain. We also found that hepatic and renal nitric oxide levels or renal TNF-α concentrations were lower in the ΔhtrA mutant as compared to WT strain infected mice. In conclusion, we show here that the C. jejuni HtrA protein plays a pivotal role in inducing host cell apoptosis and immunopathology during murine campylobacteriosis in the gut in vivo.

HtrA proteases and chaperones exhibit important roles in periplasmic protein quality control and stress responses. The genetic inactivation of htrA has been described for many bacterial pathogens. However, in some cases such as the gastric pathogen Helicobacter pylori, HtrA is secreted where it cleaves the tumour-suppressor E-cadherin interfering with gastric disease development, but the generation of htrA mutants is still lacking. Here, we show that the htrA gene locus is highly conserved in worldwide strains. HtrA presence was confirmed in 992 H. pylori isolates in gastric biopsy material from infected patients. Differential RNA-sequencing (dRNA-seq) indicated that htrA is encoded in an operon with two subsequent genes, HP1020 and HP1021. Genetic mutagenesis and complementation studies revealed that HP1020 and HP1021, but not htrA, can be mutated. In addition, we demonstrate that suppression of HtrA proteolytic activity with a newly developed inhibitor is sufficient to effectively kill H. pylori, but not other bacteria. We show that Helicobacter htrA is an essential bifunctional gene with crucial intracellular and extracellular functions. Thus, we describe here the first microbe in which htrA is an indispensable gene, a situation unique in the bacterial kingdom. HtrA can therefore be considered a promising new target for anti-bacterial therapy.

Neuroplastin cell recognition molecules have been implicated in synaptic plasticity. Polymorphisms in the regulatory region of the human neuroplastin gene (NPTN) are correlated with cortical thickness and intellectual abilities in adolescents and in individuals with schizophrenia.

Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin(-/-), integrin beta1(-/-), and focal adhesion kinase (FAK)(-/-) deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin(-/-), integrin beta1(-/-), and FAK(-/-) knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells.

Nuclear factor kappa-B (NF-κB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-κB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-κB activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-κB and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-κB p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-κB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-κB to chronic inflammation and neoplasia.

Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.

Helicobacter pylori are bacteria that colonize the stomach persistently, which confers risk of serious diseases, including peptic ulceration and gastric neoplasia. Aberrant expression of cell cycle control proteins has been demonstrated in H. pylori infected gastric epithelial cells, suggesting that perturbation of the cell cycle plays a role in the pathogenesis of various H. pylori associated diseases. In this study, we investigate the modulation of the cell cycle control protein p21(WAF1) by H. pylori in the gastric carcinoma cell line NCI-N87 and in primary gastric cells derived from healthy tissue. We observed an up-regulation of p21(WAF1) in both NCI-N87 and primary cells. Chromatin immunoprecipitation analysis revealed that the increased expression of p21(WAF1) induced by H. pylori is associated with the release of HDAC-1 from the p21(WAF1) promoter and hyper-acetylation of histone H4. The elucidation of the epigenetic regulation of p21(WAF1) by H. pylori may help to dissect the pathogenetic mechanisms underlying the development and progression of H. pylori associated diseases.

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.

Deubiquitinylases (DUBs) are highly specialized enzymes which are responsible for removal of covalently attached ubiquitin(s) from the targeted proteins. DUBs play an important role in maintaining the protein homeodynamics. Recently, DUBs have emerged as novel therapeutic targets in cancer, inflammation, diabetes, and neurodegeneration. Among the different families of DUBs, the metalloprotease group or JAB1/MOV34/MPR1 (JAMMs) proteases are unique in terms of catalytic mechanism. JAMMs exhibit a Zn2+-dependent deubiquitinylase activity. Within the JAMM family, deubiquitinylases Rpn11 and CSN5 are constituents of large bimolecular complexes, namely the 26S proteasome and COP9 signalosome (CSN), respectively. Rpn11 and CSN5 are potential drug targets in cancer and selective inhibitors of both proteins have been reported in the literature. However, the selectivity of JAMM inhibitors (capzimin for RPN11 and CSN5i-3 for CSN5) has not been structurally resolved yet. In the present work, we have explored the binding modes of capzimin and CSN5i-3 and rationalize their selectivity for Rpn11 and CSN5 targets. We found that capzimin interacts with the active site Zn+2 of Rpn11 in a bidentate manner and also interacts with the residues in the distal ubiquitin binding site. MD simulations studies and binding energy analysis revealed that the selective binding of the inhibitors can be only explained by the consideration of larger heterodimeric complexes of Rpn11 (Rpn8-Rpn11) and CSN5 (CSN5-CSN6). Simulation of these protein-protein complexes is necessary to avoid unrealistic large conformational changes. The selective binding of inhibitors is mainly governed by residues in the distal ubiquitin binding site. This study demonstrates that selective inhibitor binding design for Rpn11 and CSN5 JAMM proteases requires consideration of heterodimeric protein-protein target structures.

Helicobacter (H.) pylori is an important risk factor for gastric malignancies worldwide. Its outer membrane proteome takes an important role in colonization of the human gastric mucosa. However, in zoonotic non-H. pylori helicobacters (NHPHs) also associated with human gastric disease, the composition of the outer membrane (OM) proteome and its relative contribution to disease remain largely unknown. By means of a comprehensive survey of the diversity and distribution of predicted outer membrane proteins (OMPs) identified in all known gastric Helicobacter species with fully annotated genome sequences, we found genus- and species-specific families known or thought to be implicated in virulence. Hop adhesins, part of the Helicobacter-specific family 13 (Hop, Hor and Hom) were restricted to the gastric species H. pylori, H. cetorum and H. acinonychis. Hof proteins (family 33) were putative adhesins with predicted Occ- or MOMP-family like 18-stranded β-barrels. They were found to be widespread amongst all gastric Helicobacter species only sporadically detected in enterohepatic Helicobacter species. These latter are other members within the genus Helicobacter, although ecologically and genetically distinct. LpxR, a lipopolysaccharide remodeling factor, was also detected in all gastric Helicobacter species but lacking as well from the enterohepatic species H. cinaedi, H. equorum and H. hepaticus. In conclusion, our systemic survey of Helicobacter OMPs points to species and infection-site specific members that are interesting candidates for future virulence and colonization studies.

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.

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.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrA Hp ) belongs to the well conserved family of serine proteases. HtrA Hp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrA Hp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrA Hp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrA Hp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrA Hp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrA Hp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrA Hp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.

The stomach bacterium Helicobacter pylori is one of the most prevalent pathogens in humans, closely linked with serious diseases such as gastric cancer. The microbe has been associated with its host for more than 100,000 years and escorted modern humans out of Africa. H. pylori is predominantly transmitted within families and dispersed globally, resulting in distinct phylogeographic patterns, which can be utilized to investigate migrations and bioturbation events in human history. Latin America was affected by several human migratory waves due to the Spanish colonisation that drastically changed the genetic load and composition of the bacteria and its host. Genetic evidence indicates that independent evolutionary lines of H. pylori have evolved in mestizos from Colombia and other countries in the region during more than 500 years since colonisation. The vacuolating cytotoxin VacA represents a major virulence factor of the pathogen comprising two domains, p33 and p55, the latter of which is essential for binding to the host epithelial cell. The evolution of the VacA toxin in Colombia has been strongly biased due to the effects of Spanish colonization. However, the variation patterns and microevolution of the p55 domain have not yet been described for this population. In the present study, we determined the genetic polymorphisms and deviations in the neutral model of molecular evolution in the p55 domain of 101 clinical H. pylori isolates collected in Bogotá, a city located in Andean mountains characterized by its high gastric cancer risk and its dominant mestizo population. The microevolutionary patterns of the p55 domain were shaped by recombination, purifying and episodic diversifying positive selection. Furthermore, amino acid positions 261 and 321 in the p55 domain of VacA show a high variability among mestizos clinical subsets, suggesting that natural selection in H. pylori may operate differentially in patients with different gastric diseases.

The cell adhesion molecule neuroplastin (Np) is a novel candidate to influence human intelligence. Np-deficient mice display complex cognitive deficits and reduced levels of Plasma Membrane Ca2+ ATPases (PMCAs), an essential regulator of the intracellular Ca2+ concentration ([iCa2+]) and neuronal activity. We show abundant expression and conserved cellular and molecular features of Np in glutamatergic neurons in human hippocampal-cortical pathways as characterized for the rodent brain. In Nptn lox/loxEmx1Cre mice, glutamatergic neuron-selective Np ablation resulted in behavioral deficits indicating hippocampal, striatal, and sensorimotor dysfunction paralleled by highly altered activities in hippocampal CA1 area, sensorimotor cortex layers I-III/IV, and the striatal sensorimotor domain detected by single-photon emission computed tomography. Altered hippocampal and cortical activities correlated with reduction of distinct PMCA paralogs in Nptn lox/loxEmx1Cre mice and increased [iCa2+] in cultured mutant neurons. Human and rodent Np enhanced the post-transcriptional expression of and co-localized with PMCA paralogs in the plasma membrane of transfected cells. Our results indicate Np as essential for PMCA expression in glutamatergic neurons allowing proper [iCa2+] regulation and normal circuit activity. Neuron-type-specific Np ablation empowers the investigation of circuit-coded learning and memory and identification of causal mechanisms leading to cognitive deterioration.

Campylobacter jejuni is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA Cj ), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA Cj using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA Cj by comparison to the HtrA counterparts from Helicobacter pylori and Escherichia coli. We defined the architecture of HtrA Cj at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA Cj . Together, this is the first report on the in-depth functional and structural analysis of HtrA Cj , which may allow the construction of therapeutically relevant HtrA Cj inhibitors in the near future.

The gastric pathogen Helicobacter pylori colonizes approximately half of the human world population. The bacterium injects the effector protein cytotoxin associated gene A (CagA) via a type-IV secretion system into host epithelial cells, where the protein becomes phosphorylated at specific EPIYA-motifs by cellular kinases. Inside the host cell, CagA can interact with over 25 different proteins in both phosphorylation-dependent and phosphorylation-independent manners, resulting in manipulation of host-cell signaling pathways. During the course of an H. pylori infection, certain host-cell proteins undergo tyrosine dephosphorylation in a CagA-dependent manner, including the actin-binding proteins cortactin and vinculin. A predominant response of intracellular CagA is the binding and activation of tyrosine phosphatase, the human Src-homology-region-2-domain-containing-phosphatase-2 (SHP2). Here, we considered the possibility that activated SHP2 might be responsible for the dephosphorylation of cortactin and vinculin. To investigate this, phosphatase inhibitor studies were performed. Additionally, a complete knockout mutant of SHP2 in AGS cells was created by CRISPR/Cas9 technology, and these cells were infected with H. pylori. However, neither the presence of an inhibitor nor the inactivation of SHP2 prevented the dephosphorylation of cortactin and vinculin upon CagA delivery. Tyrosine dephosphorylation of these proteins is therefore independent of SHP2 and instead must be caused by another, as yet unidentified, protein tyrosine phosphatase.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5. Here, we show that increased autophagy in Bassoon knockout neurons depends on poly-ubiquitination and that the loss of Bassoon leads to elevated levels of ubiquitinated synaptic proteins per se. Our data show that Bassoon knockout neurons have a smaller SV pool size and a higher turnover rate as indicated by a younger pool of SV2. The E3 ligase Parkin is required for increased autophagy in Bassoon-deficient neurons as the knockdown of Parkin normalized autophagy and SV protein levels and rescued impaired SV recycling. These data indicate that Bassoon is a key regulator of SV proteostasis and that Parkin is a key E3 ligase in the autophagy-mediated clearance of SV proteins.