Search

H elicobacter pylori establishes a chronic lifelong infection in the human gastric mucosa, which may lead to peptic ulcer disease or gastric adenocarcinoma. The human beta-defensins (hβ Ds) are antimicrobial peptides, hβ D1 being constitutively expressed in the human stomach. We hypothesized that H . pylori may persist, in part, by downregulating gastric hβ D1 expression. We measured hβ D1 and hβ D2 expression in vivo in relation to the presence, density and severity of H . pylori infection, investigated differential effects of H . pylori virulence factors, and studied underlying signalling mechanisms in vitro. Significantly lower hβ D1 and higher hβ D2 mRNA and protein concentrations were present in gastric biopsies from infected patients. Those patients with higher-level bacterial colonization and inflammation had significantly lower hβ D1 expression, but there were no differences in hβ D2. H . pylori infection of human gastric epithelial cell lines also downregulated hβ D1. Using wild-type strains and isogenic mutants, we showed that a functional cag pathogenicity island-encoded type IV secretion system induced this downregulation. Treatment with chemical inhibitors or siRNA revealed that H . pylori usurped NF-κ B signalling to modulate hβ D1 expression. These data indicate that H . pylori downregulates hβ D1 expression via NF-κ B signalling, and suggest that this may promote bacterial survival and persistence in the gastric niche. [ABSTRACT FROM AUTHOR]

Abstract Background 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. Results In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria. Conclusion These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. Conclusion Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.

Abstract Helicobacter pylori is a highly successful pathogen uniquely adapted to colonize humans. Gastric infections with this bacterium can induce pathology ranging from chronic gastritis and peptic ulcers to gastric cancer. More virulent H. pylori isolates harbour numerous well-known adhesins (BabA/B, SabA, AlpA/B, OipA and HopZ) and the cag (cytotoxin-associated genes) pathogenicity island encoding a type IV secretion system (T4SS). The adhesins establish tight bacterial contact with host target cells and the T4SS represents a needle-like pilus device for the delivery of effector proteins into host target cells such as CagA. BabA and SabA bind to blood group antigen and sialylated proteins respectively, and a series of T4SS components including CagI, CagL, CagY and CagA have been shown to target the integrin β1 receptor followed by injection of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine may also play a role in the delivery process. While substantial progress has been made in our current understanding of many of the above factors, the host cell receptors for OipA, HopZ and AlpA/B during infection are still unknown. Here we review the recent progress in characterizing the interactions of the various adhesins and structural T4SS proteins with host cell factors. The contribution of these interactions to H. pylori colonization and pathogenesis is discussed.

Abstract Background To simplify the methodology for the isolation of Campylobacter spp. from retail broiler meat, we evaluated 108 samples (breasts and thighs) using an unpaired sample design. The enrichment broths were incubated under aerobic conditions (subsamples A) and for comparison under microaerobic conditions (subsamples M) as recommended by current reference protocols. Sensors were used to measure the dissolved oxygen (DO) in the broth and the percentage of oxygen (O2) in the head space of the bags used for enrichment. Campylobacter isolates were identified with multiplex PCR assays and typed using pulsed-field gel electrophoresis (PFGE). Ribosomal intergenic spacer analyses (RISA) and denaturing gradient gel electrophoresis (DGGE) were used to study the bacterial communities of subsamples M and A after 48 h enrichment. Results The number of Campylobacter positive subsamples were similar for A and M when all samples were combined (P = 0.81) and when samples were analyzed by product (breast: P = 0.75; thigh: P = 1.00). Oxygen sensors showed that DO values in the broth were around 6 ppm and O2 values in the head space were 14-16% throughout incubation. PFGE demonstrated high genomic similarity of isolates in the majority of the samples in which isolates were obtained from subsamples A and M. RISA and DGGE results showed a large variability in the bacterial populations that could be attributed to sample-to-sample variations and not enrichment conditions (aerobic or microaerobic). These data also suggested that current sampling protocols are not optimized to determine the true number of Campylobacter positive samples in retail boiler meat. Conclusions Decreased DO in enrichment broths is naturally achieved. This simplified, cost-effective enrichment protocol with aerobic incubation could be incorporated into reference methods for the isolation of Campylobacter spp. from retail broiler meat.

The role of microbiota in human health and disease is becoming increasingly clear as a result of modern microbiome studies in recent decades. The gastrointestinal tract is the major habitat for microbiota in the human body. This microbiota comprises several trillion microorganisms, which is equivalent to almost ten times the total number of cells of the human host. Helicobacter pylori is a known pathogen that colonizes the gastric mucosa of almost half of the world population. H. pylori is associated with several gastric diseases, including gastric cancer (GC) development. However, the impact of the gastric microbiota in the colonization, chronic infection, and pathogenesis is still not fully understood. Several studies have documented qualitative and quantitative changes in the microbiota's composition in the presence or absence of this pathogen. Among the diverse microflora in the stomach, the Firmicutes represent the most notable. Bacteria such as Prevotella sp., Clostridium sp., Lactobacillus sp., and Veillonella sp. were frequently found in the healthy human stomach. In contrast, H . pylori is very dominant during chronic gastritis, increasing the proportion of Proteobacteria in the total microbiota to almost 80%, with decreasing relative proportions of Firmicutes. Likewise, H. pylori and Streptococcus are the most abundant bacteria during peptic ulcer disease. While the development of H. pylori -associated intestinal metaplasia is accompanied by an increase in Bacteroides, the stomachs of GC patients are dominated by Firmicutes such as Lactobacillus and Veillonella , constituting up to 40% of the total microbiota, and by Bacteroidetes such as Prevotella , whereas the numbers of H. pylori are decreasing. This review focuses on some of the consequences of changes in the gastric microbiota and the function of probiotics to modulate H. pylori infection and dysbiosis in general.

Background: Helicobacter pylori is a persistent pathogen in the human stomach. However, the proposed transmission route via the oral cavity is not understood and under intense debate. While dozens of studies have shown by PCR that H. pylori DNA is frequently present in the oral cavity, data on the growth and characterization of viable H. pylori from this compartment are very scarce, and it is unclear whether the bacteria can survive in the oral cavity for longer time periods or even colonize it., Methods: Selective growth methods, scanning electron microscopy, urease assay, Western blotting, PCR, and gene sequencing were applied to identify and examine viable H. pylori in decayed milk teeth., Results: Here, we studied viable H. pylori in the plaque and root canals of 170 endodontically infected deciduous teeth that were extracted from 54 children. While H. pylori DNA was detected in several plaque and many root canal samples by PCR, live bacteria could only be cultivated from 28 root canals, but not from plaque. These 28 isolates have been identified as H. pylori by PCR and sequencing of vacA, cagA and htrA genes, phylogenetic analyses, protein expression of major H. pylori virulence factors, and by signal transduction events during infection of human cell lines., Conclusions: Thus, the microaerobic environment in the root canals of endodontically infected teeth may represent a protected and transient reservoir for live H. pylori, especially in individuals with poor dental hygiene, which could serve as a potential source for re-infection of the stomach after antibiotic therapy or for transmission to other individuals., Competing Interests: Declarations. Ethics approval and consent to participate: The study protocol was approved by the Ethics Committee of the University of Leipzig under the number 019/13-ff. Written informed consent was obtained from the patients' parents regarding the planned extraction of primary teeth and the medical necessity of the procedure. Consent for publication: The patient’s parents signed informed consent regarding publishing the picture from their child (Fig. 1). Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) that is associated with increased risk of developing colitis-associated carcinoma (CAC). The genetic profile of CACs is fairly similar to the sporadic colorectal carcinomas (sCRCs), although showing certain differences in the timing and sequence of alterations that contribute to carcinogenesis. Also, both cancer types typically show a strong histological resemblance, which complicates the pathologists' diagnosis. Due to the different clinical consequences, it is of utmost importance to categorize the corresponding cancer type correctly., Methods: In this study, we determined the mutation profiles of 64 CACs and sCRCs in the hotspot regions of 50 cancer-associated genes and compared them to 29 controls to identify genetic gene variants that can facilitate the pathologists' diagnosis. Pearson Chi-Square or Fisher's exact tests were used for statistical analyses., Results: We found that sCRCs tend to mutate more frequently in APC and PIK3CA genes than CACs and that mainly males were affected. Our CAC cohort identified the KRAS G12D mutation as group-specific variant that was not detected in the sCRCs. When separating conventional from non-conventional CACs, it was discovered that the conventional type shows significantly more mutations for ATM., Conclusions: Taken together, our data highlights genetic differences between sCRC and CAC and enables the possibility to utilize specific gene alterations to support the pathologist's diagnosis., (© 2024. The Author(s).)

Several single-nucleotide polymorphisms (SNPs) in human chromosomes are known to predispose to cancer. However, cancer-associated SNPs in bacterial pathogens were unknown until discovered in the stomach pathogen Helicobacter pylori. Those include an alanine-threonine polymorphism in the EPIYA-B phosphorylation motif of the injected effector protein CagA that affects cancer risk by modifying inflammatory responses and loss of host cell polarity. A serine-to-leucine change in serine protease HtrA is associated with boosted proteolytic cleavage of epithelial junction proteins and introduction of DNA double-strand breaks (DSBs) in host chromosomes, which co-operatively elicit malignant alterations. In addition, H. pylori genome-wide association studies (GWAS) identified several other SNPs potentially associated with increased gastric cancer (GC) risk. Here we discuss the clinical importance, evolutionary origin, and functional advantage of the H. pylori SNPs. These exciting new data highlight cancer-associated SNPs in bacteria, which should be explored in more detail in future studies., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many viral, protozoal, and fungal pathogens represent major human and animal health problems due to their great potential of causing infectious diseases. Research on these pathogens has contributed substantially to our current understanding of both microbial virulence determinants and host key factors during infection. Countless studies have also shed light on the molecular mechanisms of host-pathogen interactions that are employed by these microbes. For example, actin cytoskeletal dynamics play critical roles in effective adhesion, host cell entry, and intracellular movements of intruding pathogens. Cortactin is an eminent host cell protein that stimulates actin polymerization and signal transduction, and recently emerged as fundamental player during host-pathogen crosstalk. Here we review the important role of cortactin as major target for various prominent viral, protozoal and fungal pathogens in humans, and its role in human disease development and cancer progression. Most if not all of these important classes of pathogens have been reported to hijack cortactin during infection through mediating up- or downregulation of cortactin mRNA and protein expression as well as signaling. In particular, pathogen-induced changes in tyrosine and serine phosphorylation status of cortactin at its major phospho-sites (Y-421, Y-470, Y-486, S-113, S-298, S-405, and S-418) are addressed. As has been reported for various Gram-negative and Gram-positive bacteria, many pathogenic viruses, protozoa, and fungi also control these regulatory phospho-sites, for example, by activating kinases such as Src, PAK, ERK1/2, and PKD, which are known to phosphorylate cortactin. In addition, the recruitment of cortactin and its interaction partners, like the Arp2/3 complex and F-actin, to the contact sites between pathogens and host cells is highlighted, as this plays an important role in the infection process and internalization of several pathogens. However, there are also other ways in which the pathogens can exploit the function of cortactin for their needs, as the cortactin-mediated regulation of cellular processes is complex and involves numerous different interaction partners. Here, the current state of knowledge is summarized., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Single nucleotide polymorphisms (SNPs) account for significant genomic variability in microbes, including the highly diverse gastric pathogen Helicobacter pylori. However, data on the effects of specific SNPs in pathogen-host interactions are scarce. Recent functional studies unravelled how a serine/leucine polymorphism in serine protease HtrA affects the formation of proteolytically active trimers and modulates cleavage of host cell-to-cell junction proteins during infection. A similar serine/leucine mutation in the carbohydrate binding domain of the adhesin BabA controls binding of ABO blood group antigens, enabling binding of either only the short Lewis b/H antigens of blood group O or also the larger antigens of blood groups A and B. Here we summarize the functional importance of these two remarkable bacterial SNPs and their effect on the outcome of pathogen-host interactions., (© 2024. The Author(s).)

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 ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni .

Helicobacter pylori is a paradigm of chronic bacterial infection and is associated with peptic ulceration and malignancies. H. pylori uses specific masking mechanisms to avoid canonical ligands from activating Toll-like receptors (TLRs), such as lipopolysaccharide (LPS) modification and specific flagellin sequences that are not detected by TLR4 and TLR5, respectively. Thus, it was believed for a long time that H. pylori evades TLR recognition as a crucial strategy for immune escape and bacterial persistence. However, recent data indicate that multiple TLRs are activated by H. pylori and play a role in the pathology. Remarkably, H. pylori LPS, modified through changes in acylation and phosphorylation, is mainly sensed by other TLRs (TLR2 and TLR10) and induces both pro- and anti-inflammatory responses. In addition, two structural components of the cag pathogenicity island-encoded type IV secretion system (T4SS), CagL and CagY, were shown to contain TLR5-activating domains. These domains stimulate TLR5 and enhance immunity, while LPS-driven TLR10 signaling predominantly activates anti-inflammatory reactions. Here, we discuss the specific roles of these TLRs and masking mechanisms during infection. Masking of typical TLR ligands combined with evolutionary shifting to other TLRs is unique for H. pylori and has not yet been described for any other species in the bacterial kingdom. Finally, we highlight the unmasked T4SS-driven activation of TLR9 by H. pylori, which mainly triggers anti-inflammatory responses., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Single-nucleotide polymorphisms (SNPs) in various human genes are key factors in carcinogenesis. However, whether SNPs in bacterial pathogens are similarly crucial in cancer development is unknown. Here, we analyzed 1,043 genomes of the stomach pathogen Helicobacter pylori and pinpointed a SNP in the serine protease HtrA (position serine/leucine 171) that significantly correlates with gastric cancer. Our functional studies reveal that the 171S-to-171L mutation triggers HtrA trimer formation and enhances proteolytic activity and cleavage of epithelial junction proteins occludin and tumor-suppressor E-cadherin. 171L-type HtrA, but not 171S-HtrA-possessing H. pylori, inflicts severe epithelial damage, enhances injection of oncoprotein CagA into epithelial cells, increases NF-κB-mediated inflammation and cell proliferation through nuclear accumulation of β-catenin, and promotes host DNA double-strand breaks, collectively triggering malignant changes. These findings highlight the 171S/L HtrA mutation as a unique bacterial cancer-associated SNP and as a potential biomarker for risk predictions in H. pylori infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Fusobacterium nucleatum is supposed to play a critical role in the development of colorectal cancer. The species has also been associated with ulcerative colitis (UC) that can progress into colorectal cancer, however, the involvement of bacteria in this process remains unclear. We analysed 177 colon biopsies obtained from patients during screening, including 20 healthy controls, 56 UC cases and 69 cases at different stages of progression to colitis-associated cancer (CAC); 32 samples of sporadic colorectal carcinoma (sCRC) were also included. The presence of F. nucleatum was detected by quantitative real-time PCR (qPCR). Our data show an association between the presence of the bacteria and the progression of carcinogenesis in UC patients. In 39.5% of CAC samples F. nucleatum was detected, compared to only 1.8% in UC cases. The bacteria were detected in 6.3% of samples with initial neoplastic transformation, so-called low-grade dysplasia (LGD), whereas high-grade dysplasia (HGD) resulted in 33.3% of samples positive for F. nucleatum. The fraction of F. nucleatum-positive samples from sCRC cases was 56.3%, which was not significantly different to the CAC group. We conclude that F. nucleatum is associated with the occurrence and progression of colon carcinogenesis, rather than with UC itself., (© 2023. The Author(s).)