CagY Is an Immune-Sensitive Regulator of the Helicobacter pylori Type IV Secretion System.

BACKGROUND & AIMS: Peptic ulcer disease and gastric cancer are caused most often by Helicobacter pylori strains that harbor the cag pathogenicity island, which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host cells. cagY is an essential gene in the T4SS and has an unusual DNA repeat structure that predicts in-frame insertions and deletions. These cagY recombination events typically lead to a reduction in T4SS function in mouse and primate models. We examined the role of the immune response in cagY-dependent modulation of T4SS function. METHODS: H pylori T4SS function was assessed by measuring CagA translocation and the capacity to induce interleukin (IL)8 in gastric epithelial cells. cagY recombination was determined by changes in polymerase chain reaction restriction fragment-length polymorphisms. T4SS function and cagY in H pylori from C57BL/6 mice were compared with strains recovered from Rag1-/- mice, T- and B-cell-deficient mice, mice with deletion of the interferon gamma receptor (IFNGR) or IL10, and Rag1-/- mice that received adoptive transfer of control or Ifng-/- CD4+ T cells. To assess relevance to human beings, T4SS function and cagY recombination were assessed in strains obtained sequentially from a patient after 7.4 years of infection. RESULTS: H pylori infection of T-cell-deficient and Ifngr1-/- mice, and transfer of CD4+ T cells to Rag1-/- mice, showed that cagY-mediated loss of T4SS function requires a T-helper 1-mediated immune response. Loss of T4SS function and cagY recombination were more pronounced in Il10-/- mice, and in control mice infected with H pylori that expressed a more inflammatory form of cagY. Complementation analysis of H pylori strains isolated from a patient over time showed changes in T4SS function that were dependent on recombination in cagY. CONCLUSIONS: Analysis of H pylori strains from mice and from a chronically infected patient showed that CagY functions as an immune-sensitive regulator of T4SS function. We propose that this is a bacterial adaptation to maximize persistent infection and transmission to a new host under conditions of a robust inflammatory response.

Implementing an Undergraduate Learning Assistant Program Tailored for Remote Instruction.

The inclusion of undergraduate learning assistants (LAs) on the teaching team of a course is a high-impact practice that benefits the teaching team, students, and LAs themselves. LAs are undergraduate students who have taken the course previously and support student learning through facilitated discussion and problem-solving. Unfortunately, in the quick pivot to emergency remote instruction and lacking an online model for LA programs, some instructors temporarily discontinued or scaled down their LA programs. This report describes the recruitment, training, and roles of LAs who were engaged to support a high-enrollment, interactive lecture course delivered by emergency remote instruction. This case study can serve as a model to inform the implementation of a robust online LA program.

Maintenance of Type IV Secretion Function During Helicobacter pylori Infection in Mice.

The Helicobacter pylori type IV secretion system (T4SS) encoded on the cag pathogenicity island (cagPAI) secretes the CagA oncoprotein and other effectors into the gastric epithelium. During murine infection, T4SS function is lost in an immune-dependent manner, typically as a result of in-frame recombination in the middle repeat region of cagY, though single nucleotide polymorphisms (SNPs) in cagY or in other essential genes may also occur. Loss of T4SS function also occurs in gerbils, nonhuman primates, and humans, suggesting that it is biologically relevant and not simply an artifact of the murine model. Here, we sought to identify physiologically relevant conditions under which T4SS function is maintained in the murine model. We found that loss of H. pylori T4SS function in mice was blunted by systemic Salmonella coinfection and completely eliminated by dietary iron restriction. Both have epidemiologic parallels in humans, since H. pylori strains from individuals in developing countries, where iron deficiency and systemic infections are common, are also more often cagPAI+ than strains from developed countries. These results have implications for our fundamental understanding of the cagPAI and also provide experimental tools that permit the study of T4SS function in the murine model.IMPORTANCE The type IV secretion system (T4SS) is the major Helicobacter pylori virulence factor, though its function is lost during murine infection. Loss of function also occurs in gerbils and in humans, suggesting that it is biologically relevant, but the conditions under which T4SS regulation occurs are unknown. Here, we found that systemic coinfection with Salmonella and iron deprivation each promote retention of T4SS function. These results improve our understanding of the cag pathogenicity island (cagPAI) and provide experimental tools that permit the study of T4SS function in the murine model.

CagY-Dependent Regulation of Type IV Secretion in Helicobacter pylori Is Associated with Alterations in Integrin Binding.

Strains of Helicobacter pylori that cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4+ T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human α5ß1 integrin, we tested the hypothesis that recombination in the CagY MRR regulates T4SS function by modulating binding to α5ß1 integrin. Using a cell-free microfluidic assay, we found that H. pylori binding to α5ß1 integrin under shear flow is dependent on the CagY MRR, but independent of the presence of the T4SS pili, which are only formed when H. pylori is in contact with host cells. Similarly, expression of CagY in the absence of other T4SS genes was necessary and sufficient for whole bacterial cell binding to α5ß1 integrin. Bacteria with variant cagY alleles that reduced T4SS function showed comparable reduction in binding to α5ß1 integrin, although CagY was still expressed on the bacterial surface. We speculate that cagY-dependent modulation of H. pylori T4SS function is mediated by alterations in binding to α5ß1 integrin, which in turn regulates the host inflammatory response so as to maximize persistent infection.IMPORTANCE Infection with H. pylori can cause peptic ulcers and is the most important risk factor for gastric cancer, the third most common cause of cancer death worldwide. The major H. pylori virulence factor that determines whether infection causes disease or asymptomatic colonization is the type IV secretion system (T4SS), a sort of molecular syringe that injects bacterial products into gastric epithelial cells and alters host cell physiology. We previously showed that recombination in CagY, an essential T4SS component, modulates the function of the T4SS. Here we found that these recombination events produce parallel changes in specific binding to α5ß1 integrin, a host cell receptor that is essential for T4SS-dependent translocation of bacterial effectors. We propose that CagY-dependent binding to α5ß1 integrin acts like a molecular rheostat that alters T4SS function and modulates the host immune response to promote persistent infection.

Evidence for a primate origin of zoonotic Helicobacter suis colonizing domesticated pigs.

Helicobacter suis is the second most prevalent Helicobacter species in the stomach of humans suffering from gastric disease. This bacterium mainly inhabits the stomach of domesticated pigs, in which it causes gastric disease, but it appears to be absent in wild boars. Interestingly, it also colonizes the stomach of asymptomatic rhesus and cynomolgus monkeys. The origin of modern human-, pig- or non-human primate-associated H. suis strains in these respective host populations was hitherto unknown. Here we show that H. suis in pigs possibly originates from non-human primates. Our data suggest that a host jump from macaques to pigs happened between 100 000 and 15 000 years ago and that pig domestication has had a significant impact on the spread of H. suis in the pig population, from where this pathogen occasionally infects humans. Thus, in contrast to our expectations, H. suis appears to have evolved in its main host in a completely different way than its close relative Helicobacter pylori in humans.

Host Determinants of Expression of the Helicobacter pylori BabA Adhesin.

Expression of the Helicobacter pylori blood group antigen binding adhesin A (BabA) is more common in strains isolated from patients with peptic ulcer disease or gastric cancer, rather than asymptomatic colonization. Here we used mouse models to examine host determinants that affect H. pylori BabA expression. BabA expression was lost by phase variation as frequently in WT mice as in RAG2-/- mice that do not have functional B or T cells, and in MyD88-/-, TLR2-/- and TLR4-/- mice that are defective in toll like receptor signaling. The presence of other bacteria had no effect on BabA expression as shown by infection of germ free mice. Moreover, loss of BabA expression was not dependent on Leb expression or the capacity of BabA to bind Leb. Surprisingly, gender was the host determinant most associated with loss of BabA expression, which was maintained to a greater extent in male mice and was associated with greater bacterial load. These results suggest the possibility that loss of BabA expression is not driven by adaptive immunity or toll-like receptor signaling, and that BabA may have other, unrecognized functions in addition to serving as an adhesin that binds Leb.

The gastric microbial community, Helicobacter pylori colonization, and disease.

Long thought to be a sterile habitat, the stomach contains a diverse and unique community of bacteria. One particular inhabitant, Helicobacter pylori, colonizes half of the world's human population and establishes a decades-long infection that can be asymptomatic, pathogenic, or even beneficial for the host. Many host and bacterial factors are known to influence an individual's risk of gastric disease, but another potentially important determinant has recently come to light: the host microbiota. Although it is unclear to what extent H. pylori infection perturbs the established gastric microbial community, and H. pylori colonization seems generally resistant to disturbances in the host microbiota, it can modulate H. pylori pathogenicity. Interactions between H. pylori and bacteria at non-gastric sites are likely indirect--via programming of the pro-inflammatory vs. regulatory T lymphocytes--which may have a significant impact on human health.

The impact of Helicobacter pylori infection on the gastric microbiota of the rhesus macaque.

Helicobacter pylori colonization is highly prevalent among humans and causes significant gastric disease in a subset of those infected. When present, this bacterium dominates the gastric microbiota of humans and induces antimicrobial responses in the host. Since the microbial context of H. pylori colonization influences the disease outcome in a mouse model, we sought to assess the impact of H. pylori challenge upon the pre-existing gastric microbial community members in the rhesus macaque model. Deep sequencing of the bacterial 16S rRNA gene identified a community profile of 221 phylotypes that was distinct from that of the rhesus macaque distal gut and mouth, although there were taxa in common. High proportions of both H. pylori and H. suis were observed in the post-challenge libraries, but at a given time, only one Helicobacter species was dominant. However, the relative abundance of non-Helicobacter taxa was not significantly different before and after challenge with H. pylori. These results suggest that while different gastric species may show competitive exclusion in the gastric niche, the rhesus gastric microbial community is largely stable despite immune and physiological changes due to H. pylori infection.

Predicting the outcome of infectious diseases: variability among inbred mice as a new and powerful tool for biomarker discovery.

Individuals respond differently to infectious diseases. Even among inbred mice that are presumed to be genetically identical, the response to a microbial pathogen is variable, which is generally thought to reflect experimental inconsistencies, technical errors, and stochastic processes. Here we describe the remarkable observation that the variability of Helicobacter pylori colonization density in the stomachs of experimentally infected C57BL/6J mice is tightly correlated with weight loss and viral load after a challenge with influenza virus, though H. pylori infection per se does not affect influenza and vice versa. Since these two infectious agents are found in different tissue compartments and are detected using unrelated methods, the correlation in microbial burden must represent a biological measure of disease susceptibility among genetically nearly identical individuals and not technical or stochastic factors. We hypothesize that inbred mice represent a powerful new tool for the identification of biomarkers to predict the outcome of infectious diseases.

Global gene expression patterns of Nostoc punctiforme in steady-state dinitrogen-grown heterocyst-containing cultures and at single time points during the differentiation of akinetes and hormogonia.

The vegetative cells of the filamentous cyanobacterium Nostoc punctiforme can differentiate into three mutually exclusive cell types: nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogomium filaments. A DNA microarray consisting of 6,893 N. punctiforme genes was used to identify the global transcription patterns at single time points in the three developmental states, compared to those in ammonium-grown time zero cultures. Analysis of ammonium-grown cultures yielded a transcriptome of 2,935 genes, which is nearly twice the size of a soluble proteome. The NH(4)(+)-grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N(2)-grown (heterocyst-containing) culture showed differential transcription of 495 genes, 373 of which were up-regulated. The majority of the up-regulated genes were predicted from studies of heterocyst differentiation and N(2) fixation; other genes are candidates for more detailed genetic analysis. Three days into the developmental process, akinetes showed a similar number of differentially expressed genes (497 genes), which were equally up- and down-regulated. The down-regulated genes were enriched in core metabolic functions, consistent with entry into a nongrowth state. There were relatively few adaptive genes up-regulated in 3-day akinetes, and there was little overlap with putative heterocyst developmental genes. There were 1,827 differentially transcribed genes in 24-h hormogonia, which was nearly fivefold greater than the number in akinete-forming or N(2)-fixing cultures. The majority of the up-regulated adaptive genes were genes encoding proteins for signal transduction and transcriptional regulation, which is characteristic of a motile filament that is poised to sense and respond to the environment. The greatest fraction of the 883 down-regulated genes was involved in core metabolism, also consistent with entry into a nongrowth state. The differentiation of heterocysts (steady state, N(2) grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.

Cell cycle-dependent abundance, stability and localization of FtsA and FtsQ in Caulobacter crescentus.

Coordination between cell division and DNA replication is ensured by checkpoints that act through proteins required for cell division. Following a block in DNA replication, transcription of the cell division progression genes ftsA and ftsQ is prevented in Caulobacter crescentus. One requirement for this checkpoint is that FtsA and/or FtsQ should be limiting for division in the next cell cycle. We show that the number of FtsA and FtsQ molecules fluctuates such that their concentration is low in swarmer and stalked cells, peaks in pre-divisional cells, and then dramatically decreases after cell division. Despite constitutive expression from an inducible promoter, FtsA and FtsQ levels still vary during the cell cycle, and the half-life of FtsA increases from 13 min in swarmer cells to 55 min in stalked cell types, confirming cell type-specific degradation. The post-division degradation of FtsA and FtsQ in swarmer cells reduces their concentration to 7% and 10% of their maximal level, respectively, strongly suggesting that de novo synthesis of both proteins is required for each division cycle. The localization of FtsA and FtsQ is also cell type-specific. FtsA and FtsQ are recruited to the midcell during a short period in late pre-divisional cells, consistent with the demonstrated requirement of FtsA for late stages of cell division. As previously reported for FtsZ, constitutive expression of FtsA causes cell division defects. These results indicate that the tight control of FtsA, and probably FtsQ, by cell cycle transcription, proteolysis, and localization are critical for optimal cell division and the coordination of cell division with the DNA replication cycle.