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Jeffrey T Loh,1 Andrea L Nicol,1 David Elashoff,2 F Michael Ferrante,1 1Department of Anesthesiology, David Geffen School of Medicine, 2Department of Biomathematics, University of California Los Angeles, Los Angeles, CA, USA Background: Many studies have assessed the efficacy of radiofrequency ablation to denervate the facet joint as an interventional means of treating axial low-back pain. In these studies, varying procedural techniques were utilized to ablate the nerves that innervate the facet joints. To date, no comparison studies have been performed to suggest superiority of one technique or even compare the prevalence of side effects and complications. Materials and methods: A retrospective chart review was performed on patients who underwent a lumbar facet denervation procedure. Each patient's chart was analyzed for treatment technique (early versus advanced Australian), preprocedural visual numeric scale (VNS) score, postprocedural VNS score, duration of pain relief, and complications. Results: Pre- and postprocedural VNS scores and change in VNS score between the two groups showed no significant differences. Patient-reported benefit and duration of relief was greater in the advanced Australian technique group (P=0.012 and 0.022, respectively). The advanced Australian technique group demonstrated a significantly greater median duration of relief (4 months versus 1.5 months, P=0.022). Male sex and no pain-medication use at baseline were associated with decreased postablation VNS scores, while increasing age and higher preablation VNS scores were associated with increased postablation VNS scores. Despite increasing age being associated with increased postablation VNS scores, age and the advanced Australian technique were found to confer greater patient self-reported treatment benefit. Conclusion: The advanced Australian technique provides a significant benefit over the early Australian technique for the treatment of lumbar facet pain, both in magnitude and duration of pain relief. Keywords: low-back pain, lumbar facet arthropathy, interventional pain management, radiofrequency ablation

The article presents a case study of a 77-year-old woman who was admitted to a hospital for iron deficiency anemia and had a history of herpes simplex virus (HSV) encephalitis three years earlier complicated with post-encephalitic syndrome. Topics discussed include HSV esophagitis (HSVE) in the immunocompetent host, antiviral treatment, and the diagnostic role of esophageal biopsy in severe esophagitis.

Ischemic cholangiopathy (IC) is the leading cause of inferior long-term outcomes following donation after circulatory death (DCD) liver transplant. Biliary strictures related to IC are nonanastomotic strictures (NASs) by definition and involve the donor hepatic ducts proximal to the anastomosis, compared with postsurgical anastomotic strictures that form due to fibrotic healing. IC-related NASs can be microangiopathic with patent hepatic artery or macroangiopathic with occluded or stenotic hepatic artery. Recently, IC with NASs have been described to have four distinct patterns at imaging: diffuse necrosis, multifocal progressive, confluence dominant, and minor form, which correlate clinically with graft prognosis. Severe IC can lead to ductal wall breakdown with subsequent bile leaks that can cause significant patient morbidity, with imaging playing a vital role in diagnosis and guiding intervention. IC also predisposes the transplanted liver to biliary stasis and subsequent formation of stones, casts, and sludge. Some cases of posttransplant biliary stricturing are not IC but are a sequela of reflux cholangitis seen with choledochojejunal anastomosis. Other biliary findings in the posttransplant liver can be explained by sphincter of Oddi dysfunction that results from denervation. The authors describe and comprehensively categorize the various IC types and their imaging patterns at MRI and MR cholangiopancreatography, review the prognostic significance of these imaging patterns, and discuss imaging features of additional biliary complications associated with IC after DCD liver transplant. © RSNA, 2024 Supplemental material is available for this article.

The scaffolding protein CARD11 is a critical mediator of antigen receptor signaling in lymphocytes. Hypomorphic (partial loss-of-function) mutations in CARD11 are associated with the development of severe atopic dermatitis, in which T cell receptor signaling is reduced and helper T cell differentiation is skewed to an allergy-associated type 2 phenotype. Here, we found that the docking protein DOK3 plays a key role in the pathogenesis of atopic dermatitis by suppressing CARD11 activity. DOK3 interacted with CARD11 and decreased its phosphorylation in T cells by recruiting the catalytic subunit of protein phosphatase 4, thereby dampening downstream signaling. Knocking out Dok3 enhanced the production of the cytokine IFN-γ by T cells, which conferred protection against experimental atopic dermatitis-like skin inflammation in mice. The expression of DOK3 was increased in T cells isolated from patients with atopic dermatitis and inversely correlated with IFNG expression. A subset of hypomorphic CARD11 variants found in patients with atopic dermatitis bound more strongly than wild-type CARD11 to DOK3. Our findings suggest that the strength of the interaction of DOK3 with CARD11 may predispose individuals to developing atopic dermatitis.

Invasive fungal disease is an emerging and serious public health threat globally. The expanding population of susceptible individuals, together with the rapid emergence of multidrug-resistant fungi pathogens, call for the development of novel therapeutic strategies beyond the limited repertoire of licensed antifungal drugs. Card9 is a critical signaling molecule involved in antifungal defense; we have previously identified Dok3 to be a key negative regulator of Card9 activity in neutrophils. In this study, we identified two synthetic peptides derived from the coiled-coil domain of Card9, which can specifically block Dok3-Card9 binding. We showed that these peptides are cell-permeable, non-toxic, and can enhance antifungal cytokine production and the phagocytosis of human neutrophils upon fungal infection. Collectively, these data provide a proof of concept that disrupting the Dok3-Card9 interaction can boost the antifungal effector functions of neutrophils; they further suggest the potential utility of these peptide inhibitors as an immune-based therapeutic to fight fungal infection.

Mesenchymal stem/stromal cell (MSC) exosomes have been shown to alleviate immune dysfunction and inflammation in preclinical animal models. This therapeutic effect is attributed, in part, to their ability to promote the polarization of anti-inflammatory M2-like macrophages. One polarization mechanism has been shown to involve the activation of the MyD88-mediated toll-like receptor (TLR) signaling pathway by the presence of extra domain A-fibronectin (EDA-FN) within the MSC exosomes. Here, we uncovered an additional mechanism where MSC exosomes mediate M2-like macrophage polarization through exosomal CD73 activity. Specifically, we observed that polarization of M2-like macrophages by MSC exosomes was abolished in the presence of inhibitors of CD73 activity, adenosine receptors A 2A and A 2B , and AKT/ERK phosphorylation. These findings suggest that MSC exosomes promote M2-like macrophage polarization by catalyzing the production of adenosine, which then binds to adenosine receptors A 2A and A 2B to activate AKT/ERK-dependent signaling pathways. Thus, CD73 represents an additional critical attribute of MSC exosomes in mediating M2-like macrophage polarization. These findings have implications for predicting the immunomodulatory potency of MSC exosome preparations.

Invasive fungal infection is an under recognized and emerging global health threat. Recently, the World Health Organization (WHO) released the first ever list of health-threatening fungi to guide research and public health interventions to strengthen global response to fungi infections and antifungal resistance. Currently, antifungal drugs only demonstrate partial success in improving prognosis of infected patients, and this is compounded by the rapid evolution of drug resistance among fungi species. The increased prevalence of fungal infections in individuals with underlying immunological deficiencies reflects the importance of an intact host immune system in controlling mycoses, and further highlights immunomodulation as a potential new avenue for the treatment of disseminated fungal diseases. In this review, we will summarize how host innate immune cells sense invading fungi through their pattern recognition receptors, and subsequently initiate a series of effector mechanisms and adaptive immune responses to mediate fungal clearance. In addition, we will discuss emerging preclinical and clinical data on antifungal immunotherapies and fungal vaccines which can potentially expand our antifungal armamentarium in future., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Both Helicobacter pylori infection and a high-salt diet are risk factors for gastric cancer. We previously showed that a mutation in fur (encoding the ferric uptake regulator variant Fur-R88H) was positively selected in H. pylori strains isolated from experimentally infected Mongolian gerbils receiving a high-salt diet. In the present study, we report that continuous H. pylori growth in high-salt conditions in vitro also leads to positive selection of the fur -R88H mutation. Competition experiments with strains containing wild-type fur or fur -R88H, each labeled with unique nucleotide barcodes, showed that the fur -R88H mutation enhances H. pylori fitness under high-salt conditions but reduces H. pylori fitness under routine culture conditions. The fitness advantage of the fur -R88H mutant under high-salt conditions was abrogated by the addition of supplemental iron. To test the hypothesis that the fur -R88H mutation alters the regulatory properties of Fur, we compared the transcriptional profiles of strains containing wild-type fur or fur -R88H. Increased transcript levels of fecA2 , which encodes a predicted TonB-dependent outer membrane transporter, were detected in the fur -R88H variant compared to those in the strain containing wild-type fur under both high-salt and routine conditions. Competition experiments showed that fecA2 contributes to H. pylori fitness under both high-salt and routine conditions. These results provide new insights into mechanisms by which the fur -R88H mutation confers a selective advantage to H. pylori in high-salt environments.

Rheumatic diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), are a group of auto-inflammatory disorders associated with substantial morbidity and mortality. One unifying feature of these diseases is the presence of abnormal neutrophils exhibiting dysregulated neutrophil extracellular trap (NET) release, reactive oxygen species (ROS) production, degranulation, and pro-inflammatory cytokines secretion. Moreover, the release of autoantigens associated with NETs promotes the generation of autoantibodies and a breakdown of self-tolerance, thereby perpetuating inflammation and tissue injury in these patients. In recent years, targeted therapies directed at neutrophilic effector functions have shown promising results in the management of rheumatic diseases. In this review, we will highlight the emerging roles of neutrophils in the onset and progression of rheumatic diseases, and further discuss current and future therapeutic approaches targeting the pathogenic functions of neutrophils, which can modulate inflammation and hence improve patients' survival and quality of life., Competing Interests: Conflict of Interest Kong-Peng Lam is an Editorial Board Member of the journal. This article was subject to the journal's standard procedures, with peer review handled independently of this member and his research group., (© 2022 Jia Tong Loh et al., published by Sciendo.)

Increased neutrophils and elevated level of circulating calprotectin are hallmarks of severe COVID-19 and they contribute to the dysregulated immune responses and cytokine storm in susceptible patients. However, the precise mechanism controlling calprotectin production during SARS-CoV-2 infection remains elusive. In this study, we showed that Dok3 adaptor restrains calprotectin production by neutrophils in response to SARS-CoV-2 spike (S) protein engagement of TLR4. Dok3 recruits SHP-2 to mediate the de-phosphorylation of MyD88 at Y257, thereby attenuating downstream JAK2-STAT3 signaling and calprotectin production. Blocking of TLR4, JAK2 and STAT3 signaling could prevent excessive production of calprotectin by Dok3 -/- neutrophils, revealing new targets for potential COVID-19 therapy. As S protein from SARS-CoV-2 Delta and Omicron variants can activate TLR4-driven calprotectin production in Dok3 -/- neutrophils, our study suggests that targeting calprotectin production may be an effective strategy to combat severe COVID-19 manifestations associated with these emerging variants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Loh, Teo and Lam.)

To evaluate potential effects of gastric inflammation on Helicobacter pylori diversification and evolution within the stomach, we experimentally infected Mongolian gerbils with an H. pylori strain in which Cag type IV secretion system (T4SS) activity is controlled by a TetR/ tetO system. Gerbils infected with H. pylori under conditions in which Cag T4SS activity was derepressed had significantly higher levels of gastric inflammation than gerbils infected under conditions with repressed Cag T4SS activity. Mutations in the 5' untranslated region (UTR) of katA (encoding catalase) were detected in strains cultured from 8 of the 17 gerbils infected with Cag T4SS-active H. pylori and none of the strains from 17 gerbils infected with Cag T4SS-inactive H. pylori. Catalase enzymatic activity, steady-state katA transcript levels, and katA transcript stability were increased in strains with these single nucleotide polymorphisms (SNPs) compared to strains in which these SNPs were absent. Moreover, strains harboring these SNPs exhibited increased resistance to bactericidal effects of hydrogen peroxide, compared to control strains. Experimental introduction of the SNPs into the wild-type katA 5' UTR resulted in increased katA transcript stability, increased katA steady-state levels, and increased catalase enzymatic activity. Based on site-directed mutagenesis and modeling of RNA structure, increased katA transcript levels were correlated with higher predicted thermal stability of the katA 5' UTR secondary structure. These data suggest that high levels of gastric inflammation positively select for H. pylori strains producing increased levels of catalase, which may confer survival advantages to the bacteria in an inflammatory gastric environment.

Complements and neutrophils are two key players of the innate immune system that are widely implicated as drivers of severe COVID-19 pathogenesis, as evident by the direct correlation of respiratory failure and mortality with elevated levels of terminal complement complex C5b-9 and neutrophils. In this study, we identified a feed-forward loop between complements and neutrophils that could amplify and perpetuate the cytokine storm seen in severe SARS-CoV-2-infected patients. We observed for the first time that the terminal complement activation complex C5b-9 directly triggered neutrophil extracellular trap (NET) release and interleukin (IL)-17 production by neutrophils. This is also the first report that the production of NETs and IL-17 induced by C5b-9 assembly on neutrophils could be abrogated by mesenchymal stem cell (MSC) exosomes. Neutralizing anti-CD59 antibodies abolished this abrogation. Based on our findings, we hypothesize that MSC exosomes could alleviate the immune dysregulation in acute respiratory failure, such as that observed in severe COVID-19 patients, by inhibiting complement activation through exosomal CD59, thereby disrupting the feed-forward loop between complements and neutrophils to inhibit the amplification and perpetuation of inflammation during SARS-CoV-2 infection., Competing Interests: Declaration of Competing Interests SKL holds founding shares in Paracrine Therapeutics., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

How pathogenesis of inflammatory bowel disease (IBD) depends on the complex interplay of host genetics, microbiome and the immune system is not fully understood. Here, we showed that Downstream of Kinase 3 (DOK3), an adapter protein involved in immune signaling, confers protection of mice from dextran sodium sulfate (DSS)-induced colitis. DOK3-deficiency promotes gut microbial dysbiosis and enhanced colitis susceptibility, which can be reversed by the transfer of normal microbiota from wild-type mice. Mechanistically, DOK3 exerts its protective effect by suppressing JAK2/STAT3 signaling in colonic neutrophils to limit their S100a8/9 production, thereby maintaining gut microbial ecology and colon homeostasis. Hence, our findings reveal that the immune system and microbiome function in a feed-forward manner, whereby DOK3 maintains colonic neutrophils in a quiescent state to establish a gut microbiome essential for intestinal homeostasis and protection from IBD., (© 2021. The Author(s).)

Helicobacter pylori genomes encode over 60 predicted outer membrane proteins (OMPs). Several OMPs in the Hop family act as adhesins, but the functions of most Hop proteins are unknown. To identify hop mutant strains exhibiting differential fitness in vivo compared to in vitro , we used a genetic barcoding method that allowed us to track changes in the proportional abundance of H. pylori strains within a mixed population. We generated a library of hop mutant strains, each containing a unique nucleotide barcode, as well as a library of control strains, each containing a nucleotide barcode in an intergenic region predicted to be a neutral locus unrelated to bacterial fitness. We orogastrically inoculated each of the libraries into mice and analyzed compositional changes in the populations over time in vivo compared to changes detected in the populations during library passage in vitro . The control library proliferated as a relatively stable community in vitro , but there was a reduction in the population diversity of this library in vivo and marked variation in the dominant strains recovered from individual animals, consistent with the existence of a nonselective bottleneck in vivo . We did not identify any OMP mutants exhibiting fitness defects exclusively in vivo without corresponding fitness defects in vitro . Conversely, a babA mutant exhibited a strong fitness advantage in vivo but not in vitro . These findings, when taken together with results of other studies, suggest that production of BabA may have differential effects on H. pylori fitness depending on the environmental conditions.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma. The H. pylori cancer-associated cag pathogenicity island (cag-PAI) encodes a type IV secretion system (T4SS), which translocates microbial DNA and activates TLR9; however, most cag-PAI+-infected persons do not develop cancer and cag-PAI-independent regulators of pathogenesis, including strain-specific adhesins, remain understudied. We defined the relationships between H. pylori HopQ adhesin allelic type, gastric injury, and TLR9 activation. Type I hopQ alleles were significantly associated with magnitude of injury, cag-T4SS function, and TLR9 activation. Genetic deletion of hopQ significantly decreased H. pylori-induced TLR9 activation, implicating this adhesin in H. pylori-mediated disease., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Innate immunity is important for host defense by eliciting rapid anti-viral responses and bridging adaptive immunity. Here, we show that endogenous lipids released from virus-infected host cells activate lung γδ T cells to produce interleukin 17 A (IL-17A) for early protection against H1N1 influenza infection. During infection, the lung γδ T cell pool is constantly supplemented by thymic output, with recent emigrants infiltrating into the lung parenchyma and airway to acquire tissue-resident feature. Single-cell studies identify IL-17A-producing γδ T (Tγδ17) cells with a phenotype of TCRγδ hi CD3 hi AQP3 hi CXCR6 hi in both infected mice and patients with pneumonia. Mechanistically, host cell-released lipids during viral infection are presented by lung infiltrating CD1d + B-1a cells to activate IL-17A production in γδ T cells via γδTCR-mediated IRF4-dependent transcription. Reduced IL-17A production in γδ T cells is detected in mice either lacking B-1a cells or with ablated CD1d in B cells. Our findings identify a local host-immune crosstalk and define important cellular and molecular mediators for early innate defense against lung viral infection.

Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site-specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in Δ arsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization ( ureAB , amidases), oxidative stress responses ( katA , sodB ), transcriptional regulation related to iron or nickel homeostasis ( fur , nikR ), and genes encoding outer membrane proteins (including sabA , alpA , alpB , hopD [ labA ], and horA ). When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS-mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions., (Copyright © 2021 American Society for Microbiology.)

Downstream of kinase (Dok) 3 is a member of the Dok family of adaptor proteins known to regulate signaling pathways downstream of various immunoreceptors. As Dok-3 lacks intrinsic catalytic activity, it functions primarily as a molecular scaffold to facilitate the nucleation of protein complexes in a regulated manner and hence, achieve specificity in directing signaling cascades. Since its discovery, considerable progress has been made toward defining the role of Dok-3 in limiting B cell-receptor signaling. Nonetheless, Dok-3 has since been implicated in the signaling of Toll-like and C-type lectin receptors. Emerging data further demonstrate that Dok-3 can act both as an activator and inhibitor, in lymphoid and non-lymphoid cell types, suggesting Dok-3 involvement in a plethora of signal transduction pathways. In this review, we will focus on the structure and expression profile of Dok-3 and highlight its role during signal transduction in B cells, innate cells as well as in bone and lung tissues., (Copyright © 2020 Loh, Teo, Lim and Lam.)

The Helicobacter pylori Cag type IV secretion system (T4SS) translocates the effector protein CagA and nonprotein bacterial constituents into host cells. In this study, we infected Mongolian gerbils with an H. pylori strain in which expression of the cagUT operon (required for Cag T4SS activity) is controlled by a TetR/ tetO system. Transcript levels of cagU were significantly higher in gastric tissue from H. pylori -infected animals receiving doxycycline-containing chow (to derepress Cag T4SS activity) than in tissue from infected control animals receiving drug-free chow. At 3 months postinfection, infected animals receiving doxycycline had significantly increased gastric inflammation compared to infected control animals. Dysplasia (a premalignant histologic lesion) and/or invasive gastric adenocarcinoma were detected only in infected gerbils receiving doxycycline, not in infected control animals. We then conducted experiments in which Cag T4SS activity was derepressed during defined stages of infection. Continuous Cag T4SS activity throughout a 3-month time period resulted in higher rates of dysplasia and/or gastric cancer than observed when Cag T4SS activity was limited to early or late stages of infection. Cag T4SS activity for the initial 6 weeks of infection was sufficient for the development of gastric inflammation at the 3-month time point, with gastric cancer detected in a small proportion of animals. These experimental results, together with previous studies of cag mutant strains, provide strong evidence that Cag T4SS activity contributes to gastric carcinogenesis and help to define the stages of H. pylori infection during which Cag T4SS activity causes gastric alterations relevant for cancer pathogenesis. IMPORTANCE The "hit-and-run model" of carcinogenesis proposes that an infectious agent triggers carcinogenesis during initial stages of infection and that the ongoing presence of the infectious agent is not required for development of cancer. H. pylori infection and actions of CagA (an effector protein designated a bacterial oncoprotein, secreted by the Cag T4SS) are proposed to constitute a paradigm for hit-and-run carcinogenesis. In this study, we report the development of methods for controlling H. pylori Cag T4SS activity in vivo and demonstrate that Cag T4SS activity contributes to gastric carcinogenesis. We also show that Cag T4SS activity during an early stage of infection is sufficient to initiate a cascade of cellular alterations leading to gastric inflammation and gastric cancer at later time points., (Copyright © 2020 Lin et al.)

Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island ( cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori -induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagβ, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori -induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagβ was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells., (Copyright © 2020 American Society for Microbiology.)

Helicobacter pylori infection and a high salt diet are each risk factors for gastric cancer. In this study, we tested the hypothesis that environmental salt concentration influences the composition of the H. pylori exoproteome. H. pylori was cultured in media containing varying concentrations of sodium chloride, and aliquots were fractionated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified proteins that were selectively released into the extracellular space, and we identified selectively released proteins that were differentially abundant in culture supernatants, depending on the environmental salt concentration. We also used RNA-seq analysis to identify genes that were differentially expressed in response to environmental salt concentration. The salt-responsive proteins identified by proteomic analysis and salt-responsive genes identified by RNA-seq analysis were mostly non-concordant, but the secreted toxin VacA was salt-responsive in both analyses. Western blot analysis confirmed that VacA levels in the culture supernatant were increased in response to high salt conditions, and quantitative RT-qPCR experiments confirmed that vacA transcription was upregulated in response to high salt conditions. These results indicate that environmental salt concentration influences the composition of the H. pylori exoproteome, which could contribute to the increased risk of gastric cancer associated with a high salt diet. SIGNIFICANCE: Helicobacter pylori-induced alterations in the gastric mucosa have been attributed, at least in part, to the actions of secreted H. pylori proteins. In this study, we show that H. pylori growth in high salt concentrations leads to increased levels of a secreted VacA toxin. Salt-induced alterations in the composition of the H. pylori exoproteome is relevant to the increased risk of gastric cancer associated with consumption of a high salt diet., (Published by Elsevier B.V.)

Invasive fungal infection is a serious health threat with high morbidity and mortality. Current antifungal drugs only demonstrate partial success in improving prognosis. Furthermore, mechanisms regulating host defense against fungal pathogens remain elusive. Here, we report that the downstream of kinase 3 (Dok3) adaptor negatively regulates antifungal immunity in neutrophils. Our data revealed that Dok3 deficiency increased phagocytosis, proinflammatory cytokine production, and netosis in neutrophils, thereby enhancing mutant mouse survival against systemic infection with a lethal dose of the pathogenic fungus Candida albicans. Biochemically, Dok3 recruited protein phosphatase 1 (PP1) to dephosphorylate Card9, an essential player in innate antifungal defense, to dampen downstream NF-κB and JNK activation and immune responses. Thus, Dok3 suppresses Card9 signaling, and disrupting Dok3-Card9 interaction or inhibiting PP1 activity represents therapeutic opportunities to develop drugs to combat candidaemia.

Helicobacter pylori CagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation among H. pylori strains in the steady-state levels of CagA and that a strain-specific motif downstream of the cagA transcriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. The cagA 5' untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop on cagA transcript levels and cagA mRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both the cagA transcript and the CagA protein. Additionally, these mutations resulted in a decreased cagA mRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-state cagA transcript and CagA protein levels but did not affect cagA transcript stability. cagA transcript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augment cagA transcript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in the cagA 5' untranslated region influence the levels of cagA expression., (Copyright © 2019 American Society for Microbiology.)

Ezh2, a well-established epigenetic repressor, can down-regulate leukocyte inflammatory responses, but its role in cutaneous health remains elusive. Here we demonstrate that Ezh2 controls cutaneous tolerance by regulating Langerhans cell (LC) transmigration across the epidermal basement membrane directly via Talin1 methylation. Ezh2 deficiency impaired disassembly of adhesion structures in LCs, leading to their defective integrin-dependent emigration from the epidermis and failure in tolerance induction. Moreover, mobilization of Ezh2-deficient Langerin - dermal dendritic cells (dDCs) via high-dose treatment with a weak allergen restored tolerance, which is associated with an increased tolerogenic potential of Langerin - dDCs likely due to epigenetic de-repression of Aldh in the absence of Ezh2. Our data reveal novel roles for Ezh2 in governing LC- and dDC-mediated host protection against cutaneous allergen via distinct mechanisms., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Helicobacter pylori is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease., Design: Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed., Results: A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034)., Conclusion: These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Helicobacter pylori requires genetic agility to infect new hosts and establish long-term colonization of changing gastric environments. In this study, we analyzed H. pylori genetic adaptation in the Mongolian gerbil model. This model is of particular interest because H. pylori -infected gerbils develop a high level of gastric inflammation and often develop gastric adenocarcinoma or gastric ulceration. We analyzed the whole genome sequences of H. pylori strains cultured from experimentally infected gerbils, in comparison to the genome sequence of the input strain. The mean annualized single nucleotide polymorphism (SNP) rate per site was 1.5e -5 , which is similar to the rates detected previously in H. pylori- infected humans. Many of the mutations occurred within or upstream of genes associated with iron-related functions ( fur , tonB1 , fecA2 , fecA3 , and frpB3 ) or encoding outer membrane proteins ( alpA, oipA, fecA2, fecA3, frpB3 and cagY ). Most of the SNPs within coding regions (86%) were non-synonymous mutations. Several deletion or insertion mutations led to disruption of open reading frames, suggesting that the corresponding gene products are not required or are deleterious during chronic H. pylori colonization of the gerbil stomach. Five variants (three SNPs and two deletions) were detected in isolates from multiple animals, which suggests that these mutations conferred a selective advantage. One of the mutations (FurR88H) detected in isolates from multiple animals was previously shown to confer increased resistance to oxidative stress, and we now show that this SNP also confers a survival advantage when H. pylori is co-cultured with neutrophils. Collectively, these analyses allow the identification of mutations that are positively selected during H. pylori colonization of the gerbil model., Competing Interests: The authors declare there are no competing interests.

Helicobacter pylori infection and high dietary salt intake are risk factors for the development of gastric adenocarcinoma. One possible mechanism by which a high-salt diet could influence gastric cancer risk is by modulating H. pylori gene expression. In this study, we utilized transcriptome sequencing (RNA-seq) methodology to compare the transcriptional profiles of H. pylori grown in media containing different concentrations of sodium chloride. We identified 118 differentially expressed genes (65 upregulated and 53 downregulated in response to high-salt conditions), including multiple members of 14 operons. Twenty-nine of the differentially expressed genes encode proteins previously shown to undergo salt-responsive changes in abundance, based on proteomic analyses. Real-time reverse transcription (RT)-PCR analyses validated differential expression of multiple genes encoding outer membrane proteins, including adhesins (SabA and HopQ) and proteins involved in iron acquisition (FecA2 and FecA3). Transcript levels of sabA , hopA , and hopQ are increased under high-salt conditions, whereas transcript levels of fecA2 and fecA3 are decreased under high-salt conditions. Transcription of sabA , hopA , hopQ , and fecA3 is derepressed in an arsS mutant strain, but salt-responsive transcription of these genes is not mediated by the ArsRS two-component system, and the CrdRS and FlgRS two-component systems do not have any detectable effects on transcription of these genes. In summary, these data provide a comprehensive view of H. pylori transcriptional alterations that occur in response to high-salt environmental conditions., (Copyright © 2018 American Society for Microbiology.)

Epidemiological studies have indicated increased risk for breast cancer within 10 years of childbirth. Acute inflammation during mammary involution has been suggested to promote this parity-associated breast cancer. We report here that estrogen exacerbates mammary inflammation during involution. Microarray analysis shows that estrogen induces an extensive proinflammatory gene signature in the involuting mammary tissue. This is associated with estrogen-induced neutrophil infiltration. Furthermore, estrogen induces the expression of protumoral cytokines/chemokines, COX-2 and tissue-remodeling enzymes in isolated mammary neutrophils and systemic neutrophil depletion abolished estrogen-induced expression of these genes in mammary tissue. More interestingly, neutrophil depletion diminished estrogen-induced growth of ERα-negative mammary tumor 4T1 in Balb/c mice. These findings highlight a novel aspect of estrogen action that reprograms the activity of neutrophils to create a pro-tumoral microenvironment during mammary involution. This effect on the microenvironment would conceivably aggravate its known neoplastic effect on mammary epithelial cells.

Leukocytes undergo frequent phenotypic changes and rapidly infiltrate peripheral and lymphoid tissues in order to carry out immune responses. The recruitment of circulating leukocytes into inflamed tissues depends on integrin-mediated tethering and rolling of these cells on the vascular endothelium, followed by transmigration into the tissues. This dynamic process of migration requires the coordination of large numbers of cytosolic and transmembrane proteins whose functional activities are typically regulated by post-translational modifications (PTMs). Our recent studies have shown that the lysine methyltransferase, Ezh2, critically regulates integrin signalling and governs the adhesion dynamics of leukocytes via direct methylation of talin, a key molecule that controls these processes by linking integrins to the actin cytoskeleton. In this review, we will discuss the various modes of leukocyte migration and examine how PTMs of cytoskeletal/adhesion associated proteins play fundamental roles in the dynamic regulation of leukocyte migration. Furthermore, we will discuss molecular details of the adhesion dynamics controlled by Ezh2-mediated talin methylation and the potential implications of this novel regulatory mechanism for leukocyte migration, immune responses, and pathogenic processes, such as allergic contact dermatitis and tumorigenesis., Competing Interests: There is no conflict of interest.

Unlabelled: Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs., Importance: Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines., (Copyright © 2016 Shaffer et al.)

Purpose: Helicobacter pylori infection and a high dietary salt intake are each risk factors for the development of gastric cancer. We hypothesize that changes in environmental salt concentrations lead to alterations in the H. pylori membrane proteome., Experimental Design: Label-free and iTRAQ methods were used to identify H. pylori proteins that change in abundance in response to alterations in environmental salt concentrations. In addition, we biotinylated intact bacteria that were grown under high- or low-salt conditions, and thereby analyzed salt-induced changes in the abundance of surface-exposed proteins., Results: Proteins with increased abundance in response to high salt conditions included CagA, the outer membrane protein HopQ, and fibronectin domain-containing protein HP0746. Proteins with increased abundance in response to low salt conditions included VacA, two VacA-like proteins (ImaA and FaaA), outer-membrane iron transporter FecA3, and several proteins involved in flagellar activity. Consistent with the proteomic data, bacteria grown in high salt conditions exhibited decreased motility compared to bacteria grown in lower salt conditions., Conclusion and Clinical Relevance: Alterations in the H. pylori membrane proteome in response to high salt conditions may contribute to the increased risk of gastric cancer associated with a high salt diet., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Helicobacter pylori exhibits a high level of intraspecies genetic diversity. In this study, we investigated whether the diversification of H. pylori is influenced by the composition of the diet. Specifically, we investigated the effect of a high-salt diet (a known risk factor for gastric adenocarcinoma) on H. pylori diversification within a host. We analyzed H. pylori strains isolated from Mongolian gerbils fed either a high-salt diet or a regular diet for 4 months by proteomic and whole-genome sequencing methods. Compared to the input strain and output strains from animals fed a regular diet, the output strains from animals fed a high-salt diet produced higher levels of proteins involved in iron acquisition and oxidative-stress resistance. Several of these changes were attributable to a nonsynonymous mutation in fur (fur-R88H). Further experiments indicated that this mutation conferred increased resistance to high-salt conditions and oxidative stress. We propose a model in which a high-salt diet leads to high levels of gastric inflammation and associated oxidative stress in H. pylori-infected animals and that these conditions, along with the high intraluminal concentrations of sodium chloride, lead to selection of H. pylori strains that are most fit for growth in this environment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A cytosolic role for the histone methyltransferase Ezh2 in regulating lymphocyte activation has been suggested, but the molecular mechanisms underpinning this extranuclear function have remained unclear. Here we found that Ezh2 regulated the integrin signaling and adhesion dynamics of neutrophils and dendritic cells (DCs). Ezh2 deficiency impaired the integrin-dependent transendothelial migration of innate leukocytes and restricted disease progression in an animal model of multiple sclerosis. Direct methylation of talin, a key regulatory molecule in cell migration, by Ezh2 disrupted the binding of talin to F-actin and thereby promoted the turnover of adhesion structures. This regulatory effect was abolished by targeted disruption of the interactions of Ezh2 with the cytoskeletal-reorganization effector Vav1. Our studies reveal an unforeseen extranuclear function for Ezh2 in regulating adhesion dynamics, with implications for leukocyte migration, immune responses and potentially pathogenic processes.

Transition metals are necessary for all forms of life including microorganisms, evidenced by the fact that 30% of all proteins are predicted to interact with a metal cofactor. Through a process termed nutritional immunity, the host actively sequesters essential nutrient metals away from invading pathogenic bacteria. Neutrophils participate in this process by producing several metal chelating proteins, including lactoferrin and calprotectin (CP). As neutrophils are an important component of the inflammatory response directed against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was hypothesized that CP plays a role in the host response to H. pylori. Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV Secretion System (cag T4SS) was investigated. This study indicates elevated gastric levels of CP are associated with the infiltration of neutrophils to the H. pylori-infected tissue. When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice. In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion. Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS.

Persistent colonization of the human stomach with Helicobacter pylori is a risk factor for gastric adenocarcinoma, and H. pylori-induced carcinogenesis is dependent on the actions of a bacterial oncoprotein known as CagA. Epidemiological studies have shown that high dietary salt intake is also a risk factor for gastric cancer. To investigate the effects of a high-salt diet, we infected Mongolian gerbils with a wild-type (WT) cagA(+) H. pylori strain or an isogenic cagA mutant strain and maintained the animals on a regular diet or a high-salt diet. At 4 months postinfection, gastric adenocarcinoma was detected in 100% of the WT-infected/high-salt-diet animals, 58% of WT-infected/regular-diet animals, and none of the animals infected with the cagA mutant strain (P < 0.0001). Among animals infected with the WT strain, those fed a high-salt diet had more severe gastric inflammation, higher gastric pH, increased parietal cell loss, increased gastric expression of interleukin 1β (IL-1β), and decreased gastric expression of hepcidin and hydrogen potassium ATPase (H,K-ATPase) compared to those on a regular diet. Previous studies have detected upregulation of CagA synthesis in response to increased salt concentrations in the bacterial culture medium, and, concordant with the in vitro results, we detected increased cagA transcription in vivo in animals fed a high-salt diet compared to those on a regular diet. Animals infected with the cagA mutant strain had low levels of gastric inflammation and did not develop hypochlorhydria. These results indicate that a high-salt diet potentiates the carcinogenic effects of cagA(+) H. pylori strains.

Unlabelled: Helicobacter pylori contains four genes that are predicted to encode proteins secreted by the autotransporter (type V) pathway. One of these, the pore-forming toxin VacA, has been studied in great detail, but thus far there has been very little investigation of three VacA-like proteins. We show here that all three VacA-like proteins are >250 kDa in mass and localized on the surface of H. pylori. The expression of the three vacA-like genes is upregulated during H. pylori colonization of the mouse stomach compared to H. pylori growth in vitro, and a wild-type H. pylori strain outcompeted each of the three corresponding isogenic mutant strains in its ability to colonize the mouse stomach. One of the VacA-like proteins localizes to a sheath that overlies the flagellar filament and bulb, and therefore, we designate it FaaA (flagella-associated autotransporter A). In comparison to a wild-type H. pylori strain, an isogenic faaA mutant strain exhibits decreased motility, decreased flagellar stability, and an increased proportion of flagella in a nonpolar site. The flagellar localization of FaaA differs markedly from the localization of other known autotransporters, and the current results reveal an important role of FaaA in flagellar localization and motility., Importance: The pathogenesis of most bacterial infections is dependent on the actions of secreted proteins, and proteins secreted by the autotransporter pathway constitute the largest family of secreted proteins in pathogenic Gram-negative bacteria. In this study, we analyzed three autotransporter proteins (VacA-like proteins) produced by Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We demonstrate that these three proteins each enhance the capacity of H. pylori to colonize the stomach. Unexpectedly, one of these proteins (FaaA) is localized to a sheath that overlies H. pylori flagella. The absence of FaaA results in decreased H. pylori motility as well as a reduction in flagellar stability and a change in flagellar localization. The atypical localization of FaaA reflects a specialized function of this autotransporter designed to optimize H. pylori colonization of the gastric niche.

Helicobacter pylori infection and consumption of a high-salt diet are each associated with an increased risk for the development of gastric cancer. To investigate potential synergism between these factors, we used a global proteomic approach to analyze H. pylori strains cultured in media containing varying salt concentrations. Among the differentially expressed proteins identified, CagA exhibited the greatest increase in expression in response to high salt concentrations. Analysis of 36 H. pylori strains isolated from patients in two regions of Colombia with differing incidences of gastric cancer revealed marked differences among strains in salt-responsive CagA expression. Sequence analysis of the cagA promoter region in these strains revealed a DNA motif (TAATGA) that was present in either one or two copies. Salt-induced upregulation of CagA expression was detected more commonly in strains containing two copies of the TAATGA motif than in strains containing one copy. Mutagenesis experiments confirmed that two copies of the TAATGA motif are required for salt-induced upregulation of CagA expression. In summary, there is considerable heterogeneity among H. pylori strains in salt-regulated CagA expression, and these differences are attributable to variation in a specific DNA motif upstream of the cagA transcriptional start site.

Background: Helicobacter pylori infection is usually acquired in childhood, but little is known about its natural history in asymptomatic children, primarily due to the paucity of non-invasive diagnostic methods. H. pylori strains harboring cagA and specific alleles of hopQ and vacA are associated with increased risk for gastric cancer. Many studies of H. pylori virulence markers in children have the bias that symptomatic subjects are selected for endoscopy, and these children may harbor the most virulent strains. Our aim is to genotype cagA, hopQ, and vacA alleles in stool DNA samples of healthy Colombian children residing in an area with high incidence of gastric cancer, to avoid selection bias resulting from endoscopy., Methods: H. pylori status of 86 asymptomatic children was assessed by (13) C-urea breath test (UBT) and PCR. H. pylori 16S rRNA, cagA, hopQ, and vacA genes were amplified from stool DNA samples and sequenced., Results: UBT was positive in 69 (80.2%) of 86 children; in stool DNA analysis, 78.3% were positive by 16S rRNA PCR. cagA, vacA, and hopQ were detected in 66.1%, 84.6%, and 72.3% of stool DNA samples from 16S rRNA-positive children. Of the children's DNA samples, which revealed vacA and hopQ alleles, 91.7% showed vacA s1 and 73.7% showed type I hopQ. Type I hopQ alleles were associated with cagA positivity and vacA s1 genotypes (p < 0.0001)., Conclusions: Using stool DNA samples, virulence markers of H. pylori were successfully genotyped in a high percentage of the asymptomatic infected children, revealing a high prevalence of genotypes associated with virulence. Type I hopQ alleles were associated with the presence of cagA and the vacA s1 genotype., (© 2012 Blackwell Publishing Ltd.)

Background: Helicobacter pylori infection is a risk factor for the development of gastric cancer, and the bacterial oncoprotein CagA contributes to gastric carcinogenesis., Methods: We analyzed H. pylori isolates from persons in Colombia and observed that there was marked variation among strains in levels of CagA expression. To elucidate the basis for this variation, we analyzed sequences upstream from the CagA translational initiation site in each strain., Results: A DNA motif (AATAAGATA) upstream of the translational initiation site of CagA was associated with high levels of CagA expression. Experimental studies showed that this motif was necessary but not sufficient for high-level CagA expression. H. pylori strains from a region of Colombia with high gastric cancer rates expressed higher levels of CagA than did strains from a region with lower gastric cancer rates, and Colombian strains of European phylogeographic origin expressed higher levels of CagA than did strains of African origin. Histopathologic analysis of gastric biopsy specimens revealed that strains expressing high levels of CagA or containing the AATAAGATA motif were associated with more advanced precancerous lesions than those found in persons infected with strains expressing low levels of CagA or lacking the AATAAGATA motif., Conclusions: CagA expression varies greatly among H. pylori strains. The DNA motif identified in this study is associated with high levels of CagA expression, and may be a useful biomarker to predict gastric cancer risk., Impact: These findings help to explain why some persons infected with cagA-positive H. pylori develop gastric cancer and others do not., (©2011 AACR)

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.

Background: LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2); hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB), synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only., Results: We report that disruption of luxS renders H. pylori non-motile in soft agar and by microscopy, whereas disruption of mccAHp or mccBHp (other genes in the cysteine provision pathway) does not, implying that the lost phenotype is not due to disrupted cysteine provision. The motility defect of the DeltaluxSHp mutant was complemented genetically by luxSHp and also by addition of in vitro synthesised AI-2 or 4, 5-dihydroxy-2, 3-pentanedione (DPD, the precursor of AI-2). In contrast, exogenously added cysteine could not restore motility to the DeltaluxSHp mutant, confirming that AI-2 synthesis, but not the metabolic effect of LuxS was important. Microscopy showed reduced number and length of flagella in the DeltaluxSHp mutant. Immunoblotting identified decreased levels of FlaA and FlgE but not FlaB in the DeltaluxSHp mutant, and RT-PCR showed that the expression of flaA, flgE, motA, motB, flhA and fliI but not flaB was reduced. Addition of DPD but not cysteine to the DeltaluxSHp mutant restored flagellar gene transcription, and the number and length of flagella., Conclusions: Our data show that as well as being a metabolic enzyme, H. pylori LuxS has an alternative role in regulation of motility by modulating flagellar transcripts and flagellar biosynthesis through production of the signalling molecule AI-2.

Previous studies have shown that the Helicobacter pylori ArsRS two-component signal transduction system contributes to acid-responsive gene expression. To identify additional members of the ArsRS regulon and further investigate the regulatory role of the ArsRS system, we analyzed protein expression in wild-type and arsS null mutant strains. Numerous proteins were differentially expressed in an arsS mutant strain compared to a wild-type strain when the bacteria were cultured at pH 5.0 and also when they were cultured at pH 7.0. Genes encoding 14 of these proteins were directly regulated by the ArsRS system, based on observed binding of ArsR to the relevant promoter regions. The ArsRS-regulated proteins identified in this study contribute to acid resistance (urease and amidase), acetone metabolism (acetone carboxylase), resistance to oxidative stress (thioredoxin reductase), quorum sensing (Pfs), and several other functions. These results provide further definition of the ArsRS regulon and underscore the importance of the ArsRS system in regulating expression of H. pylori proteins during bacterial growth at both neutral pH and acidic pH.

Helicobacter pylori genomes contain about 30 hop genes that encode outer membrane proteins. Helicobacter pylori hopQ alleles exhibit a high level of genetic diversity, and two families of hopQ alleles have been described. Type I hopQ alleles are found more commonly in cag-positive H. pylori strains from patients with peptic ulcer disease than in cag-negative strains from patients without ulcer disease. In this study, we mutated hopQ in four H. pylori strains that each contained a type I hopQ allele, and then analyzed interactions of the wild-type and hopQ mutant strains with AGS cells. In comparison with the wild-type strains, two of the hopQ mutant strains exhibited increased adherence to AGS cells and two hopQ mutants did not exhibit any detectable differences in adherence. Higher levels of tyrosine-phosphorylated CagA were detected when AGS cells were cocultured with a hyperadherent hopQ mutant strain than when cocultured with the corresponding wild-type strain. These data indicate that in some strains of H. pylori, the HopQ protein can attenuate bacterial adherence to gastric epithelial cells.

Helicobacter pylori infection and a high dietary salt intake are risk factors for the development of gastric adenocarcinoma. In this study, we tested the hypothesis that high salt concentrations might alter gene expression in H. pylori. Transcriptional profiling experiments indicated that the expression of multiple H. pylori genes, including cagA, was regulated in response to the concentrations of sodium chloride present in the bacterial culture medium. Increased expression of cagA in response to high salt conditions was confirmed by the use of transcriptional reporter strains and by immunoblotting. H. pylori CagA is translocated into gastric epithelial cells via a type IV secretion pathway, and on entry into target cells, CagA undergoes tyrosine phosphorylation and causes multiple cellular alterations. Coculture of gastric epithelial cells with H. pylori grown under high salt conditions resulted in increased tyrosine-phosphorylated CagA and increased secretion of interleukin-8 by the epithelial cells compared with coculture of the cells with H. pylori grown under low salt conditions. Up-regulation of H. pylori cagA expression in response to high salt concentrations may be a factor that contributes to the development of gastric adenocarcinoma.

Furanone metabolites called AI-2 (autoinducer 2), used by some bacterial species for signaling and cell density-regulated changes in gene expression, are made while regenerating S-adenosyl methionine (SAM) after its use as a methyl donor. The luxS-encoded enzyme, in particular, participates in this activated methyl cycle by generating both a pentanedione, which is transformed chemically into these AI-2 compounds, and homocysteine, a precursor of methionine and SAM. Helicobacter pylori seems to contain the genes for this activated methyl cycle, including luxS, but not genes for AI-2 uptake and transcriptional regulation. Here we report that deletion of luxS in H. pylori reference strain SS1 diminished its competitive ability in mice and motility in soft agar, whereas no such effect was seen with an equivalent Delta luxS derivative of the unrelated strain X47. These different outcomes are consistent with H. pylori's considerable genetic diversity and are reminiscent of phenotypes seen after deletion of another nonessential metabolic gene, that encoding polyphosphate kinase 1. We suggest that synthesis of AI-2 by H. pylori may be an inadvertent consequence of metabolite flux in its activated methyl cycle and that impairment of this cycle and/or pathways affected by it, rather than loss of quorum sensing, is deleterious for some H. pylori strains. Also tenable is a model in which AI-2 affects other microbes in H. pylori's gastric ecosystem and thereby modulates the gastric environment in ways to which certain H. pylori strains are particularly sensitive.

In this study, we investigated a potential requirement of two-component signal transduction systems for acid resistance in Helicobacter pylori. In comparison to a wild-type strain, isogenic strains with null mutations in either HP0165 or HP1364 histidine kinases were impaired in their ability to grow at pH 5.0. The growth of complemented mutant strains was similar to that of the wild-type strain. H. pylori DNA array analyses and transcriptional reporter assays indicated that acid-responsive gene transcription was altered in the HP0165 and HP1364 null mutant strains compared to the parental wild-type strain. These results indicate that intact HP0165 and HP1364 histidine kinases are required for acid resistance in H. pylori.

LuxS plays a role in the synthesis of an extracellular signaling molecule, autoinducer 2 (AI-2). To analyze a possible role of AI-2 in regulating Helicobacter pylori gene expression, we constructed a panel of transcriptional reporter strains. We show that the expression of H. pylori flaA is growth phase dependent and that flaA transcription increases in association with increased culture density. Mutating the luxS gene eliminates growth-phase-dependent control of flaA, and this growth phase dependence is restored when the luxS mutant strain is complemented with the wild-type luxS gene.

The nodulation genes of Bradyrhizobium japonicum are essential for infection and establishment of a nitrogen-fixing symbiosis. Here, we demonstrate that plant-produced isoflavones induce nodulation gene expression in a population density-dependent fashion. Nodulation gene induction is highest at a low population density and significantly reduced in more dense cultures. A quorum signal molecule in the conditioned medium of B. japonicum cultures mediates this repression. Repression in response to the quorum signal results from the induction of NolA which, in turn, induces NodD2 leading to inhibition of nod gene expression. Consistent with this, nolA-lacZ and nodD2-lacZ expression increased with increasing population density. Unlike the wild type, the ability to induce nodY-lacZ expression did not decline with population density in a NolA mutant. Normally, nod gene expression is repressed in planta (i.e. within nodules). However, expression of a nodY-GUS fusion was not repressed in a NolA mutant, suggesting that quorum-sensing control may mediate in planta repression of the nod genes. Addition of conditioned medium to cultures significantly reduced nod gene expression. Treatment of inoculant cultures with conditioned medium also reduced the ability of B. japonicum to nodulate soybean plants.