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Salmonellae are associated epidemiologically and experimentally with colon cancer. To understand how Salmonella induces cell transformation, we performed multi-omics and phenotypic analyses of Salmonella clinical strains isolated from patients later diagnosed with colon cancer (case strains) and control strains from patients without cancer. We show that high transformation efficiency is a frequent intrinsic feature of clinical (case and control) salmonellae, yet case strains showed higher transformation efficiency than control strains. Transformation efficiency correlates with gene expression, nutrient utilization, and intracellular virulence, but not with genetic features, suggesting a phenotypic convergence of Salmonella strains resulting in cell transformation. We show that both bacterial entry and intracellular replication are required for host cell transformation and are associated with hyperactivation of the mTOR pathway. Strikingly, transiently inactivating mTOR through chemical inhibition reverses the transformation phenotype instigated by Salmonella infection. This suggests that targeting the mTOR pathway could prevent the development of Salmonella-induced tumors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The gut microbiota produces high concentrations of antimicrobial short-chain fatty acids (SCFAs) that restrict the growth of invading microorganisms. The enteric pathogen Salmonella enterica serovar ( S. ) Typhimurium triggers inflammation in the large intestine to ultimately reduce microbiota density and bloom, but it is unclear how the pathogen gains a foothold in the homeostatic gut when SCFA-producing commensals are abundant. Here, we show that S. Typhimurium invasion of the ileal mucosa triggers malabsorption of dietary amino acids to produce downstream changes in nutrient availability in the large intestine. In gnotobiotic mice engrafted with a community of 17 human Clostridia isolates, S. Typhimurium virulence factors triggered marked changes in the cecal metabolome, including an elevated abundance of amino acids. In an ex vivo fecal culture model, we found that two of these amino acids, lysine and ornithine, countered SCFA-mediated growth inhibition by restoring S. Typhimurium pH homeostasis through the inducible amino acid decarboxylases CadA and SpeF, respectively. In a mouse model of gastrointestinal infection, S. Typhimurium CadA activity depleted dietary lysine to promote cecal ecosystem invasion in the presence of an intact microbiota. From these findings, we conclude that virulence factor-induced malabsorption of dietary amino acids in the small intestine changes the nutritional environment of the large intestine to provide S. Typhimurium with resources needed to counter growth inhibition by microbiota-derived SCFAs., Competing Interests: Competing interests statement:The authors declare no competing interest.

In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella. Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis., (© 2024. The Author(s).)

Intestinal edema is a common manifestation of numerous gastrointestinal diseases and is characterized by the accumulation of fluid in the interstitial space of the intestinal wall. Technical advances in laser capture microdissection and low-biomass proteomics now allow us to specifically characterize the intestinal edema proteome. Using advanced proteomics, we identify peptides derived from antimicrobial factors with high signal intensity, but also highlight major contributions from the blood clotting system, extracellular matrix (ECM) and protease-protease inhibitor networks. The ECM is a complex fibrillar network of macromolecules that provides structural and mechanical support to the intestinal tissue. One abundant component of the ECM observed in Salmonella-driven intestinal edema is the glycoprotein fibronectin, recognized for its structure-function interplay regulated by mechanical forces. Using mechanosensitive staining of fibronectin fibers reveals that they are tensed in the edema, despite the high abundance of proteases able to cleave fibronectin. In contrast, fibronectin fibers increasingly relax in other cecal tissue areas as the infection progresses. Co-staining for fibrin(ogen) indicates the formation of a provisional matrix in the edema, similar to what is observed in response to skin injury, while collagen staining reveals a sparse and disrupted collagen fiber network. These observations plus the absence of low tensional fibronectin fibers and the additional finding of a high number of protease inhibitors in the edema proteome could indicate a critical role of stretched fibronectin fibers in maintaining tissue integrity in the severely inflamed cecum. Understanding these processes may also provide valuable functional diagnostic markers of intestinal disease progression in the future., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The pathogen Salmonella enterica encompasses a range of bacterial serovars that cause intestinal inflammation and systemic infections in humans. Mice are a widely used infection model due to their relative simplicity and versatility. Here, we provide standardized protocols for culturing the prolific zoonotic pathogen S. enterica serovar Typhimurium for intragastric inoculation of mice to model colitis or systemic dissemination, along with techniques for direct extraintestinal infection. Furthermore, we present procedures for quantifying pathogen burden and for characterizing the immune response by analyzing tissue pathology, inflammatory markers, and immune cells from intestinal tissues. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Murine colitis model utilizing oral streptomycin pretreatment and oral S. Typhimurium administration Basic Protocol 2: Intraperitoneal injection of S. Typhimurium for modeling extraintestinal infection Support Protocol 1: Preparation of S. Typhimurium inoculum Support Protocol 2: Preparation of mixed S. Typhimurium inoculum for competitive infection Basic Protocol 3: Assessment of S. Typhimurium burden Support Protocol 3: Preservation and pathological assessment of S. Typhimurium-infected tissues Support Protocol 4: Measurement of inflammatory marker expression in intestinal tissues by qPCR Support Protocol 5: Preparation of intestinal content for inflammatory marker quantification by ELISA Support Protocol 6: Immune cell isolation from Salmonella-infected intestinal tissues., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Background: In most developing or undeveloped countries, patients are often co-infected with multiple pathogens rather than a single pathogen. While different pathogens have their impact on morbidity and mortality, co-infection of more than one pathogen usually made the disease outcome different. Many studies reported the co-infection of Schistosoma with Salmonella in pandemic areas. However, the link or the underlying mechanism in the pathogenesis caused by Schistosoma-Salmonella co-infection is still unknown., Methods: In this study, Salmonella typhimurium (S. typhimurium) was challenged to Schistosoma mansoni (S. mansoni)-infected mice. Further experiments such as bacterial culture, histopathological examination, western blotting, and flow cytometry were performed to evaluate the outcomes of the infection. Cytokine responses of the mice were also determined by ELISA and real-time quantitative PCR., Results: Our results demonstrated that co-infected mice resulted in higher bacterial excretion in the acute phase but higher bacterial colonization in the chronic phase. Lesser egg burden was also observed during chronic schistosomiasis. Infection with S. typhimurium during schistosomiasis induces activation of the inflammasome and apoptosis, thereby leading to more drastic tissue damage. Interestingly, co-infected mice showed a lower fibrotic response in the liver and spleen. Further, co-infection alters the immunological functioning of the mice, possibly the reason for the observed pathological outcomes., Conclusion: Collectively, our findings here demonstrated that S. mansoni-infected mice challenged with S. typhimurium altered their immunological responses, thereby leading to different pathological outcomes., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

During infection, Salmonella hijacks essential host signaling pathways. These molecular manipulations disrupt cellular integrity and may induce oncogenic transformation. Systemic S. Typhi infections are linked to gallbladder cancer, whereas severe non-typhoidal Salmonella (NTS) infections are associated with colon cancer (CC). These diagnosed infections, however, represent only a small fraction of all NTS infections as many infections are mild and go unnoticed. To assess the overall impact of NTS infections, we performed a retrospective serological study on NTS exposure in patients with CC. The magnitude of exposure to NTS, as measured by serum antibody titer, is significantly positively associated with CC. Repetitively infecting mice with low NTS exposure showed similar accelerated tumor growth to that observed after high NTS exposure. At the cellular level, NTS preferably infects (pre-)transformed cells, and each infection round exponentially increases the rate of transformed cells. Thus, repetitive exposure to NTS associates with CC risk and accelerates tumor growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Caffeine has been reported for its antiinflammatory properties by stimulating phagocytosis. In this study, we investigated the antiinflammatory and antiinfective potential of caffeine in murine macrophage cell cultures and Swiss mice infected with virulent Salmonella enterica serotype typhimurium. Peritoneal macrophages (pMØ) were treated with caffeine on 96-well plates for 24 hr and then infected with Salmonella for 4 hr. In another experiment, the pMØ were first infected with the bacterium for 4 hr and then treated with caffeine for 24 hr. In addition, Swiss mice were inoculated, intraperitoneally, with S. typhimurium and then received caffeine intravenously. Control groups received phosphate-buffered saline (PBS) or dexamethasone. We found that treatments with caffeine increased the macrophage cell viability and reduced the intracellular bacterial load. The administration of caffeine to Swiss mice reduced the infiltration of leukocytes into the peritoneal cavity after the bacterial challenge. Furthermore, the bacterial burdens in the peritoneal fluid, bloodstream, spleen, and liver were decreased by caffeine treatment. The expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, and inducible nitric oxide synthase (iNOs) were down-regulated after infection in caffeine-treated mice. We can conclude that caffeine has both antiinflammatory and antiinfective properties that can be useful for management of bacterial infections along with antibiotics., (© 2021 John Wiley & Sons Ltd.)

Microbial infections are controlled by host inflammatory responses that are initiated by innate immune receptors after recognition of conserved microbial products. As inflammation can also lead to disease, tissues that are exposed to microbial products such as the intestinal epithelium are subject to stringent regulatory mechanisms to prevent indiscriminate signalling through innate immune receptors. The enteric pathogen Salmonella enterica subsp. enterica serovar Typhimurium, which requires intestinal inflammation to sustain its replication in the intestinal tract, uses effector proteins of its type III secretion systems to trigger an inflammatory response without the engagement of innate immune receptors. Furthermore, S. Typhimurium uses a different set of effectors to restrict the inflammatory response to preserve host homeostasis. The S. Typhimurium-host interface is a remarkable example of the unique balance that emerges from the co-evolution of a pathogen and its host., (© 2021. Springer Nature Limited.)

Salmonella Typhimurium is a human pathogen associated with eggs and egg-derived products. In Australia, it is recommended that eggs should be refrigerated to prevent condensation that can enhance bacterial penetration across the eggshell. Except for the United States, the guidelines on egg refrigeration are not prescriptive. In the current study, in-vitro and in-vivo experiments were conducted to understand the role of egg storage temperatures (refrigerated vs ambient) on bacterial load and the virulence genes expression of Salmonella Typhimurium. The in-vitro egg study showed that the load of Salmonella Typhimurium significantly increased in yolk and albumen stored at 25 °C. The gene expression study showed that ompR, misL, pefA, spvA, shdA, bapA, and csgB were significantly up-regulated in the egg yolk stored at 5 °C and 25 °C for 96 h; however, an in-vivo study revealed that mice infected with egg yolk stored at 25 °C, developed salmonellosis from day 3 post-infection (p.i.). Mice fed with inoculated egg yolk, albumen, or eggshell wash stored at refrigerated temperature did not show signs of salmonellosis during the period of the experiment. Data obtained in this study highlighted the importance of egg refrigeration in terms of improving product safety., (© 2021. The Author(s).)

Background: Iron deficiency anaemia (IDA) is a major health concern. However, preventive iron supplementation in regions with high burden of infectious diseases resulted in an increase of infection related morbidity and mortality., Methods: We fed male C57BL/6N mice with either an iron deficient or an iron adequate diet. Next, they received oral iron supplementation or placebo followed by intraperitoneal infection with Salmonella Typhimurium (S.Tm)., Findings: We found that mice with IDA had a poorer clinical outcome than mice on an iron adequate diet. Interestingly, iron supplementation of IDA mice resulted in higher bacterial burden in organs and shortened survival. Increased transferrin saturation and non-transferrin bound iron in the circulation together with low expression of ferroportin facilitated the access of the pathogen to iron and promoted bacterial growth. Anaemia, independent of iron supplementation, was correlated with reduced neutrophil counts and cytotoxic T cells. With iron supplementation, anaemia additionally correlated with increased splenic levels of the cytokine IL-10, which is suggestive for a weakened immune control to S.Tm infection., Interpretation: Supplementing iron to anaemic mice worsens the clinical course of bacterial infection. This can be traced back to increased iron delivery to bacteria along with an impaired anti-microbial immune response. Our findings may have important implications for iron supplementation strategies in areas with high endemic burden of infections, putting those individuals, who potentially profit most from iron supplementation for anaemia, at the highest risk for infections., Funding: Financial support by the Christian Doppler Laboratory for Iron Metabolism and Anemia Research., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells., Competing Interests: The authors have declared that no competing interests exist.

Scope: Salmonella is the main food-borne pathogen, which can infect intestinal epithelial cells and causes colitis. Genistein has a variety of biological activities that alleviates colitis induced by sodium dextran sulfate in a variety of ways, but its protective effects on colitis caused by pathogenic bacteria are still unknown., Methods and Results: This study explores the protective effect of genistein in reducing colitis caused by Salmonella infection. Salmonella causes colon inflammation through activating cyclooxygenase-2/prostaglandin E2, and genistein inhibits colitis caused by Salmonella typhimurium infection. Salmonella infection increases colonic mucosal damage, proliferating cells, and goblet cell loss, while the administration of genistein solves these pathological changes. In addition, it is further proved that Salmonella causes severe colitis related to goblet cell loss and activates the host crypt stem cells to repair the damaged epithelium. Salmonella infection inhibites the host mammalian target of rapamycin, activates light chain 3 II pathways to induce autophagy to eliminate pathogenic bacteria. Genistein increases Lactobacillus in feces and reduces Salmonella colonization to inhibit colitis induces by Salmonella infection., Conclusion: This study demonstrates genistein alleviated colitis and inhibites the goblet cell loss causes by Salmonella infection through regulating the gut bacteria and intestinal stem cell development., (© 2021 Wiley-VCH GmbH.)

Introduction: Salmonella foodborne disease during pregnancy causes a significant fetal loss in domestic livestock and preterm birth, chorioamnionitis and miscarriage in humans. These complications could be associated with alterations in placental structure. This study was aimed to determine how a low dose of Salmonella Enteritidis during late gestation affects placental histomorphometric in mice., Methods: We used a self-limiting enterocolitis murine model. BALB/c pregnant animals received a low dose of Salmonella Enteritidis (3-4 x 10 2  CFU/mouse) on gestational day (GD) 15. At day 3 post infection bacterial loads, serum cytokines expression and placental histomorphometrics parameters were analyzed., Results: We found that a sub-lethal infection with Salmonella induced a significant drop in fetal weight -to-placental weight-ratio and an increase in the placental coefficient. After bacterial inoculation maternal organs were colonized, inducing placental morphometric alterations, including increased placental thickness, reduced surface area, and diminished major and minor diameters. Also, foci of necrosis accompanied by acute leukocyte infiltration in decidual zone, reduction of vascular spaces and vascular congestion in labyrinth zone, were also evident in placentas from infected females on GD 18. Our data shows that placentas from infected mothers are phenotypically different from control ones. Furthermore, expression of IFN-gamma and IL-6 was up regulated in response to Salmonella in maternal serum., Discussion: Our findings demonstrate that a low dose of Salmonella during late gestation alters the placental morphometry leading to negative consequences on pregnancy outcome such as significant reduction in fetal body weight., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, for which there is a lack of a clinically approved vaccine in humans. As an intracellular pathogen, Salmonella impacts many cellular pathways. However, the intercellular communication mechanism facilitated by host-derived small extracellular vesicles (EVs), such as exosomes, is an overlooked aspect of the host responses to this infection. We used a comprehensive proteome-based network analysis of exosomes derived from Salmonella-infected macrophages to identify host molecules that are trafficked via these EVs. This analysis predicted that the host-derived small EVs generated during macrophage infection stimulate macrophages and promote activation of T helper 1 (Th1) cells. We identified that exosomes generated during infection contain Salmonella proteins, including unique antigens previously shown to stimulate protective immune responses against Salmonella in murine studies. Furthermore, we showed that host EVs formed upon infection stimulate a mucosal immune response against Salmonella infection when delivered intranasally to BALB/c mice, a route of antigen administration known to initiate mucosal immunity. Specifically, the administration of these vesicles to animals stimulated the production of anti-Salmonella IgG antibodies, such as anti-OmpA antibodies. Exosomes also stimulated antigen-specific cell-mediated immunity. In particular, splenic mononuclear cells isolated from mice administered with exosomes derived from Salmonella-infected antigen-presenting cells increased CD4+ T cells secreting Th1-type cytokines in response to Salmonella antigens. These results demonstrate that small EVs, formed during infection, contribute to Th1 cell bias in the anti-Salmonella responses. Collectively, this study helps to unravel the role of host-derived small EVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria. Understanding the EV-mediated defense mechanisms will allow the development of future approaches to combat bacterial infections., Competing Interests: The authors have declared that no competing interests exist.

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella INtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence., Competing Interests: The authors have declared that no competing interests exist.

National-based prospective surveillance of all-age patients with acute diarrhea was conducted in China between 2009‒2018. Here we report the etiological, epidemiological, and clinical features of the 152,792 eligible patients enrolled in this analysis. Rotavirus A and norovirus are the two leading viral pathogens detected in the patients, followed by adenovirus and astrovirus. Diarrheagenic Escherichia coli and nontyphoidal Salmonella are the two leading bacterial pathogens, followed by Shigella and Vibrio parahaemolyticus. Patients aged <5 years had higher overall positive rate of viral pathogens, while bacterial pathogens were more common in patients aged 18‒45 years. A joinpoint analysis revealed the age-specific positivity rate and how this varied for individual pathogens. Our findings fill crucial gaps of how the distributions of enteropathogens change across China in patients with diarrhea. This allows enhanced identification of the predominant diarrheal pathogen candidates for diagnosis in clinical practice and more targeted application of prevention and control measures.

Background: Anti-inflammatory properties have been attributed to latex proteins of the medicinal plant Calotropis procera., Purpose: A mixture of cysteine peptidases (LPp2) from C. procera latex was investigated for control of inflammatory mediators and inflammation in a mouse model of Salmonella infection., Methods: LPp2 peptidase activity was confirmed by the BANA assay. Cytotoxicity assays were conducted with immortalized macrophages. Peritoneal macrophages (pMØ) from Swiss mice were stimulated with lipopolysaccharide (LPS) in 96-well plates and then cultured with nontoxic concentrations of LPp2. Swiss mice intravenously received LPp2 (10 mg/kg) and then were challenged intraperitoneally with virulent Salmonella enterica Ser. Typhimurium., Results: LPp2 was not toxic at dosages lower than 62.2 μg/mL. LPp2 treatments of pMØ stimulated with LPS impaired mRNA expression of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-10. LPp2 increased the intracellular bacterial killing in infected pMØ. Mice given LPp2 had a lower number of leukocytes in the peritoneal cavity in comparison to control groups 6 h after infection. The bacterial burden and histological damage were widespread in target organs of mice receiving LPp2., Conclusion: We conclude that LPp2 contains peptidases with strong anti-inflammatory properties, which may render mice more susceptible to early disseminated infection caused by Salmonella., 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 © 2020. Published by Elsevier B.V.)

Successful intestinal infection by Salmonella requires optimized invasion of the gut epithelium, a function that is energetically costly. Salmonella have therefore evolved to intricately regulate the expression of their virulence determinants by utilizing specific environmental cues. Here we show that a powerful repressor of Salmonella invasion, a cis-2 unsaturated long chain fatty acid, is present in the murine large intestine. Originally identified in Xylella fastidiosa as a diffusible signal factor for quorum sensing, this fatty acid directly interacts with HilD, the master transcriptional regulator of Salmonella, and prevents hilA activation, thus inhibiting Salmonella invasion. We further identify the fatty acid binding region of HilD and show it to be selective and biased in favour of signal factors with a cis-2 unsaturation over other intestinal fatty acids. Single mutation of specific HilD amino acids to alanine prevented fatty acid binding, thereby alleviating their repressive effect on invasion. Together, these results highlight an exceedingly sensitive mechanism used by Salmonella to colonize its host by detecting and exploiting specific molecules present within the complex intestinal environment., Competing Interests: The authors have declared that no competing interests exist.

The Enteritidis and Dublin serovars of Salmonella enterica are phylogenetically closely related yet differ significantly in host range and virulence. S Enteritidis is a broad-host-range serovar that commonly causes self-limited gastroenteritis in humans, whereas S Dublin is a cattle-adapted serovar that can infect humans, often resulting in invasive extraintestinal disease. The mechanism underlying the higher invasiveness of S Dublin remains undetermined. In this work, we quantitatively compared the proteomes of clinical isolates of each serovar grown under gut-mimicking conditions. Compared to S Enteritidis, the S Dublin proteome was enriched in proteins linked to response to several stress conditions, such as those encountered during host infection, as well as to virulence. The S Enteritidis proteome contained several proteins related to central anaerobic metabolism pathways that were undetected in S Dublin. In contrast to what has been observed in other extraintestinal serovars, most of the coding genes for these pathways are not degraded in S Dublin. Thus, we provide evidence that S Dublin metabolic functions may be much more affected than previously reported based on genomic studies. Single and double null mutants in stress response proteins Dps, YciF, and YgaU demonstrate their relevance to S Dublin invasiveness in a murine model of invasive salmonellosis. All in all, this work provides a basis for understanding interserovar differences in invasiveness and niche adaptation, underscoring the relevance of using proteomic approaches to complement genomic studies., (Copyright © 2021 American Society for Microbiology.)

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli . The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function., 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 © 2021 den Hartog, Butcher, Ablack, Pace, Ablack, Xiong, Das, Stappenbeck, Eckmann, Ernst and Crowe.)

Baicalin has been reported to protect mice against Salmonella typhimurium (S. typhimurium) infection, while its molecular mechanisms are unclear. In this study, multiplicity of infection (MOI) and observation time were measured. Cell viability and LDH levels were examined in RAW264.7 cells and H9 cells. RAW264.7 cells were stimulated with S typhimurium in the presence or absence of Baicalin, and the levels of pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The changes in reactive oxygen species (ROS) production were determined by fluorescence microscopy and ELISA. The autophagy and TLR4/MAPK/NF-κB signalling pathway were examined by immunofluorescence microscopy, quantitative reverse transcription-polymerase chain reaction and Western blotting. The results indicated that MOI of 30 and duration of autophagy evident at 5 h were applicable to this study. Baicalin prevented death of macrophages, promoted bactericidal activity, decreased the levels of pro-inflammatory cytokines and ROS and reduced the changes of key biomarkers in autophagy and TLR4/MAPK/NF-κB signalling pathway infected by S typhimurium. TLR4-overexpressed cells, autophagy and TLR4/MAPK/NF-κB signalling pathway were activated by S typhimurium, which was suppressed by Baicalin. Our findings indicated that Baicalin exerts anti-inflammatory and cell-protective effects, and it mediates autophagy by down-regulating the activity of TLR4 infected by S typhimurium., (© 2020 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from mouse bone marrow are commonly used models for the study of Salmonella infection. However, these models raise technical or ethical issues that highlight the need for alternative methods. This chapter describes a technique for immortalizing early hematopoietic progenitor cells derived from wild-type or transgenic mice and using them to produce macrophages. It validates, through a specific example, the interest of this cellular approach for the study of Salmonella infection.

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S . Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella -containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S . Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S . Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.

In contrast to enteric fever, reports of secondary hemophagocytic lymphohistiocytosis (HLH) in invasive non-typhoidal salmonellosis are scarce. We report a child with ceftriaxone-resistant invasive Salmonella Enteritidis infection with secondary HLH, who was successfully managed with intravenous meropenem. Secondary HLH in the context of S . Enteritidis has not been described before.

Ethnopharmacological Relevance: Typhoid fever treatment remains a challenge in endemic countries. Detarium microcarpum is traditionally used to manage typhoid., Aim of the Study: The study aims to explore the efficacy of hydroethanolic extract of Detarium microcarpum root bark in rats infected with salmonella., Material and Methods: The phytochemical profile of the extract was obtained by UHPLC-MS analysis in an attempt of standardization. The in vitro antimicrobial activity was determined using broth dilution method. Salmonella infection was induced by oral administration of S. thyphimurium to immunosuppressed rats. Infected rats were then treated 2 h later with the extract (75, 150 and 300 mg/kg), distilled water (normal and salmonella control) and ciprofloxacin (8 mg/kg) for control. Body weight was monitored and stools were cultured to determine the number of colony-forming units. At the end of treatment, animals were sacrificed, blood and organs were collected for hematological, biochemical and histopathological analyses., Results: Detarium microcarpum extract as well as the isolated compound (rhinocerotinoic acid) exhibited good antimicrobial activity in vitro with bacteriostatic effects. The plant extract significantly (p < 0.05) inhibited the bacterial development in infected animals with an effective dose (ED 50 ) of 75 mg/kg. In addition, the extract prevented body weight loss, hematological, biochemical and histopathological damages in treated rats., Conclusion: Detarium microcarpum extract possesses antisalmonella properties justifying its traditional use for the typhoid fever management., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background & Aims: Loss-of-function variants in the laccase domain containing 1 (LACC1) gene are associated with immune-mediated diseases, including inflammatory bowel disease. It is not clear how LACC1 balances defenses against intestinal bacteria vs intestinal inflammation or what cells are responsible for this balance in humans or mice., Methods: Lacc1 -/- mice and mice with myeloid-specific disruption of Lacc1 (Lacc1 Δmye ) were given oral Salmonella Typhimurium or dextran sodium sulfate. CD45RB hi CD4 + T cells were transferred to Lacc1 -/- Rag2 -/- mice to induce colitis. Organs were collected and analyzed by histology and protein expression. Bone marrow-derived macrophages and dendritic cells, lamina propria macrophages, and mesenteric lymph node dendritic cells were examined. We performed assays to measure intestinal permeability, cell subsets, bacterial uptake and clearance, reactive oxygen species, nitrite production, autophagy, signaling, messenger RNA, and cytokine levels., Results: Lacc1 -/- mice developed more severe T-cell transfer colitis than wild-type mice and had an increased burden of bacteria in intestinal lymphoid organs, which expressed lower levels of T helper (Th) 1 and Th17 cytokines and higher levels of Th2 cytokines. Intestinal lymphoid organs from mice with deletion of LACC1 had an increased burden of bacteria after oral administration of S Typhimurium and after administration of dextran sodium sulfate compared with wild-type mice. In macrophages, expression of LACC1 was required for toll-like receptor-induced uptake of bacteria, which required PDK1, and of mitogen-activated protein kinase (MAPK)- and nuclear factor κB-dependent induction of reactive oxygen species, reactive nitrogen species, and autophagy. Expression of LACC1 by dendritic cells was required for increasing expression of Th1 and Th17 cytokines and reducing expression of Th2 cytokines upon coculture with CD4 + T cells. Mice with LACC1-deficient myeloid cells had an increased burden of bacteria and altered T-cell cytokines in intestinal lymphoid organs, similar to Lacc1 -/- mice. Complementation of cytokines produced by myeloid cells to cocultures of LACC1-deficient myeloid cells and wild-type CD4 + T cells restored T-cell cytokine regulation. When S Typhimurium-infected Lacc1 Δmye mice were injected with these myeloid cell-derived cytokines, intestinal tissues increased production of Th1 and Th17 cytokines, and bacteria were reduced., Conclusions: Disruption of Lacc1 in mice increases the burden of bacteria in intestinal lymphoid organs and intestinal inflammation after induction of chronic colitis. LACC1 expression by myeloid cells in mice is required to clear bacteria and to regulate adaptive T-cell responses against microbes., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Salmonella Typhimurium is a Gram-negative bacterium that causes enterocolitis in humans and pigs. Lipopolysaccharide (LPS) is a component of the outer leaflet of Gram-negative bacteria that provokes endotoxin shock. LPS can be synthesized completely or incompletely and creates S (smooth) or R (rough) chemotypes. Toll-like receptors (TLR) 2, 4, and 9 initiate an inflammatory reaction to combat bacterial infections. We associated/challenged one-week-old gnotobiotic piglets with wild-type S. Typhimurium with S chemotype or its isogenic ∆ rfa mutants with R chemotype LPS. The wild-type S. Typhimurium induced TLR2 and TLR4 mRNA expression but not TLR9 mRNA expression in the ileum and colon of one-week-old gnotobiotic piglets 24 h after challenge. The TLR2 and TLR4 stimulatory effects of the S. Typhimurium ∆ rfa mutants were related to the completeness of their LPS chain. The transcription of IL-12/23 p40, IFN-γ, and IL-6 in the intestine and the intestinal and plasmatic levels of IL-12/23 p40 and IL-6 but not IFN-γ were related to the activation of TLR2 and TLR4 signaling pathways. The avirulent S . Typhimurium ∆ rfa mutants are potentially useful for modulation of the TLR2 and TLR4 signaling pathways to protect the immunocompromised gnotobiotic piglets against subsequent infection with the virulent S. Typhimurium.

The pathogenesis of Salmonella Typhimurium depends on the bacterium's ability to survive and replicate within host cells. The formation and maintenance of a unique membrane-bound compartment, termed the Salmonella-containing vacuole (SCV), is essential for S. Typhimurium pathogenesis. SCV-bound S. Typhimurium induces formation of filamentous tubules that radiate outwards from the SCV, termed Salmonella-induced filaments (SIFs). SIF formation is concomitant with the onset of replication within host epithelial cells. SIF biogenesis, formation and maintenance of the SCV, and the intracellular positioning of the SCV within the host cell requires translocation of bacterial proteins (effectors) into the host cell. Effectors secreted by the type III secretion system encoded on Salmonella pathogenicity island 2 (T3SS2) function to interfere with host cellular processes and promote both intracellular survival and replication of S. Typhimurium. Seven T3SS2-secreted effectors, SifA, SopD2, PipB2, SteA, SseJ, SseF, and SseG have previously been implicated to play complementary, redundant, and/or antagonistic roles with respect to SIF biogenesis, intracellular positioning of the SCV, and SCV membrane dynamics modulation during infection. We undertook a systematic study to delineate the contribution of each effector to these processes by (i) deleting all seven of these effectors in a single S. Typhimurium strain; and (ii) deleting combinations of multiple effectors based on putative effector function. Using this deletion mutant library, we show that each of SIF biogenesis, intracellular SCV localization, intramacrophage replication, colonization, and virulence depends on the activities of multiple effectors. Together, our data demonstrates the complex interplay between these seven effectors and highlights the necessity to study T3SS2-secreted effectors as groups, rather than studies of individual effectors., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: I have read the journal's policy and have the following conflicts: SEW is listed as an inventor on patent application WO2014200929A1, which describes a treatment to prevent the inflammation-associated expansion of Enterobacteriaceae. The other author SJT has declared that no competing interests exist.

Whether cadmium ingestion affects the susceptibility to infection and the detailed mechanism have not been investigated. We aimed to evaluate the effects of cadmium on the intestinal mucosal barrier and Salmonella infection. We found that oral administration of cadmium caused damage to the intestinal mucosal barrier, with body weight loss, an increase in inflammation, significantly reduced Muc2 expression and goblet cell loss in the intestine. The effect of cadmium on secretory cell differentiation was further demonstrated to be regulated by the overactivation of the Notch signaling pathway by increased production of ROS both in mice and in intestinal organoids. The damage of cadmium to the intestinal barrier, and goblet cell and Paneth cells loss, dramatically increased susceptibility to enteropathogensinfection at a low dose (10 2 CFU), with a high death ratio, body weight loss and severe intestinal inflammation. However, enteropathogens susceptibility and intestinal barrier damage enhanced by cadmium was alleviated by inhibiting ROS production and Notch pathway activation, with reversion of goblet cell loss. This study indicated cadmium didn't only affect the integrity of intestinal barrier and epithelial differentiation, but also increased the risk of enteropathogenic infection from food contamination or environmental pollution, which signals an alarm for public health., 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 © 2020 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context., Competing Interests: The authors have declared that no competing interests exist.

Probiotics are recognized for outcompeting pathogenic bacteria by competitive receptor-mediated colonization and secretion of functional metabolites which are antimicrobial against certain microbes as well as improving host's gut health and immunity. Recently, we have constructed a bioactive Lactobacillus casei (LC) strain, LC + mcra , by inserting mcra (myosin cross-reactive antigen) gene, which stimulates the conversion of conjugated linoleic acids. In this study, we evaluated the modulation of gut microbiome and protective roles of LC + mcra against pathogenic Salmonella enterica serovar Typhimurium (ST) and enterohemorrhagic E. coli (EHEC) infections in BALB/cJ mice. We observed that LC + mcra colonized efficiently in mice gut intestine and competitively reduced the infection with ST and EHEC in various locations of small and large intestine, specifically cecum, jejunum, and ileum ( p < 0.05). Positive modulation of the cecal microbiota, for example, higher relative abundances of Firmicutes, lower relative abundances of Proteobacteria, and increased bacterial species diversity/richness, was detected in ST-challenged mice pretreated with LC + mcra based on 16S metagenomic sequencing. Cytokine gene expression analysis indicated that mice pretreated with LC + mcra associated with attenuated bacterial pathogen-induced gut inflammation. Furthermore, mice fed daily with LC + mcra for one week could protect themselves from the impairments caused by enteric infections with ST or EHEC. These impairments include weight loss, negative hematological changes, intestinal histological alterations, and potential death. This in vivo study suggests that daily consumption of novel conjugated linoleic acids over-producing probiotic effectively improves intestinal microbiota composition and prevents/combats foodborne enteric bacterial infections with pathogenic Salmonella and diarrheagenic E. coli .

Misregulation of gut function and homeostasis impinges on the overall well-being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria-mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function., (© 2019 Federation of European Biochemical Societies.)

Bacterial pathogens employ diverse fitness and virulence mechanisms to gain an advantage in competitive niches. These lifestyle-specific traits require integration into the regulatory network of the cell and are often controlled by pre-existing transcription factors. In this review, we highlight recent advances that have been made in characterizing this regulatory flexibility in prominent members of the Enterobacteriaceae. We focus on the direct global interactions between transcription factors and their target genes in pathogenic Escherichia coli and Salmonella revealed using chromatin immunoprecipitation coupled with next-generation sequencing. Furthermore, the implications and advantages of such regulatory adaptations in benefiting distinct pathogenic lifestyles are discussed., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Salmonella causes intracellular infections in humans. Besides quinolones, third generation cephalosporins are first line drugs used for salmonellosis therapy. An unresolved anomaly of this practice involves high relapse rates associated to quinolone- or cephalosporin-susceptible Salmonella isolates in patients that are discharged clinically following initial recovery. Reduced drug accessibility to intracellular locations has been hypothesized to impair pathogen eradication although supporting evidence is lacking in vivo. Here, we uncover a novel penicillin-binding protein as the first Salmonella factor likely contributing to relapse following beta-lactam, mainly ceftriaxone, therapy., Methods: We used Salmonella enterica serovar Typhimurium mutants lacking the alternative penicillin-binding proteins PBP2 SAL or PBP3 SAL . Affinity of PBP2 SAL and PBP3 SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model., Findings: S. Typhimurium does not express PBP2 SAL or PBP3 SAL in the Mueller-Hinton medium used for susceptibility testing. The pathogen produces these PBPs in response to acidic pH and nutrient limitation, conditions found in phagosomes of mammalian cells. PBP3 SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3 SAL was unable to cause relapse in mice following ceftriaxone therapy., Interpretation: The reduced capacity of ceftriaxone to clear S. Typhimurium in vivo is favoured by a switch in beta-lactam targets. This switch, involving production of the less-susceptible PBP3 SAL , remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3 SAL with novel drugs., Competing Interests: Declarations of Competing Interest The authors declare no conflict of interest. Dr. García-del Portillo, Dr. Pucciarelli, Dr. Castanheira, Dr. Cestero and, David López-Escarpa have a patent EP20382036 pending to Spanish Office of Patents, which is relevant to this work., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Intestinal epithelial cells (IECs) are at the forefront of host-pathogen interactions, coordinating a cascade of immune responses to protect against pathogens. Here we show that IEC-intrinsic vitamin A signaling restricts pathogen invasion early in the infection and subsequently activates immune cells to promote pathogen clearance. Mice blocked for retinoic acid receptor (RAR) signaling selectively in IECs (stopΔIEC) showed higher Salmonella burden in colonic tissues early in the infection that associated with higher luminal and systemic loads of the pathogen at later stages. Higher pathogen burden in stopΔIEC mice correlated with attenuated mucosal interferon gamma (IFNγ) production by underlying immune cells. We found that, at homeostasis, the intestinal epithelium of stopΔIEC mice produced significantly lower amounts of interleukin 18 (IL-18), a potent inducer of IFNγ. Regulation of IL-18 by vitamin A was also observed in a dietary model of vitamin A supplementation. IL-18 reconstitution in stopΔIEC mice restored resistance to Salmonella by promoting epithelial cell shedding to eliminate infected cells and limit pathogen invasion early in infection. Further, IL-18 augmented IFNγ production by underlying immune cells to restrict pathogen burden and systemic spread. Our work uncovers a critical role for vitamin A in coordinating a biphasic immune response to Salmonella infection by regulating IL-18 production by IECs., Competing Interests: The authors have declared that no competing interests exist.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

The prophage BTP1 is highly conserved among strains of the pathogenic lineage Salmonella Typhimurium ST313. We aimed to analyze the role of BTP1 and the gene bstA(BTP1-encoded) in virulence of S. Typhimurium D23580, the ST313 lineage 2 reference strain. The deletion mutant D23580ΔbstA showed significantly higher replication and survival rates within human-derived THP-1 macrophages than the wild-type (WT) strain, while the mutant isolate ΔBTP1, lacking the full prophage, did not significantly differ from the WT. Interestingly, during mice infection, ΔBTP1 yielded significantly higher counts in all tested organs [spleens, livers and mesenteric lymph nodes (MLN)] than the WT, and organs were significantly enlarged compared to WT-infected animals. D23580ΔbstA significantly outcompeted the WT during competitive infection of mice, and yielded significantly enlarged spleens and MLN compared to WT-infected animals during single strain infection. Moreover, increased cellular infiltration and focal necrosis were observed in the liver samples of mice infected with D23580ΔbstA and ΔBTP1 compared to WT-infected animals. In conclusion, removal of the gene bstA and the prophage BTP1 in S. Typhimurium D23580 led to increased virulence in mice, demonstrating that bstA is an antivirulence gene., (© FEMS 2020.)

Scope: The influence of the intestinal microbiota, such as Lactobacillus, on the intestinal mucosa, particularly intestinal stem cells, remains incompletely understood. In this study, mice and intestinal organoids are used to explore the regulatory effect of Lactobacillus on the proliferation and differentiation of intestinal epithelial cells., Methods and Results: This study demonstrates that S. typhimurium causes intestinal epithelial damage and affected growth of intestinal organoids. S. typhimurium also colonizes the intestine and then causes pathological changes to the intestinal epithelium, intestinal inflammation, and even death. However, L. acidophilus alleviates damage to intestinal organoids, increases the survival ratio of mice infected with S. typhimurium, and reduces tumor necrosis factor-α (TNF-α) secretion. Moreover, L. acidophilus affects the differentiation of epithelial cells through inhibition of the excessive expansion of goblet cells and Paneth cells induced by S. typhimurium to avoid over-exhaustion. Finally, it is also demonstrated that L. acidophilus ameliorates overactivation of Wnt/β-catenin pathway by Salmonella, depending on the contact with toll-like receptor 2 (TLR2), to affect the proliferation of the intestinal epithelium., Conclusions: This study demonstrates that L. acidophilus protects the intestinal mucosa against S. typhimurium infection through not only the inhibition of pathogen invasion but also determination of the fate of the intestinal epithelium., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Inflammasomes are intracellular multiple protein complexes that mount innate immune responses to tissue damage and invading pathogens. Their excessive activation is crucial in the development and pathogenesis of inflammatory disorders. Microtubules have been reported to provide the platform for mediating the assembly and activation of NLRP3 inflammasome. Recently, we have identified the microtubule-associated immune molecule guanine nucleotide exchange factor-H1 (GEF-H1) that is crucial in coupling microtubule dynamics to the initiation of microtubule-mediated immune responses. However, whether GEF-H1 also controls the activation of other immune receptors that require microtubules is still undefined. Here we employed GEF-H1-deficient mouse bone marrow-derived macrophages (BMDMs) to interrogate the impact of GEF-H1 on the activation of NLRP3 inflammasome. NLRP3 but not NLRC4 or AIM2 inflammasome-mediated IL-1β production was dependent on dynamic microtubule network in wild-type (WT) BMDMs. However, GEF-H1 deficiency did not affect NLRP3-driven IL-1β maturation and secretion in macrophages. Moreover, α-tubulin acetylation and mitochondria aggregations were comparable between WT and GEF-H1-deficient BMDMs in response to NLRP3 inducers. Further, GEF-H1 was not required for NLRP3-mediated immune defense against Salmonella typhimurium infection. Collectively, these findings suggest that the microtubule-associated immune modulator GEF-H1 is dispensable for microtubule-mediated NLRP3 activation and host defense in mouse macrophages., Competing Interests: The authors declare no conflict of interest.

Autophagy is a crucial cellular degradation and recycling pathway. During autophagy double-membrane vesicles, called autophagosomes, encapsulate cellular components and deliver their cargo to the lytic compartment for degradation. Formation of autophagosomes is regulated by the Atg1 kinase complex in yeast and the homologous ULK1 kinase complex in mammals. While research on Atg1 and ULK1 has advanced our understanding of how these protein kinases function in autophagy, the other Atg1/ULK1 kinase complex members have received much less attention. Here, we focus on the functions of the Atg1 kinase complex members Atg11 and Atg17 as well as the ULK1 kinase complex member FIP200 in autophagy. These three proteins act as scaffolds in their respective complexes. Recent studies have made it evident that they have similar but also distinct functions. In this article, we review our current understanding of how these scaffold proteins function from autophagosome formation to fusion and also discuss their possible roles in diseases., (© 2020 Elsevier Inc. All rights reserved.)

Salmonella is an intracellular bacterium found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. Mouse models have been extensively used to model in vivo distinct aspects of human Salmonella infections and have led to the identification of several host susceptibility genes. We have investigated the susceptibility of Collaborative Cross strains to intravenous infection with Salmonella enterica serovar Typhimurium as a model of human systemic invasive infection. In this model, strain CC042/GeniUnc (CC042) mice displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weights and decreased splenocyte numbers before and after infection, affecting mostly CD8 + T cells, B cells, and all myeloid cell populations, compared with control C57BL/6J mice. CC042 mice also had lower thymus weights with a reduced total number of thymocytes and double-negative and double-positive (CD4 + , CD8 + ) thymocytes compared to C57BL/6J mice. Analysis of bone marrow-resident hematopoietic progenitors showed a strong bias against lymphoid-primed multipotent progenitors. An F2 cross between CC042 and C57BL/6N mice identified two loci on chromosome 7 ( Stsl6 and Stsl7 ) associated with differences in bacterial loads. In the Stsl7 region, CC042 carried a loss-of-function variant, unique to this strain, in the integrin alpha L ( Itgal ) gene, the causative role of which was confirmed by a quantitative complementation test. Notably, Itgal loss of function increased the susceptibility to S. Typhimurium in a (C57BL/6J × CC042)F1 mouse background but not in a C57BL/6J mouse inbred background. These results further emphasize the utility of the Collaborative Cross to identify new host genetic variants controlling susceptibility to infections and improve our understanding of the function of the Itgal gene., (Copyright © 2019 American Society for Microbiology.)

Salmonella is a human and animal pathogen that causes gastro-enteric diseases. The key to Salmonella infection is its entry into intestinal epithelial cells, where the bacterium resides within a Salmonella-containing vacuole (SCV). Salmonella entry also induces the formation of empty macropinosomes, distinct from the SCV, in the vicinity of the entering bacteria. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. Salmonella also induces membrane tubules that emanate from the SCV and lead to SCV shrinkage. Here, we show that these antipodal events are utilized by Salmonella to either establish a vacuolar niche or to be released into the cytosol by SCV rupture. We identify the molecular machinery underlying dynamic SCV growth and shrinkage. In particular, the SNARE proteins SNAP25 and STX4 participate in SCV inflation by fusion with macropinosomes. Thus, host compartment size control emerges as a pathogen strategy for intracellular niche regulation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: The editors declare that they have no conflicts of interest regarding the publication of this special issue. Acknowledgments The authors gratefully acknowledge the Espirito Santo Research Foundation (FAPES) (Grant CNPq/FAPES No. 24/2018; Termo Outorga 569/2018) and the Brazilian National Council for Research and Development (CNPq) (CNPq Grant Bolsa Produtividade 2015-2019) for the financial support to our research project. Elisardo C. Vasquez Thiago M. C. Pereira Manuel Campos-Toimil Marcelo P. Baldo Veronica A. Peotta

Hematopoietic stem cells (HSCs) undergo rapid expansion in response to stress stimuli. Here we investigate the bioenergetic processes which facilitate the HSC expansion in response to infection. We find that infection by Gram-negative bacteria drives an increase in mitochondrial mass in mammalian HSCs, which results in a metabolic transition from glycolysis toward oxidative phosphorylation. The initial increase in mitochondrial mass occurs as a result of mitochondrial transfer from the bone marrow stromal cells (BMSCs) to HSCs through a reactive oxygen species (ROS)-dependent mechanism. Mechanistically, ROS-induced oxidative stress regulates the opening of connexin channels in a system mediated by phosphoinositide 3-kinase (PI3K) activation, which allows the mitochondria to transfer from BMSCs into HSCs. Moreover, mitochondria transfer from BMSCs into HSCs, in the response to bacterial infection, occurs before the HSCs activate their own transcriptional program for mitochondrial biogenesis. Our discovery demonstrates that mitochondrial transfer from the bone marrow microenvironment to HSCs is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Non-typhoidal Salmonella (NTS) spp. causes about 40% of all infective aortitis and it is characterized by high morbidity and mortality. Human infection occurs by fecal-oral transmission through ingestion of contaminated food, milk, or water (inter-human or zoonotic transmission). Approximately 5% of patients with NTS gastroenteritis develop bacteremia and the incidence of extra-intestinal focal infection in NTS bacteremia is about 40%. The organism can reach an extra-intestinal focus through blood dissemination, direct extension from the surrounding organs and direct bacterial inoculation (e.g. invasive medical procedures). Medical and surgical interventions are both needed to successfully control the infection. Here, we report a case of abdominal sub-renal aortitis caused by Salmonella enterica serovar Enteritidis in an 80-year-old man.

Several commensal and pathogenic Gram-negative bacteria produce DNA-damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin-producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin-producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC-deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3-kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin-producing bacteria in promoting a microenvironment conducive to malignant transformation., (© 2019 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Iron is a necessary nutrient for humans and nearly all bacterial species. During Salmonella infection, macrophages limit the availability of iron to intracellular pathogens in one of the central components of nutritional immunity. However, Salmonella also have mechanisms to interfere with the antimicrobial effect of host iron withdrawal and meet their own nutrient requirements by scavenging iron from the host. Here, we provide what is, to our knowledge, the first report that SpvB, a pSLT-encoded cytotoxic protein whose function is associated with the intracellular stage of salmonellosis, perturbs macrophage iron metabolism, thereby facilitating Salmonella survival and intracellular replication. In investigating the underlying mechanism, we observed that the Salmonella effector SpvB down-regulated nuclear factor erythroid-derived 2-related factor 2 (NRF2), and its C-terminal domain was necessary and sufficient for NRF2 degradation via the proteasome pathway. Decreased NRF2 expression in the nucleus resulted in a decrease in its transcriptional target ferroportin, encoding the sole macrophage iron exporter, thus ultimately decreasing iron efflux and increasing the intracellular iron content. Additionally, SpvB contributes to the pathogenesis of Salmonella including severe serum hypoferremia, increased splenic and hepatic bacterial burden, and inflammatory injury in vivo . Together, our observations uncovered a novel contribution of SpvB to Salmonella pathology via interference with host intracellular iron metabolism.-Yang, S., Deng, Q., Sun, L., Dong, K., Li, Y., Wu, S., Huang, R. Salmonella effector SpvB interferes with intracellular iron homeostasis via regulation of transcription factor NRF2.