ABSTRACT
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.
Subject(s)
Cell Differentiation , ErbB Receptors , Organoids , Salmonella Infections , Salmonella typhimurium , Stem Cells , Animals , Organoids/microbiology , Stem Cells/metabolism , Mice , Salmonella typhimurium/pathogenicity , Salmonella typhimurium/physiology , Salmonella Infections/microbiology , Salmonella Infections/pathology , ErbB Receptors/metabolism , ErbB Receptors/genetics , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Cell Proliferation , Disease Models, Animal , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Edema , Fibronectins , Animals , Fibronectins/metabolism , Mice , Edema/metabolism , Edema/pathology , Edema/microbiology , Extracellular Matrix/metabolism , Proteomics/methods , Mice, Inbred C57BL , Salmonella Infections/microbiology , Salmonella Infections/pathology , Salmonella Infections/metabolism , Intestines/microbiology , Intestines/pathology , Salmonella typhimurium/pathogenicity , Salmonella typhimurium/metabolismABSTRACT
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.
Subject(s)
Colitis , Salmonella Infections , Humans , Mice , Animals , Salmonella typhimurium , Disease Models, Animal , Salmonella Infections/complications , Salmonella Infections/pathology , Intestines/pathology , Colitis/microbiology , Colitis/pathologyABSTRACT
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.
Subject(s)
Coinfection , Salmonella Infections , Schistosomiasis mansoni , Schistosomiasis , Animals , Mice , Schistosomiasis mansoni/complications , Schistosomiasis mansoni/pathology , Salmonella typhimurium , Spleen/pathology , Coinfection/microbiology , Liver/pathology , Schistosoma mansoni/physiology , Salmonella Infections/pathology , FibrosisABSTRACT
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.
Subject(s)
Colonic Neoplasms , Salmonella Infections , Animals , Mice , Retrospective Studies , Salmonella , Salmonella Infections/pathology , Risk FactorsABSTRACT
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-1ß (IL-1ß), 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.
Subject(s)
Caffeine , Salmonella Infections , Animals , Anti-Inflammatory Agents/therapeutic use , Caffeine/pharmacology , Caffeine/therapeutic use , Disease Models, Animal , Macrophages, Peritoneal , Mice , Nitric Oxide Synthase Type II/metabolism , Salmonella Infections/drug therapy , Salmonella Infections/microbiology , Salmonella Infections/pathology , Salmonella typhimuriumABSTRACT
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.
Subject(s)
Anemia, Iron-Deficiency/drug therapy , Bacteremia/complications , Iron/blood , Salmonella Infections/complications , Anemia, Iron-Deficiency/blood , Anemia, Iron-Deficiency/complications , Animals , Bacteremia/blood , Bacteremia/pathology , Bacterial Load , Iron/administration & dosage , Iron/adverse effects , Male , Mice , Mice, Inbred C57BL , Salmonella Infections/blood , Salmonella Infections/pathologyABSTRACT
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.
Subject(s)
Eggs/microbiology , Food Safety/methods , Refrigeration/methods , Salmonella Food Poisoning/prevention & control , Salmonella Infections/prevention & control , Salmonella typhimurium/pathogenicity , Animals , Australia , Chickens , Colony Count, Microbial , Female , Food Microbiology , Gene Expression Regulation, Bacterial , Genes, Bacterial , Humans , Mice , Mice, Inbred BALB C , Salmonella Food Poisoning/microbiology , Salmonella Food Poisoning/pathology , Salmonella Infections/microbiology , Salmonella Infections/pathology , Salmonella typhimurium/genetics , Salmonella typhimurium/growth & development , Temperature , VirulenceABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Intestinal Mucosa/microbiology , Membrane Proteins/metabolism , Salmonella typhimurium/metabolism , Type III Secretion Systems/metabolism , Vacuoles/microbiology , Animals , Bacterial Proteins/genetics , Cells, Cultured , Enterocytes/metabolism , Enterocytes/microbiology , Intestinal Mucosa/cytology , Macrophages/immunology , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , RNA-Seq , Salmonella Infections/pathology , Tight Junctions/microbiology , Type III Secretion Systems/genetics , Vacuoles/metabolismABSTRACT
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 ß-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.
Subject(s)
Actins/chemistry , Cytoskeleton/chemistry , Salmonella Infections/microbiology , Salmonella enterica/physiology , p21-Activated Kinases/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Phosphorylation , Rho Guanine Nucleotide Exchange Factors/genetics , Rho Guanine Nucleotide Exchange Factors/metabolism , Salmonella Infections/metabolism , Salmonella Infections/pathology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , p21-Activated Kinases/geneticsABSTRACT
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.
Subject(s)
Colitis/drug therapy , Genistein/pharmacology , Goblet Cells/pathology , Salmonella Infections/pathology , Stem Cells/cytology , Animals , Autophagy/drug effects , Colitis/microbiology , Colon/drug effects , Colon/pathology , Cyclooxygenase 2 , Dinoprostone , Gastrointestinal Microbiome , Inflammation , Male , Mice, Inbred C57BL , Salmonella Infections/drug therapy , Salmonella typhimurium , Wnt Signaling Pathway/drug effectsABSTRACT
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.
Subject(s)
Inflammation/microbiology , Salmonella Infections/microbiology , Salmonella typhimurium/metabolism , Animals , Host-Pathogen Interactions , Humans , Inflammation/pathology , Salmonella Infections/pathologyABSTRACT
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.
Subject(s)
Antigen-Presenting Cells/immunology , Extracellular Vesicles/immunology , Immunity, Cellular/immunology , Macrophages/immunology , Salmonella Infections/immunology , Salmonella typhimurium/immunology , Th1 Cells/immunology , Animals , Female , Macrophages/pathology , Mice , Mice, Inbred BALB C , Salmonella Infections/microbiology , Salmonella Infections/pathologyABSTRACT
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 102 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.
Subject(s)
Placenta/pathology , Pregnancy Complications, Infectious/pathology , Salmonella Infections/pathology , Salmonella enteritidis/physiology , Animals , Chorioamnionitis/microbiology , Chorioamnionitis/pathology , Disease Models, Animal , Female , Fetal Growth Retardation/etiology , Fetal Growth Retardation/pathology , Male , Mice , Mice, Inbred BALB C , Placenta/microbiology , Placenta Diseases/microbiology , Placenta Diseases/pathology , Pregnancy , Pregnancy Complications, Infectious/microbiology , Salmonella Food Poisoning/complications , Salmonella Food Poisoning/pathology , Salmonella Infections/complications , Systemic Inflammatory Response Syndrome/complications , Systemic Inflammatory Response Syndrome/microbiology , Systemic Inflammatory Response Syndrome/pathologyABSTRACT
National-based prospective surveillance of all-age patients with acute diarrhea was conducted in China between 2009â2018. 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 18â45 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.
Subject(s)
Diarrhea/epidemiology , Diarrhea/pathology , Gastroenteritis/epidemiology , Gastroenteritis/pathology , Adolescent , Adult , Age Factors , Caliciviridae Infections/epidemiology , Caliciviridae Infections/pathology , Child , Child, Preschool , China/epidemiology , Diarrhea/microbiology , Escherichia coli/isolation & purification , Escherichia coli Infections/epidemiology , Escherichia coli Infections/pathology , Gastroenteritis/microbiology , Humans , Middle Aged , Norovirus/isolation & purification , Rotavirus/isolation & purification , Rotavirus Infections/epidemiology , Rotavirus Infections/pathology , Salmonella/isolation & purification , Salmonella Infections/epidemiology , Salmonella Infections/pathology , Shigella/isolation & purification , Vibrio Infections/epidemiology , Vibrio Infections/pathology , Vibrio parahaemolyticus/isolation & purification , Young AdultABSTRACT
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.
Subject(s)
Epithelial Cells/microbiology , Salmonella Infections/microbiology , Salmonella typhimurium/physiology , Virus Latency/physiology , 3T3 Cells , Animals , Caco-2 Cells , Epithelial Cells/pathology , Genomic Islands/genetics , HeLa Cells , Humans , Mice , Salmonella Infections/pathology , Salmonella typhimurium/genetics , Salmonella typhimurium/pathogenicity , THP-1 Cells , Vacuoles/microbiology , Vacuoles/pathology , Virulence Factors/genetics , Virus Latency/geneticsABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Fatty Acids, Unsaturated/metabolism , Intestines/microbiology , Laryngeal Neoplasms/microbiology , Salmonella Infections/microbiology , Salmonella typhimurium/pathogenicity , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Bacterial Proteins/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/microbiology , Carcinoma, Squamous Cell/pathology , Gene Expression Regulation, Bacterial , Humans , Intestines/physiology , Laryngeal Neoplasms/metabolism , Laryngeal Neoplasms/pathology , Mice , Mice, Inbred C57BL , Salmonella Infections/metabolism , Salmonella Infections/pathology , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Tumor Cells, Cultured , VirulenceABSTRACT
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-1ß, 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.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Calotropis/chemistry , Peptide Hydrolases/pharmacology , Plant Proteins/pharmacology , Salmonella Infections/drug therapy , Salmonella typhimurium/drug effects , Animals , Anti-Inflammatory Agents/isolation & purification , Gene Expression Regulation , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Latex/chemistry , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Mice , Peptide Hydrolases/isolation & purification , Plant Components, Aerial/chemistry , Plant Extracts/chemistry , Plant Proteins/isolation & purification , Plants, Medicinal , Primary Cell Culture , Salmonella Infections/immunology , Salmonella Infections/microbiology , Salmonella Infections/pathology , Salmonella typhimurium/immunology , Salmonella typhimurium/pathogenicity , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Autophagy/drug effects , Flavonoids/pharmacology , Human Embryonic Stem Cells/drug effects , Inflammation/prevention & control , Macrophages/drug effects , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Salmonella Infections/prevention & control , Salmonella typhimurium/pathogenicity , Toll-Like Receptor 4/metabolism , Animals , Cytokines/metabolism , Human Embryonic Stem Cells/enzymology , Human Embryonic Stem Cells/microbiology , Human Embryonic Stem Cells/pathology , Humans , Inflammation/enzymology , Inflammation/microbiology , Inflammation/pathology , Inflammation Mediators/metabolism , Macrophages/enzymology , Macrophages/microbiology , Macrophages/pathology , Mice , NF-kappa B/genetics , RAW 264.7 Cells , Reactive Oxygen Species/metabolism , Salmonella Infections/enzymology , Salmonella Infections/microbiology , Salmonella Infections/pathology , Signal Transduction , Toll-Like Receptor 4/geneticsABSTRACT
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.