Murine AML12 hepatocytes allow Salmonella Typhimurium T3SS1-independent invasion and intracellular fate.
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ABSTRACT:
SUBMITTER: Holbert S
PROVIDER: S-EPMC8611075 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC7224572 | biostudies-literature
Project description:Salmonella typhimurium invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI1). TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1. Two unique loci, sirB and sirC, which contribute to SPI1 gene transcription were defined. sirC is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype. sirB is required for maximal expression of sirC and consists of two open reading frames located near kdsA, a gene involved in lipopolysaccharide biosynthesis. sirC expression, unlike expression of other SPI1 genes, does not require HilA. Overexpression of sirC or sirA restores expression of a subset of SPI1 genes, including invF and sspC, in the absence of HilA. These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression. We postulate that HilA-independent activation of inv expression is important for efficient assembly and function of the SPI1 TTSS.
| S-EPMC93764 | biostudies-literature
Project description:BackgroundInvasive infections due to foodborne pathogens, including Salmonella enterica serovar Typhimurium, are prevalent and life-threatening. This study aimed to evaluate the effects of ginsenoside Rg3 (Rg3) on the adhesion, invasion, and intracellular survival of S. Typhimurium.MethodsThe impacts of Rg3 on bacterial growth and host cell viability were determined using the time kill and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assays, respectively. Gentamicin assay and confocal microscopic examination were undertaken to determine the effects of Rg3 on the adhesive and invasive abilities of S. Typhimurium to Caco-2 and RAW 264.7 cells. Quantitative reverse transcription polymerase chain reaction was performed to assess the expression of genes correlated with the adhesion, invasion, and virulence of S. Typhimurium.ResultsSubinhibitory concentrations of Rg3 significantly reduced (p < 0.05) the adhesion, invasion, and intracellular survival of S. Typhimurium. Rg3 considerably reduced (p < 0.05) the bacterial motility as well as the release of nitrite from infected macrophages in a concentration-dependent manner. The expression of genes related to the adhesion, invasion, quorum sensing, and virulence of S. Typhimurium including cheY, hilA, OmpD, PrgK, rsgE, SdiA, and SipB was significantly reduced after Rg3 treatment. Besides, the compound downregulated rac-1 and Cdc-42 that are essential for actin remodeling and membrane ruffling, thereby facilitating Salmonella entry into host cells. This report is the first to describe the effects of Rg3 on "trigger" entry mechanism and intracellular survival S. Typhimurium.ConclusionRg3 could be considered as a supplement agent to prevent S. Typhimurium infection.
| S-EPMC7790883 | biostudies-literature
Project description:Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S Typhimurium delays epithelial cell turnover in the intestine.
| S-EPMC4936369 | biostudies-literature
Project description:Flagella are multiprotein complexes necessary for swimming and swarming motility. In Salmonella enterica serovar Typhimurium, flagella-mediated motility is repressed by the PhoP/PhoQ regulatory system. We now report that Salmonella can move on 0.3% agarose media in a flagella-independent manner when experiencing the PhoP/PhoQ-inducing signal low Mg(2+). This motility requires the PhoP-activated mgtA, mgtC, and pagM genes, which specify a Mg(2+) transporter, an inhibitor of Salmonella's own F1Fo ATPase, and a small protein of unknown function, respectively. The MgtA and MgtC proteins are necessary for pagM expression because pagM mRNA levels were lower in mgtA and mgtC mutants than in wild-type Salmonella, and also because pagM expression from a heterologous promoter rescued motility in mgtA and mgtC mutants. PagM promotes group motility by a surface protein(s), as a pagM-expressing strain conferred motility upon a pagM null mutant, and proteinase K treatment eliminated motility. The pagM gene is rarely found outside subspecies I of S. enterica and often present in nonfunctional allelic forms in organisms lacking the identified motility. Deletion of the pagM gene reduced bacterial replication on 0.3% agarose low Mg(2+) media but not in low Mg(2+) liquid media. Our findings define a form of motility that allows Salmonella to scavenge nutrients and to escape toxic compounds in low Mg(2+) semisolid environments.
| S-EPMC4330729 | biostudies-literature
Project description:Salmonella enterica serovar Typhimurium infects intestinal epithelia and macrophages, which is prevented by inhibiting adhesion and cell invasion. This study aimed to investigate the role of methyl gallate (MG) in adhesion, invasion, and intracellular survival of Salmonella Typhimurium in Caco-2 and RAW 264.7 cells via a gentamicin protection assay, confocal microscopy, and quantitative reverse-transcription polymerase chain reaction. MG (30 µg/mL) inhibited adhesion and invasion of Salmonella Typhimurium by 54.01% and 60.5% in RAW 264.7 cells, respectively. The combination of MG with sub-minimum inhibitory concentration (MIC) of marbofloxacin (MRB) inhibited the adhesion, invasion, and intracellular survival by 70.49%, 67.36%, and 74%, respectively. Confocal microscopy further revealed reductions in bacterial count in Caco-2 cells treated with MG alone or with sub-MIC of MRB. Furthermore, MG alone or in combination with sub-MIC of MRB decreased the motility of Salmonella Typhimurium. Quorum sensing genes including sdiA, srgE, and rck were downregulated by 52.8%, 61.7%, and 22.2%, respectively. Moreover, rac-1 was downregulated by 56.9% and 71.9% for MG alone and combined with sub-MIC of MRB, respectively, in mammalian cells. Furthermore, MG downregulated virulence genes of Salmonella Typhimurium including cheY, ompD, sipB, lexA, and ompF by 59.6%, 60.2%, 20.5%, 31.4%, and 16.2%, respectively. Together, the present results indicate that MG alone or in combination with a sub-MIC of MRB effectively inhibited the adhesion, invasion, and intracellular survival of Salmonella Typhimurium in vitro by downregulating quorum sensing and virulence genes.
| S-EPMC6389159 | biostudies-literature
Project description:Penetration of intestinal epithelial cells by Salmonella enterica serovar Typhimurium requires the expression of invasion genes, found in Salmonella pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of Escherichia coli csrA, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of csrA was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes, hilA, invF, sipC, and prgH. Overexpression of csrA from an induced araBAD promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected hilA, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate hilA. We also found that both the loss and the overexpression of csrA reduced the expression of two regulators of hilA, hilC and hilD, suggesting that csrA exerts its control of hilA through one or both of these regulators. We further found, however, that CsrA could affect the expression of both invF and sipC independent of its effects on hilA. One additional striking phenotype of the csrA mutant, not observed in a comparable E. coli mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the csrA mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the csrA-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the csrA mutant arise from effects of CsrA on different targets.
| S-EPMC97782 | biostudies-literature
Project description:UnlabelledSalmonella enterica pathogenicity island 1 (SPI-1) encodes proteins required for invasion of gut epithelial cells. The timing of invasion is tightly controlled by a complex regulatory network. The transcription factor (TF) HilD is the master regulator of this process and senses environmental signals associated with invasion. HilD activates transcription of genes within and outside SPI-1, including six other TFs. Thus, the transcriptional program associated with host cell invasion is controlled by at least 7 TFs. However, very few of the regulatory targets are known for these TFs, and the extent of the regulatory network is unclear. In this study, we used complementary genomic approaches to map the direct regulatory targets of all 7 TFs. Our data reveal a highly complex and interconnected network that includes many previously undescribed regulatory targets. Moreover, the network extends well beyond the 7 TFs, due to the inclusion of many additional TFs and noncoding RNAs. By comparing gene expression profiles of regulatory targets for the 7 TFs, we identified many uncharacterized genes that are likely to play direct roles in invasion. We also uncovered cross talk between SPI-1 regulation and other regulatory pathways, which, in turn, identified gene clusters that likely share related functions. Our data are freely available through an intuitive online browser and represent a valuable resource for the bacterial research community.ImportanceInvasion of epithelial cells is an early step during infection by Salmonella enterica and requires secretion of specific proteins into host cells via a type III secretion system (T3SS). Most T3SS-associated proteins required for invasion are encoded in a horizontally acquired genomic locus known as Salmonella pathogenicity island 1 (SPI-1). Multiple regulators respond to environmental signals to ensure appropriate timing of SPI-1 gene expression. In particular, there are seven transcription regulators that are known to be involved in coordinating expression of SPI-1 genes. We have used complementary genome-scale approaches to map the gene targets of these seven regulators. Our data reveal a highly complex and interconnected regulatory network that includes many previously undescribed target genes. Moreover, our data functionally implicate many uncharacterized genes in the invasion process and reveal cross talk between SPI-1 regulation and other regulatory pathways. All datasets are freely available through an intuitive online browser.
| S-EPMC5013294 | biostudies-literature
Project description:BackgroundCritical to the development of Salmonellosis in humans is the interaction of the bacterium with the epithelial lining of the gastrointestinal tract. Traditional scientific reasoning held type III secretion system (T3SS) as the virulence factor responsible for bacterial invasion. In this study, field-isolated Salmonella enterica serovar Kentucky and a known human pathogen Salmonella enterica serovar Typhimurium were mutated and evaluated for the invasion of human colorectal adenocarcinoma epithelial cells.ResultsS. enterica serovar Kentucky was shown to actively invade a eukaryotic monolayer, though at a rate that was significantly lower than Typhimurium. Additionally, strains mutated for T3SS formation were less invasive than the wild-type strains, but the decrease in invasion was not significant in Kentucky.ConclusionsStrains mutated for T3SS formation were able to initiate invasion of the eukaryotic monolayer to varying degrees based on strain, In the case of Kentucky, the mutated strain initiated invasion at a level that was not significantly different from the wild-type strain. A different result was observed for Typhimurium as the mutation significantly lowered the rate of invasion in comparison to the wild-type strain.
| S-EPMC5382418 | biostudies-literature
Project description:Recent evidence demonstrated that chronic intake of quercetin attenuated hepatic fat accumulation in various animal models of obesity and diabetes. However, whether quercetin has the ability to enhance energy metabolism in hepatocytes and its exact mechanisms have yet to be identified. In the present study, we investigated whether quercetin directly enhanced the energy metabolism of cultured hepatocytes by focusing on lipophagy, involving selective autophagic degradation of lipid droplets. As an indicator of mitochondrial respiration, oxygen consumption was measured following 12-h treatment with quercetin or its related flavonoids, isorhamnetin and rutin (10 μM) using an extracellular flux analyzer. Treatment of alpha mouse liver 12 (AML12) hepatocytes with quercetin enhanced mitochondrial respiration, but isorhamnetin and rutin did not. Results of a palmitate-bovine serum albumin fatty acid oxidation assay showed that quercetin significantly increased the oxygen consumption of AML12 hepatocytes, suggesting enhanced fatty acid β-oxidation. However, as expression levels of mitochondrial oxidative phosphorylation proteins were unaltered by quercetin, we explored whether lipophagy contributed to enhanced fatty acid β-oxidation. Increased colocalization of lipid droplets and lysosomes confirmed that quercetin promoted lipophagy in AML12 hepatocytes. Furthermore, pharmacological inhibition of the autophagy-lysosomal pathway abolished the enhancement of fatty acid β-oxidation induced by quercetin in AML12 hepatocytes, suggesting that the enhancement of lipophagy by quercetin contributed to increased fatty acid β-oxidation. Finally, we showed that quercetin could activate AMPK signaling, which regulates autophagy even under nutrient-sufficient conditions. Our findings indicate that quercetin enhanced energy metabolism by a potentially novel mechanism involving promotion of lipophagy to produce the substrate for fatty acid β-oxidation in mitochondria through activation of AMPK signaling. Our results suggest the possibility that nutrient-induced lipophagy might contributes to the reduction of fat in hepatocytes.
| S-EPMC8233147 | biostudies-literature