scholarly journals Flagellin Is Required for Host Cell Invasion and Normal Salmonella Pathogenicity Island 1 Expression by Salmonella enterica Serovar Paratyphi A

2015 ◽  
Vol 83 (9) ◽  
pp. 3355-3368 ◽  
Author(s):  
Dana Elhadad ◽  
Prerak Desai ◽  
Galia Rahav ◽  
Michael McClelland ◽  
Ohad Gal-Mor
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Salmonella Enterica ◽  
Pathogenicity Island ◽  
Dependent Manner ◽  
Content Type ◽  
Type Three Secretion System ◽  
Serovar Typhimurium ◽  
System 1 ◽  
Type Three Secretion

Salmonella entericaserovar Paratyphi A is a human-specific serovar that, together withSalmonella entericaserovar Typhi andSalmonella entericaserovar Sendai, causes enteric fever. Unlike the nontyphoidalSalmonella entericaserovar Typhimurium, the genomes ofS. Typhi andS. Paratyphi A are characterized by inactivation of multiple genes, including in the flagellum-chemotaxis pathway. Here, we explored the motility phenotype ofS. Paratyphi A and the role of flagellin in key virulence-associated phenotypes. Motility studies established that the human-adapted typhoidalS. Typhi,S. Paratyphi A, andS. Sendai are all noticeably less motile thanS. Typhimurium, and comparative transcriptome sequencing (RNA-Seq) showed that inS. Paratyphi A, the entire motility-chemotaxis regulon is expressed at significantly lowers levels than inS. Typhimurium. Nevertheless,S. Paratyphi A, likeS. Typhimurium, requires a functional flagellum for epithelial cell invasion and macrophage uptake, probably in a motility-independent mechanism. In contrast, flagella were found to be dispensable for host cell adhesion. Moreover, we demonstrate that inS. Paratyphi A, but not inS. Typhimurium, the lack of flagellin results in increased transcription of the flagellar and theSalmonellapathogenicity island 1 (SPI-1) regulons in a FliZ-dependent manner and in oversecretion of SPI-1 effectors via type three secretion system 1. Collectively, these results suggest a novel regulatory linkage between flagellin and SPI-1 inS. Paratyphi A that does not occur inS. Typhimurium and demonstrate curious distinctions in motility and the expression of the flagellum-chemotaxis regulon between these clinically relevant pathogens.

scholarly journals Differences in Host Cell Invasion and Salmonella Pathogenicity Island 1 Expression between Salmonella enterica Serovar Paratyphi A and NontyphoidalS. Typhimurium

2016 ◽  
Vol 84 (4) ◽  
pp. 1150-1165 ◽  
Author(s):  
Dana Elhadad ◽  
Prerak Desai ◽  
Guntram A. Grassl ◽  
Michael McClelland ◽  
Galia Rahav ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Salmonella Enterica ◽  
Intestinal Inflammation ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Host Cells ◽  
Host Cell Invasion ◽  
Content Type ◽  
Microaerobic Conditions

Active invasion into nonphagocytic host cells is central toSalmonella entericapathogenicity and dependent on multiple genes withinSalmonellapathogenicity island 1 (SPI-1). Here, we explored the invasion phenotype and the expression of SPI-1 in the typhoidal serovarS. Paratyphi A compared to that of the nontyphoidal serovarS. Typhimurium. We demonstrate that whileS. Typhimurium is equally invasive under both aerobic and microaerobic conditions,S. Paratyphi A invades only following growth under microaerobic conditions. Transcriptome sequencing (RNA-Seq), reverse transcription-PCR (RT-PCR), Western blot, and secretome analyses established thatS. Paratyphi A expresses much lower levels of SPI-1 genes and secretes lesser amounts of SPI-1 effector proteins thanS. Typhimurium, especially under aerobic growth. Bypassing the native SPI-1 regulation by inducible expression of the SPI-1 activator, HilA, considerably elevated SPI-1 gene expression, host cell invasion, disruption of epithelial integrity, and induction of proinflammatory cytokine secretion byS. Paratyphi A but not byS. Typhimurium, suggesting that SPI-1 expression is naturally downregulated inS. Paratyphi A. Using streptomycin-treated mice, we were able to establish substantial intestinal colonization byS. Paratyphi A and showed moderately higher pathology and intestinal inflammation in mice infected withS. Paratyphi A overexpressinghilA. Collectively, our results reveal unexpected differences in SPI-1 expression betweenS. Paratyphi A andS. Typhimurium, indicate thatS. Paratyphi A host cell invasion is suppressed under aerobic conditions, and suggest that lower invasion in aerobic sites and suppressed expression of immunogenic SPI-1 components contributes to the restrained inflammatory infection elicited byS. Paratyphi A.

scholarly journals Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis

2016 ◽  
Vol 84 (7) ◽  
pp. 2149-2158 ◽  
Author(s):  
António J. M. Santos ◽  
Charlotte H. Durkin ◽  
Sophie Helaine ◽  
Emmanuel Boucrot ◽  
David W. Holden
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Host Cell ◽  
Salmonella Enterica ◽  
Host Cells ◽  
Dependent Manner ◽  
Microtubule Organizing Center ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Chromosomal Passenger

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 intracellularSalmonella entericaserovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intactSalmonellapathogenicity 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 ofSalmonella-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 byS. Typhimurium delays epithelial cell turnover in the intestine.

scholarly journals Dormant Intracellular Salmonella enterica Serovar Typhimurium Discriminates among Salmonella Pathogenicity Island 2 Effectors To Persist inside Fibroblasts

2013 ◽  
Vol 82 (1) ◽  
pp. 221-232 ◽  
Author(s):  
Cristina Núñez-Hernández ◽  
Ana Alonso ◽  
M. Graciela Pucciarelli ◽  
Josep Casadesús ◽  
Francisco García-del Portillo
Keyword(s):  
Salmonella Enterica ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Intracellular Bacteria ◽  
Secretion Systems ◽  
Cultured Fibroblasts ◽  
Content Type ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium

ABSTRACTSalmonella entericauses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recentin vivostudies have shown that intracellular bacteria activate the TTSS encoded bySalmonellapathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observedin vivoin bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bacteria persisting inside fibroblasts, but its induction mode and the effectors involved remain unknown. Here, we show that nongrowing dormant intracellular bacteria use the two-component system OmpR-EnvZ to induce SPI-2 expression and the PhoP-PhoQ system to regulate the time at which induction takes place, 2 h postentry. Dormant bacteria were shown to discriminate the usage of SPI-2 effectors. Among the effectors tested, SseF, SseG, and SseJ were required for survival, while others, such as SifA and SifB, were not. SifA and SifB dispensability correlated with the inability of intracellular bacteria to secrete these effectors even when overexpressed. Conversely, SseJ overproduction resulted in augmented secretion and exacerbated bacterial growth. Dormant bacteria produced other effectors, such as PipB and PipB2, that, unlike what was reported for epithelial cells, did not to traffic outside the phagosomal compartment. Therefore, permissiveness for secreting only a subset of SPI-2 effectors may be instrumental for dormancy. We propose that theS. entericaserovar Typhimurium nonproliferative intracellular lifestyle is sustained by selection of SPI-2 effectors that are produced in tightly defined amounts and delivered to phagosome-confined locations.

scholarly journals Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Maria Letizia Di Martino ◽  
Viktor Ek ◽  
Wolf-Dietrich Hardt ◽  
Jens Eriksson ◽  
Mikael E. Sellin
Keyword(s):  
Host Cell ◽  
Salmonella Enterica ◽  
Cell Types ◽  
Secretion System ◽  
Host Cells ◽  
Host Cell Invasion ◽  
Cell Type ◽  
Content Type ◽  
Invasion Mechanisms ◽  
Serovar Typhimurium

ABSTRACT Bacterial host cell invasion mechanisms depend on the bacterium’s virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (S.Tm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. S.Tm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative S.Tm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how S.Tm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map S.Tm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant S.Tm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found S.Tm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells. IMPORTANCE Salmonella enterica serovar Typhimurium (S.Tm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.

scholarly journals Repression of Salmonella Host Cell Invasion by Aromatic Small Molecules from the Human Fecal Metabolome

2017 ◽  
Vol 83 (19) ◽  
Author(s):  
Rafael J. M. Peixoto ◽  
Eduardo S. Alves ◽  
Melody Wang ◽  
Rosana B. R. Ferreira ◽  
Alessandra Granato ◽  
...  
Keyword(s):  
Biological Activity ◽  
Small Molecules ◽  
Host Cell ◽  
Small Molecule ◽  
Cell Invasion ◽  
Salmonella Enterica ◽  
Chemical Diversity ◽  
Host Cell Invasion ◽  
Human Gut ◽  
Content Type

ABSTRACT The human microbiome is a collection of microorganisms that inhabit every surface of the body that is exposed to the environment, generally coexisting peacefully with their host. These microbes have important functions, such as producing vitamins, aiding in maturation of the immune system, and protecting against pathogens. We have previously shown that a small-molecule extract from the human fecal microbiome has a strong repressive effect on Salmonella enterica serovar Typhimurium host cell invasion by modulating the expression of genes involved in this process. Here, we describe the characterization of this biological activity. Using a series of purification methods, we obtained fractions with biological activity and characterized them by mass spectrometry. These experiments revealed an abundance of aromatic compounds in the bioactive fraction. Selected compounds were obtained from commercial sources and tested with respect to their ability to repress the expression of hilA, the gene encoding the master regulator of invasion genes in Salmonella. We found that the aromatic compound 3,4-dimethylbenzoic acid acts as a strong inhibitor of hilA expression and of invasion of cultured host cells by Salmonella. Future studies should reveal the molecular details of this phenomenon, such as the signaling cascades involved in sensing this bioactive molecule. IMPORTANCE Microbes constantly sense and adapt to their environment. Often, this is achieved through the production and sensing of small extracellular molecules. The human body is colonized by complex communities of microbes, and, given their biological and chemical diversity, these ecosystems represent a platform where the production and sensing of molecules occur. In previous work, we showed that small molecules produced by microbes from the human gut can significantly impair the virulence of the enteric pathogen Salmonella enterica. Here, we describe a specific compound from the human gut that produces this same effect. The results from this work not only shed light on an important biological phenomenon occurring in our bodies but also may represent an opportunity to develop drugs that can target these small-molecule interactions to protect us from enteric infections and other diseases.

scholarly journals Targeting Essential Genes in Salmonella enterica Serovar Typhimurium with Antisense Peptide Nucleic Acid

2012 ◽  
Vol 56 (12) ◽  
pp. 6407-6409 ◽  
Author(s):  
Muhammad A. Soofi ◽  
Mohamed N. Seleem
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Essential Genes ◽  
Dependent Manner ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Gene Inhibition ◽  
Dose Dependent ◽  
Antisense Peptide ◽  
Great Potency

ABSTRACTWe investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)3K cell-penetrating peptide to target possible essential genes (ligA,rpoA,rpoD,engA,tsf, andkdtA) inSalmonella entericaserovar Typhimurium and inhibit bacterial growthin vitro and in cell culture. All targeted PNA-based gene inhibition has shown great potency in gene expression inhibition in a sequence-specific and dose-dependent manner at micromolar concentrations. Among tested PNAs, the anti-rpoAand -rpoDPNAs showed the greatest potency.

scholarly journals Codependent and Independent Effects of Nitric Oxide-Mediated Suppression of PhoPQ and Salmonella Pathogenicity Island 2 on Intracellular Salmonella enterica Serovar Typhimurium Survival

2009 ◽  
Vol 77 (11) ◽  
pp. 5107-5115 ◽  
Author(s):  
Travis J. Bourret ◽  
Miryoung Song ◽  
Andrés Vázquez-Torres
Keyword(s):  
Nitric Oxide ◽  
Salmonella Enterica ◽  
Nitrosative Stress ◽  
Pathogenicity Island ◽  
High Order ◽  
Dependent Manner ◽  
Activated Macrophages ◽  
Serovar Typhimurium ◽  
Ifn Γ ◽  
Independent Effects

ABSTRACT Here we show that the Salmonella enterica serovar Typhimurium PhoQ sensor kinase lessens the cytotoxicity of reactive nitrogen species (RNS) generated by inducible nitric oxide synthase (iNOS) in the innate response of mononuclear phagocytic cells. This observation is consistent with the expression patterns of PhoP-activated genes during moderate nitrosative stress in the innate host response. In contrast, RNS synthesized during high-NO fluxes of gamma interferon (IFN-γ)-activated macrophages repress PhoP-activated lpxO, pagP, and phoP gene transcription. Because PhoP-regulated Salmonella pathogenicity island 2 (SPI2) genes are also repressed by high-order RNS (39), we investigated whether the NO-mediated inhibition of PhoPQ underlies the repression of SPI2. Our studies indicate that a third of the expression of the SPI2 spiC gene recorded in nonactivated macrophages depends on PhoQ. Transcription of spiC is repressed in IFN-γ-primed macrophages in an iNOS-dependent manner, irrespective of the phoQ status of the bacteria. Transcription of spiC is restored in IFN-γ-treated, iNOS-deficient macrophages to levels sustained by a phoQ mutant in nonactivated phagocytes, suggesting that most NO-dependent repression of spiC is due to the inhibition of PhoPQ-independent targets. Comparison of the intracellular fitness of spiC, phoQ, and spiC phoQ mutants revealed that PhoPQ and SPI2 have codependent and independent effects on S. Typhimurium survival during innate nitrosative stress. However, the intracellular survival of most S. Typhimurium bacteria is conferred by the PhoPQ two-component regulator, and the SPI2 type III secretion system is repressed by high-order RNS of IFN-γ-activated macrophages.

scholarly journals Salmonella entericaSerovar Typhimurium Binds to HeLa Cells via Fim-Mediated Reversible Adhesion and Irreversible Type Three Secretion System 1-Mediated Docking

2010 ◽  
Vol 79 (1) ◽  
pp. 330-341 ◽  
Author(s):  
Benjamin Misselwitz ◽  
Saskia K. Kreibich ◽  
Samuel Rout ◽  
Bärbel Stecher ◽  
Balamurugan Periaswamy ◽  
...  
Keyword(s):  
Host Cell ◽  
Hela Cells ◽  
Secretion System ◽  
Type I ◽  
Wild Type ◽  
Cell Binding ◽  
Reversible Adhesion ◽  
Type Three Secretion System ◽  
System 1 ◽  
Type Three Secretion

ABSTRACTThe food-borne pathogenSalmonella entericaserovar Typhimurium invades mammalian epithelial cells. This multistep process comprises bacterial binding to the host cell, activation of theSalmonellatype three secretion system 1 (T1), injection of effector proteins, triggering of host cell actin rearrangements, andS. Typhimurium entry. While the latter steps are well understood, much less is known about the initial binding step. Earlier work had implicated adhesins (but not T1) or T1 (but not other adhesins). We have studied here theSalmonellavirulence factors mediatingS. Typhimurium binding to HeLa cells. Using an automated microscopy assay and isogenicS. Typhimurium mutants, we analyzed the role of T1 and of several known adhesins (Fim, Pef, Lpf, Agf, and Shd) in host cell binding. In wild-typeS. Typhimurium, host cell binding was mostly attributable to T1. However, in the absence of T1, Fim (but not Pef, Lpf, Agf, and Shd) also mediated HeLa cell binding. Furthermore, in the absence of T1 and type I fimbriae (Fim), we still observed residual binding, pointing toward at least one additional, unidentified binding mechanism. Dissociation experiments established that T1-mediated binding was irreversible (“docking”), while Fim-mediated binding was reversible (“reversible adhesion”). Finally, we show that noninvasive bacteria docking via T1 or adhering via Fim can efficiently invade HeLa cells, if actin rearrangements are triggeredin transby a wild-typeS. Typhimurium helper strain. Our data show that binding to HeLa cells is mediated by at least two different mechanisms and that both can lead to invasion if actin rearrangements are triggered.

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