The Actin-Polymerizing Activity of SipA Is Not Essential for Salmonella enterica Serovar Typhimurium-Induced Mucosal Inflammation

ABSTRACTSalmonella entericaserovar Typhimurium depends on type III secretion systems to inject effector proteins into host cells to promote bacterial invasion and to induce intestinal inflammation. SipA, a type III effector, is known to play important roles in both the invasion and the elicitation of intestinal inflammation. The actin-modulating activity of SipA has been shown to promoteSalmonellaentry into epithelial cells. To investigate whether the actin-modulating activity of SipA is required for its ability to induce an inflammatory responsein vivo, we generated the SipAK635A E637Wmutant, which is deficient in actin-modulating activity.Salmonellastrains expressing the chromosomal SipAK635A E637Wpoint mutation had reduced invasion abilities but still caused colitis similar to that caused by the wild-type strain in a mouse model of infection. Our data indicate that the SipA actin-polymerizing activity is not essential for the SipA-induced inflammatory response in the mouse model of infection.

Download Full-text

Cytosporone B, an Inhibitor of the Type III Secretion System of Salmonella enterica Serovar Typhimurium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02421-12 ◽

2013 ◽

Vol 57 (5) ◽

pp. 2191-2198 ◽

Cited By ~ 40

Author(s):

Jianfang Li ◽

Chao Lv ◽

Weiyang Sun ◽

Zhenyu Li ◽

Xiaowei Han ◽

...

Keyword(s):

Type Iii Secretion ◽

Salmonella Enterica ◽

Bacterial Resistance ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Content Type ◽

Type Iii ◽

Serovar Typhimurium

ABSTRACTBacterial virulence factors have been increasingly regarded as attractive targets for development of novel antibacterial agents. Virulence inhibitors are less likely to generate bacterial resistance, which makes them superior to traditional antibiotics that target bacterial viability.Salmonella entericaserovar Typhimurium, an important food-borne human pathogen, has type III secretion system (T3SS) as its major virulence factor. T3SS secretes effector proteins to facilitate invasion into host cells. In this study, we identified several analogs of cytosporone B (Csn-B) that strongly block the secretion ofSalmonellapathogenicity island 1 (SPI-1)-associated effector proteins, without affecting the secretion of flagellar protein FliCin vitro. Csn-B and two other derivatives exhibited a strong inhibitory effect on SPI-1-mediated invasion to HeLa cells, while no significant toxicity to bacteria was observed. Nucleoid proteins Hha and H-NS bind to the promoters of SPI-1 regulator geneshilD,hilC, andrtsAto repress their expression and consequently regulate the expression of SPI-1 apparatus and effector genes. We found that Csn-B upregulated the transcription ofhhaandhns, implying that Csn-B probably affected the secretion of effectors through the Hha–H-NS regulatory pathway. In summary, this study presented an effective SPI-1 inhibitor, Csn-B, which may have potential in drug development against antibiotic-resistantSalmonella.

Download Full-text

Identification of Salmonella Pathogenicity Island-2 Type III Secretion System Effectors Involved in Intramacrophage Replication of S. enterica Serovar Typhimurium: Implications for Rational Vaccine Design

mBio ◽

10.1128/mbio.00065-13 ◽

2013 ◽

Vol 4 (2) ◽

Cited By ~ 69

Author(s):

Rita Figueira ◽

Kathryn G. Watson ◽

David W. Holden ◽

Sophie Helaine

Keyword(s):

Type Iii Secretion ◽

Salmonella Enterica ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Content Type ◽

Type Iii ◽

Serovar Typhimurium ◽

Bacterial Replication ◽

Heterologous Antigens

ABSTRACT Salmonella enterica serovars cause severe diseases in humans, such as gastroenteritis and typhoid fever. The development of systemic disease is dependent on a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). Translocation of effector proteins across the Salmonella-containing vacuole, via the SPI-2 T3SS, enables bacterial replication within host cells, including macrophages. Here, we investigated the contribution of these effectors to intramacrophage replication of Salmonella enterica serovar Typhimurium using Fluorescence Dilution, a dual-fluorescence tool which allows direct measurement of bacterial replication. Of 32 strains, each carrying single mutations in genes encoding effectors, 10 (lacking sifA, sseJ, sopD2, sseG, sseF, srfH, sseL, spvD, cigR, or steD) were attenuated in replication in mouse bone marrow-derived macrophages. The replication profiles of strains combining deletions in effector genes were also investigated: a strain lacking the genes sseG, sopD2, and srfH showed an increased replication defect compared to single-mutation strains and was very similar to SPI-2 T3SS-deficient bacteria with respect to its replication defect. This strain was substantially attenuated in virulence in vivo and yet retained intracellular vacuole integrity and a functional SPI-2 T3SS. Moreover, this strain was capable of SPI-2 T3SS-mediated delivery of a model antigen for major histocompatibility complex (MHC) class I-dependent T-cell activation. This work establishes a basis for the use of a poly-effector mutant strain as an attenuated vaccine carrier for delivery of heterologous antigens directly into the cytoplasm of host cells. IMPORTANCE Live attenuated strains of Salmonella enterica serotype Typhi have generated much interest in the search for improved vaccines against typhoid fever and as vaccine vectors for the delivery of heterologous antigens. A promising vaccine candidate is the ΔaroC ΔssaV S. Typhi strain, which owes its attenuation mainly to lack of a type III secretion system (SPI-2 T3SS). The SPI-2 T3SS is important for bacterial proliferation inside macrophages, but not all of the effectors involved in this process have been identified. Here, we show that 10 effectors of the related strain S. Typhimurium contribute to intracellular replication in macrophages. Moreover, we establish that a poly-effector mutant strain of S. Typhimurium can have a severe replication defect and maintain a functional SPI-2 T3SS, which can be exploited for delivery of heterologous antigens.

Download Full-text

A LiveSalmonellaVaccine Delivering PcrV through the Type III Secretion System Protects againstPseudomonas aeruginosa

mSphere ◽

10.1128/msphere.00116-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 5

Author(s):

Julia Aguilera-Herce ◽

Meritxell García-Quintanilla ◽

Rocío Romero-Flores ◽

Michael J. McConnell ◽

Francisco Ramos-Morales

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Salmonella Enterica ◽

Opportunistic Pathogen ◽

Host Cells ◽

Gram Negative ◽

Content Type ◽

Specific Antibodies ◽

Type Iii ◽

Serovar Typhimurium

ABSTRACTPseudomonas aeruginosais a common Gram-negative opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. The development of a broadly protective vaccine againstP. aeruginosaremains a major challenge. Here, we used an attenuated strain ofSalmonella entericaserovar Typhimurium as a vehicle to expressP. aeruginosaantigens. A fusion between theS. entericatype III secretion effector protein SseJ and theP. aeruginosaantigen PcrV expressed under the control of thesseApromoter was translocated bySalmonellainto host cellsin vitroand elicited the generation of specific antibodies in mice. Mice immunized with attenuatedSalmonellaexpressing this fusion had reduced bacterial loads in the spleens and lungs and lower serum levels of proinflammatory cytokines than control mice afterP. aeruginosainfection. Importantly, immunized mice also showed significantly enhanced survival in this model. These results suggest that type III secretion effectors ofS. entericaare appropriate carriers in the design of a live vaccine to prevent infections caused byP. aeruginosa.IMPORTANCEThe Gram-negative bacteriumPseudomonas aeruginosais an important opportunistic pathogen that causes infections in cystic fibrosis and hospitalized patients. Therapeutic treatments are limited due to the emergence and spread of new antibiotic-resistant strains. In this context, the development of a vaccine is a priority. Here, we used an attenuated strain ofSalmonella entericaserovar Typhimurium as a vehicle to express and deliver thePseudomonasantigen PcrV. This vaccine induced the generation of specific antibodies in mice and protected them from lethal infections withP. aeruginosa. This is an important step toward the development of an effective vaccine for the prevention of infections caused byP. aeruginosain humans.

Download Full-text

A Salmonella Virulence Factor Activates the NOD1/NOD2 Signaling Pathway

mBio ◽

10.1128/mbio.00266-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 18

Author(s):

A. Marijke Keestra ◽

Maria G. Winter ◽

Daisy Klein-Douwel ◽

Mariana N. Xavier ◽

Sebastian E. Winter ◽

...

Keyword(s):

Signaling Pathway ◽

Type Iii Secretion ◽

Salmonella Enterica ◽

Intestinal Inflammation ◽

Inflammatory Responses ◽

Type Iii Secretion System ◽

Secretion System ◽

Content Type ◽

Type Iii

ABSTRACTThe invasion-associated type III secretion system (T3SS-1) ofSalmonella entericaserotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasionin vitrobut required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responsesin vitroandin vivo.IMPORTANCESalmonella entericaserotype Typhimurium (S. Typhimurium) deploys a type III secretion system (T3SS-1) to induce intestinal inflammation and benefits from the ensuing host response, which enhances growth of the pathogen in the intestinal lumen. However, the mechanisms by which the T3SS-1 triggers inflammatory responses have not been resolved. Here we show that the T3SS-1 effector protein SipA induces NF-κB activation and intestinal inflammation by activating the NOD1/NOD2 signaling pathway. These data suggest that the T3SS-1 escalates innate responses through a SipA-mediated activation of pattern recognition receptors in the host cell cytosol.

Download Full-text

MdsABC-Mediated Pathway for Pathogenicity in Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.00653-15 ◽

2015 ◽

Vol 83 (11) ◽

pp. 4266-4276 ◽

Cited By ~ 5

Author(s):

Saemee Song ◽

Boeun Lee ◽

Ji-Hyun Yeom ◽

Soonhye Hwang ◽

Ilnam Kang ◽

...

Keyword(s):

Salmonella Enterica ◽

Disulfide Bonds ◽

Efflux Pump ◽

Host Cells ◽

Deletion Strain ◽

Bacterial Number ◽

Phagocytic Cells ◽

Content Type ◽

Serovar Typhimurium ◽

Key Factor

ABSTRACTMdsABC is aSalmonella-specific tripartite efflux pump that has been implicated in the virulence ofSalmonella entericaserovar Typhimurium; however, little is known about the virulence factors associated with this pump. We observed MdsABC expression-dependent alterations in the degree of resistance to extracellular oxidative stress and macrophage-mediated killing. Thin-layer chromatography and tandem mass spectrometry analyses revealed that overexpression of MdsABC led to increased secretion of 1-palmitoyl-2-stearoyl-phosphatidylserine (PSPS), affecting the ability of the bacteria to invade and survive in host cells. Overexpression of MdsABC and external addition of PSPS similarly rendered themdsABCdeletion strain resistant to diamide. Diagonal gel analysis showed that PSPS treatment reduced the diamide-mediated formation of disulfide bonds, particularly in the membrane fraction of the bacteria.Salmonellainfection of macrophages induced the upregulation of MdsABC expression and led to an increase of intracellular bacterial number and host cell death, similar to the effects of MdsABC overexpression and PSPS pretreatment on themdsABCdeletion strain. Our study shows that MdsABC mediates a previously uncharacterized pathway that involves PSPS as a key factor for the survival and virulence ofS. Typhimurium in phagocytic cells.

Download Full-text

Neutrophils Are a Source of Gamma Interferon during Acute Salmonella enterica Serovar Typhimurium Colitis

Infection and Immunity ◽

10.1128/iai.01508-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1692-1697 ◽

Cited By ~ 28

Author(s):

Alanna M. Spees ◽

Dawn D. Kingsbury ◽

Tamding Wangdi ◽

Mariana N. Xavier ◽

Renée M. Tsolis ◽

...

Keyword(s):

Salmonella Enterica ◽

Intestinal Inflammation ◽

Gamma Interferon ◽

Content Type ◽

Inflammatory Monocytes ◽

Intestinal Lesions ◽

Serovar Typhimurium ◽

Cellular Source ◽

Ifn Γ ◽

Cecal Mucosa

ABSTRACTGamma interferon (IFN-γ) is an important driver of intestinal inflammation during colitis caused bySalmonella entericaserovar Typhimurium. Here we used the mouse colitis model to investigate the cellular sources of IFN-γ in the cecal mucosa during the acute phase of anS. Typhimurium infection. While IFN-γ staining was detected in T cells, NK cells, and inflammatory monocytes at 2 days after infection, the majority of IFN-γ-positive cells in the cecal mucosa were neutrophils. Furthermore, neutrophil depletion blunted mucosalIfngexpression and reduced the severity of intestinal lesions duringS. Typhimurium infection. We conclude that neutrophils are a prominent cellular source of IFN-γ during the innate phase ofS. Typhimurium-induced colitis.

Download Full-text

Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) Mediates Ubiquitination-Dependent STAT3 Activation upon Salmonella enterica Serovar Typhimurium Infection

Infection and Immunity ◽

10.1128/iai.00081-17 ◽

2017 ◽

Vol 85 (8) ◽

Cited By ~ 7

Author(s):

Hai-Hua Ruan ◽

Zhen Zhang ◽

Su-Ying Wang ◽

Logan M. Nickels ◽

Li Tian ◽

...

Keyword(s):

Tumor Necrosis Factor ◽

Salmonella Enterica ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Receptor ◽

Effector Proteins ◽

Host Cells ◽

Associated Factor ◽

Content Type ◽

Serovar Typhimurium ◽

Necrosis Factor

ABSTRACT Salmonella enterica serovar Typhimurium can inject effector proteins into host cells via type III secretion systems (T3SSs). These effector proteins modulate a variety of host transcriptional responses to facilitate bacterial growth and survival. Here we show that infection of host cells with S. Typhimurium specifically induces the ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). This TRAF6 ubiquitination is triggered by the Salmonella pathogenicity island 1 (SPI-1) T3SS effectors SopB and SopE2. We also demonstrate that TRAF6 is involved in the SopB/SopE2-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a signaling event conducive to the intracellular growth of S. Typhimurium. Specifically, TRAF6 mediates lysine-63 ubiquitination within the Src homology 2 (SH2) domain of STAT3, which is an essential step for STAT3 membrane recruitment and subsequent phosphorylation in response to S. Typhimurium infection. TRAF6 ubiquitination participates in STAT3 phosphorylation rather than serving as only a hallmark of E3 ubiquitin ligase activation. Our results reveal a novel strategy in which S. Typhimurium T3SS effectors broaden their functions through the activation of host proteins in a ubiquitination-dependent manner to manipulate host cells into becoming a Salmonella-friendly zone.

Download Full-text

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

mBio ◽

10.1128/mbio.00603-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 2

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.

Download Full-text

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

Infection and Immunity ◽

10.1128/iai.01461-15 ◽

2016 ◽

Vol 84 (4) ◽

pp. 1150-1165 ◽

Cited By ~ 13

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.

Download Full-text

Peroral Ciprofloxacin Therapy Impairs the Generation of a Protective Immune Response in a Mouse Model for Salmonella enterica Serovar Typhimurium Diarrhea, while Parenteral Ceftriaxone Therapy Does Not

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05819-11 ◽

2012 ◽

Vol 56 (5) ◽

pp. 2295-2304 ◽

Cited By ~ 15

Author(s):

Kathrin Endt ◽

Lisa Maier ◽

Rina Käppeli ◽

Manja Barthel ◽

Benjamin Misselwitz ◽

...

Keyword(s):

Immune Response ◽

Mouse Model ◽

Antibiotic Treatment ◽

Salmonella Enterica ◽

Adaptive Immune Response ◽

Content Type ◽

Fecal Shedding ◽

Treatment Regimens ◽

Adaptive Immune ◽

Serovar Typhimurium

ABSTRACTNontyphoidalSalmonella(NTS) species cause self-limiting diarrhea and sometimes severe disease. Antibiotic treatment is considered only in severe cases and immune-compromised patients. The beneficial effects of antibiotic therapy and the consequences for adaptive immune responses are not well understood. We used a mouse model forSalmonelladiarrhea to assess the effects ofper ostreatment with ciprofloxacin (15 mg/kg of body weight intragastrically 2 times/day, 5 days) or parenteral ceftriaxone (50 mg/kg intraperitoneally, 5 days), two common drugs used in human patients. The therapeutic and adverse effects were assessed with respect to generation of a protective adaptive immune response, fecal pathogen excretion, and the emergence of nonsymptomatic excreters. In the mouse model, both therapies reduced disease severity and reduced the level of fecal shedding. In line with clinical data, in most animals, a rebound of pathogen gut colonization/fecal shedding was observed 2 to 12 days after the end of the treatment. Yet, levels of pathogen shedding and frequency of appearance of nonsymptomatic excreters did not differ from those for untreated controls. Moreover, mice treated intraperitoneally with ceftriaxone developed an adaptive immunity protecting the mice from enteropathy in wild-typeSalmonella entericaserovar Typhimurium challenge infections. In contrast, the mice treated intragastrically with ciprofloxacin were not protected. Thus, antibiotic treatment regimens can disrupt the adaptive immune response, but treatment regimens may be optimized in order to preserve the generation of protective immunity. It might be of interest to determine whether this also pertains to human patients. In this case, the mouse model might be a tool for further mechanistic studies.

Download Full-text