The Salmonella Effector SteA Contributes to the Control of Membrane Dynamics of Salmonella-Containing Vacuoles

ABSTRACTSalmonella entericaserovar Typhimurium is a bacterial pathogen causing gastroenteritis in humans and a typhoid-like systemic disease in mice.S. Typhimurium virulence is related to its capacity to multiply intracellularly within a membrane-bound compartment, theSalmonella-containing vacuole (SCV), and depends on type III secretion systems that deliver bacterial effector proteins into host cells. Here, we analyzed the cellular function of theSalmonellaeffector SteA. We show that, compared to cells infected by wild-typeS. Typhimurium, cells infected by ΔsteAmutant bacteria displayed fewerSalmonella-induced filaments (SIFs), an increased clustering of SCVs, and morphologically abnormal vacuoles containing more than one bacterium. The increased clustering of SCVs and the appearance of vacuoles containing more than one bacterium were suppressed by inhibition of the activity of the microtubule motor dynein or kinesin-1. Clustering and positioning of SCVs are controlled by the effectors SseF and SseG, possibly by helping to maintain a balanced activity of microtubule motors on the bacterial vacuoles. Deletion ofsteAinS. Typhimurium ΔsseFor ΔsseGmutants revealed that SteA contributes to the characteristic scattered distribution of ΔsseFor ΔsseGmutant SCVs in infected cells. Overall, this shows that SteA participates in the control of SCV membrane dynamics. Moreover, it indicates that SteA is functionally linked to SseF and SseG and suggests that it might contribute directly or indirectly to the regulation of microtubule motors on the bacterial vacuoles.

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Intracellular survival of Salmonella enterica serovar Typhi in human macrophages is independent of Salmonella pathogenicity island (SPI)-2

Microbiology ◽

10.1099/mic.0.041624-0 ◽

2010 ◽

Vol 156 (12) ◽

pp. 3689-3698 ◽

Cited By ~ 36

Author(s):

Chantal G. Forest ◽

Elyse Ferraro ◽

Sébastien C. Sabbagh ◽

France Daigle

Keyword(s):

Salmonella Enterica ◽

Systemic Disease ◽

Distinct Type ◽

Intracellular Survival ◽

Effector Proteins ◽

Host Cells ◽

Secretion Systems ◽

Human Macrophages ◽

Successful Infection ◽

Serovar Typhimurium

For successful infection, Salmonella enterica secretes and injects effector proteins into host cells by two distinct type three secretion systems (T3SSs) located on Salmonella pathogenicity islands (SPIs)-1 and -2. The SPI-2 T3SS is involved in intracellular survival of S. enterica serovar Typhimurium and systemic disease. As little is known regarding the function of the SPI-2 T3SS from S. enterica serovar Typhi, the aetiological agent of typhoid fever, we investigated its role for survival in human macrophages. Mutations in the translocon (sseB), basal secretion apparatus (ssaR) and regulator (ssrB) did not result in any reduction in survival under many of the conditions tested. Similar results were obtained with another S. Typhi strain or by using human primary cells. Results were corroborated based on complete deletion of the SPI-2 T3SS. Surprisingly, the data suggest that the SPI-2 T3SS of S. Typhi is not required for survival in human macrophages.

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A Salmonella type III effector, PipA, works in a different manner than the PipA family effectors GogA and GtgA

PLoS ONE ◽

10.1371/journal.pone.0248975 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0248975

Author(s):

Momo Takemura ◽

Takeshi Haneda ◽

Hikari Idei ◽

Tsuyoshi Miki ◽

Nobuhiko Okada

Keyword(s):

Hela Cells ◽

Critical Role ◽

Effector Proteins ◽

Host Cells ◽

Microbial Pathogens ◽

Secretion Systems ◽

Type Iii ◽

Zinc Metalloprotease ◽

Type Iii Secretion Systems ◽

Serovar Typhimurium

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors—GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE—that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.

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Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

Infection and Immunity ◽

10.1128/iai.00560-17 ◽

2018 ◽

Vol 86 (4) ◽

Cited By ~ 6

Author(s):

Maarten F. de Jong ◽

Neal M. Alto

Keyword(s):

Escherichia Coli ◽

Defense Mechanisms ◽

Immune Defense ◽

Effector Proteins ◽

Innate Immune ◽

Host Cells ◽

Secretion Systems ◽

Content Type ◽

E Coli ◽

Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

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Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽

10.1128/mbio.00303-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 37

Author(s):

Erez Mills ◽

Kobi Baruch ◽

Gili Aviv ◽

Mor Nitzan ◽

Ilan Rosenshine

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Public Health Problem ◽

Effector Proteins ◽

Host Cells ◽

Global Public Health ◽

Secretion Systems ◽

Comprehensive Description ◽

Content Type ◽

Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

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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.

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

Infection and Immunity ◽

10.1128/iai.01304-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 221-232 ◽

Cited By ~ 16

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.

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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.

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The First 45 Amino Acids of SopA Are Necessary for InvB Binding and SPI-1 Secretion

Journal of Bacteriology ◽

10.1128/jb.188.7.2411-2420.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2411-2420 ◽

Cited By ~ 12

Author(s):

Wendy Higashide ◽

Daoguo Zhou

Keyword(s):

Type Iii Secretion ◽

Catalytic Domain ◽

Effector Proteins ◽

Host Cells ◽

Amino Acid Residues ◽

Secretion Systems ◽

Reporter System ◽

Type Iii ◽

Serovar Typhimurium ◽

Type Iii Protein Secretion

ABSTRACT Salmonella enterica serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE78-240, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of Salmonella SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA1-45, but not SopA1-44, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.

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Yersinia enterocolitica Inhibits Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Cellular Uptake

Infection and Immunity ◽

10.1128/iai.00984-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 174-183 ◽

Cited By ~ 3

Author(s):

Fabien Habyarimana ◽

Matthew C. Swearingen ◽

Glenn M. Young ◽

Stephanie Seveau ◽

Brian M. M. Ahmer

Keyword(s):

Listeria Monocytogenes ◽

Yersinia Enterocolitica ◽

Salmonella Enterica ◽

Effector Proteins ◽

Cell Uptake ◽

Secretion Systems ◽

Content Type ◽

T3ss Effector ◽

Luxr Homolog ◽

Serovar Typhimurium

ABSTRACTYersinia enterocoliticabiovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely,Salmonella entericaserovar Typhimurium uses a T3SS encoded bySalmonellapathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake ofS. Typhimurium andY. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other.Y. enterocoliticareducesS. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using anS. Typhimurium SPI1 mutant alone. However,Y. enterocoliticahad no effect onS. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells.Y. enterocoliticawas also able to inhibit the invasion of epithelial and macrophage-like cells byListeria monocytogenes.Y. enterocoliticamutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blockingS. Typhimurium uptake by epithelial cells.S. Typhimurium encodes a LuxR homolog, SdiA, which detectsN-acylhomoserine lactones (AHLs) produced byY. enterocoliticaand upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating theS. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed byY. enterocolitica.

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Mutational analysis of Salmonella translocated effector members SifA and SopD2 reveals domains implicated in translocation, subcellular localization and function

Microbiology ◽

10.1099/mic.0.28995-0 ◽

2006 ◽

Vol 152 (8) ◽

pp. 2323-2343 ◽

Cited By ~ 25

Author(s):

Nat F. Brown ◽

Jason Szeto ◽

Xiuju Jiang ◽

Brian K. Coombes ◽

B. Brett Finlay ◽

...

Keyword(s):

Subcellular Localization ◽

Host Cell ◽

Tertiary Structure ◽

Mutational Analysis ◽

Distinct Type ◽

Effector Proteins ◽

Host Cells ◽

Secretion Systems ◽

Serovar Typhimurium ◽

Anchoring Motif

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen causing disease in several hosts. These bacteria use two distinct type III secretion systems that inject effector proteins into the host cell for invasion and to alter maturation of the Salmonella-containing vacuole. Members of the Salmonella translocated effector (STE) family contain a conserved N-terminal translocation signal of approximately 140 aa. In this study, the STE family member SifA was examined using deletion strategies. Small deletions (approx. 20 residues long) throughout SifA were sufficient to block its secretion and/or translocation into host cells. Transfection of HeLa cells with a GFP-SifA fusion was previously shown to be sufficient to induce formation of Sif-like tubules resembling structures present in Salmonella-infected cells. The present study showed that both N- and C-terminal domains of SifA are required for this phenotype. Furthermore, both domains could induce aggregation of Lamp1-positive compartments, provided they were coupled to the minimal C-terminal membrane-anchoring motif of SifA. Mutation or deletion of the conserved STE N-terminal WEK(I/M)xxFF translocation motif of SopD2 disrupted its association with Lamp1-positive compartments, implicating these residues in both effector translocation and subcellular localization. Interestingly, one GFP-SifA deletion mutant lacking residues 42–101, but retaining the WEK(I/M)xxFF motif, targeted the Golgi apparatus. In addition, short peptides containing the signature WEK(I/M)xxFF motif derived from the N-termini of Salmonella effectors SopD2, SseJ and SspH2 were sufficient to localize GFP to the Golgi. These studies suggest that Salmonella effectors contain multifunctional motifs or domains that regulate several effector traits, including protein secretion/translocation, localization and subversion of host cell systems. Conditions that perturb the tertiary structure of effectors can influence their localization in host cells by liberating cryptic intracellular targeting motifs.

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