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

2013 ◽  
Vol 57 (5) ◽  
pp. 2191-2198 ◽  
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.

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 Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

2012 ◽  
Vol 80 (6) ◽  
pp. 1996-2007 ◽  
Author(s):  
David T. Mulder ◽  
Colin A. Cooper ◽  
Brian K. Coombes
Keyword(s):  
Salmonella Enterica ◽  
Gene Clusters ◽  
Secretion System ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Macrophage Infection ◽  
Content Type ◽  
Type Vi Secretion ◽  
Systemic Dissemination ◽  
Serovar Typhimurium

ABSTRACTThe enteropathogenSalmonella entericaserovar Typhimurium employs a suite of tightly regulated virulence factors within the intracellular compartment of phagocytic host cells resulting in systemic dissemination in mice. A type VI secretion system (T6SS) withinSalmonellapathogenicity island 6 (SPI-6) has been implicated in this process; however, the regulatory inputs and the roles of noncore genes in this system are not well understood. Here we describe four clusters of noncore T6SS genes in SPI-6 based on a comparative relationship with the T6SS-3 ofBurkholderia malleiand report that the disruption of these genes results in defects in intracellular replication and systemic dissemination in mice. In addition, we show that the expression of the SPI-6-encoded Hcp and VgrG orthologs is enhanced during late stages of macrophage infection. We identify six regions that are transcriptionally active during cell infections and that have regulatory contributions from the regulators of virulence SsrB, PhoP, and SlyA. We show that levels of protein expression are very weak underin vitroconditions and that expression is not enhanced upon the deletion ofssrB,phoP,slyA,qseC,ompR, orhfq, suggesting an unknown activating factor. These data suggest that the SPI-6 T6SS has been integrated into theSalmonellaTyphimurium virulence network and customized for host-pathogen interactions through the action of noncore genes.

scholarly journals 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 ◽  
2013 ◽  
Vol 4 (2) ◽  
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.

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 The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

2013 ◽  
Vol 81 (4) ◽  
pp. 1207-1220 ◽  
Author(s):  
Carlos J. Blondel ◽  
Juan C. Jiménez ◽  
Lorenzo E. Leiva ◽  
Sergio A. Álvarez ◽  
Bernardo I. Pinto ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Host Cells ◽  
Economic Losses ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Core Components

ABSTRACTSalmonella entericaserotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported thatS. Gallinarum harbors a type VI secretion system (T6SS) encoded inSalmonellapathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observedin vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced underin vitrobacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins.In vitrobacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon afterSalmonellauptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpVandvgrG) revealed that SPI-19 T6SS contributes toS. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked toSalmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used bySalmonellato survive within host cells.

scholarly journals MdsABC-Mediated Pathway for Pathogenicity in Salmonella enterica Serovar Typhimurium

2015 ◽  
Vol 83 (11) ◽  
pp. 4266-4276 ◽  
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.

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

2017 ◽  
Vol 85 (8) ◽  
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.

scholarly journals Salmonella enterica Serovar Typhimurium SPI-1 and SPI-2 Shape the Global Transcriptional Landscape in a Human Intestinal Organoid Model System

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Anna-Lisa E. Lawrence ◽  
Basel H. Abuaita ◽  
Ryan P. Berger ◽  
David R. Hill ◽  
Sha Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Intestinal Epithelium ◽  
Salmonella Enterica ◽  
Secretion System ◽  
Host Responses ◽  
Secretion Systems ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Type 3

ABSTRACT The intestinal epithelium is a primary interface for engagement of the host response by foodborne pathogens, like Salmonella enterica Typhimurium. While the interaction of S. Typhimurium with the mammalian host has been well studied in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestine. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells and a supporting mesenchymal cell layer. HIOs contain luminal space that supports bacterial replication, are more amenable to experimental manipulation than animals and are more reflective of physiological host responses. Here, we use the HIO model to define host transcriptional responses to S. Typhimurium infection, also determining host pathways dependent on Salmonella pathogenicity island-1 (SPI-1)- and -2 (SPI-2)-encoded type 3 secretion systems (T3SS). Consistent with prior findings, we find that S. Typhimurium strongly stimulates proinflammatory gene expression. Infection-induced cytokine gene expression was rapid, transient, and largely independent of SPI-1 T3SS-mediated invasion, likely due to continued luminal stimulation. Notably, S. Typhimurium infection led to significant downregulation of host genes associated with cell cycle and DNA repair, leading to a reduction in cellular proliferation, dependent on SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as mediators of S. Typhimurium-dependent cell cycle suppression. These findings from Salmonella-infected HIOs delineate common and distinct contributions of SPI-1 and SPI-2 T3SSs in inducing early host responses during enteric infection and reinforce host cell proliferation as a process targeted by Salmonella. IMPORTANCE Salmonella enterica serovar Typhimurium (S. Typhimurium) causes a significant health burden worldwide, yet host responses to initial stages of intestinal infection remain poorly understood. Due to differences in infection outcome between mice and humans, physiological human host responses driven by major virulence determinants of Salmonella have been more challenging to evaluate. Here, we use the three-dimensional human intestinal organoid model to define early responses to infection with wild-type S. Typhimurium and mutants defective in the SPI-1 or SPI-2 type-3 secretion systems. While both secretion system mutants show defects in mouse models of oral Salmonella infection, the specific contributions of each secretion system are less well understood. We show that S. Typhimurium upregulates proinflammatory pathways independently of either secretion system, while the downregulation of the host cell cycle pathways relies on both SPI-1 and SPI-2. These findings lay the groundwork for future studies investigating how SPI-1- and SPI-2-driven host responses affect infection outcome and show the potential of this model to study host-pathogen interactions with other serovars to understand how initial interactions with the intestinal epithelium may affect pathogenesis.

scholarly journals Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Charles L. Larson ◽  
Paul A. Beare ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Q Fever ◽  
Secretion System ◽  
Host Cells ◽  
Growth Defect ◽  
Wild Type ◽  
Signal Sequences ◽  
Content Type

ABSTRACT Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetii icmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation. IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella. Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella. Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.

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