Bacterial flagella are filamentous appendages on the cell surface that mediate bacterial motility. The filament of the flagellum is composed of ϳ20,000 flagellin subunits (1). Flagellin monomers of many bacterial species function as pathogen-associated molecular patterns and can be detected by host cells using both surface-localized toll-like receptor 5 (TLR5) 2 and cytosolic Nod-like receptors (2-6). The interaction of flagellin with TLR5 at the cell surface has been characterized in detail, identifying the structural basis for the TLR5-flagellin interaction (7,8). TLR5 sensing has been shown to be important in Legionnaires disease, a respiratory tract infection caused by the bacterium Legionella pneumophila, since human TLR5 polymorphisms correlated with disease susceptibility (7,8). In the intestine, TLR5 localizes to the basolateral surface of enterocytes and to CD11c ϩ lamina propria dendritic cells (9, 10). In these locations, TLR5 is thought to be involved in sensing a breach of the intestinal mucosa by enteroinvasive pathogens, triggering an inflammatory response that limits systemic spread of the bacteria (11).In addition to sensing by TLRs, flagellin was recently shown to be sensed by two different Nod-like receptors, Ipaf and Birc1e (also known as Naip5) (2, 4 -6, 12). Ipaf transmits a proinflammatory signal in response to flagellin by activating the inflammasome, resulting in caspase 1-dependent activation of Il-1␤, which is accompanied by cell death (pyroptosis) (13-17). Birc1e/Naip5 signals in response to flagellin, restricting intracellular growth of L. pneumophila by antagonizing the ability of the bacterium to avoid fusion with lysosomes and form a replicative phagosome (18). Several lines of evidence suggest that pathogens residing in a membrane-bound compartment of the cell, such as L. pneumophila and S. Typhimurium (Salmonella enterica serotype Typhimurium), may release flagellins from their vacuole into the host cytosol, which triggers signaling via Ipaf and Birc1e. First, S. Typhimurium and L. pneumophila mutants lacking flagellin elicit reduced caspase activation, IL-1␤ secretion, and cytotoxicity in macrophages (2, 4 -6, 19). Second, mutants of S. Typhimurium lacking the SPI-1 T3SS and L. pneumophila mutants lacking a functional Type IV secretion system (T4SS) fail to trigger Ipaf-dependent signaling (4,5,19). Third, introduction of purified flagellins into the cytosol of macrophages using pore-forming toxins or detergents or by heterologous expression in Escherichia coli expressing listeriolysin O triggers caspase-1 activation and IL-1␤ activation by an Ipaf-dependent mechanism (2, 4 -6, 20).Although injection of effector proteins into host cells by the S. Typhimurium SPI-1 T3SS has been visualized microscopically (21, 22), it has not yet been demonstrated that in infected cells, bacteria are able to translocate flagellin from a membrane-bound compartment into the cytosol. In this report, we show that S. Typhimurium translocates flagellin into the cytosol of infected cells by a process t...

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SummaryThe virB operon, encoding a Type IV secretion system (T4SS), is essential for intracellular survival and persistent infection by Brucella spp. To better understand the role of the T4SS in evading host defence mechanisms and establishing chronic infection, we compared transcriptional profiles of the host response to infection with wild-type and virB mutant Brucella strains. Analysis of gene expression profiles in murine splenocytes 3 days after inoculation with wild-type Brucella strains revealed an inflammatory response, with a prominent upregulation of genes induced by both type I and type II interferons. Real-time RT-PCR showed that a group of genes from these pathways were induced by day 3 post infection and declined to baseline levels by day 7. In contrast, neither of the two virB mutant strains elicited a proinflammatory gene expression profile, demonstrating that the T4SS was required to trigger this response. Infection studies using type I interferon receptor knockout mice showed that a lack of type I interferon signalling did not affect Brucella replication during the first 4 weeks of infection. Thus, induction of type I interferons does not appear to be an essential mechanism by which the T4SS promotes persistent infection by Brucella.

The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng ؊/؊ or ␤ 2 m ؊/؊ mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1 ؊/؊ mice and Igh6 ؊/؊ mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6 ؊/؊ mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.Brucella abortus is one of the causative agents of brucellosis, a chronic zoonotic infection characterized by bacterial persistence in the reticuloendothelial system (RES) of the liver, spleen, and lymph nodes. The chronic infection of the RES observed during human brucellosis can be studied in the murine model host. A signature-tagged transposon screen identified a type IV secretion system (T4SS) as an essential virulence factor for persistence in the murine RES (11). The T4SS is encoded by the virB locus on chromosome II of the B. abortus genome. Mutational inactivation of the T4SS reduces the ability of B. abortus to survive and/or replicate in human epithelial cell lines (HeLa cells), murine bone marrow-derived macrophages, and macrophage-like cell lines (3,5,8,14,29,30), and similar phenotypes have been described for Brucella melitensis and Brucella suis virB mutants (7,10,16,24). Within murine bone marrow-derived macrophages, B. abortus traffics to an endoplasmic reticulum (ER)-like compartment, where it localizes to ER exit sites. In contrast, virB mutants fail to acquire ER markers (3, 4). These differences in cellular trafficking are reflected in the kinetics of bacterial growth and/or killing: whereas wild-type (wt) B. abortus is able to replicate in macrophages in vitro, virB mutant bacteria are eliminated within the first 24 h after infection (3, 29, 30).These observations raise the question whether the T4SS is required for evasion of macrophage killing mechanisms in vivo. The role of the T4SS in evading two major macrophage ...

SummaryYopE, a type III secreted effector of Yersinia, is a GTPase Activating Protein for Rac1 and RhoA whose catalytic activity is critical for virulence. We found that YopE also inhibited reactive oxygen species (ROS) production and inactivated Rac2. How YopE distinguishes among its targets and which specific targets are critical for Yersinia survival in different tissues are unknown. A screen identifying YopE mutants in Yersinia pseudotuberculosis that interact with different Rho GTPases showed that YopE residues at positions 102, 106, 109 and 156 discern among switch I and II regions of Rac1, Rac2 and RhoA. Two mutants, which expressed YopE alleles with different antiphagocytic, ROS-inhibitory and cell-rounding activities, YptbL109A and YptbESptP, were studied in animal infections. Inhibition of both phagocytosis and ROS production were required for splenic colonization, whereas fewer YopE activities were required for Peyer's patch colonization. This study shows that Y. pseudotuberculosis encounters multiple host defences in different tissues and uses distinct YopE activities to disable them.

The Brucella abortus virB locus contains 12 open reading frames, termed virB1 through virB12, which encode a type IV secretion system. Polar mutations in the virB locus markedly reduce the ability of B. abortus to survive in cultured macrophages or to persist in organs of mice. While a nonpolar deletion of the virB2 gene reduces survival in cultured macrophages and in organs of mice, a nonpolar deletion of virB1 only reduces survival in macrophages, whereas virB12 is dispensable for either virulence trait. Here we investigated the role of the remaining genes in the virB locus during survival in macrophages and virulence in mice. Mutants carrying nonpolar deletions of the virB3, virB4, virB5, virB6, virB7, virB8, virB9, virB10, or virB11 gene were constructed and characterized. All mutations reduced the ability of B. abortus to survive in J774A.1 mouse macrophage-like cells to a degree similar to that caused by a deletion of the entire virB locus. Deletion of virB3, virB4, virB5, virB6, virB8, virB9, virB10, or virB11 markedly reduced the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection. Interestingly, deletion of virB7 did not reduce the ability of B. abortus to persist in spleens of mice. We conclude that virB2, virB3, virB4, virB5, virB6, virB8, virB9, virB10, and virB11 are essential for virulence of B. abortus in mice, while functions encoded by the virB1, virB7, and virB12 genes are not required for persistence in organs with this animal model. Brucellosis, also known as Malta fever or undulant fever, is the most frequent anthropozoonosis in the world, with over half a million new cases each year (26, 44). The disease is caused by gram-negative bacteria that are members of the genus Brucella. These pathogens cause abortion and sterility in animals, while in humans the disease is characterized by recurrent episodes of fever. In both cases, the pathogens are capable of surviving for extended periods of time in the organs of the reticulo-endothelial system, such as liver, spleen, lymph nodes, and bone marrow. While Brucella species lack many of the classical virulence factors present in other Proteobacteria, such as fimbriae, toxins, or virulence plasmids, their chromosomes carry the virB locus, which encodes a type IV secretion system (T4SS) that is required for infection and modulation of the host immune system (24,29,42,56). T4SSs are ancestrally related to conjugation systems and have been identified in a large number of gram-negative pathogens that cause disease in both mammals and plants (1,4,5,20,43,52,54,62,64). Based on sequence comparison of their structural components, T4SSs can be divided into two groups, the virB-like T4SSs (T4A) and the dot/icm-like T4SSs (T4B) (14). Both T4A and T4B secretion systems have been shown to translocate or secrete a range of different substrates from DNA bound to protein to single proteins to multisubunit toxins (4, 8, 11, 12, 16, 36, 37, 39-41, 43, 53, 55, 61, 64). The closest homologues of the virB locus are T4A systems encoded by ...

Summary Identifying molecular targets of Yersinia virulence effectors, or Yops, during animal infection is challenging because few cells are targeted by Yops in an infected organ and isolating these sparse effector-containing cells is difficult. YopH, a tyrosine phosphatase, is essential for full virulence of Yersinia. Investigating the YopH-targeted signal-transduction pathway(s) in neutrophils during infection of a murine host, we find that several host proteins, including the essential signaling adapter SLP-76, are dephosphorylated in the presence of YopH in neutrophils isolated from infected tissues. YopH inactivated PRAM-1/SKAP-HOM and the SLP-76/Vav/PLCγ2 signal-transduction axes, leading to an inhibition of calcium response in isolated neutrophils. Consistent with a failure to mount a calcium response, IL-10 production was reduced in neutrophils containing YopH from infected tissues. Finally, a yopH mutant survived better in the absence of neutrophils, indicating that neutrophil inactivation by YopH by targeting PRAM-1/SKAP-HOM and SLP-76/Vav/PLCγ2 signaling hubs may be critical for Yersinia survival.

A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3′ end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H2O2, decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.