Innate immune recognition of flagellin limits systemic persistence of Brucella.

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin-deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand-off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.

Innate immune encounters of the (Type) 4th kind: Brucella.

In humans, pathogenic Brucella species cause a febrile illness known as brucellosis. A key pathogenic trait of this group of organisms is their ability to survive in immune cells and persist in tissues of the reticuloendothelial system, a process that requires the function of a Type IV secretion system. In contrast to other well-studied Gram-negative bacteria, Brucella spp. do not cause inflammation at the site of invasion, but have a latency period of 2-4 weeks before the onset of symptoms. This review discusses several mechanisms that allow Brucella spp. both to evade detection by pattern recognition receptors of the innate immune system and suppress their signalling. In contrast to these stealth features, the VirB Type IV secretion system, which mediates survival within phagocytic cells, stimulates innate immune responses in vivo. The responses stimulated by this virulence factor are sufficient to check bacterial growth, but not to elicit sterilizing immunity. The result is a stand-off between host and pathogen that results in persistent infection.

ROS-inhibitory activity of YopE is required for full virulence of Yersinia in mice.

YopE, 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.

Natural antibody contributes to host defense against an attenuated Brucella abortus virB mutant.

Brucella abortus is an intracellular pathogen that persists within phagocytic cells of the reticuloendothelial system. To identify in vivo interactions between B. abortus and the host that lead to persistent infection, we studied the persistence of B. abortus and an isogenic virB mutant deficient in the VirB type IV secretion system (T4SS) in knockout mice. In contrast to control mice, mice lacking B cells (Igh6(-/-)) were permissive for infection with the attenuated virB mutant. To determine the basis for this phenotype, we characterized immune functions of Igh6(-/-) mice in the context of B. abortus infection. Igh6(-/-) mice had greater numbers of extracellular bacteria in the spleen and increased early expression of proinflammatory cytokines during B. abortus infection. Further, a virB mutant, despite its wild-type level of survival, failed to elicit microgranuloma formation in the spleens of Igh6(-/-) mice, suggesting a requirement for the T4SS to elicit this pathological change. Passive transfer of immunoglobulin G from naïve mice restored the ability of Igh6(-/-) mice to control the persistence of the virB mutant by a complement-independent mechanism. Further, adoptive transfer of CD11b(+) cells from C57BL/6 mice to Igh6(-/-) mice restored the ability of the knockout mice to limit the replication of the virB mutant in the spleen, suggesting that the Igh6(-)(/)(-) mutation affects phagocyte function and that phagocyte function can be restored by natural antibody.

VirB3 to VirB6 and VirB8 to VirB11, but not VirB7, are essential for mediating persistence of Brucella in the reticuloendothelial system.

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.

T cells help to amplify inflammatory responses induced by Salmonella enterica serotype Typhimurium in the intestinal mucosa.

Salmonella enterica serotype Typhimurium causes an acute inflammatory reaction in the ceca of streptomycin-pretreated mice. We determined global changes in gene expression elicited by serotype Typhimurium in the cecal mucosa. The gene expression profile was dominated by T-cell-derived cytokines and genes whose expression is known to be induced by these cytokines. Markedly increased mRNA levels of genes encoding gamma interferon (IFN-gamma), interleukin-22 (IL-22), and IL-17 were detected by quantitative real-time PCR. Furthermore, the mRNA levels of genes whose expression is induced by IFN-gamma, IL-22, or IL-17, including genes encoding macrophage inflammatory protein 2 (MIP-2), inducible nitric oxide synthase (Nos2), lipocalin-2 (Lcn2), MIP-1alpha, MIP-1beta, and keratinocyte-derived cytokine (KC), were also markedly increased. To assess the importance of T cells in orchestrating this proinflammatory gene expression profile, we depleted T cells by using a monoclonal antibody prior to investigating cecal inflammation caused by serotype Typhimurium in streptomycin-pretreated mice. Depletion of CD3+ T cells resulted in a dramatic reduction in gross pathology, a significantly reduced recruitment of neutrophils, and a marked reduction in mRNA levels of Ifn-gamma, Il-22, Il-17, Nos2, Lcn2, and Kc. Our results suggest that T cells play an important role in amplifying inflammatory responses induced by serotype Typhimurium in the cecal mucosa.

A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.

Identifying Yersinia YopH-targeted signal transduction pathways that impair neutrophil responses during in vivo murine infection.

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 adaptor 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.

The small protein CydX is required for function of cytochrome bd oxidase in Brucella abortus.

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 H(2)O(2), 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.

VirB12 is a serological marker of Brucella infection in experimental and natural hosts.

The Brucella species type IV secretion system, encoded by the virB1-12 locus, is required for intracellular replication and persistent infection in vivo. The requirement of VirB proteins for infection suggests that they are expressed in vivo and may therefore represent serological markers of infection. To test this idea, we purified recombinant VirB1, VirB5, VirB11, and VirB12 and tested for their recognition by antibodies in sera from experimentally infected mice and goats by using an indirect enzyme-linked immunosorbent assay. Antibody responses to VirB12 but not to VirB1, VirB5, or VirB11 were detected in 20/20 mice experimentally inoculated with Brucella abortus and 12/12 goats experimentally infected with Brucella melitensis. The potential use of VirB12 as a serological tool for the diagnosis of brucellosis was evaluated in the natural bovine host. Serum samples from 145 cattle of known serology (29% negative and 71% positive) were analyzed for the production of antibody responses to VirB12. One hundred two cattle samples (70.3%) were positive for antibodies to VirB12, while 43 samples were negative (29.7%). A positive serological response to VirB12 correlated with positive serology to whole B. abortus antigen in 99% of samples tested. These results show that VirB12 is expressed during infection of both experimental and natural hosts of Brucella species, and they suggest that VirB12 may be a useful serodiagnostic marker for brucellosis.

Brucella requires a functional Type IV secretion system to elicit innate immune responses in mice.

The 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.

Brucella abortus virB12 is expressed during infection but is not an essential component of the type IV secretion system.

The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12, showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus, B. melitensis, and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.