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Find video protocols related to scientific articles indexed in Pubmed.
Intracellular bacteria interfere with dendritic cell functions: role of the type I interferon pathway.
PLoS ONE
PUBLISHED: 01-01-2014
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Dendritic cells (DCs) orchestrate host defenses against microorganisms. In infectious diseases due to intracellular bacteria, the inefficiency of the immune system to eradicate microorganisms has been attributed to the hijacking of DC functions. In this study, we selected intracellular bacterial pathogens with distinct lifestyles and explored the responses of monocyte-derived DCs (moDCs). Using lipopolysaccharide as a control, we found that Orientia tsutsugamushi, the causative agent of scrub typhus that survives in the cytosol of target cells, induced moDC maturation, as assessed by decreased endocytosis activity, the ability to induce lymphocyte proliferation and the membrane expression of phenotypic markers. In contrast, Coxiella burnetii, the agent of Q fever, and Brucella abortus, the agent of brucellosis, both of which reside in vacuolar compartments, only partly induced the maturation of moDCs, as demonstrated by a phenotypic analysis. To analyze the mechanisms used by C. burnetii and B. abortus to alter moDC activation, we performed microarray and found that C. burnetii and B. abortus induced a specific signature consisting of TLR4, TLR3, STAT1 and interferon response genes. These genes were down-modulated in response to C. burnetii and B. abortus but up-modulated in moDCs activated by lipopolysaccharide and O. tsutsugamushi. This transcriptional alteration was associated with the defective interferon-? production. This study demonstrates that intracellular bacteria specifically affect moDC responses and emphasizes how C. burnetii and B. abortus interfere with moDC activation and the antimicrobial immune response. We believe that comparing infection by several bacterial species may be useful for defining new pathways and biomarkers and for developing new treatment strategies.
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A Toxin-Antitoxin Module of Salmonella Promotes Virulence in Mice.
PLoS Pathog.
PUBLISHED: 12-01-2013
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Toxin-antitoxin (TA) modules are widely prevalent in both bacteria and archaea. Originally described as stabilizing elements of plasmids, TA modules are also widespread on bacterial chromosomes. These modules promote bacterial persistence in response to specific environmental stresses. So far, the possibility that TA modules could be involved in bacterial virulence has been largely neglected, but recent comparative genomic studies have shown that the presence of TA modules is significantly associated with the pathogenicity of bacteria. Using Salmonella as a model, we investigated whether TA modules help bacteria to overcome the stress conditions encountered during colonization, thereby supporting virulence in the host. By bioinformatics analyses, we found that the genome of the pathogenic bacterium Salmonella Typhimurium encodes at least 11 type II TA modules. Several of these are conserved in other pathogenic strains but absent from non-pathogenic species indicating that certain TA modules might play a role in Salmonella pathogenicity. We show that one TA module, hereafter referred to as sehAB, plays a transient role in virulence in perorally inoculated mice. The use of a transcriptional reporter demonstrated that bacteria in which sehAB is strongly activated are predominantly localized in the mesenteric lymph nodes. In addition, sehAB was shown to be important for the survival of Salmonella in these peripheral lymphoid organs. These data indicate that the transient activation of a type II TA module can bring a selective advantage favouring virulence and demonstrate that TA modules are engaged in Salmonella pathogenesis.
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Bartonella and Brucella--weapons and strategies for stealth attack.
Cold Spring Harb Perspect Med
PUBLISHED: 08-03-2013
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Bartonella spp. and Brucella spp. are closely related ?-proteobacterial pathogens that by distinct stealth-attack strategies cause chronic infections in mammals including humans. Human infections manifest by a broad spectrum of clinical symptoms, ranging from mild to fatal disease. Both pathogens establish intracellular replication niches and subvert diverse pathways of the hosts immune system. Several virulence factors allow them to adhere to, invade, proliferate, and persist within various host-cell types. In particular, type IV secretion systems (T4SS) represent essential virulence factors that transfer effector proteins tailored to recruit host components and modulate cellular processes to the benefit of the bacterial intruders. This article puts the remarkable features of these two pathogens into perspective, highlighting the mechanisms they use to hijack signaling and trafficking pathways of the host as the basis for their stealthy infection strategies.
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Human plasma cells express granzyme B.
Eur. J. Immunol.
PUBLISHED: 05-16-2013
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While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.
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Neutrophils exert a suppressive effect on Th1 responses to intracellular pathogen Brucella abortus.
PLoS Pathog.
PUBLISHED: 02-14-2013
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Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-? and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.
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Brucella evasion of adaptive immunity.
Future Microbiol
PUBLISHED: 02-05-2013
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The complex immune system of mammals is the result of evolutionary forces that include battles against pathogens, as sensing and defeating intruders is a prerequisite to host survival. On the other hand, microorganisms have evolved multiple mechanisms to evade both arms of immunity: the innate and the adaptive immune systems. The successful pathogenic intracellular bacterium Brucella is not an exception to the rule: Brucella displays mechanisms that allow evasion of immune surveillance in order to establish persistent infections in mammals. In this review, we highlight some key mechanisms that pathogenic Brucella use to evade the adaptive immune system.
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Lipopolysaccharides with acylation defects potentiate TLR4 signaling and shape T cell responses.
PLoS ONE
PUBLISHED: 02-04-2013
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Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4(+) T and CD8(+) T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.
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Brucella T4SS: the VIP pass inside host cells.
Curr. Opin. Microbiol.
PUBLISHED: 01-11-2013
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For many Gram-negative bacteria, like Brucella, the type IV secretion system (T4SS) has a critical role in bacterial virulence. In Brucella, the VirB T4SS permits the injection of bacterial effectors inside host cells, leading to subversion of signaling pathways and favoring bacterial growth and pathogenesis. The virB operon promoter is tightly regulated by a combination of transcriptional activators and repressors that are expressed according to the environmental conditions encountered by Brucella. Recent advances have shed light on the Brucella T4SS regulatory mechanisms and also its substrates. Characterization of the targets and functions of these translocated effectors is underway and will help understand the role of the T4SS in the establishment of a replication niche inside host cells.
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Pathogenic brucellae replicate in human trophoblasts.
J. Infect. Dis.
PUBLISHED: 01-09-2013
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Brucellae replicate in a vacuole derived from the endoplasmic reticulum (ER) in epithelial cells, macrophages, and dendritic cells. In animals, trophoblasts are also key cellular targets where brucellae efficiently replicate in association with the ER. Therefore, we investigated the ability of Brucella spp. to infect human trophoblasts using both immortalized and primary trophoblasts. Brucella extensively proliferated within different subpopulations of trophoblasts, suggesting that they constitute an important niche in cases where the fetal-maternal barrier is breached. In extravillous trophoblasts (EVTs), B. abortus and B. suis replicated within single-membrane acidic lysosomal membrane-associated protein 1-positive inclusions, whereas B. melitensis replicated in the ER-derived compartment. Furthermore, B. melitensis but not B. abortus nor B. suis interfered with the invasive capacity of EVT-like cells in vitro. Because EVTs are essential for implantation during early stages of pregnancy, the nature of the replication niche may have a central role during Brucella-associated abortion in infected women.
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In Vivo Identification and Characterization of CD4(+) Cytotoxic T Cells Induced by Virulent Brucella abortus Infection.
PLoS ONE
PUBLISHED: 01-01-2013
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CD4(+) T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4(+) T cells (CD4(+) CTL) during Brucella abortus infection. These CD4(+) CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-?. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4(+) CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4(+) T cell population appears at early stages of the infection concomitantly with high levels of IFN-? and granzyme B expression. CD4(+) CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.
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BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.
Front Cell Infect Microbiol
PUBLISHED: 01-01-2013
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Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.
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Peyers patch dendritic cells sample antigens by extending dendrites through M cell-specific transcellular pores.
Gastroenterology
PUBLISHED: 09-28-2011
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Peyers patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo.
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In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system.
Cell. Microbiol.
PUBLISHED: 06-24-2011
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Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.
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Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2.
Cell. Microbiol.
PUBLISHED: 05-30-2011
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Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-?-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a ?virB mutant. In HeLa cells, a ?ricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortus?ricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.
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Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst.
Mol. Microbiol.
PUBLISHED: 03-21-2011
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The oxidative burst produced by the NADPH oxidase (Phox) is an essential weapon used by host cells to eradicate engulfed pathogens. In Salmonella typhimurium, oxidative stress resistance has been previously proposed to be mediated by the pathogenicity island 2 type III secretion system (T3SS-2), periplasmic superoxide dismutases and cytoplasmic catalases/peroxidases. Here, we fused an OxyR-dependent promoter to the gfp to build the ahpC-gfp transcriptional fusion. This reporter was used to monitor hydrogen peroxide levels as sensed by Salmonella during the course of an infection. We showed that the expression of this fusion was under the exclusive control of reactive oxygen species produced by the host. The ahpC-gfp expression was noticeably modified in the absence of bacterial periplasmic superoxide dismutases or cytoplasmic catalases/peroxidases. Surprisingly, inactivation of the T3SS-2 had no effect on the ahpC-gfp expression. All together, these results led to a model in which Salmonella resistance relies on its arsenal of detoxifying enzymes to cope with Phox-mediated oxidative stress.
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An evolutionary strategy for a stealthy intracellular Brucella pathogen.
Immunol. Rev.
PUBLISHED: 02-26-2011
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Brucella is an intracellular bacterial pathogen that causes abortion and infertility in mammals and leads to a debilitating febrile illness that can progress into a long lasting disease with severe complications in humans. Its virulence depends on survival and replication properties in host cells. In this review, we describe the stealthy strategy used by Brucella to escape recognition of the innate immunity and the means by which this bacterium evades intracellular destruction. We also discuss the development of adaptive immunity and its modulation during brucellosis that in course leads to chronic infections. Brucella has developed specific strategies to influence antigen presentation mediated by cells. There is increasing evidence that Brucella also modulates signaling events during host adaptive immune responses.
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Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern.
PLoS ONE
PUBLISHED: 01-07-2011
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The brucellae are ?-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-? release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in ?-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.
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Contrasting roles of macrophages and dendritic cells in controlling initial pulmonary Brucella infection.
Eur. J. Immunol.
PUBLISHED: 11-26-2010
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Control of pulmonary pathogens constitutes a challenging task as successful immune responses need to be mounted without damaging the lung parenchyma. Using immunofluorescence microscopy and flow cytometry, we analyzed in the mouse the initial innate immune response that follows intranasal inoculation of Brucella abortus. Bacteria were absent from parenchymal dendritic cells (DC) but present in alveolar macrophages in which they replicated. When the number of alveolar macrophages was reduced prior to Brucella infection, small numbers of pulmonary DC were infected and a massive recruitment of TNF-?- and iNOS-producing DC ensued. Coincidentally, Brucella disseminated to the lung-draining mediastinal lymph nodes (LN) where they replicated in both migratory DC and migratory alveolar macrophages. Together, these results demonstrate that alveolar macrophages are critical regulators of the initial innate immune response against Brucella within the lungs and show that pulmonary DC and alveolar macrophages play rather distinct roles in the control of microbial burden.
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The virulence protein SopD2 regulates membrane dynamics of Salmonella-containing vacuoles.
PLoS Pathog.
PUBLISHED: 06-16-2010
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Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV). This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs) and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA(-) mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA(-) mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA(-) mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s). Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA(-) associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s) in the biogenesis and maintenance of the Salmonella replicative niche.
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Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice.
Vaccine
PUBLISHED: 04-22-2010
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Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusDeltawbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. DeltawbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.
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SKIP, the host target of the Salmonella virulence factor SifA, promotes kinesin-1-dependent vacuolar membrane exchanges.
Traffic
PUBLISHED: 04-06-2010
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In Salmonella-infected cells, the bacterial effector SifA forms a functional complex with the eukaryotic protein SKIP (SifA and kinesin-interacting protein). The lack of either partner has important consequences on the intracellular fate and on the virulence of this pathogen. In addition to SifA, SKIP binds the microtubule-based motor kinesin-1. Yet the absence of SifA or SKIP results in an unusual accumulation of kinesin-1 on the bacterial vacuolar membrane. To understand this apparent contradiction, we investigated the interaction between SKIP and kinesin-1 and the function of this complex. We show that the C-terminal RUN (RPIP8, UNC-14 and NESCA) domain of SKIP interacted specifically with the tetratricopeptide repeat (TPR) domain of the kinesin light chain. Overexpression of SKIP induced a microtubule- and kinesin-1-dependent anterograde movement of late endosomal/lysosomal compartments. In infected cells, SifA contributed to the fission of vesicles from the bacterial vacuole and the SifA/SKIP complex was required for the formation and/or the anterograde transport of kinesin-1-enriched vesicles. These observations reflect the role of SKIP as a linker and/or an activator for kinesin-1.
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Transcriptome analysis of the Brucella abortus BvrR/BvrS two-component regulatory system.
PLoS ONE
PUBLISHED: 03-04-2010
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The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions.
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The Brucella abortus phosphoglycerate kinase mutant is highly attenuated and induces protection superior to that of vaccine strain 19 in immunocompromised and immunocompetent mice.
Infect. Immun.
PUBLISHED: 03-01-2010
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Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that can function as virulence factors to better understand the host-pathogen interplay. Herein, we identified the gene encoding the phosphoglycerate kinase (PGK) of B. abortus strain 2308. To test the role of PGK in Brucella pathogenesis, a pgk deletion mutant was constructed. Replacement of the wild-type pgk by recombination was demonstrated by Southern and Western blot analyses. The B. abortus Delta pgk mutant strain exhibited extreme attenuation in bone marrow-derived macrophages and in vivo in BALB/c, C57BL/6, 129/Sv, and interferon regulatory factor-1 knockout (IRF-1 KO) mice. Additionally, at 24 h postinfection the Delta pgk mutant was not found within the same endoplasmic reticulum-derived compartment as the wild-type bacteria, but, instead, over 60% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP1. Furthermore, the B. abortus Delta pgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Delta pgk mutant strain induced protective immunity in 129/Sv or IRF-1 KO mice that was superior to the protection conferred by commercial strain 19 or RB51. Finally, the results shown here demonstrated that Brucella PGK is critical for full bacterial virulence and that a Delta pgk mutant may serve as a potential vaccine candidate in future studies.
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Bacterial manipulation of innate immunity to promote infection.
Nat. Rev. Microbiol.
PUBLISHED: 01-16-2010
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The mammalian innate immune response provides a barrier against invading pathogens. Innate immune mechanisms are used by the host to respond to a range of bacterial pathogens in an acute and conserved fashion. Host cells express pattern recognition receptors that sense pathogen-associated molecular patterns. After detection, an arsenal of antimicrobial mechanisms is deployed to kill bacteria in infected cells. Innate immunity also stimulates antigen-specific responses mediated by the adaptive immune system. In response, pathogens manipulate host defence mechanisms to survive and eventually replicate. This Review focuses on the control of host innate immune responses by pathogenic intracellular bacteria.
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Helicobacter pylori impairs murine dendritic cell responses to infection.
PLoS ONE
PUBLISHED: 01-11-2010
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Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes.
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Interaction between the SifA virulence factor and its host target SKIP is essential for Salmonella pathogenesis.
J. Biol. Chem.
PUBLISHED: 09-28-2009
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SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.
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Pathogenic bacteria and dead cells are internalized by a unique subset of Peyers patch dendritic cells that express lysozyme.
Gastroenterology
PUBLISHED: 09-07-2009
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Lysozyme has an important role in preventing bacterial infection. In the gastrointestinal tract, lysozyme is thought to be mainly expressed by Paneth cells of the crypt epithelium. We investigated its expression in the Peyers patch, a major intestinal site of antigen sampling and pathogen entry.
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Brucella abortus induces Irgm3 and Irga6 expression via type-I IFN by a MyD88-dependent pathway, without the requirement of TLR2, TLR4, TLR5 and TLR9.
Microb. Pathog.
PUBLISHED: 07-12-2009
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The innate immune system senses bacterial pathogens by pattern recognition receptors, such as the well-characterised Toll-like Receptors (TLR). The activation of TLR signalling cascades depends on several adaptor proteins, among which MyD88 plays a key role in triggering innate immune responses. Here, we show in murine macrophages that Brucella abortus triggers expression of the interferon-inducible resistance proteins (IRGs, p47 GTPases) via type-I IFN secretion at late time points, when Brucella has reached its replication niche. This induction requires the adaptor molecule MyD88 but does not involve the TLRs normally implicated in sensing Gram-negative bacteria, namely TLR2, TLR4, TLR5 and TLR9. Brucella mutants lacking the functional VirB type-IV secretion system were not capable of inducing Irgm3 and Irga6 expression, suggesting that the type-IV secretion system is part of the triggering of the activation process. Our data suggest that Brucella is recognized intracellularly by an unknown receptor, different from the conventional ones used for Gram-negative sensing, but one that nevertheless signals through MyD88.
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The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.
PLoS Pathog.
PUBLISHED: 05-27-2009
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The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.
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The differential interaction of Brucella and ochrobactrum with innate immunity reveals traits related to the evolution of stealthy pathogens.
PLoS ONE
PUBLISHED: 04-24-2009
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During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria.
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Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep.
Vaccine
PUBLISHED: 01-11-2009
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Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.
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Lipopolysaccharide as a target for brucellosis vaccine design.
Microb. Pathog.
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The gram-negative bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a world wide-distributed zoonotic disease that represents a serious problem for animal and human health. There is no human-to-human contagion and, since there is no human vaccine, animal vaccination is essential to control brucellosis. However, current vaccines (all developed empirically) do not provide 100% protection and are infectious in humans. Attempts to generate new vaccines by obtaining mutants lacking the lipopolysaccharide O-polysaccharide, in purine metabolism or in Brucella type IV secretion system have not been successful. Here we propose a new approach to develop brucellosis vaccines based on the concept that Brucella surface molecules evade efficient detection by innate immunity, thus delaying protective Th1 responses and opening a time window to reach sheltered intracellular compartments. We showed recently that a branch of the core oligosaccharide section of Brucella lipopolysaccharide hampers recognition by TLR4-MD2. Mutation of glycosyltransferase WadC, involved in the synthesis of this branch, results in a lipopolysaccharide that, while keeping the O-polysaccharide essential for optimal protection, shows a truncated core, is more efficiently recognized by MD2 and triggers an increased cytokine response. In keeping with this, the wadC mutant is attenuated in dendritic cells and mice. In the mouse model of brucellosis vaccines, the Brucella abortus wadC mutant conferred protection similar to that provided by S19, the best cattle vaccine available. The properties of the wadC mutant provide the proof of concept for this new approach and open the way for more effective brucellosis vaccines.
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Small GTPases and Brucella entry into the endoplasmic reticulum.
Biochem. Soc. Trans.
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A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ?ricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.
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Brucella ? 1,2 cyclic glucan is an activator of human and mouse dendritic cells.
PLoS Pathog.
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Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella ? 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella ? 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella ? 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.
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The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition.
PLoS Pathog.
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Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.
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The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming.
Blood
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We recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14(+) DCs at priming naive CD8(+) T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14(+) DCs) might explain the observed functional difference. Blocking IL-15 during CD8(+) T-cell priming reduced T-cell proliferation by ? 50%. These IL-15-deprived CD8(+) T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-? and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8(+) T cells. Conversely, blocking IL-10 during cocultures of dermal CD14(+) DCs and naive CD8(+) T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8(+) T cells inhibited their induction. TGF-?1 that is transcribed by dermal CD14(+) DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14(+) DCs on CD8(+) T-cell priming.
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What have we learned from brucellosis in the mouse model?
Vet. Res.
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Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48?h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-?, TLR9, Myd88, T?? and TNF-? favor Brucella replication; whereas IL-1?, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-? and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (? 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model.
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Internal affairs: investigating the Brucella intracellular lifestyle.
FEMS Microbiol. Rev.
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Bacteria of the genus Brucella are Gram-negative pathogens of several animal species that cause a zoonotic disease in humans known as brucellosis or Malta fever. Within their hosts, brucellae reside within different cell types where they establish a replicative niche and remain protected from the immune response. The aim of this article is to discuss recent advances in the field in the specific context of the Brucella intracellular lifestyle. We initially discuss the different host cell targets and their relevance during infection. As it represents the key to intracellular replication, the focus is then set on the maturation of the Brucella phagosome, with particular emphasis on the Brucella factors that are directly implicated in intracellular trafficking and modulation of host cell signalling pathways. Recent data on the role of the type IV secretion system are discussed, novel effector molecules identified and how some of them impact on trafficking events. Current knowledge on Brucella gene regulation and control of host cell death are summarized, as they directly affect intracellular persistence. Understanding how Brucella molecules interplay with their host cell targets to modulate cellular functions and establish the intracellular niche will help unravel how this pathogen causes disease.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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