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.
Brucella; TIR domain; Btp1/BtpA; TLR; DC; NF-κB
Brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. In some cases, human brucellosis results in a persistent infection that may reactivate years after the initial exposure. The mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection and are a preferential niche for the development of the bacteria. Here, we report that in contrast to several intracellular bacteria, Brucella prevented the infected DCs from engaging in their maturation process and impaired their capacities to present antigen to naïve T cells and to secrete interleukin-12. Moreover, Brucella-infected DCs failed to release tumor necrosis factor alpha (TNF-α), a defect involving the bacterial protein Omp25. Exogenous TNF-α addition to Brucella-infected DCs restored cell maturation and allowed them to present antigens. Two avirulent mutants of B. suis, B. suis bvrR and B. suis omp25 mutants, which do not express the Omp25 protein, triggered TNF-α production upon DC invasion. Cells infected with these mutants subsequently matured and acquired the ability to present antigens, two properties which were dramatically impaired by addition of anti-TNF-α antibodies. In light of these data, we propose a model in which virulent Brucella alters the maturation and functions of DCs through Omp25-dependent control of TNF-α production. This model defines a specific evasion strategy of the bacteria by which they can escape the immune response to chronically infect their host.
Brucella species are the causative agents of one of the most prevalent zoonotic diseases: brucellosis. Infections by Brucella species cause major economic losses in agriculture, leading to abortions in infected animals and resulting in a severe, although rarely lethal, debilitating disease in humans. Brucella species persist as intracellular pathogens that manage to effectively evade recognition by the host's immune system. Sugar-modified components in the Brucella cell envelope play an important role in their host interaction. Brucella lipopolysaccharide (LPS), unlike Escherichia coli LPS, does not trigger the host's innate immune system. Brucella produces cyclic β-1,2-glucans, which are important for targeting them to their replicative niche in the endoplasmic reticulum within the host cell. This paper will focus on the role of LPS and cyclic β-1,2-glucans in Brucella-mammalian infections and discuss the use of mutants, within the biosynthesis pathway of these cell envelope structures, in vaccine development.
Brucella is a facultative intracellular pathogen of various mammals and the etiological agent of brucellosis. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection. Furthermore, Brucella prevented the infected DCs from engaging in maturation processes and impaired their capacity to present antigen to naive T cells and to secrete interleukin-12 (IL-12). The lipopolysaccharide (LPS) phenotype is largely associated with the virulence of Brucella. Depending on whether they express the O-side chain of LPS or not, the bacteria display a smooth or rough phenotype. Rough Brucella mutants are attenuated and induce a potent protective T-cell-dependent immune response. Due to the essential role of DCs in the initiation of T-cell-dependent adaptive immune responses, it seemed pertinent to study the interaction between rough Brucella strains and human DCs. In the present paper, we report that, in contrast to smooth bacteria, infection of DCs with rough mutants of Brucella suis or Brucella abortus leads to both phenotypic and functional maturation of infected cells. Rough mutant-infected DCs then acquire the capacity to produce IL-12 and to stimulate naive CD4+ T lymphocytes. Experiments with rough and smooth purified LPS of Brucella supported the hypothesis of an indirect involvement of the O-side chain. These results provide new data concerning the role of LPS in Brucella virulence strategy and illuminate phenomena contributing to immune protection conferred by rough vaccine strains.
Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.
Brucella melitensis is an intracellular bacterium that invades and replicates within macrophages and dendritic cells. With over 500,000 new infections per year, brucellosis is the most prevalent zoonosis worldwide and incurs significant human morbidity and economic loss. The intracellular location of Brucella renders the organism resistant to antibiotics. A safe and effective human vaccine does not exist. Thus, better understanding of the host-pathogen interactions supporting establishment of the intracellular replicative niche is critical. In this study, we found that infection of macrophages with Brucella induces a host stress response called the Unfolded Protein Response (UPR), a conserved stress response originating in the endoplasmic reticulum (ER). Full induction of the UPR requires live bacteria and expression of a microtubule modulating protein, TcpB. Inhibition of the UPR with the drug tauroursodeoxycholic acid significantly diminished Brucella replication. Together these results suggest Brucella induces the UPR to enable its own replication within host macrophages. Thus the UPR may represent a novel therapeutic target for the treatment of brucellosis.
Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.
Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human beings and infects hosts primarily through the oral route. The epithelium covering domed villi of jejunal-ileal Peyer's patches is an important site of entry for several pathogens, including Brucella. Here, we use the calf ligated ileal loop model to study temporal in vivo Brucella-infected host molecular and morphological responses. Our results document Brucella bacteremia occurring within 30 min after intraluminal inoculation of the ileum without histopathologic traces of lesions. Based on a system biology Dynamic Bayesian Network modeling approach (DBN) of microarray data, a very early transient perturbation of the host enteric transcriptome was associated with the initial host response to Brucella contact that is rapidly averted allowing invasion and dissemination. A detailed analysis revealed active expression of Syndecan 2, Integrin alpha L and Integrin beta 2 genes, which may favor initial Brucella adhesion. Also, two intestinal barrier-related pathways (Tight Junction and Trefoil Factors Initiated Mucosal Healing) were significantly repressed in the early stage of infection, suggesting subversion of mucosal epithelial barrier function to facilitate Brucella transepithelial migration. Simultaneously, the strong activation of the innate immune response pathways would suggest that the host mounts an appropriate protective immune response; however, the expression of the two key genes that encode innate immunity anti-Brucella cytokines such as TNF-α and IL12p40 were not significantly changed throughout the study. Furthermore, the defective expression of Toll-Like Receptor Signaling pathways may partially explain the lack of proinflammatory cytokine production and consequently the absence of morphologically detectable inflammation at the site of infection. Cumulatively, our results indicate that the in vivo pathogenesis of the early infectious process of Brucella is primarily accomplished by compromising the mucosal immune barrier and subverting critical immune response mechanisms.
Brucellosis is a chronic infectious disease caused by Brucella spp., a Gram-negative facultative intracellular pathogen that affects humans and animals, leading to significant impact on public health and animal industry. Human brucellosis is considered the most prevalent bacterial zoonosis in the world and is characterized by fever, weight loss, depression, hepato/splenomegaly, osteoarticular, and genital infections. Relevant aspects of Brucella pathogenesis have been intensively investigated in culture cells and animal models. The mouse is the animal model more commonly used to study chronic infection caused by Brucella. This model is most frequently used to investigate specific pathogenic factors of Brucella spp., to characterize the host immune response, and to evaluate therapeutics and vaccines. Other animal species have been used as models for brucellosis including rats, guinea pigs, and monkeys. This paper discusses the murine and other laboratory animal models for human and animal brucellosis.
Cells in the Brucella spp. are intracellular pathogens that survive and replicate within host monocytes. Brucella maintains persistent infections in animals despite the production of high levels of anti-Brucella-specific antibodies. To determine the effect of antibody opsonization on the ability of Brucella to establish itself within monocytes, the intracellular trafficking of virulent Brucella abortus 2308 and attenuated hfq and bacA mutants was followed in the human monocytic cell line THP-1. Early trafficking events of B. abortus 2308-containing phagosomes (BCP) were indistinguishable from those seen for control particles (heat-killed B. abortus 2308, live Escherichia coli HB101, or latex beads). All phagosomes transiently communicated the early-endosomal compartment and rapidly matured into LAMP-1+, cathepsin D+, and acidic phagosomes. By 2 h postinfection, however, the number of cathepsin D+ BCP was significantly lower for live B. abortus 2308-infected cells than for either Brucella mutant strains or control particles. B. abortus 2308 persisted within these cathepsin D−, LAMP-1+, and acidic vesicles; however, at the onset of intracellular replication, the numbers of acidic B. abortus 2308 BCP decreased while remaining cathepsin D− and LAMP-1+. In contrast to B. abortus 2308, the isogenic hfq and bacA mutants remained in acidic, LAMP-1+ phagosomes and failed to initiate intracellular replication. Notably, markers specific for the host endoplasmic reticulum were absent from the BCPs throughout the course of the infection. Thus, opsonized B. abortus in human monocytes survives within phagosomes that remain in the endosomal pathway and replication of virulent B. abortus 2308 within these vesicles corresponds with an increase in intraphagosomal pH.
Brucella strains produce abortion and infertility in their natural hosts and a zoonotic disease in humans known as undulant fever. These bacteria do not produce classical virulence factors, and their capacity to successfully survive and replicate within a variety of host cells underlines their pathogenicity. Extensive replication of the brucellae in placental trophoblasts is associated with reproductive tract pathology in natural hosts and prolonged persistence in macrophages leads to the chronic infections that are a hallmark of brucellosis in both natural hosts and humans. This review describes how Brucella strains have efficiently adapted to their intracellular lifestyle in the host.
Brucella spp. are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals. NK cells play a key role in host defense. They are implicated in an early immune response to a variety of pathogens. However, it was shown that they do not control Brucella infection in mice. On the other hand, NK cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human NK cells mediate the killing of intramacrophagic Mycobacterium tuberculosis in in vitro infection. Therefore, we have analyzed the behavior of Brucella suis infecting isolated human macrophages in the presence of syngeneic NK cells. We show that (i) NK cells impair the intramacrophagic development of B. suis, a phenomenon enhanced by NK cell activators, such as interleukin-2; (ii) NK cells cultured in the presence of infected macrophages are highly activated and secrete gamma interferon and tumor necrosis factor alpha; (iii) impairment of bacterial multiplication inside infected cells is marginally associated with the cytokines produced during the early phase of macrophage-NK cell cocultures; (iv) direct cell-to-cell contact is required for NK cells to mediate the inhibition of B. suis development; and (v) inhibition of B. suis development results from an induction of NK cell cytotoxicity against infected macrophages. Altogether, these findings show that NK cells could participate early in controlling the intramacrophagic development of B. suis in humans. It seems thus reasonable to hypothesize a role for NK cells in the control of human brucellosis. However, by impairing the activity of these cells in the acute phase of the illness, the pathogen should avoid this control.
Toll/Interleukin-1 like receptors are evolutionarily conserved proteins in eukaryotes that play crucial role in pathogen recognition and innate immune responses. Brucella are facultative intracellular bacterial pathogens causing brucellosis in animal and human hosts. Brucella behaves as a stealthy pathogen by evading the immune recognition or suppressing the TLR signaling cascades. Brucella encode a TIR domain containing protein, TcpB, which suppresses NF-κB activation as well as proinflammatory cytokine secretion mediated by TLR2 and TLR4 receptors. TcpB targets the TIRAP mediated pathway to suppress TLR signaling. With the objective of detailed characterization, we have over expressed and purified TcpB from Brucella melitensis in native condition. The purified protein exhibited lipid binding properties and cell permeability. NF-κB inhibition property of endogenous TcpB has also been demonstrated. The data provide insight into the mechanism of action of TcpB in the intracellular niche of Brucella.
Brucella; TcpB; TIR domain; NF-κB; Phosphoinositide; Inflammation
Brucellosis is an important zoonotic disease of almost worldwide distribution. One significant immune phenomenon of this disease is the ability of the pathogen to hide and survive in the host, establishing long lasting chronic infections. Brucella was found to have the ability to actively modulate the host immune response in order to establish chronic infections, but the mechanism by which the pathogen achieves this remains largely unknown. In our screening for protective antigens of Brucella abortus, 3 proteins (BAB1_0597, BAB1_0917, and BAB2_0431) were found to induce significantly higher levels of gamma interferon (IFNγ) in splenocytes of PBS immunized mice than those immunized with S19. This finding strongly implied that these three proteins inhibit the production of IFNγ. Previous studies have shown that LPS, PrpA, and Btp1/TcpB are three important immunomodulatory molecules with the capacity to interfere with host immune response. They have been shown to have the ability to inhibit the secretion of IFNγ, or to increase the production of IL-10. Due to the role of these proteins in virulence and immunomodulation, they likely offer significant potential as live, attenuated Brucella vaccine candidates. Understanding the mechanisms by which these proteins modulate the host immune responses will deepen our knowledge of Brucella virulence and provide important information on the development of new vaccines against Brucellosis.
Brucella; chronic infection; immune response; live attenuated vaccine; virulence proteins
Brucella spp. cause chronic zoonotic disease often affecting individuals and animals in impoverished economic or public health conditions; however, these bacteria do not have obvious virulence factors. Restriction of iron availability to pathogens is an effective strategy of host defense. For brucellae, virulence depends on the ability to survive and replicate within the host cell where iron is an essential nutrient for the growth and survival of both mammalian and bacterial cells. Iron is a particularly scarce nutrient for bacteria with an intracellular lifestyle. Brucella melitensis and Brucella canis share ∼99% of their genomes but differ in intracellular lifestyles. To identify differences, gene transcription of these two pathogens was examined during infection of murine macrophages and compared to broth grown bacteria. Transcriptome analysis of B. melitensis and B. canis revealed differences of genes involved in iron transport. Gene transcription of the TonB, enterobactin, and ferric anguibactin transport systems was increased in B. canis but not B. melitensis during infection of macrophages. The data suggest differences in iron requirements that may contribute to differences observed in the lifestyles of these closely related pathogens. The initial importance of iron for B. canis but not for B. melitensis helps elucidate differing intracellular survival strategies for two closely related bacteria and provides insight for controlling these pathogens.
Brucella spp. are facultative intracellular bacteria that cause brucellosis in humans and other animals. Brucella spp. are taken up by macrophages, and the outcome of the macrophage-Brucella interaction is a basis for establishment of a chronic Brucella infection. Microarrays were used to analyze the transcriptional response of the murine macrophage-like J774.A1 cell line to infection with virulent Brucella melitensis strain 16M. It was found that most significant changes in macrophage gene transcription happened early following infection, and global macrophage gene expression profiles returned to normal between 24 and 48 h postinfection. These findings support the observation that macrophages kill the majority of Brucella cells at the early infection stage, but the surviving Brucella cells are able to avoid macrophage brucellacidal activity inside replicative phagosomes at the later infection stage. At 4 h postinfection, macrophage genes involved in cell growth, metabolism, and responses to endogenous stimuli were down-regulated, while the inflammatory response (e.g., tumor necrosis factor alpha and Toll-like receptor 2), the complement system, the responses to external stimuli, and other immune responses were up-regulated. It is likely that the most active brucellacidal activity happened between 0 and 4 h postinfection. Mitochondrion-associated gene expression, which is involved in protein synthesis and transport, electron transfer, and small-molecule transfer, and many other mitochondrial functions were significantly down-regulated at 4 h postinfection. Although there were both pro- and antiapoptosis effects, B. melitensis 16M appears to inhibit apoptosis of macrophages by blocking release of cytochrome c and production of reactive oxygen species in the mitochondria, thus preventing activation of caspase cascades.
The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact β-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the “domino theory” of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.
Brucella spp. are facultative intracellular bacterial pathogens responsible for brucellosis, a worldwide zoonosis that causes abortion in domestic animals and chronic febrile disease associated with serious complications in humans. There is currently no approved vaccine against human brucellosis, and antibiotic therapy is long and costly. Development of a safe protective vaccine requires a better understanding of the roles played by components of adaptive immunity in the control of Brucella infection. The importance of lymphocyte subsets in the control of Brucella growth has been investigated separately by various research groups and remains unclear or controversial. Here, we used a large panel of genetically deficient mice to compare the importance of B cells, transporter associated with antigen processing (TAP-1), and major histocompatibility complex class II-dependent pathways of antigen presentation as well as T helper 1 (Th1), Th2, and Th17-mediated responses on the immune control of Brucella melitensis 16 M infection. We clearly confirmed the key function played by gamma interferon (IFN-γ)-producing Th1 CD4+ T cells in the control of B. melitensis infection, whereas IFN-γ-producing CD8+ T cells or B cell-mediated humoral immunity plays only a modest role in the clearance of bacteria during primary infection. In the presence of a Th1 response, Th2 or Th17 responses do not really develop or play a positive or negative role during the course of B. melitensis infection. On the whole, these results could improve our ability to develop protective vaccines or therapeutic treatments against brucellosis.
Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.
Brucella spp. are facultative intracellular pathogens that cause brucellosis in a broad range of hosts, including humans. Brucella melitensis, B. abortus, and B. suis are highly infectious and can be readily transmitted in aerosolized form, and a human vaccine against brucellosis is unavailable. Therefore, these pathogens are recognized as potential bioterror agents. Because genetic systems for studying host–Brucella interactions have been unavailable, little is known about the host factors that mediate infection. Here, we demonstrate that a Drosophila S2 cell system and RNA interference can be exploited to study the role that evolutionarily conserved Brucella host proteins play in these processes. We also show that this system provides for the identification and characterization of host factors that mediate Brucella interactions with the host cell endoplasmic reticulum. In fact, we identified 52 host factors that, when depleted, inhibited or increased Brucella infection. Among the identified Brucella host factors, 29 have not been previously shown to support bacterial infection. Finally, we demonstrate that the novel host factor inositol-requiring enzyme 1 (IRE1) and its mammalian ortholog (IRE1α) are required for Brucella infection of Drosophila S2 and mammalian cells, respectively. Therefore, this work contributes to our understanding of host factors mediating Brucella infection.
There is no safe, effective human vaccine against brucellosis. Live attenuated Brucella strains are widely used to vaccinate animals. However these live Brucella vaccines can cause disease and are unsafe for humans. Killed Brucella or subunit vaccines are not effective in eliciting long term protection. In this study, we evaluate an approach using a live, non-pathogenic bacteria (E. coli) genetically engineered to mimic the brucellae pathway of infection and present antigens for an appropriate cytolitic T cell response.
E. coli was modified to express invasin of Yersinia and listerialysin O (LLO) of Listeria to impart the necessary infectivity and antigen releasing traits of the intracellular pathogen, Brucella. This modified E. coli was considered our vaccine delivery system and was engineered to express Green Fluorescent Protein (GFP) or Brucella antigens for in vitro and in vivo immunological studies including cytokine profiling and cytotoxicity assays.
The E. coli vaccine vector was able to infect all cells tested and efficiently deliver therapeutics to the host cell. Using GFP as antigen, we demonstrate that the E. coli vaccine vector elicits a Th1 cytokine profile in both primary and secondary immune responses. Additionally, using this vector to deliver a Brucella antigen, we demonstrate the ability of the E. coli vaccine vector to induce specific Cytotoxic T Lymphocytes (CTLs).
Protection against most intracellular bacterial pathogens can be obtained mostly through cell mediated immunity. Data presented here suggest modified E. coli can be used as a vaccine vector for delivery of antigens and therapeutics mimicking the infection of the pathogen and inducing cell mediated immunity to that pathogen.
The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans. Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here we show in a model of Brucella abortus infection of murine bone marrow–derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway. The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties. The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport. A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes. Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER. Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.
macrophage; Brucella; endoplasmic reticulum; type IV secretion; trafficking
Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection.
BRUCELLA; ADHESION; VIRULENCE
A method for the in vitro study of intracellular brucella has been described. Exudative leukocytes containing intracellular brucella have been maintained in vitro in a synthetic tissue culture medium or in human or animal serum. Intracellular brucella are protected in vitro against the lethal action of therapeutic agents or the bactericidal action of serum. This protection of intracellular brucella is dependent upon the presence of an intact, viable host cell. None of the currently available therapeutic agents, whether used alone or in combinations, were capable of killing all intracellular brucella in vitro in 24 hours. A remarkable protection of intracellular brucella against streptomycin has been demonstrated. The most effective reduction in the number of viable intracellular brucella was accomplished by exposure of the host cells to streptomycin plus aureomycin, terramycin, or chloramphenicol. The available evidence suggests that the ability of brucella to localize and remain viable within the cells of an infected host is an important biologic factor in establishing and perpetuating brucella infections, despite therapeutic measures or the operation of the host's humoral defense mechanisms. Reduction of neotetrazolium by leukocytes and brucella in vitro provides a method for assessing the metabolic status of the host cell, but does not discriminate with any degree of certainty a viable from a non-viable intracellular organism.
1. Comparison of the infections of chick embryos by the chorio-allantoic route indicates that Bacterium tularense and Brucella suis, abortus, and melitensis exhibit varying degrees of facultative intracellular parasitism. Pasteurella pestis is adapted to rapid proliferation and spread in the intercellular fluids. 2. In the early stages of infection Bacterium tularense has a marked affinity for growth within ectodermal epithelial cells. Brucella suis and Brucella abortus differ in their selectivity for cells of mesodermal derivation and especially in their effect on vascular endothelium. The strain of Brucella melitensis studied is limited in its intracellular growth to ectodermal epithelium. 3. Many of the features characteristic of these infections in the natural hosts are reproduced in the chick embryo and its membranes. 4. The possible implications regarding the differences in behavior of these microorganisms in relation to the problem of infection and pathogenesis of these diseases are discussed.
Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of 10 classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics) and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning.
Brucella; pathogenesis; host immunity; vaccine; systems biology; bioinformatics
During the complex interaction between an infectious agent and a host organism, the pathogen can interfere with the host cell's programmed death to its own benefit. Induction or prevention of host cell apoptosis appears to be a critical step for determining the infection outcome. Members of the gram-negative bacterial genus Brucella are intracellular pathogens which preferentially invade monocytic cells and develop within these cells. We investigated the effect of Brucella suis infection on apoptosis of human monocytic phagocytes. The present study provides evidence that Brucella infection inhibited spontaneously occurring apoptosis in human monocytes. Prevention of monocyte apoptosis was not mediated by Brucella lipopolysaccharide and required bacterial survival within infected cells. Both invaded and noninvaded cells were protected, indicating that soluble mediators released during infection were involved in the phenomenon. Analysis of Brucella-infected monocytes revealed specific overexpression of the A1 gene, a member of the bcl-2 family implicated in the survival of hematopoietic cells. Brucella infection also rendered macrophage-like cells resistant to Fas ligand- or gamma interferon-induced apoptosis, suggesting that Brucella infection protected host cells from several cytotoxic processes occurring at different steps of the immune response. The present data clearly show that Brucella suis modulated the monocyte/macrophage's apoptotic response to the advantage of the pathogen, thus preventing host cell elimination. This might represent a strategy for Brucella development in infected hosts.