Pablo C. Baldi

Diminished Production of T Helper 1 Cytokines Correlates with T Cell Unresponsiveness to Brucella Cytoplasmic Proteins in Chronic Human Brucellosis

This study evaluated the cellular immune response against Brucella species cytoplasmic protein (CP) in peripheral blood mononuclear cells (PBMC) of 25 patients with brucellosis. In vitro proliferation and cytokine gene expression and production were investigated. PBMC from 14 patients proliferated in response to CP (responder patients [RPs]) and cells from 11 patients did not (nonresponder patients [NRPs]). CP-specific interleukin (IL)-2 and interferon-gamma were significantly induced in PBMC from RPs, compared with cells from NRPs. No significant differences were found in the production of IL-10 between the 2 groups. CP did not induce IL-4 production. A close relationship was observed between the clinical status of the patients and the T cell response against CP. Patient with acute infections responded to CP and induced production of T helper 1 (Th1) cytokines, whereas chronically infected patients did not. Diminished production of Th1 cytokines may contribute to T cell unresponsiveness in chronic human brucellosis.

The 18-kDa cytoplasmic protein of Brucella species : an antigen useful for diagnosis : is a lumazine synthase

Previous studies have shown that the detection of antibodies to an 18-kDa cytoplasmic protein of Brucella spp. is useful for the diagnosis of human and animal brucellosis. This protein has now been expressed in recombinant form in Escherichia coli. The recombinant protein is soluble only under reducing conditions, but alkylation with iodoacetamide renders it soluble in non-reducing media. As shown by gel exclusion chromatography, this soluble form arranges in pentamers of 90 kDa. The reactivity of human and animal sera against the recombinant protein was similar to that found with the native protein present in brucella cytoplasmic fraction, suggesting that the recombinant protein is correctly folded. The protein has low but significant homology (30%) with lumazine synthases involved in bacterial riboflavin biosynthesis, which also arrange as pentamers. Biological tests on the crude extract of the recombinant bacteria and on the purified recombinant protein showed that the biological activity of the Brucella spp. 18-kDa protein is that of lumazine synthase. Preliminary crystallographic analysis showed that the Brucella spp. lumazine synthase arranges in icosahedric capsids similar to those formed by the lumazine synthases of other bacteria. The high immunogenicity of this protein, potentially useful for the design of acellular vaccines, could be explained by this polymeric arrangement.

Proinflammatory Response of Human Osteoblastic Cell Lines and Osteoblast-Monocyte Interaction upon Infection with Brucella spp.

ABSTRACT The ability of Brucella spp. to infect human osteoblasts and the cytokine response of these cells to infection were investigated in vitro. Brucella abortus, B. suis, B. melitensis, and B. canis were able to infect the SaOS-2 and MG-63 osteoblastic cell lines, and the first three species exhibited intracellular replication. B. abortus internalization was not significantly affected by pretreatment of cells with cytochalasin D but was inhibited up to 92% by colchicine. A virB10 mutant of B. abortus could infect but not replicate within osteoblasts, suggesting a role for the type IV secretion system in intracellular survival. Infected osteoblasts produced low levels of chemokines (interleukin-8 [IL-8] and macrophage chemoattractant protein 1 [MCP-1]) and did not produce proinflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor alpha [TNF-α]). However, osteoblasts stimulated with culture supernatants from Brucella-infected human monocytes (THP-1 cell line) produced chemokines at levels 12-fold (MCP-1) to 17-fold (IL-8) higher than those of infected osteoblasts and also produced IL-6. In the inverse experiment, culture supernatants from Brucella-infected osteoblasts induced the production of IL-8, IL-1β, IL-6, and TNF-α by THP-1 cells. The induction of TNF-α and IL-1β was largely due to granulocyte-macrophage colony-stimulating factor produced by infected osteoblasts, as demonstrated by inhibition with a specific neutralizing antibody. This study shows that Brucella can invade and replicate within human osteoblastic cell lines, which can directly and indirectly mount a proinflammatory response. Both phenomena may have a role in the chronic inflammation and bone and joint destruction observed in osteoarticular brucellosis.

A bile salt hydrolase of Brucella abortus contributes to the establishment of a successful infection through the oral route in mice.

ABSTRACT Choloylglycine hydrolase (CGH), a bile salt hydrolase, has been annotated in all the available genomes of Brucella species. We obtained the Brucella CGH in recombinant form and demonstrated in vitro its capacity to cleave glycocholate into glycine and cholate. Brucella abortus 2308 (wild type) and its isogenic Δcgh deletion mutant exhibited similar growth rates in tryptic soy broth in the absence of bile. In contrast, the growth of the Δcgh mutant was notably impaired by both 5% and 10% bile. The bile resistance of the complemented mutant was similar to that of the wild-type strain. In mice infected through the intragastric or the intraperitoneal route, splenic infection was significantly lower at 10 and 20 days postinfection in animals infected with the Δcgh mutant than in those infected with the wild-type strain. For both routes, no differences in spleen CFU were found between animals infected with the wild-type strain and those infected with the complemented mutant. Mice immunized intragastrically with recombinant CGH mixed with cholera toxin (CGH+CT) developed a specific mucosal humoral (immunoglobulin G [IgG] and IgA) and cellular (interleukin-2) immune responses. Fifteen days after challenge by the same route with live B. abortus 2308 cells, splenic CFU counts were 10-fold lower in mice immunized with CGH+CT than in mice immunized with CT or phosphate-buffered saline. This study shows that CGH confers on Brucella the ability to resist the antimicrobial action of bile salts. The results also suggest that CGH may contribute to the ability of Brucella to infect the host through the oral route.

Antibody reactivity to Omp31 from Brucella melitensis in human and animal infections by smooth and rough Brucellae.

ABSTRACT Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.

Human Infection with M- Strain of Brucella canis

The less mucoid strain of Brucella canis or M- strain is used for the serologic diagnosis of canine brucellosis. While this strain is avirulent in dogs, we report the case of clinical brucellosis that developed in a laboratory worker a few days after handling live M- cells for antigen production.

Macrophage-elicited osteoclastogenesis in response to Brucella abortus infection requires TLR2/MyD88-dependent TNF-α production

Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. In this manuscript, we described an immune mechanism for inflammatory bone loss in response to infection by Brucella abortus. We established a requirement for MyD88 and TLR2 in TNF‐α‐elicited osteoclastogenesis in response to B. abortus infection. CS from macrophages infected with B. abortus induced BMM to undergo osteoclastogenesis. Although B. abortus‐infected macrophages actively secreted IL‐1β, IL‐6, and TNF‐α, osteoclastogenesis depended on TNF‐α, as CS from B. abortus‐infected macrophages failed to induce osteoclastogenesis in BMM from TNFRp55–/– mice. CS from B. abortus‐stimulated MyD88–/– and TLR2–/– macrophages failed to express TNF‐α, and these CS induced no osteoclast formation compared with that of the WT or TLR4–/– macrophages. Omp19, a B. abortus lipoprotein model, recapitulated the cytokine production and subsequent osteoclastogenesis induced by the whole bacterium. All phenomena were corroborated using human monocytes, indicating that this mechanism could play a role in human osteoarticular brucellosis. Our results indicate that B. abortus, through its lipoproteins, may be involved in bone resorption through the pathological induction of osteoclastogenesis.

Potential Role of Fibroblast-Like Synoviocytes in Joint Damage Induced by Brucella abortus Infection through Production and Induction of Matrix Metalloproteinases

ABSTRACT Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators.

The TolC Homologue of Brucella suis Is Involved in Resistance to Antimicrobial Compounds and Virulence

ABSTRACT Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and otherα -2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host.

Brucella-infected hepatocytes mediate potentially tissue-damaging immune responses

BACKGROUND & AIMS Hepatic involvement is frequent in human brucellosis. While different histopathological lesions have been reported in these patients, the underlying cellular and molecular mechanisms have not been addressed. METHODS This study assessed whether Brucella abortus can infect a human hepatoma cell line and induce a proinflammatory response in these cells. RESULTS The bacterium not only infected the human hepatoma cell line HepG2 but also exhibited intracellular replication. The infection induced hepatoma cells to secrete IL-8, and supernatants from Brucella-infected hepatoma cells were shown to induce the migration of human neutrophils. The infection also induced the expression of the intercellular adhesion molecule ICAM-1 on hepatoma cells, and the adhesion of neutrophils to these cells was significantly higher than to uninfected hepatoma cells. ICAM-1 expression was also induced by stimulation of hepatoma cells with supernatants from Brucella-infected neutrophils. While Brucella infection did not induce the expression of matrix metalloproteinases (MMPs) in hepatoma cells, it significantly induced MMP-9 in neutrophils. Hepatoma cell apoptosis was significantly induced by B. abortus infection and also by stimulation with supernatants from Brucella-infected neutrophils. CONCLUSIONS The present study provides clues regarding potential mechanisms of tissue damage during liver brucellosis.