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Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen.

ABSTRACT: Human brucellosis is caused mainly by Brucella melitensis, which is often acquired by ingesting contaminated goat or sheep milk and cheese. Bacterial factors required for food-borne infection of humans by B. melitensis are poorly understood. In this study, a mouse model of oral infection was characterized to assess the roles of urease, the VirB type IV secretion system, and lipopolysaccharide for establishing infection through the digestive tract. B. melitensis strain 16M was consistently recovered from the mesenteric lymph node (MLN), spleen, and liver beginning at 3 or 7 day postinfection (dpi). In the gut, persistence of the inoculum was observed up to 21 dpi. No inflammatory lesions were observed in the ileum or colon during infection. Mutant strains lacking the ureABC genes of the ure1 operon, virB2, or pmm encoding phosphomannomutase were constructed and compared to the wild-type strain for infectivity through the digestive tract. Mutants lacking the virB2 and pmm genes were attenuated in the spleen (P < 0.05) and MLN (P < 0.001), respectively. The wild-type and mutant strains had similar levels of resistance to low pH and 5 or 10% bile, suggesting that the reduced colonization of mutants was not the result of reduced resistance to acid pH or bile salts. In an in vitro lymphoepithelial cell (M-cell) model, B. melitensis transited rapidly through polarized enterocyte monolayers containing M-like cells; however, transit through monolayers containing only enterocytes was reduced or absent. These results indicate that B. melitensis is able to spread systemically from the digestive tract after infection, most likely through M cells of the mucosa-associated lymphoid tissue.

SUBMITTER: Paixao TA

PROVIDER: S-EPMC2747930 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen.

Paixão Tatiane A TA Roux Christelle M CM den Hartigh Andreas B AB Sankaran-Walters Sumathi S Dandekar Satya S Santos Renato L RL Tsolis Renée M RM

Infection and immunity 20090803 10

Human brucellosis is caused mainly by Brucella melitensis, which is often acquired by ingesting contaminated goat or sheep milk and cheese. Bacterial factors required for food-borne infection of humans by B. melitensis are poorly understood. In this study, a mouse model of oral infection was characterized to assess the roles of urease, the VirB type IV secretion system, and lipopolysaccharide for establishing infection through the digestive tract. B. melitensis strain 16M was consistently recove ...[more]

PMID: 19651862

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Project description:Brucellosis is a bacterial zoonosis of worldwide distribution caused by bacteria of the genus Brucella. In Brucella abortus and Brucella melitensis, the major species infecting domestic ruminants, the smooth lipopolysaccharide (S-LPS) is a virulence factor. This S-LPS carries a N-formyl-perosamine homopolymer O-polysaccharide that is the major antigen in serodiagnostic tests and is required for virulence. We report that the Brucella O-PS can be structurally and antigenically modified using wbdR, the acetyl-transferase gene involved in N-acetyl-perosamine synthesis in Escherichia coli O157:H7. Brucella constructs carrying plasmidic wbdR expressed a modified O-polysaccharide but were unstable, a problem circumvented by inserting wbdR into a neutral site of chromosome II. As compared to wild-type bacteria, both kinds of wbdR constructs expressed shorter O-polysaccharides and NMR analyses showed that they contained both N-formyl and N-acetyl-perosamine. Moreover, deletion of the Brucella formyltransferase gene wbkC in wbdR constructs generated bacteria producing only N-acetyl-perosamine homopolymers, proving that wbdR can replace for wbkC. Absorption experiments with immune sera revealed that the wbdR constructs triggered antibodies to new immunogenic epitope(s) and the use of monoclonal antibodies proved that B. abortus and B. melitensis wbdR constructs respectively lacked the A or M epitopes, and the absence of the C epitope in both backgrounds. The wbdR constructs showed resistance to polycations similar to that of the wild-type strains but displayed increased sensitivity to normal serum similar to that of a per R mutant. In mice, the wbdR constructs produced chronic infections and triggered antibody responses that can be differentiated from those evoked by the wild-type strain in S-LPS ELISAs. These results open the possibilities of developing brucellosis vaccines that are both antigenically tagged and lack the diagnostic epitopes of virulent field strains, thereby solving the diagnostic interference created by current vaccines against Brucella.

Project description:The intracellular bacterial pathogen Brucella causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able to withstand oxidative stresses and express the type IV secretion system (T4SS) to evade host immune responses. The extracytoplasmic function (ECF) sigma factor system is a major signal transduction mechanism in bacteria that senses environmental cues and responds by regulating gene expression. In this study, we defined an ECF σ bcrS and its cognate anti-σ factor abcS in Brucella melitensis M28 by conserved domain analysis and a protein interaction assay. BcrS directly activates an adjacent operon, bcrXQP, that encodes a methionine-rich peptide and a putative methionine sulfoxide reductase system, whereas AbcS is a negative regulator of bcrS and bcrXQP. The bcrS-abcS and bcrXQP operons can be induced by hypochlorous acid and contribute to hypochlorous acid resistance in vitro. Next, RNA sequencing analysis and genome-wide recognition sequence search identified the regulons of BcrS and AbcS. Interestingly, we found that BcrS positively influences T4SS expression in an AbcS-dependent manner and that AbcS also affects T4SS expression independently of BcrS. Last, we demonstrate that abcS is required for the maintenance of persistent infection, while bcrS is dispensable in a mouse infection model. Collectively, we conclude that BcrS and AbcS influence expression of multiple genes responsible for Brucella virulence traits. IMPORTANCE Brucella is a notorious intracellular pathogen that induces chronic infections in animals and humans. To survive and replicate within macrophages, these bacteria require a capacity to withstand oxidative stresses and to express the type IV secretion system (T4SS) to combat host immune responses. In this study, we characterized an extracytoplasmic function sigma/anti-sigma factor system that regulates resistance to reactive chlorine species and T4SS expression, thereby establishing a potential link between two crucial virulence traits of Brucella. Furthermore, the anti-sigma factor AbcS contributes to Brucella persistent infection of mice. Thus, this work provides novel insights into Brucella virulence regulation as well as a potential drug target for fighting Brucella infections.

Project description:BACKGROUND: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. METHODOLOGY/PRINCIPAL FINDINGS: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

Dataset Information

Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen.

Publications

Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen.

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