scholarly journals An extracytoplasmic function (ECF) sigma/anti-sigma factor system regulates hypochlorous acid resistance and impacts expression of the type IV secretion system in Brucella melitensis

2021 ◽  
Author(s):  
Huoming Li ◽  
Sen Hu ◽  
Xin Yan ◽  
Yan Yang ◽  
Wenxing Liu ◽  
...  
Keyword(s):  
Sigma Factor ◽  
Hypochlorous Acid ◽  
Brucella Melitensis ◽  
Acid Resistance ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Oxidative Stresses ◽  
Type Iv ◽  
Factor System

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 (ECF) 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.

scholarly journals Cytotoxicity in Macrophages Infected with Rough Brucella Mutants Is Type IV Secretion System Dependent

2007 ◽  
Vol 76 (1) ◽  
pp. 30-37 ◽  
Author(s):  
Jianwu Pei ◽  
Qingmin Wu ◽  
Melissa Kahl-McDonagh ◽  
Thomas A. Ficht
Keyword(s):  
Cell Death ◽  
Brucella Melitensis ◽  
Regulatory Element ◽  
Random Mutagenesis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
Mutant Bank

ABSTRACT Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16MΔmanBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (ΔmanBA) and the T4SS (ΔvirB). As predicted, deletion of virB from 16MΔmanBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.

scholarly journals Putative Quorum-Sensing Regulator BlxR of Brucella melitensis Regulates Virulence Factors Including the Type IV Secretion System and Flagella

2008 ◽  
Vol 190 (9) ◽  
pp. 3274-3282 ◽  
Author(s):  
Amy A. Rambow-Larsen ◽  
Gireesh Rajashekara ◽  
Erik Petersen ◽  
Gary Splitter
Keyword(s):  
Quorum Sensing ◽  
Virulence Factors ◽  
Brucella Melitensis ◽  
Regulatory Protein ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Deletion Strain ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
Systemic Spread

ABSTRACT Brucella melitensis is an intracellular pathogen that establishes a replicative niche within macrophages. While the intracellular lifestyle of Brucella is poorly understood and few virulence factors have been identified, components of a quorum-sensing pathway in Brucella have recently been identified. The LuxR-type regulatory protein, VjbR, and an N-acylhomoserine lactone signaling molecule are both involved in regulating expression of the virB-encoded type IV secretion system. We have identified a second LuxR-type regulatory protein (BlxR) in Brucella. Microarray analysis of a blxR mutant suggests that BlxR regulates the expression of a number of genes, including those encoding the type IV secretion system and flagella. Confirming these results, deletion of blxR in B. melitensis reduced the transcriptional activities of promoters for the virB operon, flagellar genes, and another putative virulence factor gene, bopA. Furthermore, our data suggested that both BlxR and VjbR are positively autoregulated and cross-regulate the expression of each other. The blxR deletion strain exhibited reduced growth in macrophages, similar to that observed for a vjbR deletion strain. However, unlike the vjbR deletion, the blxR deletion did not fully attenuate virulence in mice. More strikingly, bioluminescent imaging revealed that dissemination of the blxR mutant was similar to that of wild-type B. melitensis, while the vjbR mutant was defective for systemic spread in IRF-1−/− mice, suggesting that these regulators are not functionally redundant but that they converge in a common pathway regulating bacterial processes.

scholarly journals Establishment of Systemic Brucella melitensis Infection through the Digestive Tract Requires Urease, the Type IV Secretion System, and Lipopolysaccharide O Antigen

2009 ◽  
Vol 77 (10) ◽  
pp. 4197-4208 ◽  
Author(s):  
Tatiane A. Paixão ◽  
Christelle M. Roux ◽  
Andreas B. den Hartigh ◽  
Sumathi Sankaran-Walters ◽  
Satya Dandekar ◽  
...  
Keyword(s):  
Digestive Tract ◽  
Cell Model ◽  
Brucella Melitensis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Human Brucellosis ◽  
Type Iv Secretion System ◽  
Wild Type ◽  
Type Iv ◽  
Mutant Strains

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.

scholarly journals A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis

2005 ◽  
Vol 7 (8) ◽  
pp. 1151-1161 ◽  
Author(s):  
Rose-May Delrue ◽  
Chantal Deschamps ◽  
Sandrine Leonard ◽  
Caroline Nijskens ◽  
Isabelle Danese ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Brucella Melitensis ◽  
Flagellar Apparatus ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv

scholarly journals Brucella melitensis Invades Murine Erythrocytes during Infection

2014 ◽  
Vol 82 (9) ◽  
pp. 3927-3938 ◽  
Author(s):  
Marie-Alice Vitry ◽  
Delphine Hanot Mambres ◽  
Michaël Deghelt ◽  
Katrin Hack ◽  
Arnaud Machelart ◽  
...  
Keyword(s):  
Brucella Melitensis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Bacterial Cells ◽  
Content Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Time Point

ABSTRACTBrucellaspp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed thatBrucella melitensisis able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced followingBrucellavaccination. In contrast toBartonella, the type IV secretion system and flagellar expression are not critically required for the persistence ofBrucellain blood. ImageStream analysis of blood cells showed that following a brief extracellular phase,Brucellais associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated thatB. melitensisis able to invade erythrocytesin vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possibleBrucellatransmission by bloodsucking insects in nature.

scholarly journals An RpoH-Like Heat Shock Sigma Factor Is Involved in Stress Response and Virulence in Brucella melitensis 16M

2006 ◽  
Vol 188 (21) ◽  
pp. 7707-7710 ◽  
Author(s):  
Marie Delory ◽  
Régis Hallez ◽  
Jean-Jacques Letesson ◽  
Xavier De Bolle
Keyword(s):  
Sigma Factor ◽  
Wild Type Strain ◽  
Brucella Melitensis ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Wild Type ◽  
Phenotypic Analysis ◽  
Type Iv ◽  
Increased Sensitivity ◽  
Encoding Genes

ABSTRACT B. melitensis 16M genome analysis revealed the presence of six putative sigma factor-encoding genes: rpoD, rpoH1, rpoH2, rpoE1, rpoE2, and rpoN. We mutated all these genes except rpoD. Phenotypic analysis of the mutants reveals that a strain carrying an rpoH2 null mutation (ΔrpoH2) is impaired for growth at 21 and 42°C and shows increased sensitivity to hydrogen peroxide. Compared to the wild-type strain, the ΔrpoH2 mutant is attenuated in all virulence models tested. Three other null mutants (ΔrpoH1, ΔrpoE1, and ΔrpoE2 mutants) are also defective for survival in mice at 4 weeks postinfection. We also demonstrated that rpoH2 deletion strongly reduces the expression of two major virulence factors in B. melitensis, the type IV secretion system and the flagellum.

scholarly journals Production of the Type IV Secretion System Differs among Brucella Species as Revealed with VirB5- and VirB8-Specific Antisera

2003 ◽  
Vol 71 (3) ◽  
pp. 1075-1082 ◽  
Author(s):  
Bruno Rouot ◽  
Maria-Teresa Alvarez-Martinez ◽  
Carine Marius ◽  
Pierrette Menanteau ◽  
Laurence Guilloteau ◽  
...  
Keyword(s):  
Minimal Medium ◽  
Protein Production ◽  
Brucella Melitensis ◽  
Secretion System ◽  
Infection Process ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Prolonged Incubation ◽  
Type Iv ◽  
Acidic Conditions

ABSTRACT Expression of the virB operon, encoding the type IV secretion system required for Brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suis VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suis from which chvE was deleted was fully virulent in a mouse model. Comparative analyses of various Brucella species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canis and B. suis, as well as the Brucella abortus- and Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortus and B. melitensis strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virB expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virB operon are not uniformly regulated in different Brucella species. Since VirB proteins were shown to modulate Brucella phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.

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