Genomic insertion of a heterologous acetyltransferase generates a new lipopolysaccharide antigenic structure in brucella abortus and brucella melitensis

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

Identification and functional analysis of the cyclopropane fatty acid synthase of Brucella abortus

The brucellae are facultative intracellular pathogens of mammals that are transmitted by contact with infected animals or contaminated materials. Several major lipidic components of the brucella cell envelope are imperfectly recognized by innate immunity, thus contributing to virulence. These components carry large proportions of acyl chains of lactobacillic acid, a long chain cyclopropane fatty acid (CFA). CFAs result from addition of a methylene group to unsaturated acyl chains and contribute to resistance to acidity, dryness and high osmolarity in many bacteria and to virulence in mycobacteria. We examined the role of lactobacillic acid in Brucella abortus virulence by creating a mutant in ORF BAB1_0476, the putative CFA synthase gene. The mutant did not incorporate [(14)C]methyl groups into lipids, lacked CFAs and synthesized the unsaturated precursors, proving that BAB1_0476 actually encodes a CFA synthase. BAB1_0476 promoter-luxAB fusion studies showed that CFA synthase expression was promoted by acid pH and high osmolarity. The mutant was not attenuated in macrophages or mice, strongly suggesting that CFAs are not essential for B. abortus intracellular life. However, when the mutant was tested under high osmolarity on agar and acid pH, two conditions likely to occur on contaminated materials and fomites, they showed reduced ability to grow or survive. Since CFA synthesis entails high ATP expenses and brucellae produce large proportions of lactobacillic acyl chains, we speculate that the CFA synthase has been conserved because it is useful for survival extracellularly, thus facilitating persistence in contaminated materials and transmission to new hosts

WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization

Brucellosis, an infectious disease caused by Brucella, is one of the most extended bacterial zoonosis in the world and an important cause of economic losses and human suffering. The lipopolysaccharide (LPS) of Brucella plays a major role in virulence as it impairs normal recognition by the innate immune system and delays the immune response. The LPS core is a branched structure involved in resistance to complement and polycationic peptides, and mutants in glycosyltransferases required for the synthesis of the lateral branch not linked to the O-polysaccharide (O-PS) are attenuated and have been proposed as vaccine candidates. For this reason, the complete understanding of the genes involved in the synthesis of this LPS section is of particular interest. The chemical structure of the Brucella LPS core suggests that, in addition to the already identified WadB and WadC glycosyltransferases, others could be implicated in the synthesis of this lateral branch. To clarify this point, we identified and constructed mutants in 11 ORFs encoding putative glycosyltransferases in B. abortus. Four of these ORFs, regulated by the virulence regulator MucR (involved in LPS synthesis) or the BvrR/BvrS system (implicated in the synthesis of surface components), were not required for the synthesis of a complete LPS neither for virulence or interaction with polycationic peptides and/or complement. Among the other seven ORFs, six seemed not to be required for the synthesis of the core LPS since the corresponding mutants kept the O-PS and reacted as the wild type with polyclonal sera. Interestingly, mutant in ORF BAB1_0953 (renamed wadD) lost reactivity against antibodies that recognize the core section while kept the O-PS. This suggests that WadD is a new glycosyltransferase adding one or more sugars to the core lateral branch. WadD mutants were more sensitive than the parental strain to components of the innate immune system and played a role in chronic stages of infection. These results corroborate and extend previous work indicating that the Brucella LPS core is a branched structure that constitutes a steric impairment preventing the elements of the innate immune system to fight against Brucell

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

Brucellosis is a worldwide extended zoonosis caused by Brucella spp. These gram-negative bacteria are not readily detected by innate immunity, a virulence-related property largely linked to their surface lipopolysaccharide (LPS). The role of the LPS lipid A and O-polysaccharide in virulence is well known. Moreover, mutation of the glycosyltransferase gene wadC of Brucella abortus, although not affecting O-polysaccharide assembly onto the lipid-A core section causes a core oligosaccharide defect that increases recognition by innate immunity. Here, we report on a second gene (wadB) encoding a LPS core glycosyltransferase not involved in the assembly of the O-polysaccharide-linked core section. As compared to wild-type B. abortus, a wadB mutant was sensitive to bactericidal peptides and non-immune serum, and was attenuated in mice and dendritic cells. These observations show that as WadC, WadB is also involved in the assembly of a branch of Brucella LPS core and support the concept that this LPS section is a virulence-related structure

Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models

The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ppdK and mae mutants was severely impaired and growth of the double fbp- glpX mutant was reduced. In macrophages, only the ppdK and mae mutants showed reduced multiplication, and studies with the ppdK mutant confirmed that it reached the replicative niche. Similarly, only the ppdK and mae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt

Papel de una etanolamina-fosfato transferasa y una glicosil transferasa en la síntesis del lipopolisacárido de Brucella y utilización de una acetil transferaas para la modificación epitópica de la cadena O

La brucelosis es una de las zoonosis bacterianas más importantes y afecta a mamíferos terrestres y marinos. El lipopolisacárido de Brucella tiene un papel crucial en la virulencia y es considerado como un patrón asociado a patógeno alterado. En este trabajo, hemos investigado la función de dos hipotéticas glicosil transferasas de lipopolisacárido [BAB1_0351 (wadB) y BAB1_1620] y de una hipotética etanolaminafosfato transferasa (BMEI0118). El mutante wadB en B. abortus (BABwadB), B.melitensis y B. suis biovar 2 carece de parte del núcleo del lipopolisacárido, pero mantiene intacta la cadena O. BABwadB es más sensible al suero y a los péptidos bactericidas y está atenuado en células dendríticas y en ratones. Además, es capaz de inducir una respuesta inmune protectora. Por otro lado, BAB1_1620, una glicosil transferasa relacionada con otras implicadas en la síntesis del lipopolisacárido, parece no estar relacionada con este proceso en B. abortus. Así mismo, su función no es esencial para la virulencia en ratones. Por otra parte, hemos demostrado que la proteína codificada por BMEI0118 (BMElptA) actúa como una transferasa que incorpora etanolamina-fosfato al lípido A. Esta modificación contribuye al bajo reconocimiento del lipopolisacárido por parte de algunos efectores del sistema inmune innato, ya que el mutante en BMELptA es más sensible al efecto bactericida del suero normal y a los péptidos catiónicos. Esta ORF presenta ortólogos en otras -2 Proteobacteria como Ochrobactrum, Sinorhizobium y Agrobacterium. La interferencia de la vacunación en el diagnóstico es un aspecto crucial para el control de la brucelosis, ya que los anticuerpos generados, dirigidos principalmente frente al lipopolisacárido, son comunes a animales infectados y vacunados. La expresión en Brucella de wbdR (acetil transferasa de cadena O en E. coli O 157:H7) lleva a la incorporación de residuos acetilo a la perosamina, generando un nuevo epítopo inmunogénico. Esta modificación posibilitará la elaboración de un nuevo test diagnóstico.Brucellosis is a disease of terrestrial and marine mammals and an important zoonosis. Brucella lipopolysaccharide plays a major role in virulence and is considered as a molecule with a modified pathogen-associated molecular pattern. The role of two hypothetical lipopolysaccharide core glycosyltransferases [BAB1_0351 (wadB) and BAB1_1620] and a putative phosphoethanolamine transferase (BMEI0118) was investigated. A wadB mutant in B. abortus, B. melitensis and B. suis biovar 2 lacked part of the lipopolysaccharide core but kept the O-chain. The wadB B. abortus mutant was sensitive to normal serum and bactericidal peptides and attenuated in dendritic cells and mice. Moreover, it elicited a protective immunoresponse, confirming that core mutants are promising brucellosis vaccines. On the other hand, BAB1_1620, a glycosyltransferase related to others implicated in lipopolysaccharide synthesis, seems not to be related to this process in B. abortus and its function is not essential for full virulence in mice. It is demostrated that BMELptA acts as a transferase that incorporate phosphoethanolamine to the lipid A section. This modification contributes to low recognition of lipopolysaccharide by some innate immune effectors since BMELptA mutant was sensitive to normal serum and bactericidal peptides. BMELptA has orthologous in other -2 Proteobacteria such as Ochrobactrum, Sinorhizobium or Agrobacterium. A crucial aspect in the control of brucellosis is that the use of S-brucellae vaccines interferes in the diagnosis, since they generate antibodies against the O-chain of the lipopolysaccharide, which is also the dominant antigen in infected animals. In order to bypass this difficulty, we have followed a new approach. The expression of wbdR (E. coli O 157:H7 O chain acetyltransferase) in B. abortus 2308 leads to the incorporation of acetyl residues to the perosamine polysaccharide, creating a new immunogenic epitope that could be the base for the elaboration of new diagnostic tests

Papel de una etanolamina-fosfato transferasa y una glicosil transferasa en la síntesis del lipopolisacárido de Brucella y utilización de una acetil transferaas para la modificación epitópica de la cadena O

La brucelosis es una de las zoonosis bacterianas más importantes y afecta a mamíferos terrestres y marinos. El lipopolisacárido de Brucella tiene un papel crucial en la virulencia y es considerado como un patrón asociado a patógeno alterado. En este trabajo, hemos investigado la función de dos hipotéticas glicosil transferasas de lipopolisacárido [BAB1_0351 (wadB) y BAB1_1620] y de una hipotética etanolaminafosfato transferasa (BMEI0118). El mutante wadB en B. abortus (BABwadB), B.melitensis y B. suis biovar 2 carece de parte del núcleo del lipopolisacárido, pero mantiene intacta la cadena O. BABwadB es más sensible al suero y a los péptidos bactericidas y está atenuado en células dendríticas y en ratones. Además, es capaz de inducir una respuesta inmune protectora. Por otro lado, BAB1_1620, una glicosil transferasa relacionada con otras implicadas en la síntesis del lipopolisacárido, parece no estar relacionada con este proceso en B. abortus. Así mismo, su función no es esencial para la virulencia en ratones. Por otra parte, hemos demostrado que la proteína codificada por BMEI0118 (BMElptA) actúa como una transferasa que incorpora etanolamina-fosfato al lípido A. Esta modificación contribuye al bajo reconocimiento del lipopolisacárido por parte de algunos efectores del sistema inmune innato, ya que el mutante en BMELptA es más sensible al efecto bactericida del suero normal y a los péptidos catiónicos. Esta ORF presenta ortólogos en otras -2 Proteobacteria como Ochrobactrum, Sinorhizobium y Agrobacterium. La interferencia de la vacunación en el diagnóstico es un aspecto crucial para el control de la brucelosis, ya que los anticuerpos generados, dirigidos principalmente frente al lipopolisacárido, son comunes a animales infectados y vacunados. La expresión en Brucella de wbdR (acetil transferasa de cadena O en E. coli O 157:H7) lleva a la incorporación de residuos acetilo a la perosamina, generando un nuevo epítopo inmunogénico. Esta modificación posibilitará la elaboración de un nuevo test diagnóstico.Brucellosis is a disease of terrestrial and marine mammals and an important zoonosis. Brucella lipopolysaccharide plays a major role in virulence and is considered as a molecule with a modified pathogen-associated molecular pattern. The role of two hypothetical lipopolysaccharide core glycosyltransferases [BAB1_0351 (wadB) and BAB1_1620] and a putative phosphoethanolamine transferase (BMEI0118) was investigated. A wadB mutant in B. abortus, B. melitensis and B. suis biovar 2 lacked part of the lipopolysaccharide core but kept the O-chain. The wadB B. abortus mutant was sensitive to normal serum and bactericidal peptides and attenuated in dendritic cells and mice. Moreover, it elicited a protective immunoresponse, confirming that core mutants are promising brucellosis vaccines. On the other hand, BAB1_1620, a glycosyltransferase related to others implicated in lipopolysaccharide synthesis, seems not to be related to this process in B. abortus and its function is not essential for full virulence in mice. It is demostrated that BMELptA acts as a transferase that incorporate phosphoethanolamine to the lipid A section. This modification contributes to low recognition of lipopolysaccharide by some innate immune effectors since BMELptA mutant was sensitive to normal serum and bactericidal peptides. BMELptA has orthologous in other -2 Proteobacteria such as Ochrobactrum, Sinorhizobium or Agrobacterium. A crucial aspect in the control of brucellosis is that the use of S-brucellae vaccines interferes in the diagnosis, since they generate antibodies against the O-chain of the lipopolysaccharide, which is also the dominant antigen in infected animals. In order to bypass this difficulty, we have followed a new approach. The expression of wbdR (E. coli O 157:H7 O chain acetyltransferase) in B. abortus 2308 leads to the incorporation of acetyl residues to the perosamine polysaccharide, creating a new immunogenic epitope that could be the base for the elaboration of new diagnostic tests

Identification and functional analysis of the cyclopropane fatty acid synthase of Brucella abortus

The brucellae are facultative intracellular pathogens of mammals that are transmitted by contact with infected animals or contaminated materials. Several major lipidic components of the brucella cell envelope are imperfectly recognized by innate immunity, thus contributing to virulence. These components carry large proportions of acyl chains of lactobacillic acid, a long chain cyclopropane fatty acid (CFA). CFAs result from addition of a methylene group to unsaturated acyl chains and contribute to resistance to acidity, dryness and high osmolarity in many bacteria and to virulence in mycobacteria. We examined the role of lactobacillic acid in Brucella abortus virulence by creating a mutant in ORF BAB1_0476, the putative CFA synthase gene. The mutant did not incorporate [(14)C]methyl groups into lipids, lacked CFAs and synthesized the unsaturated precursors, proving that BAB1_0476 actually encodes a CFA synthase. BAB1_0476 promoter-luxAB fusion studies showed that CFA synthase expression was promoted by acid pH and high osmolarity. The mutant was not attenuated in macrophages or mice, strongly suggesting that CFAs are not essential for B. abortus intracellular life. However, when the mutant was tested under high osmolarity on agar and acid pH, two conditions likely to occur on contaminated materials and fomites, they showed reduced ability to grow or survive. Since CFA synthesis entails high ATP expenses and brucellae produce large proportions of lactobacillic acyl chains, we speculate that the CFA synthase has been conserved because it is useful for survival extracellularly, thus facilitating persistence in contaminated materials and transmission to new hosts

Genomic insertion of a heterologous acetyltransferase generates a new lipopolysaccharide antigenic structure in brucella abortus and brucella melitensis

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