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

Abstract

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 Brucella.

Comparative Transcriptome Analysis of Artificially Induced Rough-Mutant Brucella Strain RM57 and Its Parent Strain Brucella melitensis M1981

“…Similarly, there was no observable difference in survival ratio of the two strains when challenged with acidic environment (Figure 5A). Previously, the rough type Brucella strains have been described as being more sensitive to the bactericidal action of normal serum than their smooth counterparts (11). However, after incubation for 90 min in non-immune serum, we observed that the survival rate of RM57 was not significantly reduced compared to the parent strain M1981 (Figure 5A).…”

“…We evaluated the effect of bactericidal action in non-immune serum on sensitivity in Brucella strains according to a previously reported protocol (11). Briefly, cultures of RM57 and M1981 strains were adjusted to 10 4 CFUs/ml using PBS then dispensed in microtiter plates (at a volume of 30 µl/well) containing 60 µl of new-born bovine serum.…”

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus

“…We discovered 14 such synthetic lethal genes in B. abortus (Fig. B and C) that have been previously reported to play a role in O‐polysaccharide synthesis (Cloeckaert et al ., ; Ugalde et al ., ; Guzman‐Verri et al ., ; Monreal et al ., ; Manterola et al ., ; Cardoso et al ., ; Lamontagne et al ., ; Gonzalez et al ., ; Mancilla et al ., ; Zhang et al ., ; Tian et al ., ; Dabral et al ., ; Salvador‐Bescos et al ., ); mutations in this gene set result in a rough phenotype in Brucella melitensis (Gonzalez et al ., ). It is well established that B. ovis is a stably rough strain that lacks LPS O‐polysaccharide (Gonzalez et al ., ; Tsolis et al ., ; Soler‐Llorens et al ., ; Mancilla, ).…”

“…The molecular connection between O-polysaccharide biosynthesis and the B. ovis chaining phenotype upon eipA depletion remains undefined, though it is known that alteration of LPS structure can influence morphology and division in C. crescentus (Cabeen et al, 2010). It has also been reported that teichoic acids, carbohydrate components of Gram-positive envelopes, play a role in the initiation of cell division (Santa Maria et al, 2014). EipA may serve in part as a bridge that links the state of envelope carbohydrates with the cell division apparatus.…”

Periplasmic protein EipA determines envelope stress resistance and virulence inBrucella abortus

“…Specifically, we sought to identify genes that are dispensable in wild-type cells, but essential in the absence of eipA. We discovered 14 such synthetic lethal genes in B. abortus ( Figure 6B and C) that have been previously reported to play a role in O-polysaccharide synthesis (Gonzalez et al, 2008, Cardoso et al, 2006, Ugalde et al, 2000, Zhang et al, 2013, Tian et al, 2014, Mancilla et al, 2012, Cloeckaert et al, 2000, Dabral et al, 2015, Monreal et al, 2003, Guzman-Verri et al, 2002, Lamontagne et al, 2007, Manterola et al, 2005, Salvador-Bescos et al, 2018; mutations in this gene set result in a rough phenotype in Brucella melitensis (Gonzalez et al, 2008). It is well established that B. ovis is a stably rough strain that lacks LPS Opolysaccharide (Mancilla, 2015, Soler-Llorens et al, 2014, Tsolis et al, 2009, Gonzalez et al, 2008.…”