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

Miriam Salvador-Bescós,Estrella Martínez-Gómez,Ignacio Moriyón,Amaia Zúñiga-Ripa,Yolanda Gil-Ramírez,Maite Iriarte,Raquel Conde-Álvarez,María J De Miguel,Axel Cloeckaert,Pilar M Muñoz,Michel S Zygmunt

doi:10.3389/fmicb.2018.02293

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.

Highlights

Members of the genus Brucella are the etiologic agents of brucellosis, a worldwide spread zoonosis that affects ruminants, camelids, swine, dogs, and several forms of marine and terrestrial wildlife and causes abortions, infertility, and the subsequent economic losses in livestock
We excluded from further analysis BAB1_0108-cgs, which is involved in cyclic glucan synthesis (Briones et al, 2001), BAB1_1786-mtgA and BAB1_1450-murG, both related to peptidoglycan synthesis, BAB1_1171-lpxB, probably implicated in lipid A formation (Iriarte et al, 2004), and BAB1_0553-wbkA, BAB1_0563-wbkE; BAB1_1000-wboA and BAB1_1000-wboB, four genes that belong to the O-PS synthesis route (McQuiston et al, 1999; Godfroid et al, 2000; González et al, 2008)
The remaining 11 Open-reading frames (ORFs) are listed in Table 1, and data on their presence in other Brucella spp. and genetic location are in the Supplementary Material (Supplementary Table S2 and Supplementary Figure S1)

Summary

Introduction

Members of the genus Brucella are the etiologic agents of brucellosis, a worldwide spread zoonosis that affects ruminants, camelids, swine, dogs, and several forms of marine and terrestrial wildlife and causes abortions, infertility, and the subsequent economic losses in livestock. Those that have been known for a long time (often referred to as “classical” Brucella species) include B. abortus and B. melitensis (the brucellae that infect domestic ruminants), B. suis (infecting swine, reindeer, hares, and several species of wild rodents), B. canis (infecting dogs), B. ovis (not zoonotic and restricted to sheep), and B. neotomae (infecting the desert woodrat) Because of their early identification and their economic and public health importance, B. abortus, B. melitensis, and B. suis are the best-characterized members of the genus, and all of them produce smooth (S) glossy colonies, a morphology that reflects the existence of a lipopolysaccharide (LPS) carrying an O-polysaccharide (O-PS) linked to the corelipid A section that anchors the molecule to the outer membrane (OM). The Brucella S-LPS carries the most significant PAMP modifications and is a major virulence factor (Lapaque et al, 2005)

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Conclusion