Smooth to rough dissociation in Brucella: the missing link to virulence

PERSPECTIVE published: 05 January 2016 doi: 10.3389/fcimb.2015.00098 Smooth to Rough Dissociation in Brucella: The Missing Link to Virulence Marcos Mancilla * Research and Development Department, ADL Diagnostic Chile Ltd., Puerto Montt, Chile Edited by: Thomas A. Ficht, Texas A&M University, USA Reviewed by: Gregory T. Robertson, Colorado State University, USA Paul De Figueiredo, Texas A&M University, USA *Correspondence: Marcos Mancilla Received: 29 October 2015 Accepted: 10 December 2015 Published: 05 January 2016 Citation: Mancilla M (2016) Smooth to Rough Dissociation in Brucella: The Missing Link to Virulence. Front. Cell. Infect. Microbiol. 5:98. doi: 10.3389/fcimb.2015.00098 Dissociation encompasses changes in a series of phenotypes: colony and cell morphology, inmunological and biochemical reactions and virulence. The concept is generally associated to the in vitro transition between smooth (S) and rough (R) colonies, a phenotypic observation in Gram-negative bacteria commonly made since the beginning of microbiology as a science. It is also well known that the loss of the O-polysaccharide, the most external lipopolysaccharide (LPS) moiety, triggers the change in the colony phenotype. Although dissociation is related to one of the most basic features used to distinguish between species, i.e., colony morphology, and, in the case of pathogens, predict their virulence behavior, it has been considered a laboratory artifact and thus did not gain further attention. However, recent insights into genetics and pathogenesis of members of Brucella, causative agents of brucellosis, have brought a new outlook on this experimental fact, suggesting that it plays a role beyond the laboratory observations. In this perspective article, the current knowledge on Brucella LPS genetics and its connection with dissociation in the frame of evolution is discussed. Latest reports support the notion that, by means of a better understanding of genetic pathways linked to R phenotype and the biological impact of this intriguing “old” phenomenon, unexpected applications can be achieved. Keywords: Brucella, lipopolysaccharide, O-polysaccharide, rough, smooth The Brucella genus includes Gram-negative microorganisms that cause brucellosis, a major worldwide zoonosis. The taxonomical criteria used to divide the genus into several species include host preference, physiological differences, phage susceptibility and cell envelope structural features. Based on the aspect of colonies on agar plates, which is in accordance with the cell surface and lipopolysaccharide (LPS) structure, Brucella may occur either as smooth (S) or rough (R) species. The zoonotically more relevant S species B. melitensis, B. suis and B. abortus express a full LPS molecule (S-LPS) that is anchored in the outer membrane (OM) (Whatmore, 2009). This group furthermore comprises species that have been isolated from rodents (B. neotomae) and marine mammals (B. ceti and B. pinnipedialis; Foster et al., 2007). More recently, B. microti, primarily isolated from voles and red foxes, was isolated directly from soil, a fact that has not been reported for any other S species (Scholz et al., 2008). In contrast, the naturally occurring R species B. ovis and B. canis express R-LPS that lacks the O-antigen, a trait linked to their reduced virulence. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 January 2016 | Volume 5 | Article 98 Mancilla Dissociation and Virulence in Brucella BRUCELLA LPS: OVERALL STRUCTURE AND FUNCTION 2010). The genes responsible for the O-PS synthesis in Gramnegative bacteria are mainly clustered in the chromosome and often form a single transcriptional unit (Reeves and Wang, 2002). They can be categorized into three groups: nucleotide sugar pathway genes; those encoding glycosyltransferases (GT), which can also be found scattered throughout the genome; and those for processing and transport. Brucella is no exception from this rule, its O-PS genes are encoded in two main loci wbk and wbo. The O-PS synthesis depends on two GT genes carried by wbo (wboA and wboB) (McQuiston et al., 1999; González et al., 2008) that are included in the Brucella genomic island GI-2 (Rajashekara et al., 2008), an unstable genetic element of 15.1 kb (Mancilla et al., 2010). This region carries an additional gene, located close to GT genes, that encodes a hypothetical protein (BMEI0999) that apparently is part of the O-PS synthetic machinery since attempts to complement 1GI-2 mutants using a plasmid carrying only wboA-wboB have failed (Rajashekara et al., 2008). Major O-PS locus wbk is located between a ribose transport system (rbs) and an rnc gene (Moriyón et al., 2004). The low GC content of approximately 50%, a feature shared with many O-PS clusters, has been linked to its hypothetical acquisition via horizontal transfer (Cloeckaert et al., 2000; Godfroid et al., 2000). This region encodes the genes putatively necessary to synthesize perosamine (gmd, per), n-formylation of perosamine residues (wbkC), GT for polymerization (wbkE, wbkA), to prime bactoprenol (wbkD, wbkF), and ABC transporters that translocate the O-PS (wzm and wzt). Genes for mannose synthesis have also been identified in the same region (manAOAg , manBOAg, and manCOAg ), but mutational analysis of manBOAg indicated that it is not essential (González et al., 2008), since independent homologs located in the chromosome II (manBAcore ) are able to meet mannose demands (Monreal et al., 2003). To date, there is no confirmed function for wbkB (encodes a putative perosamine synthetase) because the corresponding mutant preserved the S phenotype and the hypothetical ligase that binds the amino sugar O-PS to the lipid A-core in the periplasmic interface has not yet been identified (Moriyón et al., 2004). Concerning horizontal acquisitions outside from wbk and wbo, the role of a gene cluster encoding enzymes for LPS biosynthesis has been proposed (Vizcaíno et al., 2001). The cluster was named GI-8 and can be found in the majority of “classic” Brucella species although it is absent from B. abortus (Rajashekara et al., 2004). Consistent with their functionality, the expression of several genes carried by GI-8 has been detected in B. melitensis (Rossetti et al., 2009). Moreover, an exopolysaccharide consisting of glucosamine, glucose and mostly mannose has been described in B. melitensis 16M (Godefroid et al., 2010). The annotation matches with genes expected for the biosynthesis of constituents previously mentioned, therefore the data strongly support a role of GI-8 in the production of such an exopolysaccharide. Since members of Brucella are facultative intracellular parasites that usually inhabit a constrained environment that precludes horizontal transfer with other bacteria, this ecological niche might explain the homogeneity of their O-PS structures or, in other terms, the restricted O-serotyping diversity found Similar to many LPS of Gram-ne (...truncated)