Abstract
Lipopolysaccharide-deficient mutants of smooth Brucella species (rough mutants) have been shown to arise spontaneously in culture. However, in situ analysis of Brucella infected macrophages using antibody directed against O-polysaccharide suggested a loss of reactivity of Brucella consistent with the appearance of rough organisms, and a potential contribution to infection. The experiments reported describe the direct recovery of Brucella from macrophages infected in vitro and from the spleens of infected mice at a frequency similar to that described in vitro, suggesting that Brucella dissociation is not simply an in vitro artifact. The frequency of appearance of spontaneous rough organisms deficient in O-polysaccharide expression measured in vitro is approximately 2–3 logs higher than the appearance of mutation to antibiotic resistance, purine auxotrophy, or reversion of erythritol sensitive ΔeryC mutants to tolerance. Genetic trans-complementation using a plasmid-based expression of Brucella manBA successfully restored O-polysaccharide expression in only one-third of O-polysaccharide deficient spontaneous mutants. Suggesting that the appearance of rough mutants is caused by mutation at more than one locus. In addition, Sanger sequencing of the manBA structural genes detected multiple sequence changes that may explain the observed phenotypic differences. The presence of O-polysaccharide resulted in macrophage and neutrophil infiltration into the peritoneal cavity and systemic distribution of the organism. In contrast, rough organisms are controlled by resident macrophages or by extracellular killing mechanisms and rapidly cleared from this compartment consistent with the inability to cause disease. Loss of O-polysaccharide expression appears to be stochastic giving rise to organisms with biological properties distinct from the parental smooth organism during the course of infection.
Highlights
Brucellosis continues to be a major worldwide zoonosis, despite eradication programs
Determination of the spontaneous mutation rate in B. abortus S2308 and B. melitensis 16M Conversion of wild-type Brucella to pyrimidine auxotrophy provided an estimate of background genetic mutation via selection of spontaneous mutants on 5-fluoroorotic acid (FOA)
Excluding jackpot cultures (>3000 colonies/plate), 261 mutant colonies were collected from B. abortus and 930 mutant colonies were collected from B. melitensis
Summary
Brucellosis continues to be a major worldwide zoonosis, despite eradication programs. Members of the genus Brucellae are small, non-motile, Gram-negative, non-encapsulated, intracellular zoonotic pathogens. The genus is divided into 10 species, including the 6 classical species Brucella abortus, B. suis, B. melitensis, B. neotomae, B. canis, and B. ovis and newly recognized species B. ceti, B. microti, B. pinnipedialis and B. inopinata. On the basis of LPS expression, the classical species can be broken into two broad categories: smooth (S-) or rough (R-). We are focused on the transition of B. abortus or B. melitensis from smooth LPS expression to rough LPS expression. Events leading to the transition of smooth LPS expression to rough LPS expression are poorly characterized and just beginning to be explored (Mancilla et al, 2010)
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