Lipopolysaccharide-Deficient Brucella Variants Arise Spontaneously during Infection

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.

Keywords: Brucella; O-polysaccharide; lipopolysaccharide; rough mutant.

Figure 1

Genomic variation among isolates of B. melitensis 16M in stock culture originally obtained from ATCC. A preliminary genetic screen of Brucella rough variants revealed large genomic rearrangement. (A) Schematic of the manBA locus, showing the full extent of the deletion as characterized by primer walking. Primers for amplification of manBA (TAF419 and TAF420) are indicated. Unaffected genes (dark blue), truncated genes (light blue) and completely deleted genes (red). (B) Representative figure demonstrating Pro-Q Emerald 300 glycoprotein staining of Brucella LPS separated on SDS-PAGE using manBA knockout strains developed for this work and the ability to complement the deletion with the pJET1445 expression construct (C); marker (lane M), B. melitensis 16M (lane A), B. melitensis ΔmanBA (lane B), B. melitensis 16MΔmanBA/pJET1445 (lane C), B. melitensis 16MΔmanBA/pBBR1MCS6-y (lane D), B. abortus S2308 (lane E), B. abortus ΔmanBA (lane F), B. abortus 16MΔmanBA/pJET1445 (lane G), B. abortus 16MΔmanBA/pBBR1MCS6-y (lane H), E. coli O55:B5 expressing smooth LPS (lane Ec), (rtn, BMEII0904), demonstrating naturally occurring deletion of manBA. The arrows indicate two digested fragments of the rtn locus (3471 and 684 bp, respectively) that are lost with the deletion of manBA. R1–R13 are different spontaneous rough isolates, representing two broad classes of mutants. (C) Southern blot of PstI/EcoRI-digested genomic DNA isolated from B. melitensis rough isolates probed with a randomly ³²P-labeled probe to phosphodiesterase.

Figure 2

Recovery of rough Brucella from the spleens of infected mice. Mice were infected with clonal populations of smooth or rough B. melitensis, or a mixture of smooth and rough B. melitensis. Bacterial colonization was evaluated at 1-week (A) and 8-weeks (B) post-infection. Mice infected with a clonal population of rough organisms cleared the infection as early as 1 week post-infection. In contrast, spontaneous rough mutants are recovered from mice infected with smooth strains (either clonal smooth strains or a mixture of smooth and rough strains) at both 1 and 8 weeks post-infection. The horizontal line above the x-axis represents the limit of detection. Differences between rough and smooth organisms are significant at p < 0.05.

Figure 3

Spontaneous loss of O-polysaccharide reactivity among Brucella organism growing in macrophages ex vivo. Macrophages were infected with smooth B. melitensis 16M and in vitro loss of Brucella LPS was monitored using primary mouse anti-Brucella over a 48-h time period. Presence of bacteria either expressing smooth LPS (yellow, colocalized signal) or rough LPS (red) at each time point was observed using an Olympus IX70 microscope. Results were quantified using ImageJ (http://rsbweb.nih.gov/ij/). Ten fields per time point were quantified using ImageJ, the average suggests up to of 60% of bacteria within macrophages may lose reactivity with anti-LPS antibody. Results with an asterisk (*) are significant with p < 0.05. The bar shown is 25 μm.

Figure 4

Clearance kinetics of smooth and rough Brucella from the murine peritoneal cavity and in vitro susceptibility to complement. Mice were infected with 10⁸ cfu of either smooth B. melitensis 16M or rough B. melitensis 16MΔmanBA. Bacterial recovery from the ascites over a 7-day period revealed a lower percentage of smooth organisms (A) than rough organisms (B) internalized. Systemic infection was determined by recovery of smooth and rough organisms from secondary lymphoid organs, spleen (C), and liver (D). Significant differences (p ≤ 0.05) are represented with asterisks (*). (E) Shows the differences in susceptibility/resistance to complement-mediated lysis of smooth (B. melitensis 16M) and rough organisms (B. melitensis 16M R1, B. abortus RB51, and E. coli JM101) using sera from several host species. ΔLog_cfu represents the difference in recovered colony forming units (cfu) following incubation in the presence or absence of naïve sera. Resistant organisms will exhibit very little difference in survival (values close to zero) while sensitive organisms will exhibit a difference in recovery. The inoculum reveals the cfu per milliliter used in these experiments. The error bars reveal SD of across five mice.