sodA is essential for virulence of Borrelia burgdorferi in the murine model of Lyme disease

Abstract

SummaryBorrelia burgdorferi, the causative agent of Lyme disease, has a limited set of genes to combat oxidative/nitrosative stress encountered in its tick vector or mammalian hosts. We inactivated the gene encoding for superoxide dismutase A (sodA, bb0153), an enzyme mediating the dismutation of superoxide anions and examined the in vitro and in vivo phenotype of the mutant. There were no significant differences in the in vitro growth characteristics of the sodA mutant compared with the control strains. Microscopic analysis of viability of spirochaetes revealed greater percentage of cell death upon treatment of sodA mutant with superoxide generators compared with its controls. Infectivity analysis in C3H/HeN mice following intradermal needle inoculation of 10 3 or 10 5 spirochaetes per mouse revealed complete attenuation of infectivity for the sodA mutant compared with control strains at 21 days post infection. The sodA mutant was more susceptible to the effects of activated macrophages and neutrophils, suggesting that its in vivo phenotype is partly due to the killing effects of activated immune cells. These studies indicate that SodA plays an important role in combating oxidative stress and is essential for the colonization and dissemination of B. burgdorferi in the murine model of Lyme disease.

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

“…These observations indicated that the altered morphology is essentially due to overexpression of CsrA Bb under the control of the P flgB promoter. Moreover, we have not observed such persistent changes in morphology when other borrelial determinants such as sodA or a variety of antibiotic resistance markers have been expressed under the control of P flgB (27,52,74,75). Overexpression of CsrA Bb results in reduced expression of FlaB.…”

“…Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) and Rosetta(DE3)pLysS (Novagen, Madison, MI) strains were used for all procedures involving cloning and overexpression of recombinant proteins, respectively. The E. coli strains were cultured in Luria-Bertani (LB) broth supplemented with appropriate concentrations of antibiotics (27).…”

“…Recombinant CsrA (rCsrA Bb ) with a C-terminal six-histidine tag was overexpressed by inducing E. coli strains containing pES16 (pET23a-csrA Bb ) with 1 mM IPTG for 2 h (27). The bacterial pellets were disrupted using a French press in denaturing lysis buffer (8 M urea, pH 8), and the supernatants were collected, clarified by centrifugation, and subjected to affinity purification using nickel-nitrilotriacetic acid beads (Qiagen, Valencia, CA) per the manufacturer's instructions.…”

“…The control strain (ML23/pBSV2) and overexpression strain (ES25) were grown to a density of 5 ϫ 10 7 spirochetes/ml and washed three times in Hanks balanced salt solution-5 mM CaCl 2-50 mM sucrose and evaluated using the Live/Dead BacLight bacterial viability kit (Molecular Probes, Invitrogen, Carlsbad, CA) in conjunction with confocal microscopy (27).…”

“…Differential gene expression plays a critical role in the transmission, colonization, and dissemination of B. burgdorferi in mammalian hosts. Several of these open reading frames (ORFs) include mediators that provide a selective advantage to B. burgdorferi by enhancing its ability to adhere to host matrices (24,39,64) and to evade the mediators of innate and adaptive immunity (16,26,48,89), as well as other deleterious physiological processes encountered in its hosts (27). A number of studies have determined the significance of alterations of surface lipoproteins in response to these signals, and the molecular mechanisms responsible for these changes are beginning to be understood (41,86).…”