Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis , and Generation of Mutants with Deletions at the pknG, relA , and lsr2 Loci

Abstract

Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 ؋ 10 ؊7 to 2.9 ؋ 10 ؊7 . Three genes were selected for mutagenesis: pknG and relA, which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis, and lsr2, a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis (M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.Johne's disease (paratuberculosis) is a chronic wasting disease of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis. It causes significant economic loss to animal producers, especially in the dairy industry, due to an increase in forage consumption, decreased milk production, and early culling due to poor health of affected animals (6,24,31). This disease has been difficult to control because of the lack of sensitive specific diagnostic assays and the lack of an efficacious vaccine. Available diagnostic assays, such as M. avium subsp. paratuberculosis antigen enzyme-linked immunosorbent assays and the gamma interferon assay, vary in the capacity to detect infected animals in the early stages of the disease (29). This is a major problem since infected animals begin to shed M. avium subsp. paratuberculosis in their feces early in the course of the disease before clinical signs appear. Available vaccines have been shown to reduce the severity of pathology but not to stop the shedding of bacteria (20). Consequently, there is a continuing need to develop both a better diagnostic assay and a better vaccine that, at a minimum, stops shedding of bacteria during the productive life of dairy cattle.An important prerequisite before strategies can be developed to control this disease is a better understanding of the molecular mechanisms of M. avium subsp. paratuberculosis pathogenesis. To increase our knowledge of the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines, an efficient method for generating targeted gene knockouts is urgently needed. In contrast to the successful gene disruption in fast-growing mycobacteria, such as M. smegmatis (8,10,26,36), gene disruption in slow-gro...

Lsr2 is a nucleoid-associated protein that targets AT-rich sequences and virulence genes inMycobacterium tuberculosis

“…The reasons for the relatively low degree of correlation between previously reported expression data (18) and the genes targeted by Lsr2 identified in this study (Dataset S2) are currently unknown. A comparison of ChIP data with the expression microarray data in M. tb was complicated by the fact that Lsr2 is suggested to be essential in M. tb; attempts by independent groups to delete lsr2 were unsuccessful (18,23).…”

Development of a Bovine Ileal Cannulation Model To Study the Immune Response and Mechanisms of Pathogenesis of Paratuberculosis

“…paratuberculosis K10 (animals 136 and 139) and to M. avium subsp. paratuberculosis K10 (137) with a targeted disruption in relA, a global regulator gene (27). One control animal (132) was cannulated and housed in a BSL-2 facility.…”

Evaluation of a Mycobacterium avium subsp. paratuberculosisleuDMutant as a Vaccine Candidate against Challenge in a Caprine Model

“…Several attempts have been made to successfully produce mutant strains of M. avium subsp. paratuberculosis with vaccine potential by exploiting transposon mutagenesis and allelic exchange (17)(18)(19). The latter technology affords a method to select genes associated with virulence or function for gene disruption.…”