Identification of Novel Virulence Determinants inMycobacterium paratuberculosisby Screening a Library of Insertional Mutants

Abstract

Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G؉C content (50.5 to 60.5%) lower than the average G؉C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johne's and Crohn's diseases.Mycobacterium paratuberculosis causes Johne's disease (paratuberculosis), characterized by chronic granulomatous enteritis, in dairy cattle, with an estimated loss of $220 million per year in the United States alone (22). Worldwide, the prevalence of the disease can range from 3 to 4% of herds in regions with low incidence (such as England) (6) to high levels of 50% of herds in some areas within the United States (such as Wisconsin and Alabama) (8, 16). In humans, M. paratuberculosis bacilli have been found in examined tissues from Crohn's disease patients, suggesting a possible role for M. paratuberculosis in developing Crohn's disease (7). Unfortunately, the mechanisms of virulence that control M. paratuberculosis persistence during infection are poorly understood and the key steps for developing paratuberculosis remain elusive. For example, mechanisms responsible for invasion and persistence of M. paratuberculosis inside the intestine remain undefined at the molecular level (42). Both live and dead bacilli are observed in subepithelial macrophages after uptake, most likely in M cells covering the ileum (24). Once inside the macrophages, M. paratuberculosis survives and proliferates inside the phagosomes by using unknown mechanisms (41). In one report examining intestinal invasion, it was suggested that the 35-kDa membrane protein antigen could play a possible role in bacillus invasio...

Experimental challenge models for Johne's disease: A review and proposed international guidelines

“…Also, mice with variable genetic background are readily available, allowing investigation of host-pathogen interactions on a molecular level. In numerous studies (Shin et al, 2006;Stabel and Ackermann, 2002;Tanaka et al, 1994) histological and immunological features reproduced in mice were generally similar to those in ruminants (Table 6). Generally, the degree of bacterial colonization and the type of cells recruited to granulomatous lesions depend on the mouse genetic background as well as virulence of the mycobacterial strain (Mullerad et al, 2002;Shin et al, 2006;Tanaka et al, 1994), indicating the utility of the murine model in elucidation of pathogenetic mechanisms.…”

“…An attractive aspect of this relatively inexpensive murine JD model is the ease of screening a large number of attenuated mutants as vaccine candidates. In a high throughput format, analysis is focused on liver and intestine, of which both are examined for histological lesions and colonization over 12 weeks (Shin et al, 2006). Unlike other species, liver is the most affected organ following IP injection, while only hyperplastic gut-associated lymphoid tissue is seen in intestines.…”

Assessment of Live Candidate Vaccines for Paratuberculosis in Animal Models and Macrophages

“…Mice are cost effective, present some histological and immunological features similar to those of ruminants, and can serve as a preliminary screening model for vaccine candidates (39,41), but they do not develop disease typical of that seen with ruminants with paratuberculosis (9,41). In comparison, goats are much more costly but provide a direct homologue of the disease in cattle and other ruminants, which is beneficial for pathogenesis and vaccine efficacy studies (21,32).…”

The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs

“…In addition, an M. avium subsp. paratuberculosis gcpE mutant is less able to colonize tissue during infection of mice or calves (35,36), confirming that this pathway is required for virulence. However, in both cases, it is yet to be determined whether this is a direct link between isoprenoid biosynthesis and the phenotypic consequences, since no measure of IPP synthesis was made.…”

“…Surprisingly, inactivation of gcpE by transposon mutagenesis has been achieved in M. avium subsp. paratuberculosis (35), raising the possibility that there are other pathways for isoprenoid synthesis in this species but not M. tuberculosis.…”