Key Role for the Alternative Sigma Factor, SigH, in the Intracellular Life of Mycobacterium avium subsp. paratuberculosis during Macrophage Stress

Abstract

b Mycobacterium avium subsp. paratuberculosis causes Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. avium subsp. paratuberculosis is known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding of M. avium subsp. paratuberculosis pathogenesis, we examined phagosome maturation associated with transcriptional responses of M. avium subsp. paratuberculosis during macrophage infection. Monitoring cellular markers, only live M. avium subsp. paratuberculosis bacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300 M. avium subsp. paratuberculosis genes were significantly and differentially regulated in both naive and IFN-␥-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important for M. avium subsp. paratuberculosis survival inside gamma interferon (IFN-␥)-activated bovine macrophages. Interestingly, an sigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to the sigH regulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in the sigH regulon with a putative consensus sequence for SigH binding that was recognized in a subset of these genes (n ‫؍‬ 30), suggesting direct regulation by SigH. Finally, mice infections showed a significant attenuation of the ⌬sigH mutant compared to its parental strain, suggesting a role for sigH in M. avium subsp. paratuberculosis virulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne's disease.

Virulence and Immunity Orchestrated by the Global Gene Regulator sigL in Mycobacterium avium subsp. paratuberculosis

“…M. avium subsp. paratuberculosis K10 and Mycobacterium smegmatis mc 2 155 strains were grown in Middlebrook 7H9 broth and on Middlebrook 7H10 plates as previously described (2,15). For cloning, Escherichia coli DH5␣ and HB101 were used as host cells (15,21).…”

“…Earlier reports indicated gene expression of alternative sigma factors encoded in the M. avium subsp. paratuberculosis genome following macrophage infection (14,15). Additionally, among the 19 encoded alternative sigma factors (16), only the sigL transcript was induced at an early stage (2 h) of macrophage infection, indicating its importance for M. avium subsp.…”

“…Interestingly, both M. avium subsp. paratuberculosis and Mycobacterium tuberculosis share several mechanisms, especially those involved in macrophage survival (15,18). In M. tuberculosis, only 11 alternative sigma factors are encoded (19), where sigL acts as a stress response regulator and is implicated in cell membrane protein biosynthesis as well as virulence (20).…”

Superior Protection from Live-Attenuated Vaccines Directed against Johne's Disease

“…The inactivated Mycopar vaccine was administered as a single dose, according to the manufacturer's instructions, by subcutaneous injection. The live-attenuated vaccine candidates, the pgsH and pgsN vaccine constructs, were created by homologous recombination, as previously described (16,17). The experimental vaccines were grown in 7H9 liquid medium supplemented with 0.5% glycerol, 2 g/ml mycobactin J (Allied Monitor, Fayette, MO), and 10% ADC (2% glucose, 5% bovine serum albumin fraction V, and 0.85% NaCl) (30).…”

“…Further analysis showed that a lipN deletion mutant (here referred to as pgsN) was attenuated in mice, as shown by reduced histopathological lesions and colonization of the liver (16). Subsequent studies with a sigH deletion mutant (here referred to as pgsH) showed attenuation in mice and induction of superior protective immune responses both with and without the saponin-based QuilA adjuvant (15,17). In the present study, we compared key parameters desired for a successful JD vaccine (robust immune responses, reduced fecal shedding, and reduced tissue damage) when novel LAV formulations were used in comparison to a commercially available vaccine, Mycopar, or no vaccine, in a goat challenge model of JD.…”