RskA Is a Dual Function Activator-Inhibitor That Controls SigK Activity Across Distinct Bacterial Genera

Abstract

It has been previously shown that RskA, the anti-Sigma factor K of Mycobacterium tuberculosis, inhibits SigK and that mutations in RskA promote high expression of the SigK regulon. The latter observation led us to hypothesize that RskA mutations lead to loss of the anti-Sigma factor function. In this report, we used natural and artificial mutations in RskA to determine the basis of the SigK-RskA partnership. Consistent with predictions, the N-terminal cytoplasmic portion of RskA was sufficient on its own to inhibit SigK. Unexpectedly, RskA also served as an activator of SigK. This activation depended on the same N-terminal region and was enhanced by the membraneextracellular portion of RskA. Based on this, we engineered similar truncations in a Gram-negative bacterium, namely Yersinia enterocolitica. Again, we observed that, with specific alterations of RskA, we were able to enhance SigK activity. Together these results support an alternative mechanism of anti-Sigma factor function, that we could term modulator (activator-inhibitor) in both Actinobacteria and Gram-negative bacteria, suggesting that Sigma factor activation by anti-Sigma factors could be under-recognized.

In VivoAntigen Expression Regulates CD4 T Cell Differentiation and Vaccine Efficacy against Mycobacterium tuberculosis Infection

“…To investigate whether T cell differentiation and vaccine protection is directly linked to in vivo antigen transcription, we utilized a recently engineered H37Rv strain ( 36 ) (herein called H37Rv::mpt70 high ) with significantly increased in vitro expression of MPT70 due to insertion of sigK (Rv0445c) and rskA (Rv0444c) from Mycobacterium orygis ( 6 , 36 ). In line with the previous report ( 36 ), we observed that this strain upregulated in vitro expression of MPT70 compared to wild-type (WT) H37Rv, while very little change was observed for the regulators of MPT70 transcription (SigK and RskA) ( Fig. 4a ).…”

“…Having established associations between antigen expression and T cell quality, we investigated whether there was a causal relationship. For this, we utilized a newly described H37Rv strain, which has high in vitro expression of MPT70 due to a gene insert of the regulators sigK (Rv0445c) and rskA (Rv0444c) from M. orygis ( 36 ). Importantly, we observed that infection with this strain significantly increased the differentiation state of the MPT70-specific CD4 T cells and diminished their ability to enter the infected lung tissue.…”

In vivoantigen expression regulates CD4 T cell differentiation and vaccine efficacy againstMycobacterium tuberculosisinfection

“…To investigate whether T cell differentiation and vaccine protection is directly linked to in vivo antigen transcription, we utilised a recently engineered H37Rv strain (36) (herein called H37Rv::mpt70 high ) with significantly increased in vitro expression of MPT70 due to insertion of sigK (Rv0445c) and rskA (Rv0444c) from M. orygis (6, 36). In line with the previous report (36), we observed that this strain upregulated in vitro expression of MPT70 compared to wild type (WT) H37Rv, while very little changes were observed for the regulators of MPT70 transcription (SigK and RskA) (Figure 4a) . From this, we anticipated that the H37Rv::mpt70 high strain would have an increased early in vivo expression of MPT70.…”

“…Having established associations between antigen expression and T cell quality, we investigated whether there was a causal relationship. For this, we utilised a newly described H37Rv strain, which has high in vitro expression of MPT70 due to a gene insert of the regulators sigK (Rv0445c) and rskA (Rv0444c) from M. orygis (36). Infection with this strain significantly increased the differentiation state of the MPT70-specific CD4 T cells and diminished their ability to enter the infected lung tissue.…”

“…The following strains; Mtb Pasteur H37Rv, Pasteur H37Rv::mpt70 high ( rskA and sigK of M. orygis ) (36), and Pasteur H37Rv::Rv ( rskA and sigK of M. tuberculosis ) (36) were grown in Middlebrook 7H9 medium (Difco Laboratories) supplemented with 0.05% Tween 80 (Sigma-Aldrich), 0.2% glycerol, 10% ADC Enrichment (BD) in a rolling incubator at 37°C. The bacterial cultures were passaged twice and adjusted to an OD 600 of 0.5, pelleted and resuspended in glycerol, and subsequently frozen at − 80°C.…”