Tuning the Mycobacterium tuberculosis Alternative Sigma Factor SigF through the Multidomain Regulator Rv1364c and Osmosensory Kinase Protein Kinase D

Abstract

Bacterial alternative sigma factors are mostly regulated by a partnerswitching mechanism. Regulation of the virulence-associated alternative sigma factor SigF of Mycobacterium tuberculosis has been an area of intrigue, with SigF having more predicted regulators than other sigma factors in this organism. Rv1364c is one such predicted regulator, the mechanism of which is confounded by the presence of both anti-sigma factor and anti-sigma factor antagonist functions in a single polypeptide. Using protein binding and phosphorylation assays, we demonstrate that the anti-sigma factor domain of Rv1364c mediates autophosphorylation of its antagonist domain and binds efficiently to SigF. Furthermore, we identified a direct role for the osmosensor serine/threonine kinase PknD in regulating the SigF-Rv1364c interaction, adding to the current understanding about the intersection of these discrete signaling networks. Phosphorylation of SigF also showed functional implications in its DNA binding ability, which may help in activation of the regulon. In M. tuberculosis, osmotic stress-dependent induction of espA, a SigF target involved in maintaining cell wall integrity, is curtailed upon overexpression of Rv1364c, showing its role as an anti-SigF factor. Overexpression of Rv1364c led to induction of another target, pks6, involved in lipid metabolism. This induction was, however, curtailed in the presence of osmotic stress conditions, suggesting modulation of SigF target gene expression via Rv1364c. These data provide evidence that Rv1364c acts an independent SigF regulator that is sensitive to the osmosensory signal, mediating the cross talk of PknD with the SigF regulon. IMPORTANCE Mycobacterium tuberculosis, capable of latently infecting the host and causing aggressive tissue damage during active tuberculosis, is endowed with a complex regulatory capacity built of several sigma factors, protein kinases, and phosphatases. These proteins regulate expression of genes that allow the bacteria to adapt to various host-derived stresses, like nutrient starvation, acidic pH, and hypoxia. The cross talk between these systems is not well understood. SigF is one such regulator of gene expression that helps M. tuberculosis to adapt to stresses and imparts virulence. This work provides evidence for its inhibition by the multidomain regulator Rv1364c and activation by the kinase PknD. The coexistence of negative and positive regulators of SigF in pathogenic bacteria reveals an underlying requirement for tight control of virulence factor expression.

The Partner Switching System of the SigF Sigma Factor in Mycobacterium smegmatis and Induction of the SigF Regulon Under Respiration-Inhibitory Conditions

“…However, no study has been published regarding which gene product is responsible for dephosphorylation of phosphorylated RsfB in mycobacteria. The Rv1364c gene in M. tuberculosis was found to encode a multi-domain protein consisting of the sensor, PP2C phosphatase, GHKL (gyrase, Hsp90, histidine kinase, MutL) kinase, and anti-sigma antagonist domains ( Sachdeva et al, 2008 ; Greenstein et al, 2009 ; Misra et al, 2019 ). Rv1364c was shown to interact with SigF in vitro , suggesting the possibility that it might serve as an anti-SigF along with the major anti-SigF UsfX ( Misra et al, 2019 ).…”

“…The Rv1364c gene in M. tuberculosis was found to encode a multi-domain protein consisting of the sensor, PP2C phosphatase, GHKL (gyrase, Hsp90, histidine kinase, MutL) kinase, and anti-sigma antagonist domains ( Sachdeva et al, 2008 ; Greenstein et al, 2009 ; Misra et al, 2019 ). Rv1364c was shown to interact with SigF in vitro , suggesting the possibility that it might serve as an anti-SigF along with the major anti-SigF UsfX ( Misra et al, 2019 ). Although Rv1364c was demonstrated to possess both kinase and phosphatase activities that autophosphorylate and autodephosphorylate its anti-sigma antagonist domain ( Greenstein et al, 2009 ), the question remains unanswered regarding whether it can phosphorylate and dephosphorylate RsfB to modulate the anti-SigF antagonist activity of RsfB.…”

Stringent Response in Mycobacteria: From Biology to Therapeutic Potential

“…M. tuberculosis faces several stresses, such as oxidative, nitrosative, and nutrient stress upon infecting the host. However, it overcomes these potentially fatal stresses and successfully establishes chronic infection [38,60,61]. The adaptation to such stresses requires a large-scale transcriptional reprogramming, which eventually lets M. tuberculosis not only infect the macrophages but also survive for years in granuloma [38,61,62].…”

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“…However, no study has been published regarding which gene product is responsible for dephosphorylation of phosphorylated RsfB in mycobacteria. The Rv1364c gene in M. tuberculosis was found to encode a multidomain protein consisting of the sensor, PP2C phosphatase, GHKL (gyrase, Hsp90, histidine kinase, MutL) kinase, and antisigma antagonist domains (Sachdeva et al, 2008;Greenstein et al, 2009;Misra et al, 2019). Rv1364c was shown to interact with SigF in vitro, suggesting the possibility that it might serve as an anti-SigF along with the major anti-SigF UsfX (Misra et al, 2019).…”

“…The Rv1364c gene in M. tuberculosis was found to encode a multidomain protein consisting of the sensor, PP2C phosphatase, GHKL (gyrase, Hsp90, histidine kinase, MutL) kinase, and antisigma antagonist domains (Sachdeva et al, 2008;Greenstein et al, 2009;Misra et al, 2019). Rv1364c was shown to interact with SigF in vitro, suggesting the possibility that it might serve as an anti-SigF along with the major anti-SigF UsfX (Misra et al, 2019). Although Rv1364c was demonstrated to possess both kinase and phosphatase activities that autophosphorylate and autodephosphorylate its anti-sigma antagonist domain (Greenstein et al, 2009), the question remains unanswered regarding whether it can phosphorylate and dephosphorylate RsfB to modulate the anti-SigF antagonist activity of RsfB.…”