Rv0500A is a transcription factor that links Mycobacterium tuberculosis environmental response with division and impacts host colonization

Abstract

For Mycobacterium tuberculosis (Mtb) to successfully infect a host, it must be able to adapt to changes in its microenvironment, including variations in ionic signals such as pH and chloride (Cl − ), and link these responses to its growth. Transcriptional changes are a key mechanism for Mtb environmental adaptation, and we identify here Rv0500A as a novel transcriptional regulator that links Mtb environmental response and division processes. Global transcriptional profiling revealed that Rv0500A acts as a repressor and influences the expression of genes related to division, with the magnitude of its effect modulated by pH and Cl − . Rv0500A can directly bind the promoters of several of these target genes, and we identify key residues required for its DNA-binding ability and biological effect. Overexpression of rv0500A disrupted Mtb growth morphology, resulting in filamentation that was exacerbated by high environmental Cl − levels and acidic pH. Finally, we show that perturbation of rv0500A leads to attenuation of the ability of Mtb to colonize its host in vivo. Our work highlights the important link between Mtb environmental response and growth characteristics, and uncovers a new transcription factor involved in this critical facet of Mtb biology.

Spatial relationships of intra-lesion heterogeneity in Mycobacterium tuberculosis microenvironment, replication status, and drug efficacy

“…As an initial test of the system, we infected C3HeB/FeJ mice with the Erdman (P 606 ’::mKO-tetON, smyc’ ::mCherry) reporter Mtb strain for one week, before the provision of drinking water containing 5% sucrose ± 1 mg/ml doxycycline (dox) for one additional week. As shown in Fig 4A–4C , dox treatment in this short-term infection resulted in the expected induction of mKO fluorescence in Mtb, with Mtb in the mock-treated mice displaying no mKO fluorescence, in accord with results observed with this reporter in C57BL/6J mice [ 35 ], and reinforcing the suitability of dox induction for in vivo Mtb studies [ 35 , 36 ].…”

“…A dual fluorescent system where one fluorophore is placed under the control of an inducible promoter and a second spectrally distinct fluorophore is expressed constitutively has been utilized in differentiating transcriptionally active versus non-active Mtb in cultured macrophages [ 32 – 34 ]. We thus applied this strategy here in vivo , exploiting a reporter Mtb strain that carries on the chromosome a tetracycline inducible monomeric Kusabira Orange (mKO) construct, along with a constitutively expressed mCherry (P 606 ’::mKO-tetON, smyc’ ::mCherry) [ 35 ]. As an initial test of the system, we infected C3HeB/FeJ mice with the Erdman (P 606 ’::mKO-tetON, smyc’ ::mCherry) reporter Mtb strain for one week, before the provision of drinking water containing 5% sucrose ± 1 mg/ml doxycycline (dox) for one additional week.…”

PrrA modulates Mycobacterium tuberculosis response to multiple environmental cues and is critically regulated by serine/threonine protein kinases

“…Mtb samples were collected after four hours post-exposure to the indicated environmental condition, and RNA isolation was performed as previously described [1]. For RNAseq, two biological replicates per condition were used, and library preparation and sequencing carried out as previously described [43, 67]. RNAseq data was analyzed using the SPARTA program [43, 67, 68], and qRT-PCR was performed as previously described [43, 67].…”

“…For RNAseq, two biological replicates per condition were used, and library preparation and sequencing carried out as previously described [43, 67]. RNAseq data was analyzed using the SPARTA program [43, 67, 68], and qRT-PCR was performed as previously described [43, 67].…”

“…C3HeB/FeJ mice (The Jackson Laboratory) were intranasally infected with 10 3 CFUs of Mtb (35 µl volume) while under light anesthesia with 2% isoflurane [3, 8, 71]. For infections to test how prrA knockdown affected host colonization, mice were given water supplemented with 5% sucrose ± 1 mg/ml doxycycline beginning one week after infection for one additional week [67, 72]. Water was replaced twice during the week of treatment.…”