Mycobacterium tuberculosis
PhoY Proteins Promote Persister Formation by Mediating Pst/SenX3-RegX3 Phosphate Sensing
Authors: Namugenyi Sarah B. , Aagesen Alisha M. , Elliott Sarah R. , Tischler Anna D. 
Published: July 2017

The
Mycobacterium tuberculosis
phosphate-specific transport (Pst) system controls gene expression in response to phosphate availability by inhibiting the activation of the SenX3-RegX3 two-component system under phosphate-rich conditions, but the mechanism of communication between these systems is unknown. In
Escherichia coli
, inhibition of the two-component system PhoR-PhoB under phosphate-rich conditions requires both the Pst system and PhoU, a putative adaptor protein.
E. coli
PhoU is also involved in the formation of persisters, a subpopulation of phenotypically antibiotic-tolerant bacteria.
M. tuberculosis
encodes two PhoU orthologs, PhoY1 and PhoY2. We generated
phoY
single- and double-deletion mutants and examined the expression of RegX3-regulated genes by quantitative reverse transcription-PCR (qRT-PCR). Gene expression was increased only in the Δ
phoY1
Δ
phoY2
double mutant and could be restored to the wild-type level by complementation with either
phoY1
or
phoY2
or by deletion of
regX3
. These data suggest that the PhoY proteins function redundantly to inhibit SenX3-RegX3 activation. We analyzed the frequencies of antibiotic-tolerant persister variants in the
phoY
mutants using several antibiotic combinations. Persister frequency was decreased at least 40-fold in the Δ
phoY1
Δ
phoY2
mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A Δ
pstA1
mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the Δ
phoY1
Δ
phoY2
and Δ
pstA1
mutants were more susceptible to treatment with rifampin but not isoniazid. Our data demonstrate that disrupting phosphate sensing mediated by the PhoY proteins and the Pst system enhances the susceptibility of
M. tuberculosis
to antibiotics both
in vitro
and during infection.

IMPORTANCE
Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure
Mycobacterium tuberculosis
infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the
phoY
genes or a Pst system component resulted in decreased persister formation
in vitro
. Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the
M. tuberculosis
phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment.