Mycobacterium tuberculosis PhoY Proteins Promote Persister Formation by Mediating Pst/SenX3-RegX3 Phosphate Sensing

Abstract

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 ΔphoY1 ΔphoY2 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 ΔphoY1 ΔphoY2 mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A ΔpstA1 mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the ΔphoY1 ΔphoY2 and ΔpstA1 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.

Adaptation of Mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

“…Multiple genes of diverse functional categories (like mma4 , Rv2224c, ponA2 , and pstC2A1 ) identified in our Tn-seq screen have also been implicated in maintaining intrinsic tolerance to a panel of stressors and antibiotics, including but not limited to acidic pH, lysozyme, SDS, isoniazid and rifampicin (31, 40, 41). A reasonable interpretation of this observation is that the restrictive conditions formed in biofilms may favor the growth of cells that are tolerant to both endogenous and exogenous stresses.…”

“…A corollary to this hypothesis is that monoculture biofilms of mutant cells that are intrinsically sensitive to antibiotics harbor fewer persisters than wild-type. To test this, we exploited the Δ pstC2A1 strain, which exhibits hypersensitivity to rifampicin (RIF) (40). Moreover, the mutant forms biofilms indistinguishable from wild-type, thereby allowing a direct assessment of the effect of intrinsic property of the cells without any compounding effect from an altered architecture, which is generally observed with other antibiotic sensitive mutants identified in this study (e.g.…”

“…3B-C). A mutation in pstA1 also produces fewer persisters in planktonic cells (40), suggesting that regardless of the growth condition, intrinsic drug sensitivity of the representative population is a key determinant of the persister frequency. Therefore, more drug tolerant cells in biofilms than in planktonic culture increase the frequency of persisters.…”

RbpA and σB association regulates polyphosphate levels to modulate mycobacterial isoniazid‐tolerance

“…These data suggest that some other mechanisms may also be involved in PBST‐induced INH tolerance. Besides polyP, several other proteins and molecules have been shown to be involved in stress responses and generating drug‐tolerant cells (Dahl et al ., ; Chionh et al ., ; Namugenyi et al ., ). The expression level of katG is known to be negatively correlated with INH‐tolerance in mycobacterial cells (Wakamoto et al ., ); and our study showed that the expression of katG was repressed during PBST treatment (Supporting Information Fig.…”

Phosphate transporter PstSCAB of Campylobacter jejuni is essential for lactate-dependent growth and colonization in chickens

“…Consistent with a need to balance poly-P production and metabolism for wild type fitness in C. jejuni others, have demonstrated that phoX and ppk1/ppk2 mutants also have survival defects in nutrient limiting condition (18). Increased poly-P was also observed in phosphate transport mutants of M. tuberculosis (32).…”