An exacerbated phosphate starvation response triggers <i>Mycobacterium tuberculosis</i> glycerol utilization at acidic pH

The mechanisms controlling <i>Mycobacterium tuberculosis</i> (Mtb) replication and survival inside its human host remain ill-defined. Phagosome acidification and nutrient deprivation are common mechanisms used by macrophages to restrict the replication of intracellular bacteria. Mtb stops replicating at mildly acidic pH (<pH5.8), an adaptive process called "acid growth arrest," which is thought to play an important role in Mtb virulence. Using a genome-wide mutagenesis screen, we identified 95 genes whose disruption either decreases or increases Mtb fitness during acid growth arrest. We show that the virulence-associated inorganic phosphate (Pi) uptake system (Pst-1) regulates the ability of Mtb to replicate in acidic conditions. Deletion of <i>pstA1</i>, a gene coding for a subunit of the Pst-1 system, results in the overexpression of the Pi starvation regulator <i>regX3,</i> which is sufficient to restore Mtb growth in acid conditions. Our data further support the role of limited glycerol uptake and ROS-mediated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition in causing Mtb acid growth arrest. This study reveals an unexpected role of the Pi starvation response in regulating acid growth arrest and highlights the intricacy of the mechanisms regulating redox homeostasis, acid stress response, and nutrient utilization in Mtb.IMPORTANCEDespite the availability of antibiotic treatment, <i>M. tuberculosis</i> (Mtb), the causative agent of tuberculosis (TB), remains a major infectious disease killer worldwide. A better understanding of the environments that Mtb faces during infection and the mechanisms Mtb employs to respond and adapt may help identify currently unexplored pathways and targets for the development of novel anti-TB drugs. Here, we demonstrate that Mtb growth in acid can be restored by the over-expression of the Pi starvation response regulator <i>regX3</i>. This work paves the way toward a better understanding of the mechanisms controlling Mtb growth at acidic pH and highlights the role of inorganic phosphate in this process.