Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments

Abstract

SUMMARY Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis (Mtb) in macrophages. However, mechanisms by which Mtb adapts to acidic environments are poorly understood. In this study, we analyzed the physiological functions of OmpATb, a surface-accessible protein of Mtb. OmpATb did not complement the permeability defects of an M. smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of Mtb indicating that OmpATb is not a general porin. Chemical analysis of low pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of Mtb. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which Mtb neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that Mtb has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of Mtb to acidic environments in vitro but not in mice.

Differential Detergent Extraction of Mycobacterium marinum Cell Envelope Proteins Identifies an Extensively Modified Threonine-Rich Outer Membrane Protein with Channel Activity

“…MctB is a putative porin (14) implicated to be involved in copper efflux (13), whereas OmpATb shows homology with the periplasmic domain of E. coli OmpA and has been the subject of a continuous debate about its function, localization, and structure. Originally, OmpATb has been described as a surface-accessible porin with channel activity (15), but recently these results have been questioned (16).…”

Substrate Specificity of MarP, a Periplasmic Protease Required for Resistance to Acid and Oxidative Stress in Mycobacterium tuberculosis

“…Mechanisms of pH homeostasis have been well characterized in several Gram-negative and Gram-positive bacteria (14,15), but those active in Mtb remain elusive. A small number of proteins has been implicated in the mycobacterial response to low pH including: MgtC, a predicted magnesium transporter (16); OmpA TB , an outer membrane protein (17); PhoPR, a two component regulator (18); aprABC, a gene locus regulated by PhoPR in response to acid (19); and Rv3671c, a transmembrane serine protease (3,20). A mutant with a transposon insertion in Rv3671c failed to maintain its neutral intrabacterial pH in acidic conditions both in vitro and in IFN-␥ activated macrophages and lost viability (3).…”

Uptake of Sulfate but Not Phosphate by Mycobacterium tuberculosis Is Slower than That for Mycobacterium smegmatis

“…This finding has been rationalized by the highly negatively charged constriction zone of MspA, which is constituted by aspartates 90 and 91 and thought to impede the diffusion of anions more than uncharged solutes. In contrast, the uptake of glucose at a concentration of 20 M by M. tuberculosis is almost 100-fold slower than that by M. smegmatis (42) and is so slow that a permeability coefficient cannot be reliably calculated. In fact, phosphate is taken up by M. tuberculosis almost 10 times faster than glucose (0.3 versus 0.04 nmol · mg Ϫ1 · min Ϫ1 ) (Fig.…”

“…The beneficial effects of asparagine and glutamate for the growth of M. tuberculosis were reported long ago, and they have been incorporated as nitrogen sources in standard media for mycobacteria (24). The capacity of M. tuberculosis to release ammonia from asparagine and glutamate is crucial for rapid adaptation to acidic conditions in vitro (42) and may play a role in the survival of M. tuberculosis in vivo. These findings also indicate that M. tuberculosis has uptake systems for these amino acids which enable their utilization as nitrogen sources.…”