performed NMR-based metabolomics

performed NMR-based metabolomics. reservoir of the pathogen. Granuloma formation represents the tissue hallmark of the latent mycobacterium. This bunch of immune aggregates, previously thought to be beneficial for host by curtailing bacteria in a nutritionally starved, hypoxic environment, is now being reexamined as a niche for survival and dissemination during latency and reactivation (2). is a facultative aerobe, which can survive during hypoxia only if oxygen is gradually depleted (3, 4). However, sudden anaerobiosis is lethal for the bacilli (5), highlighting the importance of sensory mechanisms necessary for the adaptation to a hypoxic environment. down-regulates transcription of key complexes of the electron transport chain in response to hypoxia, and ATP levels are reported to be approximately 5 times lower as compared with the replicating counterparts (6). Despite being metabolically slow, ATP production, NAD+/NADH balance, and maintenance of energized membrane is crucial for the persistence of hypoxic mycobacteria (6). The extracellular cues during bouts of active disease, dormancy, and resuscitation probably contribute to adaptation during various stages of infection. DosS and DosT are redox and hypoxia sensor kinases, respectively, which are up-regulated upon hypoxia and nitric oxide exposure and in turn up-regulate 50 genes through response regulator DosR (7,C9). Eukaryotic-like serine/threonine protein kinases (STPKs) are regulatory elements associated with signal recognition and adaptive responses. encodes 11 STPKs, which are involved in a myriad of functions, such as cell division, virulence, transport of metabolites, and regulation of metabolic processes (10, 11). Protein kinase G (PknG), the sole soluble STPK in BCG infection, wherein it inhibits phagolysosomal fusion by an unknown mechanism (13). However, recent reports suggests a more definitive role of PknG in regulating bacterial metabolism (14). This is supported by its ubiquitous presence across pathogenic and non-pathogenic species. PknG-mediated phosphorylation of GarA relieves its interaction with -ketoglutarate decarboxylase and glutamate dehydrogenase (15). Recently, amino acid availability was shown to act as a stimulus for PknG-mediated GarA phosphorylation (14). Furthermore, upon Folic acid oxidative stress, PknG-mediated phosphorylation of L13 promotes its interaction with RenU, which in turn regulates the NADH levels (16). Disruption of PknG enhances the susceptibility of mycobacteria toward antibiotics (17), attenuates growth during stationary phase (18), and impairs the biofilm formation (16), suggesting a plausible role in latency. However, to date, the role of PknG in latency and maintenance of redox poise has not been elucidated. In Mouse monoclonal to EGFP Tag this study, we demonstrate the role of PknG in mycobacterial survival through models of latency like hypoxia, persisters, and nutrition starvation. Real-time redox measurements demonstrated an enhanced oxidative shift in the presence of antibiotics and hypoxia, resulting in the lower survival of the mutant. The absence of PknG not only limits utilization of host-derived carbon sources but also results in lower metabolic activity compared with the wild type. Our study demonstrates that by regulating the TCA cycle through GarA phosphorylation and maintenance of the precarious redox status of Folic acid bacilli, PknG augments the survival of mycobacteria during latency-like conditions. Moreover, we establish that PknG also plays a critical role in the formation of stable granulomas in guinea pigs, repositories of dormant mycobacteria. Results PknG subverts host-induced stresses survival of the mutant in mice is more attenuated during the early phase of infection (18), indicating a role in subverting host-inflicted stresses, such as reactive oxygen species Folic acid (ROS), reactive nitrogen intermediates, and low pH. Thioredoxin motifs, present at the N terminus of PknG, are known to protect mycobacteria against oxidative defense of host innate cells. To elucidate the role of PknG in combating host-imposed defenses, mycobacterial strains were exposed to various stresses was 100- and 10-fold lower as compared with and in the presence of ROS generators like cumene hydroperoxide (CHP) and menadione, respectively (Fig. 1, and mutant had reduced survival in the presence of 0.1% SDS (Fig. 1were spotted on OADC-7H11 containing 50 m menadione. cfu obtained in OADC-7H11 plates without menadione was normalized to 100%, and survival on menadione-containing plates was calculated with respect to 100%. strains to acidic stress, single cell suspensions of bacterial strains were inoculated in sodium citrate buffer (pH 4.5), and cfu were enumerated on days 0 and 7. axis (percent survival) represents survival of bacterial strains in the presence of SDS/absence of SDS 100. The data depict mean Folic acid S.D. (= 6). **, 0.005; ***, 0.0005. PknG plays an essential role in survival of mycobacteria during hypoxia The success of as a pathogen relies on its immense ability to persist inside hypoxic granulomas for decades by undergoing transcriptional and translational remodeling of redox-responsive genes. Expression of has been shown to be up-regulated during hypoxia in both (19) and (20)..