Mycobacterium tuberculosis PknK Substrate Profiling Reveals Essential Transcription Terminator Protein Rho and Two-Component Response Regulators PrrA and MtrA as Novel Targets for Phosphorylation.

The Mycobacterium tuberculosis protein kinase K regulates growth adaptation by facilitating mycobacterial survival in response to a variety of in vitro and in vivo stress conditions. Here, we further add that pknK transcription is responsive to carbon and nitrogen starvation signals. The increased survival of an M. tuberculosis ΔpknK mutant strain under carbon- and nitrogen-limiting growth conditions compared to the wild-type (WT) H37Rv suggests an integral role of PknK in regulating growth during metabolic stress. To identify the downstream targets of PknK-mediated signaling, we compared phosphoproteomic and transcription profiles of mycobacterial strains overexpressing WT and phosphorylation-defective PknK. Results implicate PknK as a signaling protein that can regulate several enzymes involved in central metabolism, transcription regulation, and signal transduction. A key finding of this study was the identification of two essential two-component response regulator (RR) proteins, PrrA and MtrA, and Rho transcription terminator, as unique targets for PknK. We confirm that PknK interacts with and phosphorylates PrrA, MtrA, and Rho in vivo. PknK-mediated phosphorylation of MtrA appears to increase binding of the RR to the cognate probe DNA. However, dual phosphorylation of MtrA and PrrA response regulators by PknK and their respective cognate sensor kinases in vitro showed nominal additive effect on the mobility of the protein-DNA complex, suggesting the presence of a potential fine-tuning of the signal transduction pathway which might respond to multiple cues. IMPORTANCE Networks of gene regulation and signaling cascades are fundamental to the pathogenesis of Mycobacterium tuberculosis in adapting to the continuously changing intracellular environment in the host. M. tuberculosis protein kinase K is a transcription regulator that responds to diverse environmental signals and facilitates stress-induced growth adaptation in culture and during infection. This study identifies multiple signaling interactions of PknK and provides evidence that PknK can change the transcriptional landscape during growth transitions by connecting distinctly different signal transduction and regulatory pathways essential for mycobacterial survival.

Pyogenic liver abscess: Clinical features and microbiological profiles in tertiary care center.

BACKGROUND: Pyogenic liver abscess (PLA) is the end result of a number of pathologic processes that cause a suppurative infection of the liver parenchyma. MATERIALS AND METHODS: Sixty-five patients of age more than 18 years and radiologically confirmed cases of liver abscess were included in this study. Pus and blood samples were collected. Pus was processed for microscopy of trophozoite of Entamoeba histolytica and aerobic and anaerobic bacterial culture. Blood was processed for antibody ELISA for Entamoeba histolytica and aerobic bacterial culture. Identification of aerobic and anaerobic isolates was done by Vitek2 and antibiotic sensitivity test for aerobic bacterial isolates was done by Vitek2. RESULT: Out of sixty five, twenty five were confirmed as PLA. All patients were male with mean age 37.9 years. Fever and upper abdominal pain were the most common symptoms. Right lobe comprised 80% of the abscess. Pus sample was more sensitive than blood sample for diagnosis. There were a total of 33 isolates in our study. Klebsiella pneumoniae (6/33) was the most common aerobic isolate and Clostridium spp. (7/33) was the anaerobic isolate. All gram-negative bacteria were showing good sensitivity for 3rd and 4th generation cephalosporins, fluoroquinolones, amikacin, gentamicin, piperacillin-tazobactam, imipenem and meropenem. Abscess >5 cm was treated with percutaneous drainage while abscess <5 cm was treated with antibiotics only. CONCLUSION: Diagnosis should be made with the combination of clinical suspicion, radiology, and microbiology. Empirical therapy should include anaerobic coverage too. Only antibiotic therapy can be given under consideration of size of abscess, persistence of fever after giving antibiotics, and any suspected complications.

MtrA Response Regulator Controls Cell Division and Cell Wall Metabolism and Affects Susceptibility of Mycobacteria to the First Line Antituberculosis Drugs.

The biological processes regulated by the essential response regulator MtrA and the growth conditions promoting its activation in Mycobacterium tuberculosis, a slow grower and pathogen, are largely unknown. Here, using a gain-of-function mutant, MtrAY 102C, which functions in the absence of the cognate MtrB sensor kinase, we show that the MtrA regulon includes several genes involved in the processes of cell division and cell wall metabolism. The expression of selected MtrA targets and intracellular MtrA levels were compromised under replication arrest induced by genetic manipulation and under stress conditions caused by toxic radicals. The loss of the mtrA gene in M. smegmatis, a rapid grower and non-pathogen, produced filamentous cells with branches and bulges, indicating defects in cell division and cell shape. The ΔmtrA mutant was sensitized to rifampicin and vancomycin and became more resistant to isoniazid, the first line antituberculosis drug. Our data are consistent with the proposal that MtrA controls the optimal cell division, cell wall integrity, and susceptibility to some antimycobacterial drugs.

Mycobacterium tuberculosis MtrAY102C is a gain-of-function mutant that potentially acts as a constitutively active protein.

The MtrAB histidine-aspartate signal transduction of mycobacteria includes the response regulator MtrA and sensor kinase MtrB. We recently showed that Mycobacterium smegmatis ΔmtrB is filamentous, defective for cell division, cell shape maintenance and shows compromised MtrA target gene expression. Interestingly, overproduction of phosphorylation competent M. tuberculosis MtrAY102C reverses the ΔmtrB mutant phenotype, although the genetic basis of phenotype reversal is unknown. Here we show that introduction of D56N mutation in MtrAY102C completely abolished its phosphorylation potential yet the double mutant protein retained a partial ability to reverse the mtrB mutant phenotype indicating that phosphorylation activity is not necessary for the function of MtrAY102C. The phosphorylation-defective MtrAD56N-Y102C protein bound its target promoters ripA and fbpB efficiently. Together, these results support a hypothesis that the gain-of-function phenotype of MtrAY102C is in part due to its ability to function as a constitutively active protein in the absence of phosphorylation.

Suppression of TLR2-induced IL-12, reactive oxygen species, and inducible nitric oxide synthase expression by Mycobacterium tuberculosis antigens expressed inside macrophages during the course of infection.

We report the enrichment of and immune responses mediated by genes expressed by Mycobacterium tuberculosis inside macrophages as a function of time. Results indicate that M. tuberculosis expresses different genes at different times postinfection. Genes expressed early (day 1) following infection enhance M. tuberculosis-mediated activation of dendritic cells (DCs), whereas genes expressed later (day 5) in the infection prevent DC activation. However, all genes downmodulated MHC class I and II expression on infected macrophages, thus compromising their ability to interact with Ag-specific T cells. Day-1 and -5 genes downmodulated proinflammatory cytokine production from DCs, thus impairing signal 3 during DC-T cell cognate interactions. Consequently, T cells activated by Ag-experienced DCs secreted low levels of IFN-gamma and IL-17 but maintained high IL-10 secretion, thus inducing suppressor responses. Further characterization revealed that day-1 and -5 genes increased TLR2-induced expression of suppressors of cytokine signaling 1 from DCs and downmodulated IL-12 expression. In addition, day-1 and -5 genes prevented the generation of reactive oxygen species in DCs. In contrast, although day-5 genes increased TLR2-mediated suppressors of cytokine signaling 1 expression in macrophages, day-1 genes downmodulated the expression of inducible NO synthase 2. Similar downregulation of immune responses was observed upon exogenous stimulation with day-1 or -5 Ags. Finally, day-1 and -5 genes promoted enhanced survival of M. tuberculosis inside DCs and macrophages. These results indicate that M. tuberculosis genes, expressed inside infected macrophages as a function of time, collectively suppress protective immune responses by using multiple and complementary mechanisms.