Mycobacterium Inorganic Phosphate Transport Related Proteins Phoy2 Biology Function And Pathogenic Mechanism Research

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tuberculosis), causes approximately2million deaths annually and remains a threat to global health. Approximately5to10%of M.tuberculosis-infected individual develops active TB at some stage in their life. The remaining,90to95%infected people remain asymptomatic, carrying so-called latent TB infection (LTBI), which is defined solely by the evidence of immunological sensitization to mycobacterial proteins (M tuberculosis purified protein derivative, PPD) in the absence of clinical signs and symptoms of active disease. The World Health Organization estimates that nearly one third of the world population is PPD+. This vast reservoir of LTBI-infected individuals is a constant source of disease caused by reactivation.The exact underlying mechanisms of LTBI and its transition to active TB remain elusive. LTBI rely on an equilibrium in which the host is able to control the infection but does not completely eradicate the bacteria. The host could form several in vivo environmental stresses include phagocytic vacuoles of macrophages, hypoxic and nutrient-limited environments within granulomas, as well as oxygen-rich alveolar airspaces to control the replication and spread of bacteria.However, some bacteria could adapt to such diverse conditions by altering its physiology in response to changes in the environment therefore survive in a phenotype called dormancy for a long period. Immunosuppressants such as HIV infection or antitumor necrosis factor (TNF) treatment for rheumatoid arthritis may lead to the reactivation of these bacteria. Studies of M tuberculosis response to several stress conditions including hypoxia, nutrient deprivation, nitric oxide treatment and growth in acidic media have been described and genes induced by certain stress conditions such as the DosR regulon and EHR genes in response to hypoxia have been identified.Phosphate is an essential nutrient for cell functions and life. Bacteria employ a sophisticated system, encoded by the Pho regulon, to manage inorganic phosphate (Pi) acquisition and metabolism. A key component of the Pho regulon is the ABC-type phosphate specific transporter (Pst) system consisting of PstSCAB. PstS is a periplasmic protein that binds Pi with high affinity, PstC and PstA are cytoplasmic membrane transporters for Pi translocation into the cytosol, and PstB is an ATPase that provides energy for the transporter. The Pho regulon is controlled by the PhoR/PhoB two-component regulatory system. Under Pi limitation, PhoR is autophosphorylated and transfers a phosphoryl group to PhoB, which in turn activates target genes including the Pho regulon. When Pi is in excess, the activation is interrupted by PhoR acting as a phosphatase on phosphor-PhoB. PhoU, a peripheral membrane protein, is also required for dephosphorylation of PhoB and is essential for repression of Pho regulon under Pi rich condition. The precise mechanism of PhoU action is unknown, but it may interact with PhoR/PhoB proteins and interfere with their functions.There are two PhoU homologues in M.tuberculosis, PhoYl (Rv3301c) and PhoY2(RvO821c) which share40%and44%homology to E. coli PhoU. Previous studies showed that disruption of phoY2but not phoY1in M. tuberculosis resulted in increased susceptibility to antibiotics and reduced persistence of the bacterium in mice. The persistence phenotype of phoY2mutant is similar to that of phoU mutant of E. coli, suggesting that PhoY2is the functional homolog of PhoU. However, the underlying molecular mechanisms for the observed phenotypes associated with phoY2mutation in mycobacteria remain unknown. In the first part of this study, we characterized a transposon inactivated phoY2mutant of Mycobacterium marinum (M marinum). We found that the disruption of phoY2resulted in elevated levels of intracellular poly-Pi and ATP. The expression of phoY2was induced by environmental stress conditions and the phoY2::Tn mutant exhibited increased sensitivity to SDS, antibiotics and excessive levels of Pi. We also investigated the phenotypes of the phoY2::Tn mutant under hypoxia-induced dormancy. Taken together, our results indicate that PhoY2is required for maintaining metabolic homeostasis and adaption to environmental stress conditions (Part1).The transition of latent to active TB is a complex process, which depends on the interaction between host and mycobacterium. In the second part of this work, we investigate the regulated role of microRNA (miRNA) in this process. miRNAs are endogenous,22-nucleotide RNAs that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. There are currently1,000human miRNAs sequences listed in the miRNA registry which may target about60%of all mammalian genes [http://www.mirbase.org/], indicating that these small molecules play fundamental and global functions in human biology, including development, differentiation, apoptosis, metabolism, viral infection, and cancer. MiRNAs also modulate the innate and adaptive immune responses to pathogens by affecting mammalian immune cell differentiation and the development of diseases of immunological origin.The clinical application of miRNAs as diagnostic or prognostic biomarkers has already been demonstrated in various types of cancers. However, compared to their well-known role in cancer, the role of miRNAs in susceptibility and resistance to infectious disease, especially those of bacterial origin, is still poorly understood. We compared the miRNA expression profiles of PBMCs from patients with active TB, subjects with LTBI, and healthy controls in order to test the hypothesis that candidate miRNAs regulate the transition from LTBI to active TB. We used a miRNA microarray chip containing,960probes to identify the differently expressed miRNAs, and performed realtime quantitative polymerase chain reaction (qPCR) for confirmation. The putative regulatory network of miRNAs that were differentially expressed in the samples from active TB and LTBI individuals was constructed based on predicted target genes and previously published genome-wide transcriptional profiles. Our study provides a greater understanding of the role of miRNAsmediated regulated networks in the transition from latent to active TB (Part...