Characterization Of PhoY2, A Transport And Potential Regulating Protein In Mycobacterium Marinum

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), remains a threat to global health. According to WHO’s (World Health Organization) report in 2013, there are about 8.6 million new cases around the world in 2012, which causes more than 1.3 million death. Among those dead cases,320 thousand also developed acquired immune deficiency syndrome (AIDS). China is one the twenty-two high burden countries of TB where 950 thousand new cases developed during 2012. It is now still quite challenging for TB control with the emergence of drug resistance, especially multi-drug resistance and extensively drug resistance, and co-infection with HIV (human immunodeficiency virus).Most of the bacteria are killed by the treatment of antibiotics after infection, but there are small amount which can survive the drug treatment and the killing of immune system. Those bacteria become persistent cells, which were discovered by Joseph Bigger in 1944. The persistent cells are genetically the same with those who are susceptible to antibiotics, but they can avoid the damage from antibiotics by entering the non-replicating dormant state. It is know that the emergence of persisters may be related to the stomachic expression of TA (Toxin-Antitoxin) system. In the field of mycobacteria research, it has been reported that phoY2 is essential for the M. tuberculosis persistence. And this is in accordance with the research in phoU, which is the homologue of phoY2 in E. coli. But based on the fact that there are few TA systems in Mycobacterium marinum (M. marinum), the mechanism of persistence formation in mycobacteria, especially in M. marinum is still unknown.Poly phosphate (Poly Pi) exists in bacteria, archaea, fungi, protozoa, plants and animals. It is directly synthesized by polyphosphate kinase (PPK) and degraded by exopolyphosphatase (PPX) in living cells, that’s how poly Pi maintains balance. PPK exits in many bacteria but lacks in mammalian cells. Poly Pi is important for many metabolism processes in living cells, such as relating the concentration of ATP, chelating ions like magnesium and calcium, controlling the transportation of DNA. Besides, poly Pi acts as a massager for stringent response in prokaryotic cells when encountering environmental changes. Imbalance of poly Pi affects the biofilm formation and the signal reception of quorum sensing system. What is more, disordered poly Pi metabolism also leads to the attenuation of bacteria.SenX3/regX3 is one of the eleven two-component regulatory systems (2CRs) encoded by M. tuberculosis genome. Bacteria sense the changes of environment by its 2CRs and it has been reported that some of the 2CRs are involved in the bacterial virulence. The phosphate-specific transport (Pst) system is up-regulated under the control of SenX3/RegX3 during phosphate starvation and regX3 is essential for the survival of M. tuberculosis in lungs and during phosphate starvation. Besides, SenX3/RegX3 regulate the gene expression of M. tuberculosis ApstAl in phosphate rich medium and the survival of the mutant strain depends on the negative regulation of RegX3 through Pst system. But till now, the exact signal transduction and the mechanism of how bacteria react to the changing concentration of environmental phosphate through SenX3/RegX3 are not known.Previous work in our lab characterized a transposon inactivated phoY2 mutant of M. marinum. They found that the disruption of phoY2 resulted in elevated levels of intracellular poly-Pi and ATP. The phoY2::Tn mutant exhibited increased sensitivity to SDS, antibiotics and excessive levels of Pi. Besides, they also investigated the phenotypes of the phoY2::Tn mutant under hypoxia-induced dormancy. On the basis of these work, we further characterized the phoY2::Tn(05A9) mutant and found that the disruption of phoY2 leads to the changes of more than three hundred genes’ expression, including phosphate transportation related Pst system and senX3/regX3. The phenotypes of phoY2::Tn mutant containing ppk antisense RNA did not change when exposed to stress conditions mentioned above, even though the concentration of poly Pi was brought down to the wild type level in the newly generated mutant. No protein-protein interaction was able to be detected between PhoY2 and Pst system or PhoP/PhoR using bacterial two-hybrid system. The promoter activity of senX3/regX3 is higher in the phoY2::Tn mutant, and further truncation analysis reveals the activity is controlled by different regulators. What is more, the persistence is significantly damaged in phoY2::Tn mutant. (Part Ⅰ)The capacity of M. tuberculosis to initiate disease processes lies in its ability to avoid its destruction within the macrophages. Most pathogens are internalized into nascent phagosomes, from where they are transported into lysosomes for their eventual destruction. In contrast, M. tuberculosis actively blocks its transfer to lysosomes and thus can survive for prolonged periods within macrophage phagosomes. The molecular mechanisms of this survival remain still unclear.Protein Kinase G (PknG), one of the eleven eukaryotic-like serine/threonine kinases (PknA to PknL) encoded in the genome of M. tuberculosis. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. The kinase activity and autophosphorylation of PknG are essential for bacterial survival inside macrophage. PknG promotes intracellular survival by blocking phagosome-lysosome fusion. Consistently, inactivation of protein kinase G by gene disruption or chemical inhibition results in mycobacterial lysosomal localization and cell death in infected macrophages. However, the substrates of PknG are still to be identified and the mechanism of how PknG blocks the fusion of phagosome and lysosome, thus promotes the survival of mycobacteria within the host remains largely unknown.It is important to figure out how PknG is secreted into the cytosol of macrophage, thus blocks the phagosome-lysosome fusion there. Mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wildtype M. marinum, we identified putative SecA2-dependent substrates, including the virulence factor protein kinase G (PknG). Interestingly, many phenotypical defects of the secA2 mutant are highly similar to those described for ΔpknG, including phagosomal maturation, metabolic alterations and an impaired capacity to initiate granuloma formation in zebrafish embryos. But the interactions between PknG and SecA2 as well as their impacts of virulence need to be further verified.It is of interest to study whether ectopically overexpressed PknG in macrophages is sufficient to rescue a PknG deficient strain from lysosomal transport and degradation. To this end, we here propose a plasmid based expression system where PknG is recombinantly expressed in the murine J774 macrophage cell line. Following PknG transfection, the survival of PknG deficient mycobacteria within macrophages expressing PknG can be analysed by both confocal images and colony forming units (CFUs). The results indicate that the infection of PknG deficient mycobacteria into macrophages expressing PknG result in a significant decrease in lysosomal delivery and has an increased survival rate compared with the infection in wild type macrophages. Our studies provides a better understanding of the role of PknG in blocking phagosome-lysosome fusion, thus leads to the survival of the pathogen inside the host. In order to study the relationship between PknG and SecA2 secretion system as well as their impacts for mycobacterial virulence, we infected the macrophage overexpressing PknG with secA2 mutant strain. Through the analysis using confocal microscopy, we find out that the PknG-overexpression can partially restore the virulence of the secA2 mutant, which provides a better understanding of the virulence factor and the secretion system SecA2 (Part Ⅱ).Transcriptome analysis has played an essential role for revealing gene expression and the complexity of regulations at transcriptional level. RNA-seq is a powerful tool for transcriptome profiling, which uses deep-sequencing technologies to directly determine the cDNA sequence. Here, we utilized RNA-seq to explore the transcriptome of Mycobacterium marinum (M. marinum), which is a useful model to study the pathogenesis of Mycobacterium tuberculosis (Mtb). Two profiles of exponential and early stationary phase cultures were generated after a physical ribosome RNA removal step. We systematically described the transcriptome and analyzed the functions for the differentiated expressed genes between the two phases. Furthermore, we predicted 360 operons throughout the whole genome, and 13 out of 17 randomly selected operons were validated by RT-PGR. In general, our study has primarily uncovered M. marinum transcriptome, which could help to gain a better understanding of the regulation system in Mtb that underlines disease pathogenesis (Part Ⅲ).