| Tuberculosis (TB), caused by the facultative intracellular pathogen Mycobacterium tuberculosis (Mtb), remains one of the world’s deadliest communicable diseases. About one-third of the global population was infected by latent tuberculosis. In 2013, an estimated 9.0 million people developed TB and China alone accounted for 11% of total cases; and 1.5 million died from the disease,360 000 of whom were HIV-positive. Globally, an estimated 480 000 people have developed multidrug-resistant TB (MDR-TB) in 2013. On average, an estimated 9.0% of patients with MDR-TB had extensively drug resistant TB (XDR-TB). A string of shocking data abovementioned indicated that Mtb is still one of the most formidable pathogens. The pathogenesis of Mtb is largely depends on its successful intracellular survival, a process which depends on an array of effectors (or virulence factors) to colonize and replicate within the host. The success of Mtb can be attributed to these effectors which contain lipids and secreted proteins. These known effectors can be classified as (a) enhancing the resistance to host toxic compounds, such as KatG, SodC and AhpC, (b) blocking phagosomes maturation, such as PtpA, PknG and SapM, and (c) evading apoptosis through NuoG and SodA. It seems that there are still a large number of effectors to be discovered, although some have been identified. The identification and characterization of novel virulence factors can enrich our understanding of Mtb biology and facilitate better control measures. Therefore, we mainly performed several studies as follows.The present study introduces a novel pipeline to predict Mtb proteins involved in host-pathogen interaction. In brief, data from multiple scales were integrated:(a) gene expression and DNA microarray experiments in macrophages; (b) genome-wide insertional mutagenesis defining gene essentiality under different conditions; (c) genes expressed in animal models; (d) genes expressed in TB patients; (e) genes lost in clinical isolates; (f) cellular localization analysis and subcellular localization prediction tools; (g) non-homology analysis and gut flora non-homology analysis. We designed an integrative analysis pipeline including whole-genome scoring, ranking and prioritization algorithm and to screen datasets from multiple scales. Based on the criteria of essentiality and selectivity,54 promising hits were identified. These proteins satisfying the following criteria:up-regulated during infection, essential for the pathogen’s in vivo survival but not in vitro survival, conserved in clinical isolates, located outside of the pathogen and non-homologous to host proteome and gut flora proteome. It is expected that the 54 selected candidates can benefit the development of novel anti-TB drugs.Secondly, we choose one potential secreted protein Rv3402c as target for an intensive study of molecular biology and cell biology, this paper focuses on the study of Rv3402c which involved in the interaction of pathogen and host macrophage cells. The Mtb iron regulated open reading frame (ORP) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-a and IL-1(3 compared with controls. Rv3402c-induced TNF-a and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.Finally, this paper explored some important functions of protein tyrosine kinase A (PtkA) which is a regulatory protein of the famous Mtb secreted proteins protein tyrosine phosphatase A (PtpA). PtkA was originally annotated as a member of the haloacid dehalogenase (HAD) superfamily based upon sequence homology. However, biochemical analysis of its enzymatic activity demonstrated that PtkA is a genuine protein-tyro sine kinase. The unpublished data showed that Mtb H37Rv ΔptkA deletion mutant is more resistant to oxidative stress (hydrogen peroxide and cumene hydroperoxide). Therefore, we assumed that there is some unknown substrates of PtkA in Mtb. We firstly identified the substrates of PtkA by combination of in vitro kinase assay and radioactive two-dimensional electrophoresis (or named standard 2D-gel-based phosphoproteomic study). But we unexpectedly found PtkA’s upstream regulatory protein that can inhibit the kinase activity of PtkA. It is very likely that the main reason that researchers cannot find other substrates of PtkA except itself and PtpA, although we don’t know what the specific inhibitory protein(s) is/are. Besides, we have optimized the experimental conditions of purification and in vitro kinases assay of PtkA protein. Then, we maked a list of phosphotyrosine-proteins from published bacterial phosphoproteomics studies. And found out the conservation of phosphotyrosine proteins which have homologue in Mtb. We overexpressed and purified those proteins in E. coli, and do in vitro kinase assay to check which one is the substrate of PtkA. Then we found TrxB2 was a genuine substrate of PtkA by using dose-manner dependent assay, phosphor-amino acid assay and site-direct mutagenesis. Phosphorylation of TrxB2 by PtkA does not affect the enzymatic activity of TrxB2, but it was able to inhibit the secretion of TrxB2. In another word, the Mtb H37Rv ΔptkA mutants released more TrxB2 proteins out of the cell compare to Mtb wild type strain. Our unpublished data showed that Mtb H37Rv ΔptkA mutants was more resistant to oxidative stress (hydrogen peroxide and cumene hydroperoxide) compare to Mtb wild type strain. Given TrxB2 is a thioredoxin reductase which can neutralize the oxidative stress. Therefore, our results may explain this phenomenon abovementioned:Mtb H37Rv ΔptkA mutants secreted more TrxB2 proteins into the extracellular, TrxB2 alone or combined with TrxC can detoxify hydrogen peroxide and cumene hydroperoxide, so that the ΔptkA mutant strains showed more resistant to the oxidative stress.In summary, this paper predicted an array of potential anti-TB drug targets, confirmed that Rv3402c is one of secreted effectors in Mtb and expanded our awareness of Mtb secreted protein regulatory networks. |
PDF Full Text Request