nshan Sha

Kinetic properties of Mycobacterium tuberculosis bifunctional GlmU

The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors.

Development of a Colorimetric Assay and Kinetic Analysis for Mycobacterium tuberculosis D-glucose-1-phosphate Thymidylyltransferase

dTDP-L-rhamnose as a sugar donor provides L-rhamnosyl residue in the synthesis of disaccharide linker (D-N-acetylglucosamine-L-rhamnose), the key structure of the Mycobacterium tuberculosis cell wall. Four enzymes are involved in the formation of dTDP-L-rhamnose and D-glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the first step of D-glucose-1-phosphate and dTTP to dTDP-D-glucose and PPi. The previous studies on RmlA essentiality proved RmlA as a potential target for antituberculosis drugs. However, there has not been a suitable assay for RmlA to screen inhibitors currently. In this study, the authors reported a microtiter plate–based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors.

Characterization of mycobacterial UDP-N-acetylglucosamine enolpyruvyle transferase (MurA)

The mycobacterial peptidoglycan has structure and biosynthetic pathways to similar those of other bacteria. UDP-N-acetylglucosamine enolpyruvyle transferase (MurA) catalyzes the first reaction in the biosynthesis of peptidoglycan. The MurA enzyme has been identified from various bacterial species, but the in-depth biochemical properties of mycobacterial MurA have not been characterized. In this study, both Mycobacterium tuberculosis MurA protein and Mycobacterium smegmatis MurA protein were overexpressed in Escherichia coli and purified by affinity chromatography. MurA activity was detected by HPLC. A colorimetric assay of MurA activity was also developed and the kinetic properties of Mtb MurA and Msm MurA were determined using this colorimetric assay. A conditional murA gene knockout strain was constructed by DNA homologous recombination. The disruption of murA in the genome of M. smegmatis led to loss of viability at a non-permissive temperature. Drastic morphological and structural alterations in the M. smegmatis murA knockout strain were observed by scanning electron microscopy and transmission electron microscopy. These results demonstrated that murA was an essential gene for growth of M. smegmatis. Therefore, MurA is a potential target for developing new anti-tuberculosis drugs.

Mycobacterium tuberculosis Rv1987 induces Th2 immune responses and enhances Mycobacterium smegmatis survival in mice

Mycobacterium tuberculosis can interfere with host immune response and escape clearance through its specific antigens. M. tuberculosis Rv1987 encoded by region of difference (RD)-2 gene is a secretory protein with immunogenic potency. Here, we investigated the impact of Rv1987 on host cytokine responses and T cell polarization in mouse aerosol model. A recombinant M. smegmatis mc2155 strain that overexpressed Rv1987 protein (named MS1987) was constructed and used to infect C57BL/6 mice. The mc2155 harbored the empty vector (named MSVec) was as a control. The results showed that MS1987 challenged mice promoted Th2-biased cytokine responses with lower secretion of IFN-γ but higher production of IL-4 and Rv1987-specific IgG antibody compared to MSVec infected mice. Neutrophilic inflammation and high bacterial burden were observed in the lung tissues of MS1987 infected mice probably own to the failed Th1 cell immunity. Besides, subcutaneous injection of Rv1987 protein could mediate the Th1 cytokine responses caused by M. bovis BCG in mice. These results indicated that M. tuberculosis Rv1987 protein could modulate host immune response towards Th2 profile, which probably contributed to the immune evasion of bacteria from host elimination.

Perfluorooctane Sulfonate Induces Autophagy-Dependent Apoptosis through Spinster 1-Mediated lysosomal-Mitochondrial Axis and Impaired Mitophagy

Lysosomal membrane permeabilization (LMP) and subsequently impaired autophagosome degradation was induced in HepG2 cells after treatment with perfluorooctane sulfonate (PFOS) for 24 h in our previous studies. We found that treatment of HepG2 cells with PFOS-induced autophagosome formation at earlier stage (6 h) of treatment in this study. The autophagosome formation inhibitor 3-methyladenine (3-MA) was able to relieve PFOS-induced LMP and release of cathepsin D in HepG2 cells. Knockdown of Spinster 1, a lysosomal membrane permease, attenuated PFOS-induced LMP in HepG2 cells. We proposed that Spinster 1 might work as a specific molecule that linked autophagy with LMP. PFOS-induced collapse of mitochondrial transmembrane potential was cathepsin D and autophagy dependent. Addition of 3-MA relieved PFOS-induced apoptosis, which was evidenced by Hoechst assay, AV/PI staining and caspase-3 activity assay. Inhibition of autophagosome formation by Atg5 siRNA attenuated PFOS-induced apoptosis. Treatment of HepG2 cells with PFOS for 24 h impaired mitophagy, as evidenced by an increase of cells with giant mitochondria and impairment of colocalization of PINK1 with light chain 3. In summary, we report that PFOS induces autophagy-dependent apoptosis in HepG2 cells through the lysosomal-mitochondrial axis and impairment of mitophagy, suggesting that autophagy is a primary target for PFOS toxicity. These findings provide new mechanistic insights into PFOS-induced hepatotoxicity.

Viability, morphology, and proteome of Mycobacterium smegmatis MSMEG_0319 knockout strain

Mycobacterium tuberculosis Rv0228, a membrane protein, is predicted as a drug target through computational methods. MSMEG_0319 (MS0319) in Mycobacterium smegmatis mc2155 is the ortholog of Rv0228. To study the effect of MS0319 protein on M. smegmatis, an MS0319 gene knockout strain (ΔMS0319) was generated via a homologous recombination technique in this study. The results showed that the lack of MS0319 protein in mc2155 cells led to the loss of viability at nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy observations showed drastic changes in cellular shape especially cell wall disruption in ΔMS0319 cells. Proteomic analysis of ΔMS0319 cells through LC‐MS/MS revealed that 462 proteins had changes in their expressions by lacking MS0319 protein. The M. tuberculosis orthologs of these 462 proteins were found through BLASTp search and functional clustering and metabolic pathway enrichment were performed on the orthologs. The results revealed that most of them were enzymes involved in metabolism of carbohydrates and amino acids, indicating that Rv0228 played an important role in cellular metabolism. All these results suggested Rv0228 as a potential target for development of antituberculosis drugs.

Mycobacterium tuberculosis Rv0431 expressed in Mycobacterium smegmatis, a potentially mannosylated protein, mediated the immune evasion of RAW 264.7 macrophages

Tuberculosis remains a global major problem. The immune responses of host against Mycobacterium tuberculosis (M. tuberculosis) are complicated. M. tuberculosis lives mainly within host cells, usually macrophages which constitute the first line of host defense. Mycobacterial proteins, especially cell wall-associated proteins, interact with macrophages of host to regulate the functions and cytokine production. Recent studies indicate that glycoproteins are involved in this process. Here, we investigated the function of Rv0431, a cell wall-associated protein in the M. tuberculosis H37Rv strain. Rv0431 protein was heterologously overexpressed in the fast-growing and nonpathogenic Mycobacterium smegmatis (M. smegmatis). Binding assay to concanavalin A (ConA) lectin was performed and the result indicated that Rv0431 protein was a potentially mannosylated protein. M. smegmatis MSMEG_5447 gene encoding a polyprenol-phosphate-mannose-protein mannosyl-transferase (PMT) which catalyzes the O-mannosylation of protein was knocked out. The Rv0431 protein overexpressed in MSMEG_5447 gene knockout stain, ΔM5447, lost its reactivity to ConA, providing evidence that Rv0431 was likely O-mannosylated. M. smegmatis overexpressed Rv0431 evaded the killing of RAW264.7 macrophages and altered the cytokine production of macrophages compared to M. smegmatis carrying empty vector. These results suggested that Rv0431, a probably mannosylated protein might promote the evasion of immune responses during mycobacterial infection.

An integrated course based on two dimensional‐electrophoresis and mass spectrometry analysis to teach proteomic techniques for undergraduate students

Here, we developed an integrated course based on two dimensional‐electrophoresis and spectrometry mass (2DE‐MS) technique for undergraduate students to help them learn proteomic techniques. The soluble proteins in wild type and gene knockout bacteria were separated by 2DE and the differently expressed proteins were identified by MS analysis. The proteomic data was finally confirmed by RT‐PCR detection. The separated experiments of 2DE, MS, RNA isolation, RT‐PCR, as well as essential bioinformatic analysis, were integrated into a one‐week course, which provided students an opportunity to systematically understand the proteomic techniques and their applications in current scientific research.

Down-regulation of N-acetylglucosamine-1-phosphate transferase (WecA) enhanced the sensitivity of Mycobacterium smegmatis against rifampin.

To construct a conditional N‐acetylglucosamine‐1‐phosphate transferase (WecA) knockdown strain of Mycobacterium smegmatis and to investigate the biological effect of WecA on mycobacterial growth, morphology and susceptibilities against anti‐tuberculosis drugs.