Arbind Kumar

Molecular characterization of oxidative stress-inducible LipD of Mycobacterium tuberculosis H37Rv.

The Mycobacterium tuberculosis has developed intricate strategies to evade the killing of microorganism and support its survival in phagocytes. The genome sequence of bacterium revealed the presence of several genes for lypolytic enzymes. Rv1923 gene, a member of Lip family in M.tuberculosis demonstrated the least sequence similarity with its counterpart in non-pathogenic strain M. smegmatis. The expression of Rv1923 gene (LipD) was not observed in in vitro growing cultures of M.tuberculosis H37Ra while an upregulation of transcription of Rv1923 gene was noticed in oxidative conditions. For detailed characterization of LipD enzyme the Rv1923 gene was cloned in pQE30-UA vector and expressed in E. coli M15 cells. LipD was purified from inclusion bodies and refolded with nearly 40xa0% protein yield. The specific activity of enzyme was calculated to be 16xa0U/mg with pNP-palmitate as a preferred substrate. Kinetic analysis showed Km 0.645xa0mM and Vmax 24.75xa0U/ml with pNP-palmitate. Ser-102, Asp-342, and His-369, predicted as the members of the catalytic triad, were confirmed by mutagenesis. Mutagenesis studies revealed that catalytic serine residues located in β-lactamase motifs (S-X-X-K) were responsible for lipolytic activity. Secondary structure analysis by CD spectroscopy demonstrated the presence of α helices and β sheets in the canonical structure of LipD. The enzyme was stable up to 50xa0°C and was active even at pH 6.0. The expression of enzyme under stress conditions and its activity and stability at high temperature and low pH suggested the possible role of LipD in the survival of mycobacterium in macrophage compartment.

Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating β-lactamase activity.

The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus β-lactamase motif SXXK in addition to a conserve pentapeptide -GXSXG-, characteristic of lipolytic enzymes, at the C-terminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and β-lactamase activities. A serine located within consensus β-lactamase motif SXXK was identified as catalytic residue in both esterase and β-lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for β-lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly up-regulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing β-lactamase activity.

Primer Based Approach for PCR Amplification of High GC Content Gene: Mycobacterium Gene as a Model.

The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene.

Functional characterization of hypothetical proteins of Mycobacterium tuberculosis with possible esterase/lipase signature: a cumulative in silico and in vitro approach

The functional aspect of several mycobacterium proteins annotated as hypothetical are yet to be discovered. In the present investigation, in silico approaches were used to predict the biological function of some of the unknown Mtb proteins, which were further validated by wet lab experiments. After screening thousands of Mtb proteins, functionally unknown hypothetical proteins Rv0421c, Rv0519c, Rv0774c, Rv1191, Rv1592c, and Rv3591c were chosen on the basis of their importance in Mtb life cycle. All these proteins posses the α/β-hydrolase topological fold, characteristic of lipases/esterases, with serine, aspartate, and histidine as the putative members of the catalytic triad. The catalytic serine is located in pentapeptide motif “GXSXG” and oxyanion residue is in dipeptide motif HG. To further support our observation, molecular docking was performed with conventional synthetic lipolytic substrates (pNP-esterss) and specific lipase/esterase inhibitors (tetrahydrolipstatin and phenylmethanesulfonyl fluoride (PMSF)). Significant docking score and strong interaction of substrates/inhibitors with these proteins revealed that these could be possible lipases/esterases. To validate the in silico studies, these genes were cloned from Mtb genome and the proteins were over-expressed in pQE-30/Escherichia coli M15 system. The expressed proteins were purified to homogeneity and enzymatic activity was determined using pNP esters as substrate. The enzyme activity of recombinant proteins was inhibited by tetrahydrolipstatin and PMSF pre-treatment. Outcome of the present investigation provided a basic platform to analyze and characterize unknown hypothetical proteins.

Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements

E. coli is most preferred system used for the production of recombinant proteins in bacteria and the availability of improved genetic tools/methods are making it more valuable than ever. Major challenges faced by this expression system are the expression of unusually difficult/complex proteins with rare codons or membrane and toxic proteins. The proteins expressed either in large amount or hydrophobic in nature tend to form insoluble mass. Despite the appropriate expression system, some proteins express at very low level or not at all. Choosing the correct expression system/protocols are obligatory for the substantial expression of protein in the native form. A number of vectors, their compatible hosts and culture conditions can be used to express recombinant proteins in large amounts and in native form. Also, vectors with the fusion tags/chaperons facilitate protein expression in soluble fraction and assist in proper protein folding besides restoring the native structure of protein. The recovery of native proteins from insoluble inclusion bodies can be achieved by optimization of refolding conditions. In the present review, we discussed recent updates on prokaryotic expression system for successful heterologous gene expression in E. coli and focused on strategies to maximize the yields of native recombinant proteins.

Rv0774c, an iron stress inducible, extracellular esterase is involved in immune-suppression associated with altered cytokine and TLR2 expression

Tuberculosis, one of the leading cause of death from infectious diseases, is caused by Mycobacterium tuberculosis. The genome of M. tuberculosis has been sequenced and nearly 40% of the whole genome sequence was categorized as hypothetical. Rv0774c was annotated as membrane exported hypothetical protein in TB database. In silico analysis revealed that Rv0774c is a paralog of PE-PGRS multi gene family with 100 aa N-terminal domain similar to PE domain of PE-PGRS proteins. Its C-terminal domain is quite different from PGRS domain, having characteristic lipase signature GXSXG & HG and catalytic residues predicted for lipolytic activity. Therefore, DNA coding for Rv0774c (303 aa), its N-terminal (1-100 aa) and C- terminal domain (100-303 aa) were separately cloned from M. tuberculosis and were over expressed in E. coli. Rv0774c gene and its C-terminal lipolytic domain preferably hydrolyzed short chain esters. Though no enzyme activity was observed in N-terminus PE like domain, it was demonstrated to enhance the thermostability of full length Rv0774c. Tetrahydrolipstatin inhibited the enzyme activity and predicted catalytic residues (Ser-185, Asp-255 and His-281) were confirmed by site directed mutagenesis. Rv0774c was secreted out in culture media by M. tuberculosis and was up-regulated in iron limiting conditions. Treatment of THP-1 cells with rRv0774c resulted in a decline in the LPS induced production of NO and expression of iNOS. rRv0774c treated THP-1 cells also showed an enhanced expression of IL-10 and TLR2. On contrary, it suppressed the LPS induced production of IL-12, chemokines MCP-1 and IL-8. Rv0774c inhibited the LPS induced phosphorylation of p38. These observations suggested that Rv0774c could modulate the pro-inflammatory immune response to support intracellular survival of the mycobacterium.

Disruption of N terminus long range non covalent interactions shifted temp.opt 25 °C to cold: Evolution of point mutant Bacillus lipase by error prone PCR

Cold adapted enzymes have applications in detergent, textile, food, bioremediation and biotechnology processes. Bacillus lipases are generally recognized as safe (GRAS) and hence are industrially attractive. Bacillus lipase of 1.4 subfamily are of lowest molecular weight and are reversibly unfolded due to absence of disulphide bonds. Therefore these are largely used to study energetic of protein stability that represents unfolding of native protein to fully unfolded state. In present study, metagenomically isolated Bacillus LipJ was laboratory evolved for cold adaptation by error Prone PCR. Library of variants were screened for high relative activity at low temperature of 10°C compared to native protein LipJ. Point mutant sequenced as Phe19→Leu was determined to be active at cold and was selected for extensive biochemical, biophysical characterization. Variant F19L showed its maximum activity at 10°C where parent protein LipJ had 20% relative activity. Psychrophilic nature of F19L was established with about 50% relative active at 5°C where native protein was frozen to act. Variant F19L showed no activity at temperature 40°C and above, establishing its thermolabile nature. Thermostability studies determined mutant to be unstable above 20°C and three fold decrease in its half life at 30°C compared to native protein. Far UV-CD and intrinsic fluorescence study demonstrated unstable tertiary structure of point variant F19L leading to its unfolding at low temperature of 20°C. Cold adaptation of mutant F19L is accompanied with increased specific activity. Mutant was catalytically more efficient with 1.3 fold increase in kcat. Homologue structure modelling predicted disruption of intersecondary hydrophobic core formed by aromatic ring of Phe19 with non polar residues placed at β3, β4, β5, β6, αF. Increased local flexibility of variant F19L explains molecular basis of its psychrophilic nature.

Point Mutation Ile137-Met Near Surface Conferred Psychrophilic Behaviour and Improved Catalytic Efficiency to Bacillus Lipase of 1.4 Subfamily

Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline optimum pH and broad substrate specificity. The activity and stability of these enzymes for a limited temperature range (30–50xa0°C) need attention for its industrial application. In the present study, Bacillus subtilis LipJ was rationally designed for low-temperature adaptation. Small amino acids with lower volume and without side chain branches have high occurrence among psychrophilic proteins. Met residue is reported to be preferred for cold adaptation as it is thermolabile in nature and undergoes oxidation at high temperature. Therefore, the Ile137 residue, three residues downstream the catalytic residue Asp133, was substituted by Met. Biochemical study demonstrated that variant Ile137Met was optimally active at 20xa0°C whereas parent enzyme was most active at 37xa0°C. The variant retained 70–80xa0% relative activity at 10xa0°C where parent enzyme demonstrated low activity. Ile137Met was observed to be unstable at and above 30xa0°C. Kinetic study demonstrated increased Km and kcat values for variant referring improved catalytic efficiency but poor substrate affinity. Homolog modelling predicted lowered number of weak interactions by substituted Met137 as molecular basis of increased flexibility of variant. Hence, increased structure flexibility might be responsible for poor substrate affinity but increased molecular motion for higher catalysis at cold.

Modulation of Trehalose Dimycolate and Immune System by Rv0774c Protein Enhanced the Intracellular Survival of Mycobacterium smegmatis in Human Macrophages Cell Line

Mycobacterium tuberculosis Rv0774c protein was reported previously to express under stress conditions. Therefore, Rv0774c gene was cloned and expressed in Mycobacterium smegmatis, a surrogate host, to determine its role in bacterial persistence and immune modulation in natural environment. The bacterial colonies expressing Rv0774c (Ms_rv0774c) were larger, smoother, more moist, and flatter than the control ones (Ms_ve). Enhanced survival of Ms_rv0774c after treatment with streptomycin was observed when compared with control. The cell envelope of Ms_rv0774c was demonstrated to have more trehalose di-mycolate (TDM) and lesser amount of mycolylmannosylphosphorylheptaprenol (Myc-PL) in comparison to control. Higher intracellular survival rate was observed for Ms_rv0774c as compared to Ms_ve in the THP-1 cells. This could be correlated to the reduction in the levels of reactive NO and iNOS expression. Infection of macrophages with Ms_rv0774c resulted in significantly increased expression of TLR2 receptor and IL-10 cytokines. However, it lowered the production of pro-inflammatory cytokines such as IL-12, TNF-α, IFN-γ, and MCP-1 in Ms_rv0774c infected macrophages in comparison to the control and could be associated with decreased phosphorylation of p38 MAPK. Though, predicted with high antigenicity index bioinformatically, extracellular in nature and accessible to host milieu, Rv0774c was not able to generate humoral response in patient samples. Overall, the present findings indicated that Rv0774c altered the morphology and streptomycin sensitivity by altering the lipid composition of M. smegmatis as well as modulated the immune response in favor of bacterial persistence.

Retracted: Ebola virus: an introduction and its pathology

The Ebola viruses are causative agent of a severe Ebola virus disease (EVD) or Ebola hemorrhagic fever (EHF) in human and other primates. Transmission of EVD occurs through the contact of body fluids from infected persons or animals, making it one of the most epidemic diseases worldwide. Underestimating the Ebola virus has cost loss of precious human lives in recent years. Ebola virus outbreak in year 2014 created a history, affecting a larger population in a wide geographical region of African sub‐continent. EVD outbreaks have a case fatality rate of up to 70%. Ebola viruses are endemic in regions of Africa. Ebola viruses mainly target the hepatocytes, endothelial, and macrophage‐rich lymphoid tissues and are characterized by immune suppression and a systemic inflammatory response that causes impairment of the vascular, coagulation, and immune systems. This impairment leads to multifocal necrosis and multi organ failure, and thus, in some ways, resembling septic shock. Currently, neither a specific treatment nor a vaccine licensed for use in humans is available. This review is focused on general characteristic of Ebola viruses, its pathogenesis, immunological response of host, and recent approaches for vaccine development against EVD. Copyright