Neelja Singhal

Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

BackgroundTuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.ResultsOn comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.ConclusionsMass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.

Proteomic analysis of streptomycin resistant and sensitive clinical isolates of Mycobacterium tuberculosis.

BackgroundStreptomycin (SM) is a broad spectrum antibiotic and is an important component of any anti-tuberculosis therapy regimen. Several mechanisms have been proposed to explain the emergence of resistance but still our knowledge is inadequate. Proteins form a very complex network and drugs are countered by their modification/efflux or over expression/modification of targets. As proteins manifest most of the biological processes, these are attractive targets for developing drugs, immunodiagnostics or therapeutics. The aim of present study was to analyze and compare the protein profile of whole cell extracts from Mycobacterium tuberculosis clinical isolates susceptible and resistant to SM.ResultsTwo-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was employed for analyzing the protein profiles. Homology and in silico characterization for identified proteins was assessed using BLAST, InterProScan and KEGG database searches. Computational studies on the possible interactions between SM and identified proteins were carried out by a battery of online servers and softwares, namely, CLUSTALW (KEGG), I-TASSER, VMD, PatchDock and FireDock. On comparing 2DE patterns, nine proteins were found consistently overexpressed in SM resistant isolates and were identified as Rv0350, Rv0440, Rv1240, Rv3075c, Rv2971, Rv3028c, Rv2145c, Rv2031c and Rv0569. In silico docking analysis showed significant interactions of SM with essential (Rv0350, Rv0440 and Rv2971) and non essential (Rv1240, Rv3075c and Rv2031c) genes.ConclusionsThe computational results suggest high protein binding affinity of SM and suggested many possible interactions between identified proteins and the drug. Bioinformatic analysis proves attributive for analysis of diversity of proteins identified by whole proteome analysis. In-depth study of the these proteins will give an insight into probable sites of drug action other than established primary sites and hence may help in search of novel chemotherapeutic agents at these new sites as inhibitors.

An improved sample preparation method for analyzing mycobacterial proteins in two-dimensional gels

Two-dimensional gel electrophoresis (2-DE) is currently a widely used analytical method for resolving complex mixtures of proteins. Sample preparation has a marked influence on 2-DE pattern. To reduce impurities and to increase the low-abundance proteins, protein precipitation is often used for the preparation of samples before 2-DE. In this study, we revealed that addition of SDS prior to TCA precipitation of mycobacterial cell extract proteins increases the resolution of the 2-D gel pattern.

MALDI-TOF MS in clinical parasitology: applications, constraints and prospects.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is currently being used for rapid and reproducible identification of bacteria, viruses and fungi in clinical microbiological laboratories. However, some studies have also reported the use of MALDI-TOF MS for identification of parasites, like Leishmania, Giardia, Cryptosporidium, Entamoeba, ticks and fleas. The present review collates all the information available on the use of this technique for parasites, in an effort to assess its applicability and the constraints for identification/diagnosis of parasites and diseases caused by them. Though MALDI-TOF MS-based identification of parasites is currently done by reference laboratories only, in future, this promising technology might surely replace/augment molecular methods in clinical parasitology laboratories.

Molecular Analysis of β-Lactamase Genes to Understand their Differential Expression in Strains of Yersinia enterocolitica Biotype 1A

Beta-lactams are used as major therapeutic agents against a number of infectious agents. Due to widespread use of β-lactams, β-lactamases have evolved at a rapid pace leading to treatment failures. Yersinia enterocolitica causes many gastrointestinal problems. It is an extremely heterogeneous species comprising more than fifty serotypes and six biotypes which differ in their ecological niches, geographical distribution and pathogenic potential. Though biotype 1A strains have been associated with outbreaks of Yersiniosis, there has been a controversy regarding their pathogenicity. The strains of Y. enterocolitica isolated from India belonged to biotype 1A and possessed genes for two β-lactamases namely, blaA and blaB. An earlier study by us reported differential expression of blaA by strains of Y. enterocolitica biotype 1A. The present study has been carried out to understand the molecular bases which regulate the expression of blaA in Y. enterocolitica biotype 1A. We concluded that six types of blaA variants were present in strains of biotype 1A. Neither amino acid substitutions in blaA nor mutations in promoter regions of blaA contributed to differential expression of blaA in Y. enterocolitica biotype 1A. Rather, the secondary structures attained by mRNA of blaA might underlie the differential expression of blaA in Y. enterocolitica.

Identification of Family Specific Fingerprints in β-Lactamase Families

Beta-lactamases are a superfamily of enzymes which degrade the β-lactam class of antibiotics. They are produced endogenously by the bacterial cells, which when exposed to the β-lactam class of antibiotics inactivate them by cleaving the β-lactam ring. Based on the presence or absence of metallic ligand, β-lactamases have been divided into two broad functional classes. β-Lactamases are a constitutively evolving and expanding superfamily of enzymes, which could be further subdivided on the basis of presence/absence of conserved motifs. In the present study we have used the MEME/MAST suit to identify the patterns/motifs which are specific to a particular family or subfamily of β-lactamases. The family specific patterns/motifs can be also useful in recognizing and assigning newly discovered β-lactamases to one or the other family or subfamily. Cross-validation showed that the proposed method is highly sensitive and specific. We have also designed a webserver, LactFP, for this purpose.

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Immunoprophylaxis of Tuberculosis: An Update of Emerging Trends

Developing effective prophylactics to combat tuberculosis is currently in an exploratory stage. The HIV pandemic and emergence of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis indicate that the current preventive measures against this ever-evolving pathogen are inadequate. The currently available vaccine BCG in its present form affords variable protection which usually wanes with aging. Various reasons have been cited to explain the discrepancies in the efficacy of BCG, including generic differences in the different BCG vaccine strains used in immunization program throughout the world. The low efficacy of BCG vaccine has promoted the search for novel vaccines for tuberculosis. The search strategies aim at completely replacing the existing vaccine and/or augmenting/improving the current BCG vaccine. Among new vaccine candidates are live attenuated M. tuberculosis vaccines, recombinant BCG, DNA vaccines, subunit vaccine, and fusion protein-based vaccines. More than 200 new vaccine candidates have been developed as a result of research work over the past few years. To date, at least eight vaccine candidates are undergoing clinical evaluation, with a few of them successfully qualifying in the first phase of clinical testing. These recent advances present an optimistic insight whereby a new tuberculosis vaccine might be expected to be available for public use in the next few years.

Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment

The presence of antibiotic resistance genes (ARGs) including those expressing ESBLs and AmpC-β-lactamases in Escherichia coli inhabiting the aquatic environments is a serious health problem. The situation is further complicated by the fact that ARGs can be easily transferred among bacterial species with the help of mobile genetic elements – plasmids, integrons, insertion sequences (IS), and transposons. Therefore, the analysis of genetic environment and mobile genetic elements associated with ARGs is important as these provide useful information about the epidemiology of these genes. In our previous study, we had reported presence of various β-lactam resistance genes present in E. coli strains inhabiting the river Yamuna traversing the National Capital Territory of Delhi (India). In the present study, we have analyzed the genetic environment of three ARGs blaTEM-1, blaCTX-M-15, and blaCMY -42 of those E. coli strains. The structure of class 1 integrons and their gene cassettes was also analyzed. Insertion sequence IS26 was present upstream of blaTEM-1, ISEcp1 was present upstream of blaCTXM-15 gene and orf477 was present downstream of blaCTXM-15. ISEcp1 was also present upstream of blaCMY -42 and, blc and sugE genes were present in the downstream region of this gene. Thus, the overall genetic environment surrounding these genes was similar to that reported from E. coli strains isolated globally. Conjugation assays, isolation and analysis of plasmid DNA of the transconjugants indicated that blaTEM-1, blaCTX-M-15, blaCMY -42 and class 1 integron were plasmid-mediated and possibly transmit between genera through horizontal gene transfer (HGT). This might lead to dissemination of antimicrobial resistance genes in aquatic environment. The work embodied in this paper is the first describing the genetic environment of bla and integrons in aquatic E. coli isolated from India.

A simple and rapid method of sample preparation from culture filtrate of M. tuberculosis for two-dimensional gel electrophoresis

Sample preparation for Two-dimensional gel electrophoresis (2DE) is tedious and not sufficient to provide a comparative profile of secreted proteins for various strains of M. tuberculosis. High lipid content in mycobacteria limits the use of common methods as it can hinder the 2DE run. This study highlights the significance of SDS-TCA procedure over common used methods for the preparation of sample from culture filtrate as well as other proteinaceous fluids.