Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

<p>Abstract</p> <p>Background</p> <p>Tuberculosis (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 <it>M. tuberculosis </it>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 <it>M.tuberculosis </it>clinical isolates.</p> <p>Results</p> <p>On 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.</p> <p>Conclusions</p> <p>Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of <it>M. tuberculosis </it>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 <it>M. tuberculosis</it>.</p

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses and fungi

Comprehensive genomic analysis of hypocholesterolemic probiotic Enterococcus faecium LR13 reveals unique proteins involved in cholesterol-assimilation

Hypercholesterolemia is a major risk factor for cardiovascular diseases (CVDs). Chemotherapeutic agents for CVDs exhibit several side effects. Specific probiotics with hypocholesterolemic effects can be safe and effective alternatives to chemotherapeutics. Here, we have analyzed and compared the genome of a novel rhizospheric Enterococcus faecium LR13 cholesterol-assimilating probiotic with other probiotic/pathogenic E. faecium strains to discern genetic factors underlying probiotic efficacy and cholesterol-assimilation. Genomic analyses of E. faecium probiotic strains revealed that LR13 and WEFA23 (cholesterol-assimilating probiotics) harbored 21 unique proteins absent in non-cholesterol-assimilating probiotics. Of these, 14 proteins could directly help in cholesterol-assimilation by producing short chain fatty acids, lipid (sterol) transport and membrane stabilization, and bile salt hydrolase activity. This suggests that cholesterol-assimilation is an intrinsic, strain-specific trait exhibited by probiotics with a specific genetic constitution. Moreover, the unique proteins identified in this study can serve as biomarkers for discerning/characterizing cholesterol-assimilating probiotics as novel biotherapeutics against CVDs

A case of Dentigerous Cyst in Association of Impacted Mandibular Canine

Dentigerous cyst being the second most common odontogenic cyst which constitutes around 20-24% of all the odontogenic cysts involving the jaws. Usually, these cysts remain asymptomatic and rarely cause enlargement and displacement of associated tooth. It develops by an accumulation of fluid between reduced enamel epithelium and the tooth crown of an unerupted tooth. This case report presents conservative treatment modality for 17-year-old female patient, diagnosed with a dentigerous cyst in association of impacted mandibular canine. The patient was treated with a minimal invasive approach, enucleation followed by marsupialization under local anesthesia, preserving all the teeth in occlusion

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

<p>Abstract</p> <p>Background</p> <p>Streptomycin (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 <it>Mycobacterium tuberculosis </it>clinical isolates susceptible and resistant to SM.</p> <p>Results</p> <p>Two-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 <it>in silico </it>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. <it>In silico </it>docking analysis showed significant interactions of SM with essential (Rv0350, Rv0440 and Rv2971) and non essential (Rv1240, Rv3075c and Rv2031c) genes.</p> <p>Conclusions</p> <p>The 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.</p

Prospects of comparative genomics of β-lactamase genes in rapid antimicrobial resistance (AMR) detection and newer β-lactamase inhibitors

Beta-lactam antibiotics have been a prime choice for treating a number of infectious diseases. However, their widespread &amp; indiscriminate use has resulted in microbial resistance towards this important class of antibiotics. Bacteria hydrolyze these antibiotics using their intrinsic/acquired antibiotic modifying enzymes, the β-lactamases. Studies from our laboratory using comparative genomics of β-lactamase genes and their promoters in a large number of Y.enterocolitica and E.coli strains revealed that, though the promoters were conserved, point mutations were present in different β-lactamase genes. Similar observations were also made while compiling a database of β-lactamase genes. Identification of consensus sequences among the β-lactamase genes of different bacteria could be useful for developing rapid and simple methods for detection of pathogens harbouring these genes. Our studies also revealed that mutations at sites other than active site of the enzyme may create diverse local changes in the 3D structure of the enzyme which might affect its binding affinity with β-lactam antibiotics as well as β-lactamase inhibitors. These findings might be useful for designing better β-lactamase inhibitors with improved efficiencies in future. Currently, we are working on developing a rapid and simple Loop Mediated Isothermal Amplification (LAMP) test using β-lactamase genes for detection of Y.enterocolitica. We are also working to identify novel sequences in β-lactamase genes which can be used as ideal targets for designing newer β-lactamase inhibitors. These studies would surely help us make assessments of the true potential of β-lactamase genes to serve as markers for rapid detection of AMR and salvaging several β-lactam antibiotics by designing novel β-lactamase inhibitors

Structural Variabilities in β-Lactamase (blaA) of Different Biovars of Yersinia enterocolitica: Implications for β-Lactam Antibiotic and β-Lactamase Inhibitor Susceptibilities.

Yersiniosis caused by Yersinia enterocolitica has been reported from all continents. The bacterial species is divided into more than fifty serovars and six biovars viz. 1A, 1B, 2, 3, 4 and 5 which differ in geographical distribution, ecological niches and pathogenicity. Most Y.enterocolitica strains harbor chromosomal genes for two β-lactamases, blaA an Ambler class A penicillinase and blaB an Ambler class C inducible cephalosporinase. In the present study, susceptibility to b-lactam antibiotics and β-lactamase inhibitor was studied for Y. enterocolitica strains of biovars 1A, 1B, 2 and 4. We observed that β-lactamases were expressed differentially among strains of different biovars. To understand the molecular mechanisms underlying such differential expression, the sequences of genes and promoters of blaA were compared. Also, the variants of blaA present in different biovars were modeled and docked with amoxicillin and clavulanic acid. The mRNA secondary structures of blaA variants were also predicted in-silico. Our findings indicated that neither variations in the promoter regions, nor the secondary structures of mRNA contributed to higher/lower expression of blaA in different biovars. Analysis of H-bonding residues of blaA variants with amoxicillin and clavulanic acid revealed that if amino acid residues of a β-lactamase interacting with amoxicillin and the clavulanic acid were similar, clavulanic acid was effective in engaging the enzyme, accounting for a significant reduction in MIC of amoxicillin-clavulanate. This finding might aid in designing better β-lactamase inhibitors with improved efficiencies in future

Identification of Family Specific Fingerprints in β

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