Vishvanath Tiwari

Comparative Proteomics of Inner Membrane Fraction from Carbapenem-Resistant Acinetobacter baumannii with a Reference Strain

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A. baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A. baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A. baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A. baumannii are: i) β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A. baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A. baumannii to carbapenem.

Carbapenem-hydrolyzing oxacillinase in high resistant strains of Acinetobacter baumannii isolated from India

Acinetobacter baumannii, a Gram negative bacterium causes nosocomial infections including bacteremia, secondary meningitis and urinary tract infections. Increased resistance of A. baumannii has been global concern. Till recently, carbapenems, latest generation of β-lactams are used for treating infections caused by A. baumannii. Emerging resistance to carbapenem class is an immediate threat to mankind. The objective of present study is to understand the growing carbapenem resistance of A. baumannii. By using iso-electric focusing followed by (in-gel) nitrocefin assay of carbapenem resistant strains of A. baumannii, we could identify three β-lactamases with pIs in the range 5.4-9.5. Expression of the β-lactamase with a pI ≈ 8.5, was found only in very high carbapenem resistant (MIC for imipenem 128 μg/ml) strains. On PCR analysis and sequencing of PCR product, this β-lactamase was confirmed to be OXA-51. Identification of this protein from IEF gel was reconfirmed with the help of Liquid chromatography and Tandem mass spectrometry (LC-MS/MS). Based on the amino acid sequence, OXA-51 found to be a 30 kDa β-lactamase containing conserved functional motifs of class D serine β-lactamase. In the present study, we have established the emergence of OXA-51 in clinical strains of A. baumannii in India which suggests its role in carbapenem resistance.

Conformational stability of OXA-51 β-lactamase explains its role in carbapenem resistance of Acinetobacter baumannii

Acinetobacter baumannii, an important nosocomial pathogen, is increasingly becoming resistant to antibiotics including recent β-lactam like imipenem. Production of different types of β-lactamases is one of the major resistance mechanisms which bacteria adapt. We recently reported the presence of a β-lactamase, OXA-51, in clinical strains of A. baumannii in ICUs of our hospital. This study is an attempt to understand the structure–function relationship of purified OXA-51 in carbapenem resistance in A. baumannii. The OXA-51 was cloned, expressed in E. coli Bl-21(DE3) and further purified. The in vitro enzyme activity of purified OXA-51 was confirmed by two independent techniques; in-gel assay and spectrophotometric method using nitrocefin. Further in vivo effect of OXA-51 was followed by transmission electron microscopy of bacterium. Biophysical and biochemical investigations of OXA-51 were done using LC-MS/MS, UV–Vis absorption, fluorescence, circular dichroic spectroscopy and isothermal calorimetry. Native OXA-51 was characterized as 30.6 kDa, pI 8.43 with no disulphide bonds and comprising of 30% α-helix, 27% β-sheet. Secondary structure of OXA-51 was significantly unchanged in broad pH (4–10) and temperature (30–60 °C) range with only local alterations at tertiary structural level. Interestingly, enzymatic activity up to 75% was retained under above conditions. Hydrolysis of imipenem by OXA-51 (km,1 μM) was found to be thermodynamically favourable. In the presence of imipenem, morphology of sensitive strain of A. baumannii was drastically changed, while OXA-51-transformed sensitive strain retained the stable coccobacillus shape, which demonstrates that imipenem is able to kill sensitive strain but is unable to do so in OXA-51-transformed strain. Hence the production of pH- and temperature-stable OXA-51 appears to be a major determinant in the resistance mechanisms adopted by A. baumannii in order to evade even the latest β-lactams, imipenem. It can be concluded from the study that OXA-51 plays a vital role in the survival of the pathogen under stress conditions and thus poses a major threat.

In-silico modeling of a novel OXA-51 from β-lactam-resistant Acinetobacter baumannii and its interaction with various antibiotics

Acinetobacter baumannii, one of the major Gram negative bacteria, causes nosocomial infections such as pneumonia, urinary tract infection, meningitis, etc. β-lactam-based antibiotics like penicillin are used conventionally to treat infections of A. baumannii; however, they are becoming progressively less effective as the bacterium produces diverse types of β-lactamases to inactivate the antibiotics. We have recently identified a novel β-lactamase, OXA-51 from clinical strains of A. baumannii from our hospital. In the present study, we generated the structure of OXA-51 using MODELLER9v7 and studied the interaction of OXA-51 with a number of β-lactams (penicillin, oxacillin, ceftazidime, aztreonam and imipenem) using two independent programs: GLIDE and GOLD. Based on the results of different binding parameters and number of hydrogen bonds, interaction of OXA-51 was found to be maximum with ceftazidime and lowest with imipenem. Further, molecular dynamics simulation results also support this fact. The lowest binding affinity of imipenem to OXA-51 indicates clearly that it is not efficiently cleaved by OXA-51, thus explaining its high potency against resistant A. baumannii. This finding is supported by experimental results from minimum inhibitory concentration analysis and transmission electron microscopy. It can be concluded that carbapenems (imipenem) are presently effective β-lactam antibiotics against resistant strains of A. baumannii harbouring OXA-51. The results presented here could be useful in designing more effective derivatives of carbapenem.

Structural studies on New Delhi Metallo-β-lactamase (NDM-2) suggest old β-lactam, penicillin to be better antibiotic for NDM-2-harbouring Acinetobacter baumanni

Acinetobacter baumannii, a Gram-negative pathogen causes nosocomial infections including pneumonia, urinary tract and respiratory infections. Carbapenem group of β-lactam antibiotics are routinely used to treat A. baumannii including multidrug-resistant clinical strains. The emergence of New Delhi Metallo-β-lactamase (NDM-2), a new type of β-lactamase and one of the major resistant determinants in A. baumannii, opened up challenges in the treatment of resistant strains. Thus, understanding the structure–function relationship of NDM-2 with different analogues of β-lactams becomes crucial. We carried out in silico studies on the interaction of various β-lactams with NDM-2 and with OXA-24, a carbapenem hydrolyzing non-NDM type β-lactamase. The binding affinity of the β-lactams to NDM-2 was found to be in the order: ceftazidime ≈ imipenem ≈ doripenem > oxacillin > aztreonam > penicillin; however, the order of their affinity to OXA-24 was quite different: ceftazidime > aztreonam > penicillin > oxacillin > doripenem > imipenem. Further, NDM-2 in comparison to OXA-24 showed stronger interaction (less X-score) with most of the β-lactams except penicillin. This suggests higher lethality posed by clinical strains expressing NDM-2 than those without NDM-2. Weak interaction between NDM-2 and penicillin clearly points out that penicillin is perhaps better option in treating A. baumannii harbouring NDM-2. Present findings provide new insights in drug resistance at the molecular level of NDM-2 and can help in designing structure-based drugs.

Differential anti-microbial secondary metabolites in different ESKAPE pathogens explain their adaptation in the hospital setup

Nosocomial infections are caused by ESKAPE (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae) pathogens, and their co-existence is associated with their ability to survive in the hospital setup. They may produce molecules, which helps in the better survival of one ESKAPE pathogens over other. We have identified all secondary metabolite gene clusters in six ESKAPE pathogens and predicted antimicrobial and anti-biofilm properties of their product secondary metabolites. To validate our model, we have taken the secondary metabolites of ESKAPE pathogens and studied their interaction with diguanylate cyclase (involved in quorum sensing) and biofilm-associated protein (involved in biofilm formation) of Acinetobacter baumannii. Results suggest the presence of differential secondary metabolites in all ESKAPE pathogens with only three common non-antimicrobial secondary metabolites. Out of twenty-three antimicrobial secondary metabolites, TP-1161, nosiheptide and meilingmycin, showed the best antimicrobial activity and nineteen showed high anti-biofilm activity. Interaction study showed that secondary metabolites produced by other ESKAPE pathogens (non-Acinetobacter) have very good interaction with diguanylate cyclase and biofilm-associated protein of A. baumannii. This concludes that better survival of these ESKAPE pathogens in hospital setup can be correlated with differential production of antimicrobial secondary metabolites. The present study also investigates the molecular mechanism of the competition of different pathogens living in similar hospital setup (similar habitat). Therefore, the present study will initiate research that might lead to the discovery of antibiotics from one ESKAPE pathogen that controls the infection of other ESKAPE pathogens or other pathogens.

Membrane proteins- key players of carbapenem resistance in acinetobacter

Objective : To determine whether or not Bonny Light Crude Oil (BLCO), when administered to albino rats for six consecutive days at 48 hours interval would result in a dose-related alteration in chromatin structure as obtained in its absorbance ratio, and subsequent impairment of its function such as DNA synthesis. Methodology and results : Twelve albino rats ( Rattus norvegicus ) were divided into four groups with group one serving as control, and group two to group four were administered with 2.5, 5.0 and 10.0 ml/kg bw of Bonny Light Crude Oil (BLCO) by intraperitoneal injection for six consecutive days. All the rats were sacrificed on the eighth day and their liver excised. The livers were all homogenized, and through differential and fractional centrifugation, the nuclei containing the chromatin were obtained. The chromatin DNA and protein absorbance ratio was determined at 260nm and 280nm by UV spectrophotometry. The results show that the chromatin ratio for control (untreated) rats was 0.95 while there were moderate increases in the ratio for treated rats. Significantly, the 260nm/280nm absorbance ratio increases occurred at 260nm and not 280nm, showing that chromatin DNA was more altered than chromatin protein. Conclusion and application of findings : Bonny light crude oil probably induced DNA polymerization by unscheduled DNA synthesis in chromatin, which suggests genotoxicity especially carcinogenicity. This demonstrates probable adverse impact to human health on exposure to crude oil spillage and pollution in air, land and water bodies.U blood and urine collected from prepubertal girls, we investigated proteomic biomarkers of cancer susceptibility. Proteomic analysis of blood serum utilized immunodepletion of the seven most abundant proteins using the Seppro IGY-R7 column system (Sigma). The flow-through fractions were digested, labelled, and combined with unique Isobaric Tandem Mass Tags (TMTs), and analyzed by an on-line automated 14 Fraction nano-LC-ESI-MS (SCX/ RP) MuDPIT (PQDLTQXL ThermoFinnigan). From the serum of prepubertal girls containing high urine concentrations of BPA and genistein, we were able to identify 1992 and 1364 peptides of which 97 and 59 were unique, respectively. In blood of girls with high concentrations of BPA in the urine, the proteins KI-67 and Intersectin (ITSN1) were up-regulated, and Deleted in Liver Cancer (DLC1) was down-regulated. Ki-67 is a cancer antigen presently being used as a tumor marker. ITSN1 is a scaffolding protein that regulates phosphotidyl-inositol 3-kinase and proliferation. Gene expression of DLC1 has been reported to be lower in breast carcinomas. In blood of girls with high urinary concentrations of genistein, Ribosomal L29 (RPL29), Eukaryotic Initiation Factor 3a (eIF-3a) and Endothelium-Converting Enzyme (ECE-1) were down regulated. Up-regulated RPL29 has been associated with a poor outcome for carcinomas of the breast, colo-rectum and esophagus. Expression of eIF-3a was elevated in breast cancer tissues compared with paired normal mammary tissues. Over expression of ECE-1 is associated with unfavorable outcome for breast cancer treatment. These data demonstrate that TMT-MS technology can be used for the identification of biomarkers of cancer susceptibility in human serum.I DUI species, two, potentially very divergent, mitochondrial DNA lineages exist within single male individuals. Contrary to the common, strictly maternal inheritance of, males in these species transmit their mtDNA to the male offspring. Despite more than two decades of research the phenomenon remains a mystery and even the very taxonomic distribution is unclear, although it seems to be limited to some dioecious bivalves. Its potential association with sex determination system(s) is to be confirmed. PCR with “universal” primers amplifying genomic DNA was employed so far in screening for DUI. This approach suffers from certain technical limitations: it depends on the apriori knowledge of sequences and is very sensitive to large sequence divergences: the DUI can easily be missed or falsely implied in the context of numts. The next generation transcriptome sequencing technology is capable of overcoming these problems. Raw data obtained from male gonadal tissues can be assembled and searched for mitochondrial transcripts using HMM profiles. This allowed identification of divergent sequences without any prior knowledge; the presence of two highly divergent sets of transcripts hinted at DUI. Obtaining the complete mtDNA sequences was then relatively straight forward, using combination of Long Range PCR and direct sequencing of re-PCR products. Here we present the preliminary analysis of transcriptomic data from several important bivalve species, showing the usefulness of the approach. The interplay of the two approaches: comparative transcriptomics and mitogenomics will hopefully lead to elucidation of the nature of DUI in particular and better understanding of mitochondrial inheritance in general.