Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae

Background

Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance.

Principal Findings

Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology.

Conclusions and Significance

The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.     

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

Background

Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/Principal Findings

Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/Significance

The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.     

Hydrogen sulfide protects SH-SY5Y neuronal cells against d-galactose induced cell injury by suppression of advanced glycation end products formation and oxidative stress

d-Galactose is widely used as an agent to cause aging effects in experimental animals. The present study aims to investigate the effects of hydrogen sulfide (H₂S) in human neuroblastoma SH-SY5Y cells exposed to d-galactose. Cells were pretreated with NaHS, an H₂S donor, and then exposed to d-galactose (25–400 mM for 48 h). We found that NaHS pretreatment significantly reversed the d-galactose-induced cell death and cellular senescence. MTT assay shows that NaHS significantly increased cell viability from 62.31 ± 1.29% to 72.34 ± 0.46% compared with d-galactose (200 mM) treatment group. The underlying mechanism appeared to involve a reduction by NaHS in the formation of advanced glycation end products (AGEs), which are known to contribute to the progression of age-related diseases. In addition, NaHS decreased the elevation of reactive oxygen species from 151.17 ± 2.07% to 124.8 ± 2.89% and malondialdehyde from 1.72 ± 0.07 to 1.10 ± 0.08 (nmol/mg protein) in SH-SY5Y cells after d-galactose exposure. NaHS also stimulated activities of superoxide dismutase from 0.42 ± 0.05 to 0.73 ± 0.04 (U/mg protein) and glutathione peroxidase from 3.98 ± 0.73 to 14.73 ± 0.77 (nmol/min/mg protein) and upregulated the gene expression levels of copper transport protein ATOX1, glutathione synthetase (GSS) and thioredoxin reductase 1 (TXNRD1) while down-regulated aldehyde oxidase 1 (AOX1). In summary, our data indicate that H₂S may have potentially anti-aging effects through the inhibition of AGEs formation and reduction of oxidative stress.     

Analysis of genetic diversity and population structure in <Emphasis Type="Italic">Capsicum</Emphasis> landraces from North Eastern India using TE-AFLP markers

North eastern (NE) India harbours a precious germplasm repository of Capsicum in the form of various landraces. The present study was undertaken to characterise the extent of genetic variation present in different Capsicum landraces from north eastern India. A set of 171 Capsicum accessions were characterised using three-endonuclease amplified fragment length polymorphism (AFLP) markers. Out of 416 bands obtained from six primer combinations, 254 (61 %) were polymorphic. The pairwise genetic dissimilarity among accessions ranged from 0.03 to 0.97. Cluster analysis based on neighbour joining showed two major clusters. Cluster I contained most of the bhut jolokia accessions whereas cluster II contained all of the Capsicum annuum genotypes. Similar grouping was observed with population STRUCTURE analysis as well as principle coordinate analysis. Analysis of molecular variance (AMOVA) revealed 45 and 54 % variation among and within populations, respectively. This information on population structure analysis and molecular characterisation will be helpful for effective utilisation of this germplasm in Capsicum improvement programs.     

Studies on the dephosphorylation of phytic acid in livestock feed using phytase from Aspergillus niger van Teighem

Phytase from Aspergillus niger van Teighem efficiently hydrolyses phytate phosphorus present in various commercial live stock feeds and was not inactivated by various formulations and antibiotics present. The enzyme retained 90-95% phytase activity at 55 degrees C, pH 2.5 after 72 h of incubation with all the commercial feeds tested, thus indicating its suitability in feed application. The phytase hydrolysis increased with the increase in temperature and a significant release of 41 nmols P(i)/ml in phytase-treated feed over control sample was observed at 55 degrees C after 48 h. Besides this, the enzyme was maximally effective when used under acidic condition, releasing 21 and 42 nmols P(i)/ml at pH 1.5 and 2.5, respectively. As the pH shifted towards 5.5, significant decline in phosphorus release was observed. However, the enzyme was able to retain almost complete phytase activity in the presence of feed constituent even after 48 h over various pH tested. Thus it can be a potential candidate in animal nutrition where the ability of present phytase to retain activity over period of time in the presence of feed constituent is desired.     

Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp. BG-11

An alkalophilic, chitinase-producing Bacillus sp. BG-11 was isolated which produced an extracellular chitinase and which was purified 16.5-fold, using standard purification techniques. The purified chitinase exhibited a broad pH and temperature optima of 7.5-9.0 and 45 deg C-55 deg C, respectively. The chitinase was stable between pH 6.0-9.0 and 50°C for more than 2 h. Half lives of enzyme at 60 deg C, 70 deg C and 80 deg C were 90 min, 30 min and 20 min respectively. Km value was 12 mg chitin per ml. Shelf life was 60 days at 4°C. Ca2+, Ni2+ and Triton-X-100 stimulated the activity up to 20% whereas Ag+, Hg2+, dithiothreitol, -mercaptoethanol, glutathione, iodoacetic acid and iodoacetamide inhibited the activity up to 50%.     

Oral Delivery of Peptide Formulations and Their Cellular Evaluation

International Journal of Peptide Research and Therapeutics - The development of peptide-based formulations presents numerous challenges to the formulator due to their complexity, delicate structure...     

Reconstitution of rapeseed high molecular weight protein from isolated subunits

The high molecular weight protein was isolated from rapeseed and characterised. Six subunits were isolated in SDS (0.01%) solution on polyacrylamide-gel electrophoresis and by gel filtration on Sephadex G-100. Reassociation by removing SDS by co-dialysis, against 10 mM sodium phosphate buffer (pH 7.9) was done and the yield was about 90%. The reconstituted protein was indistinguishable from the intact protein in all respects. The subunits isolated from the native protein and the reconstituted protein were found to have identical molecular weights and N-terminal amino acids. No disulphide bonds were observed in the subunit association. Amino acid analysis of the proteins and the six subunits was performed and the number of each amino acid residue calculated.     

Effect of amino acids on production of xylanase and pectinase from Streptomyces sp. QG-11-3

Xylanase and pectinase production by Streptomyces sp. QG-11-3 was stimulated by DL-norleucine, L-leucine, DL-isoleucine, L-lysine monohydrochloride and DL--phenylalanine by up to 3.72- and 2.78-fold, respectively, whereas the combination of DL-norleucine, L-leucine and DL-isoleucine synergistically stimulated the xylanase and pectinase production by up to 6.72- and 5.62-fold, respectively. Glycine, DL-norvaline, DL-methionine, and DL-aspartic acid showed no significant stimulatory effect on enzyme production.