Superantigen profiles of emm and emm-like typeable and nontypeable pharyngeal streptococcal isolates of South India

<p>Abstract</p> <p>Background</p> <p>The major virulence factors determining the pathogenicity of streptococcal strains include M protein encoded by <it>emm </it>and <it>emm</it>-like (<it>emmL</it>) genes and superantigens. In this study, the distribution of <it>emm, emmL </it>and superantigen genes was analyzed among the streptococcal strains isolated from the patients of acute pharyngitis.</p> <p>Methods</p> <p>The streptococcal strains were isolated from the throat swabs of 1040 patients of acute pharyngitis. The <it>emm </it>and <it>emmL </it>genes were PCR amplified from each strain and sequenced to determine the <it>emm </it>types. The dot-blot hybridization was performed to confirm the pathogens as true <it>emm </it>nontypeable strains. The presence of eleven currently known superantigens was determined in all the strains by multiplex PCR.</p> <p>Results</p> <p>Totally, 124 beta-hemolytic streptococcal strains were isolated and they were classified as group A streptococcus (GAS) [15.3% (19/124)], group C streptococcus (GCS) [59.7% (74/124)] and group G streptococcus (GGS) [25.0% (31/124)]. Among 124 strains, only 35 strains were <it>emm </it>typeable and the remaining 89 strains were <it>emm </it>nontypeable. All GAS isolates were typeable, whereas most of the GCS and GGS strains were nontypeable. These nontypeable strains belong to <it>S. anginosus </it>[75.3% (67/89)] and <it>S. dysgalactiae </it>subsp. <it>equisimilis </it>[24.7% (22/89)]. The <it>emm </it>and <it>emmL </it>types identified in this study include <it>emm12.0 </it>(28.6%), <it>stG643.0 </it>(28.6%), <it>stC46.0 </it>(17.0%), <it>emm30.11 </it>(8.5%), <it>emm3.0 </it>(2.9%), <it>emm48.0 </it>(5.7%), <it>st3343.0 </it>(2.9%), <it>emm107.0 </it>(2.9%) and <it>stS104.2 </it>(2.9%). Various superantigen profiles were observed in typeable as well as nontypeable strains.</p> <p>Conclusions</p> <p>Multiplex PCR analysis revealed the presence of superantigens in all the typeable strains irrespective of their <it>emm </it>types. However, the presence of superantigen genes in <it>emm </it>and <it>emmL </it>nontypeable strains has not been previously reported. In this study, presence of at least one or a combination of superantigen coding genes was identified in all the <it>emm </it>and <it>emmL </it>nontypeable strains. Thus, the superantigens may inevitably play an important role in the pathogenesis of these nontypeable strains in the absence of the primary virulence factor, M protein.</p

Evaluation of INSeq To Identify Genes Essential for

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P aeruginosa PGPR2 for root colonization

A simple method for selective removal of human DNA from oral microbiome for functional metagenomic applications

572-576This study presents a simple filtration based technique to reduce human DNA contamination in mouthwash samples. Quantitative PCR evaluated efficiency of proposed method in removal of human DNA and selective extraction of bacterial DNA. The b-globin and 16S rDNA were targeted to measure the level of human and bacterial DNA respectively. When compared to unfiltered metagenomic DNA, there was 89% reduction in human DNA along with proportionate enrichment of bacterial DNA was obtained in filtered DNA. This strategy can serve as an efficient method for removal of human DNA and enrichment of microbial DNA in mouth wash samples usefull for functional metagenomic applications. </span

Mechanisms of the antifungal action of marine metagenome-derived peptide, MMGP1, against Candida albicans.

BackgroundDevelopment of resistant variants to existing antifungal drugs continues to be the serious problem in Candida albicans-induced fungal pathogenesis, which has a considerable impact on animal and human health. Identification and characterization of newer drugs against C. albicans is, therefore, essential. MMGP1 is a direct cell-penetrating peptide recently identified from marine metagenome, which was found to possess potent antifungal activity against C. albicans.Methodology/principal findingsIn this study, we investigated the mechanism of antifungal action of MMGP1 against C. albicans. Agarose gel shift assay found the peptide to be having a remarkable DNA-binding ability. The modification of the absorption spectra and fluorescence quenching of the tryptophyl residue correspond to the stacking between indole ring and nucleotide bases. The formation of peptide-DNA complexes was confirmed by fluorescence quenching of SYTO 9 probe. The interaction of peptide with plasmid DNA afforded protection of DNA from enzymatic degradation by DNase I. In vitro transcription of mouse β-actin gene in the presence of peptide led to a decrease in the level of mRNA synthesis. The C. albicans treated with MMGP1 showed strong inhibition of biosynthetic incorporation of uridine analog 5-ethynyluridine (EU) into nascent RNA, suggesting the peptide's role in the inhibition of macromolecular synthesis. Furthermore, the peptide also induces endogenous accumulation of reactive oxygen species (ROS) in C. albicans. MMGP1 supplemented with glutathione showed an increased viability of C. albicans cells. The hyper-produced ROS by MMGP1 leads to increased levels of protein carbonyls and thiobarbituric acid reactive substances and it also causes dissipation of mitochondrial membrane potential and DNA fragmentation in C. albicans cells.ConclusionAnd Significance: Therefore, the antifungal activity of MMGP1 could be attributed to its binding with DNA, causing inhibition of transcription followed by endogenous production of ROS, which triggers cascade of events that leads to cell death

Biosynthesis and characterization of mercury sulphide nanoparticles produced by <i style="mso-bidi-font-style:normal">Bacillus cereus</i> MRS-1

973-978Mercury is a highly toxic heavy metal accumulated in the environment, which can be detoxified by reducing Hg2+ to non toxic form. Bacteria resistant to toxic metals and capable of converting them into non toxic forms have a direct application in the bioremediation of contaminated sites. In this study, mercury resistant strain Bacillus cereus MRS-1 was isolated from electroplating industrial effluent. This strain exhibited the ability to convert mercury into extracellular sulphide nanoparticles of mercury. The recovered HgS nanoparticles have been characterized by <span style="mso-bidi-font-size:9.0pt;font-style:normal;mso-bidi-font-style: italic" lang="EN-GB">UV-<span style="mso-bidi-font-size:9.0pt;font-style:normal;mso-bidi-font-style: italic" lang="EN-GB">VIS<span style="font-size:12.0pt;font-style: normal;mso-bidi-font-style:italic" lang="EN-GB"> spectrophotometer, FT-IR, atomic force microscopy, transmission electron microscopy, scanning electron microscopy, energy dispersive X-ray analysis, powder X-ray diffraction pattern and thermogravimetric analysis. The synthesized nanoparticles were spherical with a size range of 10–100 nm. This strain can be potentially exploited for the production of HgS nanoparticles as well as for detoxification of mercury in the environment without producing secondary pollution of mercury methylation or Hg (0) volatilization. </span

Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2).

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction

MMGP1-induced intracellular oxidation of proteins and lipids in <i>C. albicans</i>.

<p>(a) Time-dependent measurement of protein carbonyls in MMGP1 treated <i>C. albicans</i> cells by DNPH assay. (b) Time-dependent measurement of TBARS production in MMGP1 treated <i>C. albicans</i> cells by TBA assay.</p

Effect of proteinase K and DNase I on DNA–MMGP1 complexes.

<p>(a) Treatment of DNA–MMGP1 complexes with proteinase K resulted in detection of plasmid DNA band on the agarose gel. C-Control SK+ plasmid DNA; 1-DNA–MMGP1 complex formed at 0.576 µM concentration of peptide; 2-DNA–MMGP1 complex treated with proteinase K (b) DNase I protection assay. The plasmid DNA (100 ng) was preincubated with the varying concentrations peptides such as, C+-no peptide; 1-0.576 µM; 2-0.288 µM; 3- 0.144 μM; 4-0.072 µM; 5-0.036 µM, respectively followed by treatment with DNase I. The DNase treated plasmid DNA was used as negative control (C-).</p

<i>In vitro</i> transcription inhibition by MMGP1.

<p>(a) Expression of mouse β-actin gene in the presence of different concentrations of MMGP1 C-no peptide, 1-0.576 µM, 2-0.288 µM, 3- 0.144 μM, 4- 0.072; 5- 0.036 at 37 ^°C for 1 h under <i>in vitro</i> condition. The transcribed product of 304 bases were analysed on 5% denaturing polyacrylamide gel. (b) Quantitative measurement of mouse β-actin gene expression in the presence of varying concentrations of MMGP1 by gel densitometry analysis.</p