Cloning, characterization and expression of a novel metallothionein gene from triticum and theoretical and experimental structure-function relationship prediction

Metallothioneins (MTs) are small, cystein rich, low molecular mass polypeptides found in almost all organisms. They are thought to be involved in heavy-metal detoxification and metabolism of essential trace elements like copper and zinc. Unlike their mammalian counterparts, plant MTs have not been thoroughly characterized in terms of cellular regulation and function. A novel gene, from Triticum durum (pasta wheat), coding for plant MT type 1 protein was isolated and characterized. The durum mt gene was cloned in E. coli for solution X-ray scattering studies to obtain the first experimental structural data on a plant MT in the literature. Triticum durum mt gene was shown to contain 2 exons and a non-coding intron region. The coded MT protein, showing high similarity to mammalian MTs in its cystein residue distribution pattern, forms two metal binding domains bridged with an exceptionally long connecting region. This hinge region was shown to be highly conserved among plant MTs using sequence alignment algorithms on data available in the literature. Homology modeling and heuristic fragment assembly approaches were used to predict a 3D structure for the durum MT (dMT). Guided by the predicted structures, functional motif and structure searches were performed yielding a possible DNA binding and/or protein interaction function for dMT. High probability of wMT to form dimers or trimers inside the solution was also speculated. dMT was expressed in E. coli cells as a fusion protein with GST and preliminary X-ray solution scattering measurements were carried out on the purified recombinant v protein. These measurements indicated the high tendency of the protein to form aggregates in solution. Theoretical predictions and solution scattering measurements were also supported by the results of polyacrylamide gel electrophoresis and size exclusion chromatography analysis of expressed and purified recombinant dMT and GSTdMT proteins. Further, sequence and structure analyses showed a high structure and sequence similarity between dMT hinge region and the DNA binding domain of a cyanobacterial metallothionein suppressor protein (SmtB). Indeed, the results indicate that dMT metal binding domains would also bind to DNA with very high probability. These results, altogether, point to a new role for plant MTs other than metal scavenging such as being a transcription factor or a gene suppressor

Performances and Reliability of Bruker Microflex LT and VITEK MS MALDI-TOF Mass Spectrometry Systems for the Identification of Clinical Microorganisms

In clinical microbiology laboratories, routine microbial identification is mostly performed using culture based methodologies requiring 24 to 72 hours from culturing to identification. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology has been established as a cost effective, reliable, and faster alternative identification platform. In this study, we evaluated the reliability of the two available MALDI-TOF MS systems for their routine clinical level identification accuracy and efficiency in a clinical microbiology laboratory setting. A total of 1,341 routine phenotypically identified clinical bacterial and fungal isolates were selected and simultaneously analyzed using VITEK MS (bioMérieux, France) and Microflex LT (Bruker Diagnostics, Germany) MALDI-TOF MS systems. For any isolate that could not be identified with either of the systems and for any discordant result, 16S rDNA gene or ITS1/ITS2 sequencing was used. VITEK MS and Microflex LT correctly identified 1,303 (97.17%) and 1,298 (96.79%) isolates to the species level, respectively. In 114 (8.50%) isolates initial phenotypic identification was inaccurate. Both systems showed a similar identification efficiency and workflow robustness, and they were twice as more accurate compared to routine phenotypic identification in our sample pool. MALDITOF systems with their accuracy and robustness offer a good identification platform for routine clinical microbiology laboratories

Antimicrobial aptamers for detection and inhibition of microbial pathogen growth

Discovery of alternative sources of antimicrobial agents are essential in the ongoing battle against microbial pathogens. Legislative and scientific challenges considerably hinder the discovery and use of new antimicrobial drugs, and new approaches are in urgent demand. On the other hand, rapid, specific and sensitive detection of airborne pathogens is becoming increasingly critical for public health. In this respect affinity oligonucleotides, aptamers, provide unique opportunities for the development of nanotechnological solutions for such medical applications. In recent years, aptamers specifically recognizing microbial cells and viruses showed great potential in a range of analytical and therapeutic applications. This article describes the significant advances in the development of aptamers targeting specific pathogens. Therapeutic application of aptamers as neutralizing agents demonstrates great potential as a future source of antimicrobial agent

Vibrio cholerae hemolysin is required for lethality, developmental delay, and intestinal vacuolation in Caenorhabditis elegans.

BACKGROUND: Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations. CONCLUSIONS/SIGNIFICANCE: Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes

Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

Two-component systems play important roles in the physiology of many bacterial pathogens. Vibrio cholerae’s CarRS two-com-ponent regulatory system negatively regulates expression of vps (Vibrio polysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of the almEFG genes, whose prod-ucts are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory re-gion of the almEFG operon. Similarly to a carR mutant, strains lacking almE, almF, and almG exhibited enhanced polymyxin B sensitivity. We also observed that strains lacking almE or the almEFG operon have enhanced biofilm formation. Our results re-veal that CarR regulates biofilm formation and antimicrobial peptide resistance in V. cholerae. Vibrio cholerae, a Gram-negative enteric pathogen, is the caus-ative agent of the diarrheal disease cholera. To establish infec-tion,V. cholerae senses and responds to host defenses encountered during the infection cycle. As an enteric pathogen, V. cholerae needs to launch a defense against antimicrobial peptides (APs), such as bactericidal permeability-increasing cationic protein (BPI), -defensins, -defensins, and cathelicidin (LL-37) pro-duced in the human intestine (1, 2). It was shown that the outer membrane protein OmpU confers resistance to the P2 peptid

<i>hlyA</i> is required for killing during <i>V. cholerae</i> infection in <i>C. elegans</i>.

<p>Lethality analysis was performed in <i>glp-4(bn2)</i> worms that were fed with indicated bacterial strains (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011558#pone-0011558-t001" target="_blank">Table 1</a>). Agar plates were kept at 25°C and scored for survivors at 24–48 hour intervals. Data were plotted according to a Kaplan-Meier method and survival curves were compared using the logrank test. <i>p</i><0.005. CVD110 and CVD109 represent the <i>hlyA</i> deficient and <i>hlyA</i> containing vaccine strains respectively, and they are isogenic with E7946. strR, streptomycin resistance; Δ<i>hlyA, hlyA</i> deletion; CVD110 strR/pHNC44:<i>hlyA</i> is the complementation strain. A) Comparison of lethality caused by vaccine strains CVD110 (<i>hlyA</i>-), CVD109 (<i>hlyA</i>+) and <i>V. cholerae</i> WT strain E7946. CVD110 exposed worms: median survival–10 days, CVD109 exposed worms: median survival–5 days, E7946 exposed worms: median survival-5 days. p<0.0001 for CVD110 versus CVD109 and for CVD110 versus E7946. p = 0.3455 for E7946 versus CVD109. B) Comparison of lethality caused by <i>hlyA</i> deletion mutant, WT strain E7946 and CVD110. p<0.0001 for E7946 strR versus E7946 strR <i>ΔhlyA</i> and for E7946 strR versus CVD110 strR. p = 0.1383 for E7946 strR Δ<i>hlyA</i> versus CVD110 strR. C) Comparison of lethality caused by CVD110, CVD110 with <i>hlyA</i> expressing plasmid and CVD109. p<0.0001 for each curve comparison in this graph.</p

<i>V. cholerae</i> causes growth retardation of worm cultures via <i>hlyA</i> gene.

<p>Synchronized L1 stage N2 worms were fed on indicated bacterial strains on agar plates for 72 hours at 22°C. Worms washed into M9 buffer were sorted using COPAS (n = 1000). Images in A to E indicate the composition of worm cultures prior to sorting. Black arrows indicate eggs, white arrows indicate adult nematodes. (F) Curve representing the optical density (EXT) distribution of sorted worms was plotted. <i>E. coli</i> OP50 fed worm populations showed two separable peak domains (indicated with arrows), <i>V. cholerae</i> A1552 fed worms showed a single peak (indicated with an arrowhead) falling between <i>E. coli</i> OP50 induced peaks that is consistent with a population of worms mostly larger than eggs but fail to reach adult sizes EXT, extinction; strR, streptomycin resistance. (G) Population growth of <i>C. elegans</i> fed with <i>hlyA</i> deleted, <i>hlyA</i> intact <i>V. cholerae</i> strains and OP50. Student's <i>t</i> test was used to compare growth. ** denotes a statistical significance of P<0.001 according to Student's t test; *** P<0.0001. Total EXT represents the sum of EXT values for sorted worms per condition.</p