Steps toward broad-spectrum therapeutics: discovering virulence-associated genes present in diverse human pathogens.

© 2009 Stubben et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: New and improved antimicrobial countermeasures are urgently needed to counteract increased resistance to existing antimicrobial treatments and to combat currently untreatable or new emerging infectious diseases. We demonstrate that computational comparative genomics, together with experimental screening, can identify potential generic (i.e., conserved across multiple pathogen species) and novel virulence-associated genes that may serve as targets for broad-spectrum countermeasures. Results: Using phylogenetic profiles of protein clusters from completed microbial genome sequences, we identified seventeen protein candidates that are common to diverse human pathogens and absent or uncommon in non-pathogens. Mutants of 13 of these candidates were successfully generated in Yersinia pseudotuberculosis and the potential role of the proteins in virulence was assayed in an animal model. Six candidate proteins are suggested to be involved in the virulence of Y. pseudotuberculosis, none of which have previously been implicated in the virulence of Y. pseudotuberculosis and three have no record of involvement in the virulence of any bacteria. Conclusion: This work demonstrates a strategy for the identification of potential virulence factors that are conserved across a number of human pathogenic bacterial species, confirming the usefulness of this tool

Dependence of Dam activity on substrate concentration. (A) AdoMet and (B) oligonucleotide 1.

<p>Dependence of Dam activity on substrate concentration. (A) AdoMet and (B) oligonucleotide 1.</p

A comparison of <i>Y. pestis</i> and <i>E. coli</i> Dam kinetic parameters.

<p>Kinetic data for <i>Y. pestis</i> Dam was obtained from a SigmaPlot fitted hyperbola of the form <i>v</i> = <i>V</i>_max [S]/<i>K</i>_M+[S].</p

High throughput assay validation.

<p>Rate of fluorescence increase was monitored over 96 wells (wells 1–48 contained positive control assays, wells 49–96 contained negative control assays with no Dam). The solid trend lines indicate the mean rate of fluorescence increase for the positive/negative control datasets; the broken lines indicate the ±3 standard deviations from the mean, which is the 99.7% confidence limit.</p

Break light Dam activity assay.

<p>The fluorescence of the hemimethylated substrate oligonucleotide 1 is internally quenched by the dabcyl group. It is a substrate for Dam and yields the fully methylated product 2, which is rapidly cleaved by DpnI, thus forming fluorescent oligonucleotide 3.</p

Inhibition of Dam by <i>S</i>-adenosylhomocysteine.

<p>(A) A double reciprocal plot of methylation rate against AdoMet concentration. <i>S</i>-adenosylhomocysteine concentrations were; 0 (•), 5 (○), 10 (▾), 15 (Δ), 20 (▪), 30 (□) and 40 (♦) µM. (B) Apparent <i>K</i>_M plotted against the concentration of <i>S</i>-adenosylhomocysteine.</p

Kinetic analysis of yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide

Background: DNA adenine methylation plays an important role in several critical bacterial processes including mismatchrepair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulenceon DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broadspectrum antibiotics. Methodology/Principal Findings: herein we report the expression and purification of Yersinia pestisDam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that issuitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Daminhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site.When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting inseparation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays weremonitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisationof Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated forhigh throughput screening, giving a Z-factor of 0.7160.07 indicating that it is a sensitive assay for the identification ofinhibitors. Conclusions/Significance: the assay is therefore suitable for high throughput screening for inhibitors of DNAadenine methyltransferases and the kinetic characterisation of the inhibitio

Immunodominant Francisella tularensis antigens identified using proteome microarray.

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens