Novel targets for antimicrobial molecules in pseudomonas aeruginosa : identification and characterization of an essential membrane eukaryotic-like transglutaminase

Pseudomonas aeruginosa is a highly adaptable bacterium which thrives in a broad range of ecological niches and can infect multiple hosts as diverse as plants, nematodes and mammals. In humans, it is an important opportunistic pathogen in compromised individuals, such as patients with cysticfibrosis, severe burns and impaired immunity. This bacterium is also noted for its resistance, often multiple, to many antibiotics. This urgently calls for novel strategies for treatment and prevention of Pseudomonas aeruginosa infections. In this context, the discovery of novel essential genes or pathways, not yet targeted by common antibiotics, play a key role in the development of new antimicrobial molecules. We used the technology of interfering antisense RNAs to screen novel essential functions in Pseudomonas aeruginosa. Among several positives to our screenings, we focused on a protein of 688 aa, that we named PtgC. Firstly, the essential role of the ptgC gene was validated both by insertional and conditional mutagenesis. Then, we concentrated on functional aspects of PtgC, for which bioinformatic analysis, listed in the Pseudomonas Genome Database (PGD) (www.pseudomonas.com), indicated is an inner membrane protein endowed with 6 transmembrane helices and a large periplasmic domain encompassing a highly recognizable structural TG subdomain belonging to the transglutaminase-like superfamily. Unfortunately, PtgC was annotated in PGD as “hypothetical protein,” i.e. no experimental evidence of in vivo expression was available and thus its existence had only been predicted during bioinformatic genome analysis. To provide evidence of PtgC expression, we isolated membrane fractions, digested them with trypsin and identified the resulting tryptic peptides through Multidimensional Protein Identification Technology (MudPIT). Among the peptides identified by this approach, we found 6 peptides belonging to the PtgC periplasmic domain. These results validate PtgC expression and strongly suggest that PtgC localizes in membrane compartments. To assess whether the conservation of the structural TG domain correlated with transglutaminase activity, an N-(His)10-PtgC periplasmic domain (PtgCPD) was purified and tested through a colorimetric microassay, using purified guinea pig transglutaminase as positive control. PtgCPD displayed a positive transglutaminase test, with a specific activity that was about 45% of that of the guinea pig counterpart. We plan to design and test inhibitors of PtgCPD transglutaminase activity, which will be further assayed for antimicrobial effects on P. aeruginosa, both on cultured bacteria and in animal models of lung infection. Moreover, we plan to test in animal models the effectiveness of peptide nucleic acids (PNAs), targeting the ptgC mRNA, which were shown to elicit growth inhibition of cultured Pseudomonas aeruginosa