Characterization of RTX toxins in Pasteurella pneumotropica and development of methods for detection and identification of the RTX toxins.

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This study was conducted on identification of the proteins similar to RTX toxins and characterization of virulence properties of these toxins in rodent pathogen Pasteurella pneumotropica. Further, the methodology of detection and identification of these toxins was developed and evaluated. In genomic DNA of P. pneumotropica reference strain, total three proteins similar to RTX exoproteins-coding genes were identified. These RTX-like proteins were termed as PnxIA, PnxIIA, and PnxIIIA. All the recombinant proteins exhibited the cytotoxicity toward J774A.1 mouse macrophage cells. In particular, recombinant PnxIA and PnxIIA showed weak hemolytic activity toward sheep and mouse erythrocytes, otherwise recombinant PnxIIIA (rPnxIIIA) showed insignificant hemolytic activity. PnxIIIA has bacterial Ig-like domain and hemagglutinin repeat in primary structure. Thus, rPnxIIIA allowed to further evaluate extracellular matrices (ECMs) binding assay and hemagglutination assay. As results obtained from those experiments, rPnxIIIA showed significant binding ability toward rat collagen type I, and more than 30 ng/ml of rPnxIIIA showed hemagglutination of washed sheep erythrocytes. For the detection and identification of three RTX proteins in wild-type P. pneumotropica strains, genetic techniques and immunological methods were experimentally compared. However, these RTX proteins and -coding genes were highly diversified than those in reference strains. Therefore, it needs further evaluation and clarification of the molecular features of the RTX proteins in wild-type P. pneumotropica strains in future studies.