Identification and Characterization of Hemolysin-Like Proteins Similar to RTX Toxin in Pasteurella pneumotropica

Abstract

Pasteurella pneumotropica is an opportunistic pathogen that causes lethal pneumonia in immunodeficient rodents. The virulence factors of this bacterium remain unknown. In this study, we identified the genes encoding two RTX toxins, designated as pnxI and pnxII, from the genomic DNA of P. pneumotropica ATCC 35149 and characterized with respect to hemolysis. The pnxI operon was organized according to the manner in which the genes encoded the structural RTX toxin (pnxIA), the type I secretion systems (pnxIB and pnxID), and the unknown orf. The pnxII gene was involved only with the pnxIIA that coded for a structural RTX toxin. Both the structural RTX toxins of deduced PnxIA and PnxIIA were involved in seven of the RTX repeat and repeat-like sequences. By quantitative PCR analysis of the structural RTX toxin-encoding genes in P. pneumotropica ATCC 35149, the gene expression of pnxIA was found to have increased from the early log phase, while that of pnxIIA increased from the late log to the early stationary phase. As expressed in Escherichia coli, both the recombinant proteins of PnxIA and PnxIIA showed weak hemolytic activity in both sheep and murine erythrocytes. On the basis of the results of the Southern blotting analysis, the pnxIA gene was detected in 82% of the isolates, while the pnxIIA gene was detected in 39%. These results indicate that the products of both pnxIA and pnxIIA were putative associations of virulence factors in the rodent pathogen P. pneumotropica.Pasteurella pneumotropica is a gram-negative, rod-shaped bacterium that is frequently isolated from the upper respiratory tracts, digestive tracts, and vaginas of rodents (2,26). This bacterium is the major cause of pasteurellosis in immunodeficient and immunosuppressed animals, while certain effects on health are not observed in immunocompetent animals (2, 26). For the microbiological control of laboratory rodents, P. pneumotropica is one of the pathogens that must be prevented in rodent colonies. In P. pneumotropica infections, clinical diseases have generally presented with skin lesions, ophthalmitis, conjunctivitis, and otitis media (2); furthermore, clinical diseases have also been known to lead to fatal pneumonia in immunodeficient animals (15,24,26).Although details of the virulence factors of P. pneumotropica are still unavailable, predicted virulence associations have been reported (4,15,16,26). Of these, hemolysis is one of the phenotypic characteristics widely distributed in pathogenic bacteria. In particular, many pathogens that belong to the family Pasteurellaceae possess one of the pore-forming protein toxins occurring concurrently with hemolysis-a toxin determined to be a repeat in the structural toxin (RTX toxin) (42). RTX toxins are recognized as members of the type I exoprotein secretion system and were first characterized as hemolysins and leukotoxins produced by Actinobacillus pleuropneumoniae, Aggregatibacter (Actinobacillus) actinomycetemcomitans, Bordetella pertussis, enterohemorrhagic Escherichia coli, and Mannheimia haemolyti...

Pathogenicity of Pasteurella pneumotropica in Immunodeficient NOD/ShiJic-scid/Jcl and Immunocompetent Crlj:CD1 (ICR) Mice

“…However, the virulence factors of bacterial infection in laboratory animals have not been thoroughly studied. Recently, we described the identification and characterization of the RTX toxins [19,21]. The P. pneumotropica animal model developed in this study will be useful for investigating virulence factors such as RTX toxin.…”

Experimental Contact Infection of NOD/ShiJic-scid Mice with Pasteurella pneumotropica

“…We are currently investigating the virulence factors of P. pneumotropica. In our previous studies, several RTX toxins have been identified in these bacteria [19,20]. Furthermore, several candidate virulence-associated genes such as RTX toxins were identified from a draft genome sequence of P. pneumotropica [21,22].…”

“…The strain possessed outermost surface materials, which may be pili, on the bacterial cells and belonged to group two based on biochemical and genetic characteristics [17,18]. This strain produces RTX toxins PnxIA, IIA, and IIIA, and its draft genome sequence has been revealed [19,20]. The strain was thawed from frozen stocks (-80°C) and grown overnight on 5% horse blood agar plates (Sanko Junyaku Co. Ltd, Tokyo, Japan) and checked for purity.…”

Intranasal immunization with a non-adjuvanted adhesive protein descended from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection in mice

“…In current studies, several RTX (repeats in toxin) toxins were identified in this species, and of these, 250-kDa PnxI-IIA contains bacterial Ig-like domains and hemagglutinin repeats that are thought to be indispensable for the protein's attachment and invasion of bacterial cells into host organs [15,16]. In fact, the recombinant PnxIIIA that lacks these domains failed to bind to the extracellular matrix (ECM) [16].…”

“…In fact, the recombinant PnxIIIA that lacks these domains failed to bind to the extracellular matrix (ECM) [16]. PnxIIIA is reportedly less cytotoxic toward leukocytes as compared to other RTX family proteins, and therefore, PnxIIIA is one of the candidate antigens for immunization against P. pneumotropica infection in rodents [15,16]. In this regard, ECM adhesive proteins help long-term retention in the nasal cavity, which results in enhanced opportunities for NALT cell recognition when PnxIIIA is used in a vaccine for intranasal immunization.…”