Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica

Abstract

BackgroundPasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system.ResultsThe RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA) was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs) and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA.ConclusionsThe results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

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 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]. 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].…”