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Journal of Bacteriology, June 2009, p. 3698-3705, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01527-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Hemolysin-Like Proteins Similar to RTX Toxin in Pasteurella pneumotropica{triangledown} ,{dagger}

Hiraku Sasaki,1* Eiichi Kawamoto,1 Yoshikazu Tanaka,2 Takuo Sawada,3 Satoshi Kunita,4 and Ken-ichi Yagami4

Animal Research Center, Tokyo Medical University, Shinjuku, Tokyo, Japan,1 Department of Veterinary Hygiene,2 Department of Veterinary Microbiology, Nippon Veterinary and Life Science University, Tokyo, Japan,3 Graduate School of Comprehensive Human Science and Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki, Japan4

Received 29 October 2008/ Accepted 29 March 2009

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.

* Corresponding author. Mailing address: Animal Research Center, Tokyo Medical University, 6-1-1, Shinjuku, Tokyo, Japan. Phone: 81-3-3351-6141. Fax: 81-3-3350-8659. E-mail: h-sasaki{at}tokyo-med.ac.jp

{triangledown} Published ahead of print on 10 April 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, June 2009, p. 3698-3705, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01527-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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