Characterization of PaxA and its operon: a cohemolytic RTX toxin determinant from pathogenic Pasteurella aerogenes

Abstract

Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times.

FIG. 1

Southern blot of P. aerogenes and A. pleuropneumoniae with apxIIICA as probe. Genomic DNA of P. aerogenes JF1319 from a swine abortion case and A. pleuropneumoniae serotype 2 reference strain ATCC 27089 (S1536) was digested with EcoRI. After electrophoresis on a 1% agarose gel and transfer to nylon membrane, the filter was hybridized with the digoxigenin-labeled apxIIICA-derived probe.

FIG. 2

Restriction map of pax operon from P. aerogenes JF 1319 and positions of the different clones used for determination of its sequence. The black arrows represent the four genes in the pax operon, with the arrowheads showing the direction of transcription.

FIG. 3

Amino acid sequence of PaxA. The seven consensus glycine-rich nonapeptide sequences are double underlined. The four preceding similar nonapeptide repeats are underlined.

FIG. 4

Southern blot of P. aerogenes strains. Genomic DNA of the type strain ATCC 27883^T and 12 clinical isolates was digested with PstI, electrophoresed on 1% agarose gel, and transferred to nylon membranes. (A) Hybridization with digoxigenin-labeled paxCA derived probe. (B) Hybridization with digoxigenin-labeled paxBD-derived probe. M, λHindIII marker, showing the 2.3- and the 2.0-kb fragments.

FIG. 5

CAMP test with P. aerogenes isolates and recombinant E. coli K-12 strains. Strains were grown in the vicinity of S. aureus (b). The diffusion zone of the sphingomyelinase is also indicated (a). The four pax-positive P. aerogenes isolates JF1319 (lane 3), JF2006 (lane 4), JF2032 (lane 5), and JF2118 (lane 6) show a distinct zone with complete hemolysis, as does A. pleuropneumoniae serotype 3 reference strain ATCC 27090 (lane 1). The P. aerogenes type strain (lane 2), which does not contain the pax operon, shows no CAMP effect. The CAMP effect in P. aerogenes is due to the presence of pax, as shown with an E. coli K-12 DH5α transformed with the plasmid pPaxCABD containing the complete functional operon (lane 9). Neither the DH5α wild-type strain (lane 7) nor the strain containing only paxCA genes on plasmid pPaxCA (lane 8) show the CAMP reaction. A mild CAMP effect is also seen in E. coli 5K strain harboring the hlyBD genes on plasmid pLG575 and plasmid pPaxCA (lane 11) but not in the control strain containing only hlyBD (lane 10).