In vivo-expressed genes of Pasteurella multocida

Abstract

Pasteurella multocida is the causative agent of infectious diseases of economic importance such as fowl cholera, bovine hemorrhagic septicemia, and porcine atrophic rhinitis. However, knowledge of the molecular mechanisms and determinants that P. multocida requires for virulence and pathogenicity is still limited. To address this issue, we developed a genetic expression system, based on the in vivo expression technology approach first described by Mahan et al. (Science 259:686--688, 1993), to identify in vivo-expressed genes of P. multocida. Numerous genes, such as those encoding outer membrane lipoproteins, metabolic and biosynthetic enzymes, and a number of hypothetical proteins, were identified. These may prove to be useful targets for attenuating mutation and/or warrant further investigation for their roles in immunity and/or pathogenesis.

FIG. 1

Construction of the PmIVET in vivo promoter-probe vector pMKΩ. The P. multocida plasmid origin of replication region (ori) is shown on each plasmid as a black box. The open circle at the 5′ end of the kan gene indicates the ribosome binding site, AGGA. The oligonucleotide primers BAP-03 and BAP-04, used for inverse PCR to introduce a unique BamHI restriction site, are shown as bent arrows above and below the sequence in the expanded boxed section. The ribosome binding site preceding the start of the kan gene is circled. The positions of two sequencing primers, BAP-503 and BAP-1056, are denoted by the small arrows. Restriction site abbreviations: A, AvaI; B, BamHI; Bg, BglII; C, ClaI; EV, EcoRV; H, HindIII; HII, HindII; K, KpnI. #, the approximate size of pPMK1, published as 4.5 kb by Bills et al. (6), has been subsequently reestimated to be 4.9 kb as shown here.

FIG. 2

Survival of P. multocida strain X-73(pPMK1) (▴, ×) and X-73(pMKΩ) (▪, ♦) in KAN-treated mice. The arrows indicate the times at which a KAN dose of 100 μg g of body weight⁻¹ was injected. The value given on the y axis at 0 h is the total CFU administered to each mouse to initiate infection. All other values plotted are CFU milliliter of blood⁻¹. Where a y value of 10⁰ is given, no bacteria were recovered.