SUMMARY In a world where most emerging and reemerging infectious diseases are zoonotic in nature and our contacts with both domestic and wild animals abound, there is growing awareness of the potential for human acquisition of animal diseases. Like other Pasteurellaceae , Pasteurella species are highly prevalent among animal populations, where they are often found as part of the normal microbiota of the oral, nasopharyngeal, and upper respiratory tracts. Many Pasteurella species are opportunistic pathogens that can cause endemic disease and are associated increasingly with epizootic outbreaks. Zoonotic transmission to humans usually occurs through animal bites or contact with nasal secretions, with P. multocida being the most prevalent isolate observed in human infections. Here we review recent comparative genomics and molecular pathogenesis studies that have advanced our understanding of the multiple virulence mechanisms employed by Pasteurella species to establish acute and chronic infections. We also summarize efforts being explored to enhance our ability to rapidly and accurately identify and distinguish among clinical isolates and to control pasteurellosis by improved development of new vaccines and treatment regimens.

Pasteurella multocida toxin (PMT) has been hypothesized to cause activation of a GTP-binding protein (G-protein)-coupled phosphatidylinositol-specific phos-Overexpression of G q ␣ in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense G q ␣ cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing G q ␣.

The intracellularly acting protein toxin of Pasteurella multocida (PMT) causes numerous effects in cells, including activation of inositol 1,4,5-trisphosphate (IP 3 ) signaling, Ca 2؉ mobilization, protein phosphorylation, morphological changes, and DNA synthesis. The direct intracellular target of PMT responsible for activation of the IP 3 pathway is the G q/11 ␣-protein, which stimulates phospholipase C (PLC) ␤1. The relationship between PMT-mediated activation of the G q/11 -PLC-IP 3 pathway and its ability to promote mitogenesis and cellular proliferation is not clear. PMT stimulation of p42/p44 mitogen-activated protein kinase occurs upstream via G q/11 -dependent transactivation of the epidermal growth factor receptor. We have further characterized the effects of PMT on the downstream mitogenic response and cell cycle progression in Swiss 3T3 and Vero cells. PMT treatment caused dramatic morphological changes in both cell lines. In Vero cells, limited multinucleation, nuclear fragmentation, and disruption of cytokinesis were also observed; however, a strong mitogenic response occurred only with Swiss 3T3 cells. Significantly, this mitogenic response was not sustained. Cell cycle analysis revealed that after the initial mitogenic response to PMT, both cell types subsequently arrested primarily in G 1 and became unresponsive to further PMT treatment. In Swiss 3T3 cells, PMT induced up-regulation of c-Myc; cyclins D1, D2, D3, and E; p21; PCNA; and the Rb proteins, p107 and p130. In Vero cells, PMT failed to up-regulate PCNA and cyclins D3 and E. We also found that the initial PMT-mediated up-regulation of several of these signaling proteins was not sustained, supporting the subsequent cell cycle arrest. The consequences of PMT entry thus depend on the differential regulation of signaling pathways within different cell types.The protein toxin from Pasteurella multocida (PMT) is the primary etiological agent of progressive atrophic rhinitis (11), and purified PMT experimentally induces all of the major symptoms of atrophic rhinitis in animals (2, 23). PMT appears to bind to and enter mammalian cells via receptor-mediated endocytosis (30, 33), although the details of this process are still unclear. In fibroblasts and osteoblasts, PMT acts intracellularly to enhance inositolphospholipid hydrolysis (25, 26), mobilize intracellular Ca 2ϩ pools (40), increase protein phosphorylation (41), stimulate cytoskeletal changes such as stress fiber formation and focal adhesion assembly (5, 21), and initiate DNA synthesis (33). In contrast to the case for fibroblast cells, PMT exerts cytotoxic or cytopathic effects on Vero cells (2, 28), embryonic bovine lung cells (34), and canine osteosarcoma cells (30).PMT exerts its effects on these cellular processes by acting on the free, monomeric ␣ subunit of the G q/11 family of heterotrimeric G-proteins (46), which stimulates phospholipase C (PLC) ␤1 to hydrolyze phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. Accordingly, the releas...

Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

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The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

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