Comparative genome analysis of an avirulent and two virulent strains of avian Pasteurella multocida reveals candidate genes involved in fitness and pathogenicity.

BACKGROUND: Pasteurella multocida is the etiologic agent of fowl cholera, a highly contagious and severe disease of poultry causing significant mortality and morbidity throughout the world. All types of poultry are susceptible to fowl cholera. Turkeys are most susceptible to the peracute/acute forms of the disease while chickens are most susceptible to the acute and chronic forms of the disease. The whole genome of the Pm70 strain of P. multocida was sequenced and annotated in 2001. The Pm70 strain is not virulent to chickens and turkeys. In contrast, strains X73 and P1059 are highly virulent to turkeys, chickens, and other poultry species. In this study, we sequenced the genomes of P. multocida strains X73 and P1059 and undertook a detailed comparative genome analysis with the avirulent Pm70 strain. The goal of this study was to identify candidate genes in the virulent strains that may be involved in pathogenicity of fowl cholera disease. RESULTS: Comparison of virulent versus avirulent avian P. multocida genomes revealed 336 unique genes among the P1059 and/or X73 genomes compared to strain Pm70. Genes of interest within this subset included those encoding an L-fucose transport and utilization system, several novel sugar transport systems, and several novel hemagglutinins including one designated PfhB4. Additionally, substantial amino acid variation was observed in many core outer membrane proteins and single nucleotide polymorphism analysis confirmed a higher dN/dS ratio within proteins localized to the outer membrane. CONCLUSIONS: Comparative analyses of highly virulent versus avirulent avian P. multocida identified a number of genomic differences that may shed light on the ability of highly virulent strains to cause disease in the avian host, including those that could be associated with enhanced virulence or fitness.

Bivalent vaccination against pneumonic pasteurellosis in domestic sheep and goats with modified-live in-frame lktA deletion mutants of Mannheimia haemolytica.

A temperature-sensitive shuttle vector, pBB80C, was utilized to generate in-frame deletion mutants of the leukotoxin structural gene (lktA) of Mannheimia haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, and 12. Culture supernatants from the mutants contained a truncated protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Sheep and goats vaccinated intramuscularly with a mixture of serotypes 5 and 6 mutants were resistant to virulent challenge with a mixture of the wild-type parent strains. These vaccinates responded serologically to both vaccine serotypes and exhibited markedly-reduced lung lesion volume and pulmonary infectious load compared to control animals. Control animals yielded a mixture of serotypes from lung lobes, but the proportion even within an individual animal varied widely from 95% serotype 5-95% serotype 6. Cultures recovered from liver were homogeneous, but two animals yielded serotype 5 and the other two yielded serotype 6 in pure culture.

Integrin-EGF-3 domain of bovine CD18 is critical for Mannheimia haemolytica leukotoxin species-specific susceptibility.

Mannheimia haemolytica leukotoxin (LktA) is the primary virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Results from the authors' previous studies demonstrated that the site required for LktA binding leading to susceptibility to its biological effects resides within amino acid residues 500-600 of the extracellular region of bovine CD18. Experiments were designed to identify a much smaller functional domain within this 100 amino acid region of bovine CD18 that is critically required for species-specific susceptibility to LktA effects. Chimeric bovine X human CD18 with different integrin epidermal growth factor(I-EGF) like domains switched between bovine and human CD18 were generated and coexpressed with bovine CD11a in the human K562 cell line. The resulting chimeric transductants were tested for susceptibility to LktA-induced effects. The results demonstrate unequivocally that the I-EGF-3 domain of bovine CD18 (amino acid residues 541-581) is critical for conferring species-specific susceptibility to M. haemolytica leukotoxin.

Genome Sequences of Serotype A6 Mannheimia haemolytica Isolates D174 and D38 Recovered from Bovine Pneumonia.

Here, we report two genomes, one complete and one draft, from virulent bovine strains of Mannheimia haemolytica serotype A6 recovered prior to the field usage of modern antimicrobial drugs.

Genome Sequences of Mannheimia haemolytica Serotype A2 Isolates D171 and D35, Recovered from Bovine Pneumonia.

Here, we report two genomes, one complete and one draft, from isolates of serotype A2 Mannheimia haemolytica recovered from pneumonic bovine lung.

Draft Genome Sequence of Pasteurella multocida Isolate P1062, Isolated from Bovine Respiratory Disease.

Here, we report the draft genome of Pasteurella multocida isolate P1062 recovered from pneumonic bovine lung in the United States in 1959.

Evaluation of cytokine gene expression in porcine spleen cells, peripheral blood mononuclear cells, and alveolar macrophages by competitive RT-PCR.

Cytokines act as an important regulator of immune responses. Since cytokine expression levels are generally very low, more accurate and reliable methods of measuring their expression are needed. In this study, a modified competitive reverse transcription-polymerase chain reaction assay was developed to determine the expression levels and patterns of porcine IFN-gamma, IL-2, IL-4, IL-10, IL-12 p 35, and IL-12 p40 in spleen cells, peripheral blood mononuclear cells (PBMC), and alveolar macrophages that were stimulated for 4 h by lipopolysaccharide or phytohemagglutinin. Of these cytokines, the expression level of IFN-gamma was the highest in all examined cells. Constitutive expression of IL-2 and IL-4 was demonstrated in spleen cells and PBMC stimulated with phytohemagglutinin. However, their expression extent was not determinable or extremely low in the lipopolysaccharide-stimulated spleen cells and alveolar macrophages. Moderately high IL-10 expression was observed in all examined cells. IL-12 p 35 expression in alveolar macrophages was always higher than in spleen cells and PBMC. IL-12 p40 expression in alveolar macrophages was higher than in PBMC, but was lower than in spleen cells. In spleen cells, the expression of IL-12 p40 was higher than that of IL-12 p 35. In alveolar macrophages and PBMC, however, IL-12 p 35 showed a higher expression than IL-12 p40. These results indicate that each cytokine has its own characteristic expression profile in different immune cells.

Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis.

Bovine pneumonic pasteurellosis continues to be a major respiratory disease in feedlot cattle despite the recent advances in our understanding of the underlying complexities of causation. The etiological agent, Mannheimia haemolytica, possesses several virulence factors, including capsule, outer membrane proteins, adhesins, neuraminidase, endotoxin and exotoxic leukotoxin. Accumulating scientific evidence implicates leukotoxin as the primary factor contributing to clinical presentation and lung injury associated with this disease. Unlike other virulence factors, leukotoxin shows cell-type- and species-specific effects on bovine leukocytes. Recent investigations have delineated the mechanisms underlying the target-cell-specificity of leukotoxin and how this contributes to the pathogenesis of lung damage. This review summarizes current understanding of the secretion, regulation, mechanisms of action and evolutionary diversity of leukotoxin of M. haemolytica. Understanding the precise molecular mechanisms of leukotoxin is critical for the development of more effective prophylactic and therapeutic strategies to control this complex disease.

Genome Sequences of Mannheimia haemolytica Serotype A1 Strains D153 and D193 from Bovine Pneumonia.

Here we report two genome sequences, one complete and one draft, from virulent bovine strains of Mannheimia haemolytica serotype A1 recovered prior to the field usage of modern antimicrobial drugs.

Draft Genome Sequences of Two Virulent Serotypes of Avian Pasteurella multocida.

Here we report the draft genome sequences of two virulent avian strains of Pasteurella multocida. Comparative analyses of these genomes were done with the published genome sequence of avirulent P. multocida strain Pm70.

Monomeric expression of bovine beta2-integrin subunits reveals their role in Mannheimia haemolytica leukotoxin-induced biological effects.

The ruminant-specific leukotoxin (Lkt) of Mannheimia haemolytica is the key virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies by us and others indicate that M. haemolytica Lkt binds to CD18, the beta subunit of bovine beta(2)-integrins on leukocytes, and that the species specificity of Lkt-induced effects is resident in the beta subunit CD18 and not in the alpha subunit CD11. However, Lkt also binds to the CD11a subunit of LFA-1. Furthermore, antibodies specific for CD18 or CD11a inhibit signaling events leading to elevation of intracellular [Ca(2+)], tyrosine phosphorylation of the cytosolic domain of CD18, and cytolysis of bovine leukocytes. These observations underscore the need for further investigation to identify the precise subunit of bovine LFA-1 utilized by M. haemolytica Lkt as the functional receptor. For this purpose, monomeric bovine CD18 and CD11a and heterodimeric LFA-1 were expressed in the HEK-293 cell line by transfection, and the resulting transfectants were tested for susceptibility to Lkt-induced effects. All three transfectants effectively bound Lkt. However, Lkt-induced cytolysis was observed only with transfectants expressing monomeric bovine CD18 or LFA-1. Furthermore, intracellular [Ca(2+)] elevation following exposure to Lkt, which is a marker for postbinding signaling leading to cellular activation, was seen only with transfectants expressing monomeric bovine CD18 or LFA-1. These results clearly indicate that the bovine CD18 subunit of beta(2)-integrins is the functional receptor for M. haemolytica Lkt.

Recombinant expression of bovine LFA-1 and characterization of its role as a receptor for Mannheimia haemolytica leukotoxin.

Mannheimia (Pasteurella) haemolytica leukotoxin (LktA) is the primary virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM), a disease which causes major economic loss to the US cattle industry annually. Recent work from our laboratory using an antibody-based approach has shown that LktA binds to bovine LFA-1 in target cells. While this study suggests that LFA-1 might be a specific receptor, it remains to be conclusively shown that LFA-1 is sufficient to induce susceptibility to LktA. It was of interest to determine if functionally active bovine LFA-1 could be reconstituted on a LFA-1 negative cell line and reconstitute susceptibility to LktA. Here, we report the successful recombinant expression of bovine LFA-1 on the cell surface of the human erythroleukemic K562 cell line. The BoLFA-1 transductant expresses bovine CD18 and CD11a as a heterodimer. We found that LktA binds to both the CD18 and CD11a subunits of BoLFA-1 cells. Exposure of BoLFA-1 cells to LktA, induced tyrosine phosphorylation of the CD18 tail, elevation of intracellular calcium, and cytolysis. This is the first report on recombinant expression of functionally active bovine LFA-1 by transduction into an LktA-non-susceptible human cell line.

Mechanisms underlying Mannheimia haemolytica leukotoxin-induced oncosis and apoptosis of bovine alveolar macrophages.

Mannheimia (Pasteurella) haemolytica leukotoxin (LktA) binds to the bovine beta2 integrins (such as LFA-1-CD11a/CD18) and leads to subsequent cellular effects in a dose dependent manner. The objectives of this study were to delineate the mechanisms that underlie LktA-induced oncosis and apoptosis and to examine the role of LktA/LFA-1 interaction in these events. The results demonstrate that LktA-induced oncosis proceeds through a LFA-1 and caspase-1 dependent pathway referred to as 'pyrotosis', as well as through a LFA-1- and caspase-1-independent pathway. LktA-induced apoptosis in alveolar macrophages involves activation of caspase-3 and engages the extrinsic and intrinsic pathways of apoptosis, with the extrinsic pathway being dependent on LFA-1 signaling and TNFalpha.

Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage beta2 integrins.

Mannheimia (Pasteurella) haemolytica, the etiologic agent of bovine pneumonic mannheimiosis, produces an exotoxic leukotoxin. The leukotoxin (LktA) is a member of the RTX (repeats in toxin) family of bacterial cytolysins and is distinguished from other toxins by its unique target cell specificity to ruminant leukocytes occurring through binding to a specific receptor. We have demonstrated previously that the beta2 integrin LFA-1 is a receptor for LktA in bovine leukocytes and is involved in leukotoxin-induced biological effects. However the subunits within LFA-1 involved in binding to LktA, and post-binding signaling leading to cellular activation have not been well characterized. The purpose of our study was to pinpoint these precise subunits on bovine alveolar macrophages and to characterize their interaction with LktA. The results in this study indicate that although LktA can efficiently bind to the CD18 subunit of both LFA-1 and Mac-1, post-binding signaling events including elevation of intracellular calcium and CD18 tail phosphorylation are only observed through LFA-1. Furthermore, LktA also binds to the CD11a subunit of LFA-1. LktA binding to CD11a could be inhibited by a small molecule inhibitor of the I(inserted)-domain, the major ligand binding interface on CD11a. I-domain inhibition significantly blunts LktA-induced intracellular calcium elevation and tyrosine phosphorylation of the CD18 tail. Based on our results we suggest that LFA-1 serves as the functional leukotoxin receptor on bovine alveolar macrophages.