Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica

The molecular evolution of the leukotoxin structural gene (lktA) of Mannheimia (Pasteurella) haemolytica was investigated by nucleotide sequence comparison of lktA in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in M. haemolytica. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the lktA gene of the two related species Mannheimia glucosida and Pasteurella trehalosi, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess lktA alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of lktA in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.     

Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompA) of Mannheimia(Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing beta-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.     

Secretion of the Pasteurella leukotoxin by Escherichia coli

Nucleic acid sequence analysis has indicated that the leukotoxin determinant from Pasteurella haemolytica is related to the hemolysin determinant from E. coli. The cloning and expression in E. coli of the lktCA genes has been previously reported, but the existence of leukotoxin secretory genes equivalent to hlyBD has not been documented. In this report we demonstrate that a 4.0 kb segment of P. haemolytica genomic DNA distal to the lktA gene, when expressed in trans to the previous cloned lktCA genes, allow the synthesis and secretion of active leukotoxin from E. coli. Complementation analysis using the cloned hlyB and hlyD genes indicates that this secretory locus derived from P. haemolytica contains two genes which we designate, by analogy, lktB and lktD.     

Secretion of the Pasteurella leukotoxin by Escherichia coli

Abstract Nucleic acid sequence analysis has indicated that the leukotoxin determinant from Pasteurella haemolytica is related to the hemolysin determinant from E. coli . The cloning and expression in E. coli of the lktCA genes has been previously reported, but the existence of leukotoxin secretory genes equivalent to hlyBD has not been documented. In this report we demonstrate that a 4.0 kb segment of P. haemolytica genomic DNA distal to the lktA gene, when expressed in trans to the previous cloned lktCA genes, allow the synthesis and secretion of active leukotoxin from E. coli . Complementation analysis using the cloned hlyB and hlyD genes indicates that this secretory locus derived from P. haemolytica contains two genes which we designate, by analogy, lktB and lktD .     

Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage

The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (phiPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The phiPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.     

Serotypes A1 and A2 of Mannheimia haemolytica are susceptible to genotypic, capsular and phenotypic variations in contrast to T3 and T4 serotypes of Bibersteinia (Pasteurella) trehalosi

Mannheimia haemolytica and Bibersteinia (Pasteurella) trehalosi are the most common bacterial isolates that cause pulmonary diseases in ruminants worldwide. The disease is determined by specific serotypes found in cattle and small ruminants. The molecular epidemiology of strains involved in disease is important in the control of outbreaks as well as in the preparation of vaccines. This study aimed to detect the instability and variations of bacterial strains that may affect the analysis of epidemic strains, or the stability of vaccinal strains. Eight strains of M. haemolytica belonging to serotypes A1 and A2 and three B. trehalosi strains of the T3 and T4 serotypes were used. Strains were subjected to pulsed field gel electrophoresis (PFGE) and capsular and phenotypic typing at each round of a total of 50 successive subcultures. Remarkable stability was found in all selected strains of B. trehalosi in contrast to M. haemoltyica, in which strains of both serotypes showed pattern variations produced by PFGE and capsular and phenotypic analysis. Objective criteria for M. haemolytica and B. trehalosi typing are consequently addressed.     

Deletion analysis resolves cell-binding and lytic domains of the Pasteurella leukotoxin

A series of internal deletions in the lktA gene of Pasteurella haemolytica has been constructed. All of the deletions eliminated the lytic activity of the leukotoxin towards the bovine lymphoma cell line, BL-3. Deletions removing segments of the amino-proximal hydrophobic region, which is thought to constitute an essential membrane-spanning domain, were found to agglutinate BL-3 cells. Agglutination was similar to lysis by the wild-type toxin in that it was dependent upon the presence of calcium and required expression of the lktC gene. The agglutinating deletion proteins protected BL-3 cells from lysis by the wild-type toxin in a competitive fashion. This suggests that these mutants bind to a surface feature of the leukocyte which interacts with the native leukotoxin. These findings demonstrate that the cell-binding and lytic domains of the leukotoxin are separable.     

Proximity-Dependent Inhibition of Growth of Mannheimia haemolytica by Pasteurella multocida

Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.     

Coinfection with BHV-1 modulates cell adhesion and invasion by P. multocida and Mannheimia (Pasteurella) haemolytica

Viruses are thought to facilitate bacterial infections of the respiratory tract. The present study shows the effect of BHV-1 on Pasteurella multocida and Mannheimia haemolytica adherence and invasion of MDBK cells. The virus-infected MDBK cells become more susceptible to the adherence of both species of Pasteurella. The observed adherence increase depends on the length of virus pre-incubation time and on virus concentration. When MDBK cells are not infected with virus, they are only invaded by P. multocida, while M. haemolytica is not able to penetrate. The viral infection favours also the invasion by M. haemolytica.     

The ycf27 genes from cyanobacteria and eukaryotic algae: distribution and implications for chloroplast evolution

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxS(M.h.) was able to complement AI-2 production in the Escherichia coli DH5alpha luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions.     

Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica

Pneumonia is a leading cause of loss to the sheep and cattle industry throughout the world. Mannheimia (Pasteurella) haemolytica is one of the most important respiratory pathogens of domestic ruminants and causes serious outbreaks of acute pneumonia in neonatal, weaned and growing lambs, calves, and goats. M. haemolytica is also an important cause of pneumonia in adult animals. Transportation, viral infections with agents such as infectious bovine rhinotracheitis virus, parainfluenza-3 virus or bovine respiratory syncytial virus, overcrowding, housing of neonates and weaned animals together and other stressful conditions predispose animals to M. haemolytica infection [1, 2]. This review assimilates some of the findings key to cellular and molecular responses of the lung from a pathologist's perspective. It includes some of what is known and underscores areas that are not fully understood.     

Separable domains define target cell specificities of an RTX hemolysin from Actinobacillus pleuropneumoniae.

The leukotoxin (LktA) from Pasteurella haemolytica and the hemolysin (AppA) from Actinobacillus pleuropneumoniae are members of a highly conserved family of cytolytic proteins produced by gram-negative bacteria. Despite the extensive homology between these gene products, LktA is specific for ruminant leukocytes while AppA, like other hemolysins, lyses erythrocytes and a variety of nucleated cells, including ruminant leukocytes. Both proteins require activation facilitated by the product of an accessory repeat toxin (RTX) C gene for optimal biological activity. We have constructed six genes encoding hybrid toxins by recombining domains of ltkA and appA and have examined the target cell specificities of the resulting hybrid proteins. Our results indicate that the leukocytic potential of AppA, like that of LktA, maps to the C-terminal half of the protein and is physically separable from the region specifying erythrocyte lysis. As a consequence, we were able to construct an RTX toxin capable of lysing erythrocytes but not leukocytes. The specificity of one hybrid was found to be dependent upon the RTX C gene used for activation. With appC activation, this hybrid toxin lysed both erythrocytes and leukocytes, while lktC activation produced a toxin which could attack only leukocytes. This is the first demonstration that the specificity of an RTX toxin can be determined by the process of C-mediated activation.     

Real-time PCR assay for the detection of species of the genus Mannheimia

Infections caused by species of the genus Mannheimia cause diverse disease complexes in many wild and domestic animals worldwide. Fast and accurate detection of single species within the genus remains an unsolved problem till today. To resolve this diagnostic challenge, we developed a real-time PCR assay for the rapid and specific identification of five species of the genus Mannheimia (M. haemolytica, M. varigena, M. ruminalis, M. granulomatis and M. glucosida) from bacterial cultures and tissue samples. The assay was validated with reference strains, field isolates and bacteria spiked tissue samples. The sodA gene was used as target region for species-specific primer pairs. The real-time PCR assay demonstrated species specificity for all five examined Mannheimia spp. and a rapid test completion time of less than 5 h. This is a considerable advantage compared to the traditional phenotyping methods currently used to distinguish between the species of the genus. The assay was able to detect approximately 10(3) bacterial cells per gram lung tissue sample, as determined with spiked tissue samples. We assume that the assay could become useful for fast laboratory diagnostic assessment particularly of respiratory infections caused by Mannheimia in animals.     

Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes

Pneumonia caused by Mannheimia (Pasteurella) haemolytica is a highly fatal disease of bighorn sheep (Ovis canadensis). Leukotoxin (Lkt), secreted by M. haemolytica, is an important virulence factor of this organism, and is cytolytic to bighorn sheep leukocytes. Previously, we have shown that CD18, the beta subunit of beta2 integrins, serves as the receptor for Lkt on bovine leukocytes. Furthermore, anti-CD18 antibodies inhibit Lkt-induced cytotoxicity of bighorn sheep leukocytes. Therefore, we hypothesized that Lkt utilizes CD18 as its receptor on bighorn sheep leukocytes. Confirmation of bighorn sheep CD18 as a receptor for Lkt requires the demonstration that the recombinant expression of bighorn sheep CD18 in Lkt-nonsusceptible cells renders them susceptible to Lkt. Therefore, we transfected cDNA encoding CD18 of bighorn sheep into a Lkt-nonsusceptible murine cell line. Cell surface expression of bighorn sheep CD18 on the transfectants was tested by flow cytometry with anti-CD18 antibodies. Transfectants stably expressing bighorn sheep CD18 on their surface were subjected to flow cytometric analysis for detection of Lkt binding, and cytotoxicity assays for detection of Lkt-induced cytotoxicity. Leukotoxin bound to the transfectants. More importantly, the transfectants were effectively lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. These results clearly indicate that M. haemolytica Lkt utilizes CD18 as a receptor on bighorn sheep leukocytes. Identification of CD18 as a receptor for Lkt on bighorn sheep leukocytes should enhance our understanding of the pathogenesis of pneumonia, which in turn should help in the development of control measures against this fatal disease of bighorn sheep.     

Identification of a novel Mannheimia granulomatis lineage from lesions in roe deer (Capreolus capreolus)

Eight atypical Mannheimia isolates were isolated from lesions in roe deer (Capreolus capreolus). Traditional classification based on morphologic and physiologic traits showed that they belong to a distinct biogroup (taxon) within genus Mannheimia. Extensive phenotypic characterization suggested that the isolates should be classified as M. granulomatis, although the presence of distinct traits justified their classification into a separate biogroup within this species. Phylogenetic analyses based on 16S rRNA sequences from two roe deer isolates and 41 other Mannheimia strains supported that the roe deer isolates form a monophyletic group within M. granulomatis. The lktA genotype was present in all roe deer isolates based on Southern blot analysis, whereas the corresponding beta-hemolytic phenotype was absent in one of these isolates.