ISRAEL JOURNAL OF 

VETERINARY MEDICINE     

MANNHEIMIA HAEMOLYTICA: PHYLOGENY AND GENETIC ANALYSIS OF ITS MAJOR VIRULENCE FACTORS.

ADAMU J.Y.

Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Maiduguri, P.M.B. 1069, Borno State, Nigeria.

 

Abstract

Mannheimia haemolytica is the causative agent of several economically significant veterinary diseases occurring in ruminants and, much more rarely, in other animal species. It accounts for approximately 30% of the total cattle deaths worldwide, and is associated with an annual economic loss of over one billion dollars in North America alone. The organism seems to be

enigmatic, changing its niche from commensal to a pathogenic bacterium under conditions of stress. The bacterium was a subject of extensive reclassification in the past and is still under continuous revision. Using a molecular genetic approach, the genes that code for the various virulence factors of M. haemolytica have been cloned and characterized, and the full

genome sequence completed recently, enabling an improved understanding of its virulence and pathogenesis. The genetic tools and techniques developed for M. haemolytica will be useful in post-sequencing genetic analyses of the organism. In this brief review, the genetic basis of major virulence factors, the M. haemolytica genome and the application of molecular techniques to an understanding of Pasteurellaceae phylogeny are presented.

Keywords: Mannheimia (Pasteurella) haemolytica, pathogenicity, virulence factors, phylogeny 

INTRODUCTION

Mannheimia (Pasteurella) haemolytica is the aetiologic agent of pneumonic pasteurellosis of cattle and sheep, which is infections and may cause considerable economic losses to the cattle and sheep industries (1,2) with climatic and animal management factors being involved in its pathogenesis (2,3).

M. haemolytica is a heterogeneous bacterial pathogen. The heterogeneity of this bacterium is evident from the fact that historically, there are two recognized biotypes of M. haemolytica - biotype A consisting of isolates that ferment Larabinose, and biotype that ferments trehalose (4,5); together they are represented by 17 serovars (2,6). The 17 recognized serovars of the former M. haemolytica complex have recently been shown to represent three genetically distinct species.

Serovars 3, 4, 10 and 15 represent Pasteurella trehaosi (7), serovar 11 represents M. glucosida, while the reference strain for the other serovars belong to Mannheimia haemolytica (8). In addition, about 10% of isolates are untypable (UT) (2). The UT isolates may represent either capsule-deficient strains or distinct species (9, 10). Furthermore, extensive epidemiological studies of M. haemolytica strains using molecular typing techniques show some significant level of heterogeneity even within strains of the same serovars isolated from various hosts and also from different geographical areas (11,12,13).

Potential virulence factors of M. haemolytica have been identified and characterized by gene cloning and DNA sequence analyses (14). These factors include a ruminant-specific leukotoxin, an anti-phagocytic capsule, lipopolysaccharide, iron-restricted outer membrane proteins, a sialoglycoprotease, a neuraminidase and immunoglobulin proteases. Since M. haemolytica undergoes a niche change from commensal to pathogenic, the control of its virulence factor expression is also of significant interest. It has been shown that M. haemolytica exhibits a system 2 quorum-sensing mechanism to regulate gene expression under specific conditions (15).

M. haemolytica BAA-410, which was isolated from a calf with bovine respiratory disease complex, was sequenced to draft coverage. Annotation and analysis of the genome provided an opportunity to discover new features potentially related to virulence. It also permitted identification of possible transcriptional regulatory networks and a natural competence system. The genome sequence also allowed comparisons with other sequenced Pasteurellaceae genomes and an evaluation of M. haemolytica within the Pasteurellaceae lineage (16).

HISTORICAL BACKGROUND AND TAXONOMY OF MANNHEIMIA (PASTEURELLA) HAEMOLYTICA

Mannheimia (Pasteurella) haemolytica is a weakly haemolytic, gram-negative coccobacillus with the following complete taxonomy: Superkingdom-Bacteria; Phylum-Proteobacteria; Class-Gammaproteobacteria; Order-Pasteurellales;

Family- Pasteurellaceae; Genus-Pasteurella ( http://www.ncbi.nlm.nih.gov/taxonomy taxonomy ).

The bacterium has been a subject of extensive reclassification in the past; first called Bacterium bipolare multocidum by Theodore Kitt in 1885. The genus name Pasteurella was first suggested by Italian Count Trevisan to commemorate Louis Pasteur’s work with the causative agent of fowl cholera in turkeys (9). It was renamed Pasteurella haemolytica in 1932 (17) and classified into two biotypes A and T, based on its ability to ferment arabinose and trehalose respectively, though other biovariates have been revealed (18,19,20). There are 13 A serotypes and 4 T serotypes identified (21), the latter being reclassified as Pasteurella trehalosi in 1990 (7,22). Nine years later, studies based on DNA-DNA hybridization and 16SRNA sequencing led to renaming previous A serotypes (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16 and A17) as Mannheimia haemolytica while the remaining A11 serotype became M. glucosida (8). Studies of genomic DNA or ribosomal RNA have revealed lack of homologies acceptable for inclusion of some organisms at the genus or species level (23, 24, 25). The name Mannheimia was given in tribute to Walter Mannheim, a German biologist whose research has improved the understanding of the taxonomy of the Pasteurellaceae family (8). Although P. haemolytica is now classified as three distinct species- M. haemolytica, M. glucosida and P. trehalosi, for the purpose of identification and serotyping they are still treated as one species (26). In addition to isolates which fall into one of the above serotypes, approximately 10% of disease isolates from ruminants are untypable (27,28).

The taxonomy of Pasteurellaceae is not well settled and is under continuous revision. The taxonomy has involved mainly phenotypic characterization, including the determination of polyamine patterns (29,30). A few thorough studies have also been carried out at the genotypic level. Whole genome DNA–DNA hybridizations were carried out some time ago on Pasteurellaceae (31) and genetic relatedness was investigated by DNA–rRNA hybridization (24). Dewhirst et al. (25,32) investigated the phylogenetic relationships within the family by 16S rDNA sequence comparisons and a full phylogenetic tree was published recently (33). Hedegaard et al. (34) used infB sequences to investigate in more detail the genetic relationships of the genus Haemophilus. However, other phylogenetic marker genes are needed to clarify the taxonomy within the Pasteurellaceae, to provide additional tools for genetic identification and to give insight into the evolution of this group of bacteria. The rpoB gene has been used successfully for the elaboration of phylogenetic relationships in several groups of bacteria, and normally has a higher discriminatory power than 16S rDNA sequences (35, 36). Besides the use of partial rpoB sequences for delineating phylogenetic relationships (37, 38) the gene has also been applied for improving diagnosis (39,40). Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful in reorganizing its current taxonomy (41).

PATHOGENICITY

Mannheimia (Pasteurella) species are common commensals on mucous membranes of most domestic animals worldwide (42). Most animals are asymptomatic carriers of M. haemolytica and P. trehalosi and also carry strains of P. multocida. M. haemolytica is associated with disease in cattle causing pneumonic pasteurellosis, haemorrhagic septicaemia and abortion (43, 44); pneumonia, mastitis and septicaemia in domestic sheep (2) and isolates in domestic goats (45,46).

Biovariates of M. haemolytica, P. trehalosi and P. multocida have been isolated from American bison, bighorn sheep, Dall sheep, elk, moose, mountain goats, mule, deer and pronghorn (20). Other hosts of animals affected by species of Pasteurella include rabbits, pigs, crocodiles, dogs and cats (47). Recently M. haemolytica serotypes A2 and A12 were isolated from clinically ill poultry in Nigeria (48). This organism has a zoonotic potential (49). Man gets infected by P. multocida, and also by P. dogmatis, P. canis and M. haemolytica. Infections of Pasteurella are spread worldwide in wild and domestic animals. Man contracts it by small animals and farm animals, zoo and wild animals. People who work with animals are particularly at risk. The transmission mostly happens via wound infection, which becomes inflamed and is painful. Phlegmons or abscesses in the subdermis, infections of the tendon sheath, the tendon and bones can occur, followed by necrosis, periostitis and osteomyelitis. (http://www.veterinary-public-health.de/home_e/aufgaben/zoonosen/bakterien_e.htm).

MOLECULAR ANALYSIS OF VIRULENCE FACTORS OF M. HAEMOLYTICA     Capsular polysaccharide (CP)Capsular polysaccharides have been implicated in the pathogenesis of Mannheimiosis. The role of CPs in the virulence of a number of gram-negative pathogens has been well documented; these include adherence (50), prevention of desiccation (51), resistance to host immune defense (52,53) and masking cell surface (53).

The M. haemolytica A1 CPs is composed of disaccharide repeats of N-acetylmannosaminuronic acid (ManNAcA)β-1,4 linked with N-acetylmannosamine (ManNAc) (54). The genes nmaA and nmaB, which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, are involved in capsular polysaccharide biosynthesis in M. haemolytica A1 (55). ManNAcA is one of the sugar moieties in the enterobacterial common antigen (ECA) (56).

Acapsular isolates have occasionally been reported (57), but their genetic basis has not been examined. Now that the genes have been cloned, molecular techniques are used to create defined acapsular mutants (53). Furthermore, it was reported that the acapsular mutant of M. haemolytica was more easily phagocytized than the capsular strains. The capsular material may interact with pulmonary surfactant, thereby facilitating local adherence of the organism to different host cells (14, 58, 59, 60).

Lipopolysaccharide (LP)

Lipopolysaccharide represents 10-15% of the dry weight of M. haemolytica (61). Two genes potentially involved in LPs biosynthesis in M. haemolytica have been cloned and characterized. A 263 amino acid reading frame encoding a peptide similar to H. influenzae lipooligosaccharide biosynthetic gene, lic2 was identified immediately downstream of the gene encoding glycoprotease (62). The LPs gene, called lpsA is a potential glycosyltransferase involved in core-oligosaccharide biosynthesis (14). It has been proposed that M. haemolytica.

LPs can cause immune-mediated hypersensitivity that can exacerbate inflammation and damage by a  localized Arthus or Shwartzmann reaction in the lung (63). They have endotoxic activity and moderate leukocyte activity (64). The LPs constitute a major surface antigen and the somatic serotype was defined according to their structural antigenic components.. M. haemolytica serotypes of biotype A possess rough type of LPs while biotype T contains smooth LPs. LPs was also found to stimulate tumor necrosis factor (TNF) release from bovine alveolar macrophages as well as cytokines (14,65,66).

M. haemolytica LPs induce an inflammatory cytokine response and expression of the β2-integrin LFA-1 leukotoxin receptor in the host (14,67).

Outer membrane proteins (OMP)

Outer membrane proteins and lipoproteins of M. haemolytica may be involved in serum sensitivity (68,69) and are believed to be important protective antigens. Antibodies directed against some of these antigens are capable of inducing phagocytosis and complement-mediated killing (69), and hence are of interest as potential vaccine candidates. OMP profiles vary within and between serotypes (10). M. haemolytica produces a set of OMP in response to iron depletion (70). The iron-regulated outer membrane proteins (IROMPs) are recognized by convalescent bovine (and ovine) serum, which shows that they are antigenic and expressed in vivo (71). Three IROMPs ( 71, 77 and 100 kDa) expressed by M. haemolytica A1 grown in vitro under conditions of iron restriction, have been characterized (71, 72, 73). The 71- and 100 kD IROMPs were identified as bovinespecific transferin binding proteins (Tbps) that are responsible for iron acquisition (74). The genes that code for TbpA and TbpB have been cloned and characterized (75). The tbpA and tbpB are arranged in an operon, and tbpB precedes tbpA. By genetic manipulation of the tbpA/tbpB genes, recombinant Tbps have been produced in E. coli. These recombinant proteins were shown to be effective vaccine candidates in a vaccine trial and challenge study (76).

Fimbriae and Adhesin

Bacterial adhesions function in colonization by binding to receptor molecules on host cell surfaces (16). Many adhesions are pili, and the type iv pilus locus pilABCD was annotated in M. haemolytica. Type iv pili are known to function in DNA uptake, adhesion, and motility in H. influenzae, P. aeruginosa, and Neisseria species (77) and may perform these functions in M. haemolytica.

Several predicted non pilus adhesion proteins and additional proteins that could modify host mucosal surfaces are also present in M. haemolytica (16). A serotype A1-specific antigen (Ssa1) is also thought to function as an adhesin (78).

Two types of fimbrae were demonstrated in M. haemolytica, a large and rigid one 12nm in width, while the smaller and flexible 5nm wide (79,80). These structures have not however been characterized in detail, and their presence does not appear to be a universal feature of M. haemolytica isolates (81). A highmolecular adhesion protein (HMWAP; >230kDa) has been identified in M. haemolytica A1 (82).

Leukotoxins (LKT)

M. haemolytica secretes a 102 kD leukotoxin (lktA) that is a calcium-dependent cytotoxin belonging to the RTX (repeats in toxin) family of toxins. Leukotoxin genes and protein have been identified in all serotypes (83) and nearly all isolates examined secrete the toxin (84). LktA is species-specific, having leukotoxic activity only against ruminant lymphoid cells (85,86). It is also weakly haemolytic (87). Though the leukotoxin can bind to cells from a variety of species (88), cytolysis requires a specific interaction with the lymphocyte-function associated antigen 1 (LFA-1), or β2 integrin, on the target cell (89,90). At high concentrations, the toxin creates pores in the cell membrane that lead to swelling and lysis (91). At sub-lytic concentrations, the toxin activates neutrophils (60), induces inflammatory cytokine production (92), invokes cytoskeletal changes, and causes apoptosis (93,94). Combined, these activities are thought to impair primary lung immune defense mechanisms and participate in inflammation and tissue destruction that define pneumonic mannheimiosis. The leukotoxin is encoded by four-gene cluster that includes the 963 amino acid structure gene, lktA, and genes that are required for its post-translational acylation (lktC) and secretion (lktB and lktD) (95).

Glycoprotease

All types of M. haemolytica produce a zinc metalloglycoprotease that has activity against O-sialoglycoprotein (96,97). The gcp gene has been cloned into E. coli and sequenced (98). The protease is thought to act at the host cell surface to enhance adhesion and its activity in vitro can be potentiated by coincubation with the leukotoxin (99). One consequence of this activity is the aggregation of platelets leading to their deposition in the alveoli of the lung (99).

Neuraminidase

A neuraminidase produced by M. haemolytica has been suggested to play a role in colonization (100,101). It is produced by all 17 classical M. haemolytica serotypes (102). The production of antineuraminidase serum antibodies by cattle after transthoracic infection with the bacterium is evidence that neuraminidase is produced by M. haemolytica in vivo (103).

In other bacterial respiratory pathogens, neuraminidases are thought to desialate salivary glycoproteins, allowing pathogenic organisms to escape defenses in the oropharynx (104). Based on gel filtration, the associated enzymes are 150-200 KD in size and primarily cleave N-acetylneuramin lactose, but some also have activities that cleave fetuin, alpha-1-acid glycoprotein, and colominic acid (102). Furthermore, the production of neuraminidase by M. haemolytica A1 is maximal in vitro (105), which suggests a possible role during infection.

The MANNHEIMIA (PASTEURELLA) HAEMOLYTICA genome

The genome sequence of M. haemolytica was recently published (16) under the direction of Dr Sarah Highlander of Baylor College of Medicine, Houston, Texas with funding provided by the USDA National Research Grant Initiative 00-35204-9229.

The M. haemolytica genome consists of a DNA with an approximate G & C content of 41%. The genome length is about 2.57Mb. The annotated genome includes 2839 coding sequences, 1966 of which were assigned a function and 436 of which are unique to M. haemolytica. Through genome annotation many features of interest were identified, including bacteriophages (ФMhaMu1 and ФMhaMu2) and genes related to virulence, natural competence and transcriptional regulation. The general features of M. haemolytica are given in Table 1.

Comparison of competence loci and DNA uptake signal sequences (USS) in other species in the family Pasteurellaceae indicates that M. haemolytica, Actinobacillus pleuropneumoniae, and Haemophilus ducreyi form a lineage distinct from other Pasteurellaceae. This observation was supported by a phylogenetic analysis using sequences of predicted housekeeping genes (16).

The whole genome shortgun project has been deposited at GenBank under project accession number AASA00000000 while the draft version has accession number AASA01000000.

CONCLUSION

Mannheimia haemolytica continues to be an enigmatic organism. With the completion of the genome sequencing, our understanding of its virulence and pathogenesis will improve and the genetic tools and techniques that have been developed for M. haemolytica will be useful in postsequencing genetic analyses of the organism bearing in mind the great economic importance of this bacterium.

ACKNOWLEDGMENT

Thanks are due to Dr Sarah Highlander of the Baylor College of Medicine, Houston, Texas and Pat Blackall of the Department of Primary Industries, Yeerongpili, Victoria, Australia for providing some of the reprints used in this write-up.

 

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