Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica

Authors: Clawson, Michael - Mike; Schuller, Genevieve - Gennie; Dickey, Aaron; Bono, James - Jim; MURRAY, ROBERT - ZOETIS; SWEENEY, MICHAEL - ZOETIS; APLEY, MICHAEL - KANSAS STATE UNIVERSITY; DEDONDER, KEITH - VETERINARY AND BIOMEDICAL RESEARCH CENTER, INC.; CAPIK, SARAH - TEXAS A&M AGRILIFE; LARSON, ROBERT - KANSAS STATE UNIVERSITY; LUBBERS, BRIAN - KANSAS STATE UNIVERSITY; WHITE, BRAD - KANSAS STATE UNIVERSITY; BLOM, JOCHEN - JUSTUS-LIEBIG UNIVERSITY; Chitko-Mckown, Carol; Harhay, Dayna; Smith, Timothy - Tim

Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2020
Publication Date: 8/12/2020
Citation: Clawson, M.L., Schuller, G., Dickey, A.M., Bono, J.L., Murray, R.W., Sweeney, M.T., Apley, M.D., DeDonder, K.D., Capik, S.F., Larson, R.L., Lubbers, B.V., White, B.J., Blom, J., Chitko-McKown, C.G., Brichta-Harhay, D.M., Smith, T.P.L. 2020. Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica. BMC Microbiology. 20:250. https://doi.org/10.1186/s12866-020-01932-2.
DOI: https://doi.org/10.1186/s12866-020-01932-2

Interpretive Summary: Bovine respiratory disease (BRD) affects cattle worldwide and is a serious animal health, well-being, and economic concern. Global monetary losses from BRD are estimated to exceed three billion dollars annually, with close to a billion lost in the United States alone. Multiple environmental and host factors cause or contribute to BRD, as well as viral and bacterial pathogens. Mannheimia haemolytica is the predominant bacterial cause of BRD. Normally found in the upper respiratory tract of cattle, M. haemolytica can move into their lungs during times of stress and cause pneumonia. We previously showed that there are two major strains, or genotypes (1 and 2) of M. haemolytica in North American cattle, and that while both are found in the upper respiratory tract, genotype 2 strains are predominantly found in diseased cattle lungs over genotype 1 strains. This suggests that genotype 2 strains have biological determinants that facilitate their enhanced association with BRD which genotype 1 strains don’t have. To study that, the genomes of 69 diverse genotype 1 and 2 strains were sequenced, assembled and analyzed for their gene content. Of 3,111 genes identified across the strains, 179 were found in all of the genotype 2 strains and none of the genotype 1 strains. Of those, seven encode proteins that are each predicted to localize to the outer membrane that encircles each individual M. haemolytica bacterium, with segments protruding beyond it to the outside milieu. Pathogen outer membrane proteins can interact with host cells in a manner that facilitates their survival and colonization of the host. To that end, three of the seven outer membrane protein genes specific to genotype 2 strains encode adhesins, which are commonly found in pathogenic bacteria and facilitate their attachment to host cells. These adhesins and the other genotype 2 specific genes identified in this study, particularly others that encode outer membrane proteins, may have important roles in genotype 2 pathogenicity and are the focus of current investigations seeking to eliminate genotype 2 M. haemolytica from cattle.

Technical Abstract: Background: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains. Results: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level. Conclusion: Genotype 2M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2M. haemolytica, which is the primary bacterial cause of BRD.