Dataset Information

Comparative Genomic Analysis of Mannheimia haemolytica from Bovine Sources.

ABSTRACT: Bovine respiratory disease is a common health problem in beef production. The primary bacterial agent involved, Mannheimia haemolytica, is a target for antimicrobial therapy and at risk for associated antimicrobial resistance development. The role of M. haemolytica in pathogenesis is linked to serotype with serotypes 1 (S1) and 6 (S6) isolated from pneumonic lesions and serotype 2 (S2) found in the upper respiratory tract of healthy animals. Here, we sequenced the genomes of 11 strains of M. haemolytica, representing all three serotypes and performed comparative genomics analysis to identify genetic features that may contribute to pathogenesis. Possible virulence associated genes were identified within 14 distinct prophage, including a periplasmic chaperone, a lipoprotein, peptidoglycan glycosyltransferase and a stress response protein. Prophage content ranged from 2-8 per genome, but was higher in S1 and S6 strains. A type I-C CRISPR-Cas system was identified in each strain with spacer diversity and organization conserved among serotypes. The majority of spacers occur in S1 and S6 strains and originate from phage suggesting that serotypes 1 and 6 may be more resistant to phage predation. However, two spacers complementary to the host chromosome targeting a UDP-N-acetylglucosamine 2-epimerase and a glycosyl transferases group 1 gene are present in S1 and S6 strains only indicating these serotypes may employ CRISPR-Cas to regulate gene expression to avoid host immune responses or enhance adhesion during infection. Integrative conjugative elements are present in nine of the eleven genomes. Three of these harbor extensive multi-drug resistance cassettes encoding resistance against the majority of drugs used to combat infection in beef cattle, including macrolides and tetracyclines used in human medicine. The findings here identify key features that are likely contributing to serotype related pathogenesis and specific targets for vaccine design intended to reduce the dependency on antibiotics to treat respiratory infection in cattle.

SUBMITTER: Klima CL

PROVIDER: S-EPMC4771134 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Comparative Genomic Analysis of Mannheimia haemolytica from Bovine Sources.

Klima Cassidy L CL Cook Shaun R SR Zaheer Rahat R Laing Chad C Gannon Vick P VP Xu Yong Y Rasmussen Jay J Potter Andrew A Hendrick Steve S Alexander Trevor W TW McAllister Tim A TA

PloS one 20160229 2

Bovine respiratory disease is a common health problem in beef production. The primary bacterial agent involved, Mannheimia haemolytica, is a target for antimicrobial therapy and at risk for associated antimicrobial resistance development. The role of M. haemolytica in pathogenesis is linked to serotype with serotypes 1 (S1) and 6 (S6) isolated from pneumonic lesions and serotype 2 (S2) found in the upper respiratory tract of healthy animals. Here, we sequenced the genomes of 11 strains of M. hae ...[more]

PMID: 26926339

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Project description:BackgroundMannhemia haemolytica is a Gram-negative bacterium and the principal etiological agent associated with bovine respiratory disease complex. They transform from a benign commensal to a deadly pathogen, during stress such as viral infection and transportation to feedlots and cause acute pleuropneumonia commonly known as shipping fever. The U.S beef industry alone loses more than one billion dollars annually due to shipping fever. Despite its enormous economic importance there are no specific and accurate genetic markers, which will aid in understanding the pathogenesis and epidemiology of M. haemolytica at molecular level and assist in devising an effective control strategy.DescriptionDuring our comparative genomic sequence analysis of three Mannheimia haemolytica isolates, we identified a number of genes that are unique to each strain. These genes are "high value targets" for future studies that attempt to correlate the variable gene pool with phenotype. We also identified a number of high confidence single nucleotide polymorphisms (hcSNPs) spread throughout the genome and focused on non-synonymous SNPs in known virulence genes. These SNPs will be used to design new hcSNP arrays to study variation across strains, and will potentially aid in understanding gene regulation and the mode of action of various virulence factors.ConclusionsDuring our analysis we identified previously unknown possible type III secretion effector proteins, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas). The presence of CRISPR regions is indicative of likely co-evolution with an associated phage. If proven functional, the presence of a type III secretion system in M. haemolytica will help us re-evaluate our approach to study host-pathogen interactions. We also identified various adhesins containing immuno-dominant domains, which may interfere with host-innate immunity and which could potentially serve as effective vaccine candidates.

Project description:Mannheimia haemolytica is a commensal bacterium that resides in the upper respiratory tract of cattle that can play a role in bovine respiratory disease. Prophages are common in the M. haemolytica genome and contribute significantly to host diversity. The objective of this research was to undertake comparative genomic analysis of phages induced from strains of M. haemolytica serotype A1 (535A and 2256A), A2 (587A and 1127A) and A6 (1152A and 3927A).Overall, four P2-like (535AP1, 587AP1, 1127AP1 and 2256AP1; genomes: 34.9-35.7 kb; G+C content: 41.5-42.1 %; genes: 51-53 coding sequences, CDSs), four ?-like (535AP2, 587AP2, 1152AP2 and 3927AP1; genomes: 48.6-52.1 kb; 41.1-41.4 % mol G+C; genes: 77-83 CDSs and 2 tRNAs) and one Mu-like (3927AP2; genome: 33.8 kb; 43.1 % mol G+C; encoding 50 CDSs) phages were identified. All P2-like phages are collinear with the temperate phage ?MhaA1-PHL101 with 535AP1, 2256AP1 and 1152AP1 being most closely related, followed by 587AP1 and 1127AP1. Lambdoid phages are not collinear with any other known ?-type phages, with 587AP2 being distinct from 535AP2, 3927AP1 and 1152AP2. All ?-like phages contain genes encoding a toxin-antitoxin (TA) system and cell-associated haemolysin XhlA. The Mu-like phage induced from 3927A is closely related to the phage remnant ?MhaMu2 from M. haemolytica PHL21, with similar Mu-like phages existing in the genomes of M. haemolytica 535A and 587A.This is among the first reports of both ?- and Mu-type phages being induced from M. haemolytica. Compared to phages induced from commensal strains of M. haemolytica serotype A2, those induced from the more virulent A1 and A6 serotypes are more closely related. Moreover, when P2-, ?- and Mu-like phages co-existed in the M. haemolytica genome, only P2- and ?-like phages were detected upon induction, suggesting that Mu-type phages may be more resistant to induction. Toxin-antitoxin gene cassettes in ?-like phages may contribute to their genomic persistence or the establishment of persister subpopulations of M. haemolytica. Further work is required to determine if the cell-associated haemolysin XhlA encoded by ?-like phages contributes to the pathogenicity and ecological fitness of M. haemolytica.

Project description:The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) and is responsible for significant economic losses to livestock industries worldwide. Healthy cattle are frequently colonized by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a 5-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognized mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonization and infection of the bovine respiratory tract.

Project description:BackgroundComputational methods for structural gene annotation have propelled gene discovery but face certain drawbacks with regards to prokaryotic genome annotation. Identification of transcriptional start sites, demarcating overlapping gene boundaries, and identifying regulatory elements such as small RNA are not accurate using these approaches. In this study, we re-visit the structural annotation of Mannheimia haemolytica PHL213, a bovine respiratory disease pathogen. M. haemolytica is one of the causative agents of bovine respiratory disease that results in about $3 billion annual losses to the cattle industry. We used RNA-Seq and analyzed the data using freely-available computational methods and resources. The aim was to identify previously unannotated regions of the genome using RNA-Seq based expression profile to complement the existing annotation of this pathogen.ResultsUsing the Illumina Genome Analyzer, we generated 9,055,826 reads (average length ~76 bp) and aligned them to the reference genome using Bowtie. The transcribed regions were analyzed using SAMTOOLS and custom Perl scripts in conjunction with BLAST searches and available gene annotation information. The single nucleotide resolution map enabled the identification of 14 novel protein coding regions as well as 44 potential novel sRNA. The basal transcription profile revealed that 2,506 of the 2,837 annotated regions were expressed in vitro, at 95.25% coverage, representing all broad functional gene categories in the genome. The expression profile also helped identify 518 potential operon structures involving 1,086 co-expressed pairs. We also identified 11 proteins with mutated/alternate start codons.ConclusionsThe application of RNA-Seq based transcriptome profiling to structural gene annotation helped correct existing annotation errors and identify potential novel protein coding regions and sRNA. We used computational tools to predict regulatory elements such as promoters and terminators associated with the novel expressed regions for further characterization of these novel functional elements. Our study complements the existing structural annotation of Mannheimia haemolytica PHL213 based on experimental evidence. Given the role of sRNA in virulence gene regulation and stress response, potential novel sRNA described in this study can form the framework for future studies to determine the role of sRNA, if any, in M. haemolytica pathogenesis.

Dataset Information

Comparative Genomic Analysis of Mannheimia haemolytica from Bovine Sources.

Publications

Comparative Genomic Analysis of Mannheimia haemolytica from Bovine Sources.

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