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Title: Molecular evolution of outer membrane proteins and characterization of temperate bacteriophages of Pasteurella multocida strains from different host species
Author: Abdulrahman, Rezheen Fatah
ISNI:       0000 0004 6060 5327
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2016
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Pasteurella multocida is a Gram-negative commensal bacterium which resides in the upper respiratory tract of mammals and birds. The organism is responsible for a variety of economically important diseases in a wide range of domestic animal species. It causes fowl cholera of poultry, haemorrhagic septicaemia of cattle and water buffalo, atrophic rhinitis of pigs, and pneumonia of cattle, sheep and pigs. In the present study, P. multocida isolates were selected based on an established framework of evolutionary relationships among 123 isolates of P. multocida based on the concatenated partial sequences (3990 bp) of seven housekeeping enzyme genes (Davies et al., unpublished; The isolates were recovered from different host species (cattle, sheep, pigs and poultry) and were associated with different diseases. The isolates represented various capsular serotypes, outer membrane protein (OMP)-types, 16S rRNA types, and sequence types. Phylogenetic trees based on the concatenated partial sequences (3990 bp) of the seven housekeeping enzyme genes, complete sequences (22,371 bp) of fifteen housekeeping enzyme genes and the core genome were almost identical in their topographies. The trees represented eight major groups or clusters of isolates and these clusters could also be defined, to varying degrees, by the host species of origin and/or disease syndrome. The pattern of clustering of isolates associated with different host species also demonstrated that transmission of P. multocida has occurred between different host species. Such host-switching could play an important role in generating diversity within P. multocida. Comparative nucleotide sequence analysis of genes encoding the predicted outer membrane proteins of different functions was carried out in 40 isolates of P. multocida to investigate nucleotide diversity and to assess the roles horizontal DNA transfer and recombination in the evolution and diversification of P. multocida. Comparative nucleotide sequence analysis provided clear evidence that horizontal DNA transfer and recombination (both intragenic and assortative) have occurred within the genes encoding P. multocida OMPs. However, it was also demonstrated that this varied from gene-to-gene. Four functional groups of OMPs were predicted based on the prediction analyses and these functions include outer membrane biogenesis and integrity (12 VIII proteins), transport and receptor (25 proteins), adherence (7 proteins) and enzymatic activity (9 proteins). Thirty five OMPs were analysed in this study in detail. The results showed limited levels of nucleotide and amino acid sequence variation was found within the genes encoding selected proteins with the exception of OmpA and OmpH1. However, there was evidence of gene exchange (assortative recombination) between isolates from different host species and divergent genetic lineages. High levels of nucleotide and amino acid sequence variation was found within two major surface-exposed proteins, OmpA and OmpH1. The results indicated that the ompA and ompH1 genes have undergone extensive horizontal DNA transfer, intragenic and assortative recombination. Variation in OmpA and OmpH1 occurred predominantly in the surface-exposed loop regions. There was strong evidence that natural selection is driving diversification of the hypervariable extracellular loop regions in both proteins. The diversity and molecular evolutionary relationships of ompA were further investigated in a larger selection of 74 P. multocida isolates. Sequence analysis of the 26 different ompA-type alleles revealed that the P. multocida ompA gene has undergone multiple horizontal gene transfer and recombination events because complex mosaic structures were found between ompA alleles. The diversity of temperate bacteriophages was examined in 47 P. multocida isolates. Phage particles were induced with mitomycin C and characterised morphologically by transmission electron microscopy (TEM). The phage particles were morphologically diverse and represented both the Siphoviridae and Myoviridae families. Both Siphoviridae and Myoviridae phage types were induced in certain isolates indicating that a single host may harbour multiple prophages. Moreover, phage DNA was successfully isolated from 18 P. multocida isolates. Bacteriophage DNA from isolates PM86, PM172, PM486, PM934 and PM954 showed the presence of two bands of different molecular size. Although these phages had a distinct Myoviridae-type morphology, they possessed an unusually small capsid as identified by TEM. Taken together, these results suggest the presence of phage-inducible chromosomal islands (PICIs), in these P. multocida strains. To date, PICIs have not been described in P. multocida. Genetic diversity of temperate bacteriophages was assessed by restriction endonuclease (RE) analysis and 10 different RE types (A to J) were identified. Plaque assay appeared to be IX less sensitive than TEM for detection of temperate bacteriophages. Only 11 (38%) P. multocida phages produced signs of infection against indicator strains. Nucleotide sequence analysis of phage genomic DNA from the same isolates demonstrated that both λ-like and Mu-like phages are induced in the same isolates of P. multocida. The results also showed that more than one λ-like and Mu-like phage is induced in the majority of isolates. Annotation of the sequenced phages resulted in five different Mu-like phages, one phage-inducible chromosomal island and seven λ-like phages. Further bacterial genome analyses identified additional intact prophages within the genomes of 40 isolates. From one to five intact prophages and prophage-like elements were identified within the genomes of P. multocida strains. The annotated phage genomes were analysed since phages are known to carry virulence factors, including genes encoding OMPs and various toxins, and also mediate horizontal DNA transfer. Nucleotide sequence analysis of λ-like phage genomes induced in toxigenic porcine isolates, PM684 and PM848 of capsular types A and D, demonstrated the presence of the toxA gene which encodes the P. multocida toxin (PMT). Moreover, genomic analysis identified additional intact λ-like prophages containing toxA within the bacterial genomes of the porcine toxigenic isolates PM918, PM926, PM40 and PM696 as well as in ovine isolates of capsular type D. No genes encoding OMPs were found to be carried by any of the bacteriophages. Overall, it was concluded that strains of P. multocida recovered from different host species carry a diverse range of bacteriophages. The presence of two bands of different molecular size of phage DNA from isolates PM86, PM172, PM486, PM934 and PM954; together with the identification of small capsids by TEM, suggest that these elements represent PICIs. Interestingly, Southern blot hybridisation of phage DNA in these isolates confirmed induction of both Mu-like phages and PICIs for the first time in P. multocida. This study represents the first comparative genomic analysis of the genes encoding the outer membrane proteome of P. multocida; it also represents the first detailed characterisation of the temperate bacteriophage content of a large number of P. multocida isolates recovered from different host species (cattle, sheep, pigs and poultry) and various disease syndromes. The study has, for the first time, identified PICIs in P. multocida.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology