Predicting the outer membrane proteome of Pasteurella multocida based on consensus prediction enhanced by results integration and manual confirmation

Background Outer membrane proteins (OMPs) of Pasteurella multocida have various functions related to virulence and pathogenesis and represent important targets for vaccine development. Various bioinformatic algorithms can predict outer membrane localization and discriminate OMPs by structure or function. The designation of a confident prediction framework by integrating different predictors followed by consensus prediction, results integration and manual confirmation will improve the prediction of the outer membrane proteome. Results In the present study, we used 10 different predictors classified into three groups (subcellular localization, transmembrane β-barrel protein and lipoprotein predictors) to identify putative OMPs from two available P. multocida genomes: those of avian strain Pm70 and porcine non-toxigenic strain 3480. Predicted proteins in each group were filtered by optimized criteria for consensus prediction: at least two positive predictions for the subcellular localization predictors, three for the transmembrane β-barrel protein predictors and one for the lipoprotein predictors. The consensus predicted proteins were integrated from each group into a single list of proteins. We further incorporated a manual confirmation step including a public database search against PubMed and sequence analyses, e.g. sequence and structural homology, conserved motifs/domains, functional prediction, and protein-protein interactions to enhance the confidence of prediction. As a result, we were able to confidently predict 98 putative OMPs from the avian strain genome and 107 OMPs from the porcine strain genome with 83% overlap between the two genomes. Conclusions The bioinformatic framework developed in this study has increased the number of putative OMPs identified in P. multocida and allowed these OMPs to be identified with a higher degree of confidence. Our approach can be applied to investigate the outer membrane proteomes of other Gram-negative bacteria

Comparative bioinformatic and proteomic approaches to evaluate the outer membrane proteome of the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM) disease and is responsible for significant economic losses in farmed salmonids. Enteric redmouth disease is associated primarily with rainbow trout (Oncorhynchus mykiss, Walbaum) but its incidence in Atlantic salmon (Salmo salar) is increasing. Outer membrane proteins (OMPs) of Gram-negative bacteria are located at the host-pathogen interface and play important roles in virulence. The outer membrane of Y. ruckeri is poorly characterised and little is known about its composition and the roles of individual OMPs in virulence. Here, we employed a bioinformatic pipeline to first predict the OMP composition of Y. ruckeri. Comparative proteomic approaches were subsequently used to identify those proteins expressed in vitro in eight representative isolates recovered from Atlantic salmon and rainbow trout. One hundred and forty-one OMPs were predicted from four Y. ruckeri genomes and 77 of these were identified in three or more genomes and were considered as “core” proteins. Gel-free and gel-based proteomic approaches together identified 65 OMPs in a single reference isolate and subsequent gel-free analysis identified 64 OMPs in the eight Atlantic salmon and rainbow trout isolates. Together, our gel-free and gel-based proteomic analyses identified 84 unique OMPs in Y. ruckeri. Significance: Yersinia ruckeri is an important pathogen of Atlantic salmon and rainbow trout and is of major economic significance to the aquaculture industry worldwide. Disease outbreaks are becoming more problematic in Atlantic salmon and there is an urgent need to investigate in further detail the cell-surface (outer membrane) composition of strains infecting each of these host species. Currently, the outer membrane of Y. ruckeri is poorly characterised and very little is known about the OMP composition of strains infecting each of these salmonid species. This study represents the most comprehensive comparative outer membrane proteomic analysis of Y. ruckeri to date, encompassing isolates of different biotypes, serotypes, OMP-types and hosts of origin and provides insights into the potential roles of these diverse proteins in host-pathogen interactions. The study has identified key OMPs likely to be involved in disease pathogenesis and makes a significant contribution to furthering our understanding of the cell-surface composition of this important fish pathogen that will be relevant to the development of improved vaccines and therapeutics

Molecular evolution of outer membrane proteins and characterization of temperate bacteriophages of Pasteurella multocida strains from different host species

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; http://pubmlst.org/pmultocida_multihost). 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

Comparative phenotypic, proteomic and genomic approaches to assess lipopolysaccharide and outer membrane protein diversity among isolates of Yersinia ruckeri recovered from Atlantic salmon and rainbow trout

Yersinia ruckeri is the causative agent of enteric redmouth (ERM) disease in farmed salmonids. ERM disease is traditionally associated with rainbow trout (Oncorhynchus mykiss¸ Walbaum), but the incidence of the disease in Atlantic salmon (Salmo salar) has increased in recent years. Historically, motile (biotype 1), serotype O1 isolates of Y. ruckeri have been mostly responsible for ERM in rainbow trout worldwide but non-motile (biotype 2), serotype O1 isolates have become increasingly prevalent in this species over wide geographic areas since their first isolation in the UK in the 1980s. Yersinia ruckeri isolates responsible for infection of salmon have been less well characterised than those from rainbow trout and little is known about their diversity. The emergence of new pathogenic strains, together with vaccine breakdown in the field, has emphasised the need for greater knowledge about strain diversity which may lead to the development of improved vaccines for both species. In the present study, a unique and extensive strain collection encompassing 135 isolates of Y. ruckeri were characterised using complementary phenotypic, proteomic and genomic approaches. In the initial part of this thesis, 135 isolates recovered over a 14 year period in the UK from infected Atlantic salmon (109 isolates) and rainbow trout (26 isolates) were phenotypically characterised through biotype, serotype, and outer membrane protein (OMP) -type analysis. Atlantic salmon isolates represented a wider range of O-serotypes and associated lipopolysaccharide (LPS) types, and had more diverse OMP profiles than those from rainbow trout. Most significantly, a new O-serotype/LPS type was identified in 56 Atlantic salmon isolates; other Atlantic salmon isolates were represented by serotypes O1 (five isolates), O2 (34 isolates) and O5 (14 isolates). This new LPS type comprises a core polysaccharide region similar to that of serotype O1 but has a unique, previously unidentified O-antigen region. Atlantic salmon isolates could be assigned to one of four major OMP-types and to one of 11 OMP-sub types. Isolates recovered from rainbow trout were represented by the same non-motile clone that is responsible for the majority of ERM outbreaks in this species within the UK. This clone was not associated with any infected salmon. However, two isolates of the novel serotype O1/O5 were recovered from rainbow trout in 2010 and 2011. These data suggest that different Y. ruckeri strains are specifically adapted to cause disease in either Atlantic salmon or rainbow trout. The efficacy of current vaccine formulations against different clonal groups must be examined. Subsequently, an in-depth characterisation of the outer membrane (OM) proteome of isolates recovered from Atlantic salmon and rainbow trout was conducted. Outer membrane proteins are at the interface of host pathogen interactions, with important roles in adherence, evasion of host immune response, and transport. Using a bioinformatic prediction pipeline and four publicly available genomes, 141 proteins were confidently predicted to be OM associated. Subsequently, the OM proteomes of eight representative isolates (four from rainbow trout; four from Atlantic salmon) were analysed using a combination of gel-based and gel-free proteomic approaches. In total, 66 OMPs were identified through this combined approach, of which 28 were unique to the gel-free approach and 13 were unique to the gel-based approach. Further to this, the OM proteomes of these eight representative isolates were examined when cells were grown under conditions that aimed to mimic the in vivo and environmental conditions of Y. ruckeri. These included growing cells aerobically at 22°C and 28°C, anaerobically, under iron-depletion and in an artificial seawater medium at 22°C. In total, 76 OMPs were identified in all eight isolates under these growth conditions. Finally, a phylogenetic study was undertaken whereby the genomes of 16 representative isolates encompassing a range of biotypes, serotypes, host species (eight from rainbow trout, seven from Atlantic salmon and one from European eel), geographic locations and dates of isolation were considered. A phylogenetic species tree based on the concatenated sequences of 19 housekeeping genes revealed host specific lineages suggesting an earlier host-associated evolutionary split within Y. ruckeri. Subsequent analysis of the presence, absence and variation of the nucleotide and amino acid sequences of the 141 predicted OMPs revealed high levels of conservation (with 120 OMPs showing less than 1% nucleotide variation). One hundred and thirty proteins were identified in all 16 genomes examined. However, 11 proteins were not, and these included invasins, OmpE and proteins involved in pilus biogenesis. Further examination of the OMPs OmpA and OmpF, which were identified in the genomes of all 16 isolates, revealed variation in the surface exposed loop regions which may play a role in pathogenicity and/or host specificity. This study represents a comprehensive characterisation of Y. ruckeri isolates recovered from Atlantic salmon and rainbow trout using a range of molecular techniques, and reveals important adaptations that the bacteria may make in order to survive both inside and outside of the host. Importantly, this study provides comprehensive support for future work involving this fish pathogen

Molecular evolutionary analyses and epidemiology of Vibrio parahaemolyticus in Thailand

Vibrio parahaemolyticus is a seafood-borne pathogenic bacterium which is a major cause of gastroenteritis worldwide. In the present study, the genetic relationships and population structure of isolates originating from clinical and seafood production sources in Thailand were investigated by multilocus sequence typing (MLST). Nucleotide sequence variation of virulence-related genes including haemolysin and TTSS1 genes among Thai and worldwide isolates was also analyzed. The outer membrane proteome of V. parahaemolyticus isolate RIMD2210633 was predicted using bioinformatic approaches, and the outer membrane proteomes of eight isolates from different sources and representing different MLST sequence type characterized using proteomics. The 101 Thai V. parahaemolyticus isolates examined were recovered from clinical samples (n=15), healthy human carriers (n=18), various fresh seafood (n=18), frozen shrimps (n=16), fresh-farmed shrimp tissue (n=18) and shrimp-farm water (n=16). Phylogenetic analysis revealed a high degree of genetic diversity within the V. parahaemolyticus population, although isolates recovered from clinical samples, farmed shrimp and water samples represented five distinct clusters. The majority of clinical isolates were resolved into two genetic clusters and none of these isolates were found to share sequence types (STs) with strains isolated from human carriers, seafood, or water. Similarly, STs representing human carrier isolates differed from those of clinical, seafood and water isolates. The limited genetic diversity of the clinical isolates suggested non-random selection for pathogenic strains, but the absence of such strains in local seafood raises questions about the likely source of infection. Extensive serotypic diversity occurred among isolates representing the same STs and recovered from the same source at the same time point. Furthermore, evidence of interspecies horizontal gene transfer and intragenic recombination was observed at the recA locus in a large proportion of isolates; this has a substantial effect on the apparent phylogenetic relationships of the isolates. Notably, the majority of these recombinational exchanges occurred among clinical and carrier isolates, suggesting that the human intestinal tract is serving as a reservoir that is driving evolutionary change and leading to the emergence of new, potentially pathogenic strains. MLST was also applied to study genetic relationships between V. parahaemolyticus isolates from Thailand (n=101) and those from European countries (n=9). With the exception of the pandemic ST3 which was resolved from two isolates from Thai human carriers, two clinical isolates from England and a clinical isolate from Norway, none of the other European isolates examined in this study shared the same ST with the Thai isolates. This study demonstrated that Thai human carrier isolates are capable of harbouring virulence-related genes including the haemolysin-encoding genes tdhA, tdhS, trh1 and trh2, and the TTSS1-related genes vcrD1, vscC2 and VP1680, that are present in clinical isolates. In particular, the Thai human carrier isolate VP132 shared identical TTSS1-related gene fragments with the pandemic V. parahaemolyticus serotype O3:K6 (RIMD2210633) and related strains (AQ3810, AQ4037, Peru466, AN5034 and K5030) of worldwide distribution. A total of 117 outer membrane proteins (OMPs) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633. A total 73 OMPs proteins were identified from eight V. parahaemolyticus isolates recovered from clinical samples, human carriers, oyster, shrimp tissue and water in Thailand. Of the 117 predicted OMPS, 32 were identified in eight strains by proteomic analysis. OmpU, a non-specific porin protein, represents the most abundantly expressed protein in all eight isolates. OMPs involved in TTSSs (YscW, YscJ, YscC, PopN and VscC2) and iron uptake (IrgA, putative 83 Da decaheme outer membrane cytochrome C, PvuA1, PvuA2, LutA, FhuE, HutA and putative-regulated protein B) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633, but were not recovered from any of the eight Thai isolates. The absence of TTSS and iron uptake related OMPs in the eight representative strains that were grown under in vitro conditions may suggest an important requirement for in vivo growth conditions to induce expression of important virulence factor-related OMPs in V. parahaemolyticus. There was no clear association between OMP profile and the source of isolation, ST or serotype. However, a high degree of variation of OMP profiles was observed in isolates from different sources as well as in the isolates representing the same ST. This study demonstrated the usefulness of a multidisciplinary approach that includes MLST, virulence-related gene DNA sequence analysis, bioinformatic prediction and gel-based proteomic analyses for the study of molecular evolutionary relationships and the epidemiology of V. parahaemolyticus isolates from clinical and seafood production sources. The outcomes of this study highlight the role of human carriers as a reservoir of potentially pathogenic V. parahaemolyticus and this should be considered as one of the possible contamination sources in the surveillance of seafood safety

Functional genomic analysis of Haemophilus influenzae and application to the study of competence and transformation.

During the progression of this study, hundreds of additional bacterial genomes were sequenced, techniques have evolved, and novel approaches were developed to aid the field of functional and comparative genomics. Some of these techniques were used in this study to identify three novel competence-regulated operons in H. influenzae. The techniques included the use of advanced computer programs and algorithms to assist in predicting protein functions and to facilitate a comparative genomic analysis of H. influenzae with other species of the Pasteurellaceae family. Quantitative PCR was employed to examine the expression of putative transformation-related genes. Finally, PCR-mediated mutagenesis was used in a directed approach to generate mutations in the newly discovered competence-regulated operons to assess their involvement in uptake and transformation of exogenous DNA.The publication of the complete genomic sequence of Haemophilus influenzae Rd KW20 in 1995 was a truly monumental event in molecular biology. For the first time, all of the potential genes of an independent-living organism were known and awaiting functional characterization. This event required the development of fundamentally different methodologies to elucidate gene functions, with systematic global approaches becoming much more feasible. This study describes the development of a transposon-based mutagenesis strategy to facilitate a high-throughput functional analysis of the H. influenzae genome. Mutants created using this strategy were screened in a highly-parallel assay to identify genes mediating transformation in this organism. Additionally, analysis of the transposon insertion sites generated during this study identified a previously unrecognized Tn5 insertion bias

Identification of Legionella outer membrane proteins for the development of a biosensor

Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism

Microbial Secondary Metabolites and Biotechnology

Many research teams are working to demonstrate that microorganisms can be our daily partners, due to the great diversity of biochemical transformations and molecules they are able to produce. This Special Issue highlights several facets of the production of microbial metabolites of interest. From the discovery of new strains or new bioactive molecules issued from novel environments, to the increase in their synthesis by traditional or innovative methods, different levels of biotechnological processes are addressed. Combining the new dimensions of "Omics" sciences, such as genomics, transcriptomics or metabolomics, microbial biotechnologies are opening up incredible opportunities for discovering and improving microorganisms and their production