Bogema, DR, McKinnon, J, Liu, M, Hitchick, N, Miller, N, Venturini, C, Iredell, J, Darling, AE, Roy Chowdury, P & Djordjevic, SP 2020, 'Whole-genome analysis of extraintestinal Escherichia coli sequence type 73 from a single hospital over a 2 year period identified different circulating clonal groups.', Microbial Genomics, vol. 6, no. 1, pp. 1-18.View/Download from: Publisher's site
Sequence type (ST)73 has emerged as one of the most frequently isolated extraintestinal pathogenic Escherichia coli. To examine the localized diversity of ST73 clonal groups, including their mobile genetic element profile, we sequenced the genomes of 16 multiple-drug resistant ST73 isolates from patients with urinary tract infection from a single hospital in Sydney, Australia, between 2009 and 2011. Genome sequences were used to generate a SNP-based phylogenetic tree to determine the relationship of these isolates in a global context with ST73 sequences (n=210) from public databases. There was no evidence of a dominant outbreak strain of ST73 in patients from this hospital, rather we identified at least eight separate groups, several of which reoccurred, over a 2 year period. The inferred phylogeny of all ST73 strains (n=226) including the ST73 clone D i2 reference genome shows high bootstrap support and clusters into four major groups that correlate with serotype. The Sydney ST73 strains carry a wide variety of virulence-associated genes, but the presence of iss, pic and several iron-acquisition operons was notable.
Gruening, B, Dale, R, Sjoedin, A, Chapman, BA, Rowe, J, Tomkins-Tinch, CH, Valieris, R, Koester, J & Team, B 2018, 'Bioconda: sustainable and comprehensive software distribution for the life sciences', NATURE METHODS, vol. 15, no. 7, pp. 475-476.View/Download from: Publisher's site
Jenkins, C, Jelocnik, M, Micallef, ML, Galea, F, Taylor-Brown, A, Bogema, DR, Liu, M, O'Rourke, B, Chicken, C, Carrick, J & Polkinghorne, A 2018, 'An epizootic of Chlamydia psittaci equine reproductive loss associated with suspected spillover from native Australian parrots.', Emerging microbes & infections, vol. 7, no. 1, pp. 88-88.View/Download from: Publisher's site
Chlamydia psittaci is an avian pathogen capable of spill-over infections to humans. A parrot C. psittaci strain was recently detected in an equine reproductive loss case associated with a subsequent cluster of human C. psittaci infections. In this study, we screened for C. psittaci in cases of equine reproductive loss reported in regional New South Wales, Australia during the 2016 foaling season. C. psittaci specific-PCR screening of foetal and placental tissue samples from cases of equine abortion (n = 161) and foals with compromised health status (n = 38) revealed C. psittaci positivity of 21.1% and 23.7%, respectively. There was a statistically significant geographical clustering of cases ~170 km inland from the mid-coast of NSW (P < 0.001). Genomic analysis and molecular typing of C. psittaci positive samples from this study and the previous Australian equine index case revealed that the equine strains from different studs in regional NSW were clonal, while the phylogenetic analysis revealed that the C. psittaci strains from both Australian equine disease clusters belong to the parrot-associated 6BC clade, again indicative of spill-over of C. psittaci infections from native Australian parrots. The results of this work suggest that C. psittaci may be a more significant agent of equine reproductive loss than thought. A range of studies are now required to evaluate (a) the exact role that C. psittaci plays in equine reproductive loss; (b) the range of potential avian reservoirs and factors influencing infection spill-over; and
Yam, J, Gestier, S, Bryant, B, Campbell-Ward, M, Bogema, D & Jenkins, C 2018, 'The identification of Theileria bicornis in captive rhinoceros in Australia', INTERNATIONAL JOURNAL FOR PARASITOLOGY-PARASITES AND WILDLIFE, vol. 7, no. 1, pp. 85-89.View/Download from: Publisher's site
Bogema, DR, Micallef, ML, Liu, M, Padula, MP, Djordjevic, SP, Darling, AE & Jenkins, C 2018, 'Analysis of Theileria orientalis draft genome sequences reveals potential species-level divergence of the Ikeda, Chitose and Buffeli genotypes.', BMC genomics, vol. 19, no. 1, pp. 298-298.View/Download from: Publisher's site
BACKGROUNDTheileria orientalis (Apicomplexa: Piroplasmida) has caused clinical disease in cattle of Eastern Asia for many years and its recent rapid spread throughout Australian and New Zealand herds has caused substantial economic losses to production through cattle deaths, late term abortion and morbidity. Disease outbreaks have been linked to the detection of a pathogenic genotype of T. orientalis, genotype Ikeda, which is also responsible for disease outbreaks in Asia. Here, we sequenced and compared the draft genomes of one pathogenic (Ikeda) and two apathogenic (Chitose, Buffeli) isolates of T. orientalis sourced from Australian herds.RESULTSUsing de novo assembled sequences and a single nucleotide variant (SNV) analysis pipeline, we found extensive genetic divergence between the T. orientalis genotypes. A genome-wide phylogeny reconstructed to address continued confusion over nomenclature of this species displayed concordance with prior phylogenetic studies based on the major piroplasm surface protein (MPSP) gene. However, average nucleotide identity (ANI) values revealed that the divergence between isolates is comparable to that observed between other theilerias which represent distinct species. Analysis of SNVs revealed putative recombination between the Chitose and Buffeli genotypes and also between Australian and Japanese Ikeda isolates. Finally, to inform future vaccine studies, dN/dS ratios and surface location predictions were analysed. Six predicted surface protein targets were confirmed to be expressed during the piroplasm phase of the parasite by mass spectrometry.CONCLUSIONSWe used whole genome sequencing to demonstrate that the T. orientalis Ikeda, Chitose and Buffeli variants show substantial genetic divergence. Our data indicates that future researchers could potentially consider disease-associated Ikeda and closely related genotypes as a separate species from non-pathogenic Chitose and Buffeli.
Hammer, JF, Jenkins, C, Bogema, D & Emery, D 2016, 'Mechanical transfer of Theileria orientalis: possible roles of biting arthropods, colostrum and husbandry practices in disease transmission', PARASITES & VECTORS, vol. 9.View/Download from: Publisher's site
Jenkins, C & Bogema, DR 2016, 'Factors associated with seroconversion to the major piroplasm surface protein of the bovine haemoparasite Theileria orientalis', PARASITES & VECTORS, vol. 9.View/Download from: Publisher's site
Proctor, AKK, Ball, M, Freeman, P, Jenkins, C & Bogema, DR 2016, 'Prevalence of Theileria orientalis types in beef cattle herds on the North Coast of New South Wales', AUSTRALIAN VETERINARY JOURNAL, vol. 94, no. 4, pp. 117-120.View/Download from: Publisher's site
Tacchi, JL, Raymond, BBA, Haynes, PA, Berry, IJ, Widjaja, M, Bogema, DR, Woolley, LK, Jenkins, C, Minion, FC, Padula, MP & Djordjevic, SP 2016, 'Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae', OPEN BIOLOGY, vol. 6, no. 2.View/Download from: Publisher's site
Bogema, DR, Deutscher, AT, Fell, S, Collins, D, Eamens, GJ & Jenkins, C 2015, 'Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes', JOURNAL OF CLINICAL MICROBIOLOGY, vol. 53, no. 3, pp. 941-950.View/Download from: Publisher's site
Bogema, DR, Fell, SA, O'Rourke, BA, Collins, D, Eamens, GJ & Jenkins, C 2015, 'Development and validation of an inexpensive and efficient method for the extraction of Theileria orientalis DNA from blood', VETERINARY PARASITOLOGY, vol. 212, no. 3-4, pp. 379-381.View/Download from: Publisher's site
Hammer, JF, Emery, D, Bogema, DR & Jenkins, C 2015, 'Detection of Theileria orientalis genotypes in Haemaphysalis longicornis ticks from southern Australia', PARASITES & VECTORS, vol. 8.View/Download from: Publisher's site
Suann, M, Bogema, DR, Chen, Y, Mansfield, S, Barchia, IM & Herron, GA 2015, 'A TaqMan qPCR method for detecting kdr resistance in Aphis gossypii demonstrates improved sensitivity compared to conventional PCR-RFLP', JOURNAL OF PEST SCIENCE, vol. 88, no. 4, pp. 785-791.View/Download from: Publisher's site
Jenkins, C, Micallef, M, Alex, SM, Collins, D, Djordjevic, S & Bogema, DR 2015, 'Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle', Infection, Genetics and Evolution, vol. 32, pp. 199-207.View/Download from: Publisher's site
In Australia, outbreaks of clinical theileriosis caused by Theileria orientalis have been largely associated with the Ikeda genotype which can occur as a sole infection, or more commonly, as a mixture of genotypes. The most prevalent genotype, Chitose, frequently co-occurs with type Ikeda, however the role of this genotype in clinical disease has not been clearly established. Furthermore, the dynamics of individual genotypes in field infection of cattle have not been examined. In this study we developed quantitative PCR (qPCR) and genotyping methods to examine the role of the Chitose genotype in clinical disease and to investigate the temporal dynamics of T. orientalis Ikeda, Chitose and Buffeli genotypes in naïve animals introduced to a T. orientalis-endemic area. Analysis of the major piroplasm surface protein (MPSP) genes of Chitose isolates revealed the presence of two distinct phylogenetic clusters, Chitose A and Chitose B. A genotyping assay aimed at determining Chitose A/B allele frequency revealed that the Chitose A phylogenetic cluster is strongly associated with clinical disease but nearly always co-occurs with the Ikeda genotype. qPCR revealed that the Chitose genotype (particularly Chitose A), undergoes temporal switching in conjunction with the Ikeda genotype and contributes substantially to the overall parasite burden. The benign Buffeli genotype can also undergo temporal switching but levels of this genotype appear to remain low relative to the Ikeda and Chitose types. Interplay between vector and host immunological factors is presumed to be critical to the population dynamics observed in this study. Genotypic switching likely contributes to the persistence of T. orientalis in the host.
Chen, Y, Bogema, DR, Barchia, IM & Herron, GA 2014, 'Quantification of the pirimicarb resistance allele frequency in pooled cotton aphid (Aphis gossypii Glover) samples by TaqMan SNP genotyping assay.', PLoS ONE, vol. 9, no. 3, pp. 1-12.View/Download from: Publisher's site
Pesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Monitoring of resistance in arthropod populations is initially performed by bioassay, a method that detects a phenotypic response to pesticides. Molecular diagnostic assays, offering speed and cost improvements, can be developed when the causative mutation for resistance has been identified. However, improvements to throughput are limited as genotyping methods cannot be accurately applied to pooled DNA. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance.We demonstrate a new method to quantify a resistance allele frequency (RAF) from pooled insects via TaqMan assay by using raw fluorescence data to calculate the transformed fluorescence ratio k' at the inflexion point based on a four parameter sigmoid curve. Our results show that k' is reproducible and highly correlated with RAF (r >0.99). We also demonstrate that k' has a non-linear relationship with RAF and that five standard points are sufficient to build a prediction model. Additionally, we identified a non-linear relationship between runs for k', allowing the combination of samples across multiple runs in a single analysis.The transformed fluorescence ratio (k') method can be used to monitor pesticide resistance in IPM and to accurately quantify allele frequency from pooled samples. We have determined that five standards (0.0, 0.2, 0.5, 0.8, and 1.0) are sufficient for accurate prediction and are statistically-equivalent to the 13 standard points used experimentally.
Herron, GA, Langfield, BJ, Bogema, DR & Chen, Y 2014, 'Baseline susceptibility and cross-resistance in Aphis gossypii Glover (Aphididae: Hemiptera) to phorate and sulfoxaflor', Austral Entomology, vol. 53, no. 1, pp. 32-35.View/Download from: Publisher's site
Susceptible discriminating doses of phorate (0.2 g/L) and sulfoxaflor (0.01 g/L) against cotton aphid Aphis gossypii Glover were determined by laboratory bioassay where aphids were sprayed with insecticide with the aid of a Potter spray tower. All of the populations tested were susceptible to sulfoxaflor, and only a pirimicarb resistant strain had cross-resistance to phorate. If phorate is used as a side dressing in Australian cotton for insect control, neither pirimicarb, or any other chemical associated with insensitive acetylcholinesterase type one resistance, should be used as the first foliar spray for any subsequent aphid control.
Bogema, DR, Deutscher, AT, Woolley, LK, Seymour, LM, Raymond, BB, Tacchi, JL, Padula, M, Dixon, NE, Minion, F, Jenkins, C, Walker, MJ & Djordjevic, SP 2012, 'Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography', mBio, vol. 3, no. 2, pp. 1-11.View/Download from: Publisher's site
Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF down arrow QQ(677), consistent with a cleavage motif resembling S/T-X-F down arrow X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography.
Deutscher, AT, Tacchi, JL, Minion, F, Padula, M, Crossett, B, Bogema, DR, Jenkins, C, Kuit, TA, Walker, MJ & Djordjevic, SP 2012, 'Mycoplasma hyopneumoniae surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs', Journal of Proteome Research, vol. 11, no. 3, pp. 1924-1936.View/Download from: Publisher's site
P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F down arrow X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site L-761-N-V down arrow A-V-S-766 in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (alpha 3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.
Seymour, LM, Jenkins, C, Deutscher, AT, Raymond, BB, Padula, M, Tacchi, JL, Bogema, DR, Eamens, GJ, Woolley, LK, Dixon, NE, Walker, MJ & Djordjevic, SP 2012, 'Mhp182 (P102) binds fibronectin and contributes to the recruitment of plasmin(ogen) to the Mycoplasma hyopneumoniae cell surface', Cellular Microbiology, vol. 14, no. 1, pp. 81-94.View/Download from: Publisher's site
Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (KD similar to 76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with KDs of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.
Bogema, DR, Scott, NE, Padula, M, Tacchi, JL, Raymond, B, Jenkins, C, Cordwell, SJ, Minion, F, Walker, MJ & Djordjevic, SP 2011, 'Sequence TTKF | QE defines the site of proteolytic cleavage in Mhp683, a novel glycosaminoglycan and cilium adhesin of Mycoplasma hyopneumoniae', Journal Of Biological Chemistry, vol. 286, no. 48, pp. 41217-41229.View/Download from: Publisher's site
Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45683, P48683, and P50683. A peptide sequence (TTKF?QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48683 and P50683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45683, P48683, and P50683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1683F5683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1683F5683 also bound porcine epithelial cilia, and antisera to F2683 and F5683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45683, P48683, and P50683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.
Scott, NE, Bogema, D, Connolly, AM, Falconer, L, Djordjevic, SP & Cordwell, SJ 2009, 'Mass spectrometric characterisation of the surface-associated 42 kDa lipoprotein JIpA as a Glycosylated antigen in strains of Campylobacter jejuni', Journal Of Proteome Research, vol. 8, no. 10, pp. 4654-4664.View/Download from: Publisher's site
Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the developed world. Immunoproteomics highlighted a 42-45 kDa antigen that comigrated on two-dimensional (2-DE) gels with the C. jejuni major outer membrane protein (MOMP). Predictive analysis revealed two candidates for the identity of the antigen, the most likely of which was the surface-associated lipoprotein, JlpA. Recombinant JlpA (rJlpA) reacted with patient sera, confirming that JlpA is antigenic. Polyclonal antibodies raised against rJlpA reacted against 3 JlpA mass variants from multiple C. jejuni. These variants differed by approximately 1.5 kDa, suggesting the presence of the N-linked C. jejuni glycan on two sites. Soybean agglutinin affinity and 2-DE purified 2 JlpA glycoforms (43.5 and 45 kDa). Their identities were confirmed using mass spectrometry following trypsin digest. Glycopeptides within JlpA variants were identified by proteinase-K digestion, graphite micropurification and MS-MS. Sites of glycosylation were confirmed as asparagines 107 and 146, both of which are flanked by the N-linked sequon. Sequence analysis confirmed that the N146 sequon is conserved in all C. jejuni genomes examined to date, while the N107 sequon is absent in the reference strain NCTC 11168. Western blotting confirmed the presence of only a single JlpA glycoform in both virulent (O) and avirulent (GS) isolates of NCTC 11168. MS analysis showed that JlpA exists as 3 discrete forms, unmodified, glycosylated at N146, and glycosylated at both N146/107, suggesting glycan addition at N146 is necessary for N107 glycosylation. Glycine extracts and Western blotting revealed that doubly glycosylated JlpA was the predominant form on the C. jejuni JHH1 surface; however, glycosylation is not required for antigenicity. This is the first study to identify N-linked glycosylation of a surface-exposed C. jejuni virulence factor and to show strain variation in glycosylation sites.