73 results found
Pereira MF, Rossi CC, de Queiroz MV, et al., 2015, Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection, Microbiology, Vol: 161, Pages: 387-400, ISSN: 1350-0872
Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 104 to 107 c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.
Howell KJ, Weinert LA, Chaudhuri RR, et al., 2014, The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis., BMC Genomics, Vol: 15, Pages: 1179-1179, ISSN: 1471-2164
BACKGROUND: Haemophilus parasuis is the etiologic agent of Glasser's disease in pigs and causes devastating losses to the farming industry. Whilst some hyper-virulent isolates have been described, the relationship between genetics and disease outcome has been only partially established. In particular, there is weak correlation between serovar and disease phenotype. We sequenced the genomes of 212 isolates of H. parasuis and have used this to describe the pan-genome and to correlate this with clinical and carrier status, as well as with serotype. RESULTS: Recombination and population structure analyses identified five groups with very high rates of recombination, separated into two clades of H. parasuis with no signs of recombination between them. We used genome-wide association methods including discriminant analysis of principal components (DAPC) and generalised linear modelling (glm) to look for genetic determinants of this population partition, serovar and pathogenicity. We were able to identify genes from the accessory genome that were significantly associated with phenotypes such as potential serovar specific genes including capsule genes, and 48 putative virulence factors that were significantly different between the clinical and non-clinical isolates. We also show that the presence of many previously suggested virulence factors is not an appropriate marker of virulence. CONCLUSIONS: These genes will inform the generation of new molecular diagnostics and vaccines, and refinement of existing typing schemes and show the importance of the accessory genome of a diverse species when investigating the relationship between genotypes and phenotypes.
Bosse JT, Soares-Bazzolli DM, Li Y, et al., 2014, The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation, PLOS ONE, Vol: 9, ISSN: 1932-6203
Bosse JT, Li Y, Angen O, et al., 2014, Multiplex PCR Assay for Unequivocal Differentiation of Actinobacillus pleuropneumoniae Serovars 1 to 3, 5 to 8, 10, and 12, JOURNAL OF CLINICAL MICROBIOLOGY, Vol: 52, Pages: 2380-2385, ISSN: 0095-1137
Maglennon GA, Cook BS, Deeney AS, et al., 2013, Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations., Veterinary Research, Vol: 44, ISSN: 1297-9716
Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.
Luan S-L, Chaudhuri RR, Peters SE, et al., 2013, Generation of a Tn5 transposon library in Haemophilus parasuis and analysis by transposon-directed insertion-site sequencing (TraDIS), Veterinary Microbiology, Vol: 166, Pages: 558-566, ISSN: 0378-1135
Howell KJ, Weinert LA, Luan S-L, et al., 2013, Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis, JOURNAL OF BACTERIOLOGY, Vol: 195, Pages: 4264-4273, ISSN: 0021-9193
Maglennon GA, Cook BS, Matthews D, et al., 2013, Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae, Veterinary Research, Vol: 44, ISSN: 1297-9716
Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss topig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, butcurrent vaccines offer only partial control and there is a need for improved preventative strategies. A major barrierto advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lackof tools to genetically manipulate the organism. We describe the development and optimisation of the firstsuccessful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids containthe origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. Withthese plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generatingtetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNAover serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. Inaddition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising theconditions necessary for successful transformation, we have used this system to determine the minimum functionaloriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintainedover multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we havegenerated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a majorstep forward in the study of this important swine pathogen.
O'Neill C, Jones SCP, Bosse JT, et al., 2010, Prevalence of Actinobacillus pleuropneumoniae serovars in England and Wales, VETERINARY RECORD, Vol: 167, Pages: 661-662, ISSN: 0042-4900
O'Neill C, Jones SCP, Bosse JT, et al., 2010, Population-based analysis of Actinobacillus pleuropneumoniae ApxIVA for use as a DIVA antigen, VACCINE, Vol: 28, Pages: 4871-4874, ISSN: 0264-410X
Bossé JC, Sinha S, Li MS, et al., 2010, Regulation of pga operon expression and biofilm formation in Actinobacillus pleuropneumoniae by sigmaE and H-NS., Journal of Bacteriology, Vol: 192, Pages: 2414-2423
Clinical isolates of the porcine pathogen Actinobacillus pleuropneumoniae often form adherent colonies on agar plates due to expression of an operon, pgaABCD, encoding a poly-beta-1,6-N-acetyl-D-glucosamine (PGA) extracellular matrix. The adherent colony phenotype, which correlates with the ability to form biofilms on the surfaces of polystyrene plates, is lost following serial passage in broth culture, and repeated passage of the nonadherent variants on solid media does not result in reversion to the adherent colony phenotype. In order to investigate the regulation of PGA expression and biofilm formation in A. pleuropneumoniae, we screened a bank of transposon mutants of the nonadherent serovar 1 strain S4074(T) and identified mutations in two genes, rseA and hns, which resulted in the formation of the adherent colony phenotype. In other bacteria, including the Enterobacteriaceae, H-NS acts as a global gene regulator, and RseA is a negative regulator of the extracytoplasmic stress response sigma factor sigma(E). Transcription profiling of A. pleuropneumoniae rseA and hns mutants revealed that both sigma(E) and H-NS independently regulate expression of the pga operon. Transcription of the pga operon is initiated from a sigma(E) promoter site in the absence of H-NS, and upregulation of sigma(E) is sufficient to displace H-NS, allowing transcription to proceed. In A. pleuropneumoniae, H-NS does not act as a global gene regulator but rather specifically regulates biofilm formation via repression of the pga operon. Positive regulation of the pga operon by sigma(E) indicates that biofilm formation is part of the extracytoplasmic stress response in A. pleuropneumoniae.
Bosse JT, Sinha S, Schippers T, et al., 2009, Natural competence in strains of Actinobacillus pleuropneumoniae, FEMS MICROBIOLOGY LETTERS, Vol: 298, Pages: 124-130, ISSN: 0378-1097
Buettner FFR, Bendalla IM, Bosse JT, et al., 2009, Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor, PROTEOMICS, Vol: 9, Pages: 2383-2398, ISSN: 1615-9853
Bossé JT, Durham AL, Rycroft AN, et al., 2009, New Plasmid Tools for Genetic Analysis of Actinobacillus pleuropneumoniae and Other Pasteurellaceae, Vol: 75, Pages: 6124-6131
We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae. A tandem reporter plasmid, pMC-Tandem, carrying promoterless xylE and gfpmut3 genes downstream of a multiple-cloning site (MCS), can be used for identification of transcriptional regulators and conditions which favor gene expression from different cloned promoters. The ability to detect transcriptional regulators using the tandem reporter system was validated in A. pleuropneumoniae using the cloned rpoE (σE) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of σE, among a bank of random transposon mutants, as well as to detect induction of σE following exposure of A. pleuropneumoniae to ethanol or heat shock. pMCsodCP, carrying the cloned sodC promoter of A. pleuropneumoniae, was functional in A. pleuropneumoniae, Haemophilus influenzae, Haemophilus parasuis, Mannheimia haemolytica, and Pasteurella multocida. Two general expression vectors, pMK-Express and pMC-Express, which differ in their antibiotic resistance markers (kanamycin and chloramphenicol, respectively), were constructed for the Pasteurellaceae. Both plasmids have the A. pleuropneumoniae sodC promoter upstream of the gfpmut3 gene and an extended MCS. Replacement of gfpmut3 with a gene of interest allows complementation and heterologous gene expression, as evidenced by expression of the Haemophilus ducreyi nadV gene in A. pleuropneumoniae, rendering the latter NAD independent.
Godara G, Smith CP, Bosse J, et al., 2009, Functional Characterization of Actinobacillus pleuropneumoniae Urea Transport Protein, ApUT, Am J Physiol Regul Integr Comp Physiol
Mullen LM, Bosse JT, Nair SP, et al., 2008, Pasteurellaceae ComE1 proteins combine the properties of fibronectin adhesins and DNA binding competence proteins, PLoS ONE, Vol: 3
Buettner FFR, Bendallah IM, Bosse JT, et al., 2008, Analysis of the Actinobacillus pleuropneumoniae ArcA regulon identifies fumarate reductase as a determinant of virulence, INFECTION AND IMMUNITY, Vol: 76, Pages: 2284-2295, ISSN: 0019-9567
Zhou L, Jones SCP, Angen O, et al., 2008, PCR specific for Actinobacillus pleuropneumoniae serotype 3, VETERINARY RECORD, Vol: 162, Pages: 648-+, ISSN: 0042-4900
Zhou L, Jones SCP, Angen O, et al., 2008, Multiplex PCR that can distinguish between immunologically cross-reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains, JOURNAL OF CLINICAL MICROBIOLOGY, Vol: 46, Pages: 800-803, ISSN: 0095-1137
Foote SJ, Bosse JT, Bouvevitch AB, et al., 2008, The complete genome sequence of Actinobacillus pleuropneumoniae L20 (serotype 5b)., J Bacteriol, Vol: 190, Pages: 1495-1496
There are 16 capsule-based serotypes of Actinobacillus pleuropneumoniae, all of which are capable of causing disease in pigs. Here we report the finished and annotated genome sequence of the reference serotype 5b strain L20. This strain has a rough appearance and readily forms biofilms, as is typical for most field isolates.
Redfield RJ, Findlay WA, Bosse J, et al., 2006, Evolution of competence and DNA uptake specificity in the Pasteurellaceae, BMC EVOLUTIONARY BIOLOGY, Vol: 6, ISSN: 1471-2148
Bossé JT, Zhou L, Kroll JS, et al., 2006, High-throughput identification of conditionally essential genes in bacteria: from STM to TSM., Infect Disord Drug Targets, Vol: 6, Pages: 241-262, ISSN: 1871-5265
Signature-tagged mutagenesis (STM) provided the first widely applicable high-throughput method for detecting conditionally essential genes in bacteria by using negative selection to screen large pools of transposon (Tn) mutants. STM requires no prior knowledge of the bacterium's genome sequence, and has been used to study a large number of Gram-positive and Gram-negative species, greatly expanding the repertoires of known virulence factors for these organisms. Originally, hybridization of radiolabelled probes to colony or dot blots was used to detect differences in populations of tagged mutants before and after growth under a selective condition. Modifications of the tag detection method involving polymerase chain reaction (PCR) amplification and visualisation by gel electrophoresis have been developed and can be automated through the use of robotics. Genetic footprinting is another negative selection technique that uses PCR amplification to detect loss of mutants from a pool. Unlike PCR-STM, this technique allows direct amplification of Tn-flanking sequences. However, it requires the bacterium's whole genome sequence in order to design specific primers for every gene of interest. More recently, a number of techniques have been described that combine the negative-selection principle of STM and genetic footprinting with the genome-wide screening power of DNA microarrays. These techniques, although also requiring whole genome sequences, use either a form of linker-mediated or semi-random PCR to amplify and label Tn-flanking regions for hybridization to microarrays. The superior sensitivity microarray detection allows greater numbers of mutants to be screened per pool, as well as determination of the coverage/distribution of insertions in the library prior to screening, two significant advantages over STM.
Jacobsen I, Gerstenberger J, Gruber AD, et al., 2005, Deletion of the ferric uptake regulator fur impairs the in vitro growth and virulence of Actinobacillus pleuropneumoniae, INFECTION AND IMMUNITY, Vol: 73, Pages: 3740-3744, ISSN: 0019-9567
Hodgetts A, Bosse JT, Kroll JS, et al., 2004, Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation-ProteinChip (TM) (SELDI) technology, VETERINARY MICROBIOLOGY, Vol: 99, Pages: 215-225, ISSN: 0378-1135
Bosse JT, Nash JHE, Kroll JS, et al., 2004, Harnessing natural transformation in Actinobacillus pleuropneumoniae: a simple method for allelic replacements, FEMS MICROBIOLOGY LETTERS, Vol: 233, Pages: 277-281, ISSN: 0378-1097
Beddek AJ, Sheehan BJ, Bosse JT, et al., 2004, Two TonB systems in Actinobacillus pleuropneumoniae: Their roles in iron acquisition and virulence, INFECTION AND IMMUNITY, Vol: 72, Pages: 701-708, ISSN: 0019-9567
Bossé J T, MacInnes J I, 2004, Actinobacillus., Pathogenesis of bacterial infections of animals., Editors: Gyles L, Prescott F, Songer G, Thoen O, Ames, Iowa, Publisher: Blackwell Publishing, Pages: 225-241, ISBN: 9780813829395
Sheehan BJ, Bosse JT, Beddek AJ, et al., 2003, Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host, INFECTION AND IMMUNITY, Vol: 71, Pages: 3960-3970, ISSN: 0019-9567
Bosse JT, Janson H, Sheehan BJ, et al., 2002, Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection, MICROBES AND INFECTION, Vol: 4, Pages: 225-235, ISSN: 1286-4579
Bosse JT, Gilmour HD, MacInnes JI, 2001, Novel genes affecting urease activity in Actinobacillus pleuropneumoniae, JOURNAL OF BACTERIOLOGY, Vol: 183, Pages: 1242-1247, ISSN: 0021-9193
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