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Pathogenomics
Goal:
Identify previously unrecognized mechanisms of microbial pathogenicity using a unique combination of informatics, evolutionary biology, microbiology and genetics.
Pathogenicity
Processes of microbial pathogenicity at the molecular level are still minimally understood
Pathogen proteins identified that manipulate host cells by interacting with, or mimicking, host proteins.
Idea: Could we identify novel virulence factors by identifying pathogen genes more similar to host genes than you would expect based on phylogeny?
Eukaryotic-like pathogen genes
- YopH, a protein-tyrosine phosphatase, of Yersinia pestis
- Enoyl-acyl carrier protein reductase (involved in lipid metabolism) of Chlamydia trachomatis
0.1
Aquifex aeolicus
Haemophilus influenza
Escherichia coli
Anabaena
Synechocystis
Chlamydia trachomatis
Petunia x hybrida
Nicotiana tabacum
Brassica napus
Arabidopsis thaliana
Oryza sativa
100
100
100
96
63
64
52
83
99
Prioritize for biological study. - Previously studied biologically? - Can UBC microbiologists study it? - C. elegans homolog?
Screen for candidate genes.Search pathogen genes against sequence databases. Identify those with eukaryotic similarity/motifs
Rank candidates.- how much like host protein?- info available about protein?
Modify screening method /algorithm
Approach
Evolutionary significance. - Horizontal transfer? Similar by chance?
Pathogens Anthrax Necrotizing fasciitis Cat scratch disease Paratyphoid/enteric feverChancroid Peptic ulcers and gastritisChlamydia Periodontal diseaseCholera PlagueDental caries PneumoniaDiarrhea (E. coli etc.) SalmonellosisDiphtheria Scarlet feverEpidemic typhus ShigellosisMediterranean fever Strep throatGastroenteritis SyphilisGonorrhea Toxic shock syndromeLegionnaires' disease Tuberculosis Leprosy TularemiaLeptospirosis Typhoid feverListeriosis UrethritisLyme disease Urinary Tract InfectionsMeliodosis Whooping cough Meningitis +Hospital-acquired infections
Pathogens
Chlamydophila psittaci Respiratory disease, primarily in birdsMycoplasma mycoides Contagious bovine pleuropneumoniaMycoplasma hyopneumoniae Pneumonia in pigsPasteurella haemolytica Cattle shipping feverPasteurella multicoda Cattle septicemia, pig rhinitisRalstonia solanacearum Plant bacterial wiltXanthomonas citri Citrus cankerXylella fastidiosa Pierce’s Disease - grapevines
Bacterial wilt
World Research
Community
ApproachPrioritized candidates
Study function of similar gene in model host, C. elegans.
Study function of gene.
Investigate role of bacterial gene in disease: Infection study in model host
C. elegans
DATABASE
Contact other groups for possible collaborations.
Informatics/Bioinformatics• BC Genome Sequence Centre• Centre for Molecular
Medicine and Therapeutics
Evolutionary Theory• Dept of Zoology
• Dept of Botany
• Canadian Institute for Advanced Research
Pathogen Functions• Dept. Microbiology
• Biotechnology Laboratory
• Dept. Medicine
• BC Centre for Disease Control
Host Functions• Dept. Medical Genetics
• C. elegans Reverse Genetics Facility
• Dept. Biological Sciences SFU
Interdisciplinary group
Coordinator
• Interdisciplinary team unique ideas and collaborations
• Automated approach continually updated
• Better understanding: pathogen gene and similar host gene
• Insight into horizontal gene transfer events and the evolution of pathogen-host interactions.
• Public database
– other researchers may capitalize on the findings
– promote further collaboration
Power of the Approach
Bacterium Eukaryote Horizontal Transfer
0.1
Bacillus subtilis
Escherichia coli
Salmonella typhimurium
Staphylococcua aureus
Clostridium perfringens
Clostridium difficile
Trichomonas vaginalis
Haemophilus influenzae
Acinetobacillus actinomycetemcomitans
Pasteurella multocida
N-acetylneuraminate lyase (NanA) of the protozoan Trichomonas vaginalis is 92-95% similar to NanA of Pasteurellaceae bacteria.
N-acetylneuraminate lyase (sialic acid lyase, NanA)
Intracellular enzyme involved in sialic acid metabolism
In Bacteria: Proposed to parasitize the mucous membranes of animals for nutritional purposes
Hydrolysis of glycosidic linkages of terminal sialic residues in glycoproteins, glycolipids SialidaseFree sialic acid
Transporter
Free sialic acid NanA
N-acetyl-D-mannosamine + pyruvate
N-acetylneuraminate lyase – role in pathogenicity?
Pasteurellaceae
•Mucosal pathogens of the respiratory tract
•Intracellular NanA enzyme with sialic acid transporter
T. vaginalis
•Mucosal pathogen, causative agent of the STD Trichomonas
•Extracellular enzyme, so avoids need for transporter?
Eukaryote Bacteria Horizontal Transfer?
0.1Rat
Human
Escherichia coli
Caenorhabditis elegans
Pig roundworm
Methanococcus jannaschii
Methanobacterium thermoautotrophicum
Bacillus subtilis
Streptococcus pyogenes
Aquifex aeolicus
Acinetobacter calcoaceticus
Haemophilus influenzae
Chlorobium vibrioforme
Guanosine monophosphate reductase of E. coli is 81% similar to the corresponding enzyme studied in humans and rats, and shares a significant phylogenetic relationship with metazoans (left).
Its role in virulence has not been investigated.
Eukaryote Bacteria Horizontal Transfer?
Ralstonia solanacearum cellulase (ENDO-1,4-
BETA-GLUCANASE) is 56% similar to endoglucanase present in a number of fungi.
Demonstrated virulence factor for plant bacterial wilt
Hypocrea jecorina EGLII
Trichoderma viride EGL2
Penicillium janthinellum EGL2
Macrophomina phaseolina EGL2
Cryptococcus flavus CMC1
Ralstonia solanacearum egl
Humicola insolens CMC3
Humicola grisea CMC3
Aspergillus aculeatus CMC2
Aspergillus nidulans EGLA
Macrophomina phaseolina egl1
Aspergillus aculeatus CEL1
Aspergillus niger EGLB
Vibrio species manA
Trends in the Analysis
• Most cases of probable recent cross-domain gene transfer involve movement of a bacterial gene to a unicellular eukaryote
• Identifies the strongest cases of lateral gene transfer between bacteria and eukaryotes
• A control: The method identifies all previously reported Chlamydia trachomatis eukaryotic-like genes.
G+C Analysis: Identifying Pathogenicity Islands
%G+C S.D. Location Strand Gene Product 52.24 879443..880738 - NMB0854 histidyl-tRNA synthetase 46.42 880832..881488 - NMB0855 put. bacteriocin resist. 26.07 -2 881770..882237 - NMB0856 hypothetical protein 37.29 -1 882294..882470 - NMB0857 hypothetical protein 42.29 -1 882474..882674 - NMB0858 hypothetical protein 29.37 -2 882677..883054 - NMB0859 hypothetical protein 35.27 -2 883112..883369 - NMB0860 hypothetical protein 47.99 883459..884004 - NMB0861 hypothetical protein 35.00 -2 884001..884120 - NMB0862 hypothetical protein 26.37 -2 884167..884439 - NMB0863 hypothetical protein 33.33 -2 884705..884995 - NMB0864 hypothetical protein 47.05 885001..885474 - NMB0865 hypothetical protein 53.33 885517..886386 - NMB0866 hypothetical protein 52.38 886550..887473 + NMB0867 YabO/YceC/SfhB fam. prot. 57.63 887551..888192 - NMB0868 conserved hypothetical 54.42 888247..889038 - NMB0869 hypothetical protein 55.56 889531..890322 + NMB0870 3-methyl-2-oxobutanoate hydroxymethyltransferase
G+C of ORFs: Analysis of Variance
• Low G+C variance correlates with an intracellular lifestyle for the bacterium and a clonal nature (P = 0.004)
• This variance is similar within a given species
• Useful marker for investigating the clonality of bacteria? Relationship with intracellular lifestyle may reflect the ecological isolation of intracellular bacteria?
Future Developments
• Incorporate unfinished genomes, plasmids into analysis (including eukaryotic)
• Motif-based and domain-based analyses
• G+C analysis graphical viewer for identification of pathogenicity islands
• Functional tests
• Peter Wall Foundation• Pathogenomics group
– Ann M. Rose, Yossef Av-Gay, David L. Baillie, Fiona S. L. Brinkman, Robert Brunham, Stefanie Butland, Rachel C. Fernandez, B. Brett Finlay, Hans Greberg, Robert E.W. Hancock, Christy Haywood-Farmer, Steven J. Jones, Patrick Keeling, Audrey de Koning, Don G. Moerman, Sarah P. Otto, B. Francis Ouellette, Ivan Wan.
www.pathogenomics.bc.ca