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

Database front end

PhyloBLAST – a tool for the analysis

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