Using Bioinformatics to Investigate Evolution, Phylogeny, and Virulence in the Human Pathogen Clostridium difficile Kim R. Finer, KSU Brad Goodner, Hiram

Educational Context I,II Tiers I and II are designed to engage the allied health student in a Basic Microbiology course by emphasizing the clinical relevance of genomics data. This problem space may be introduced as the centerpiece in a discussion of pathogenicity. The questions posed will focus on virulence as determined by various genes which (may) have changed over time. These changes must be considered in light of increased public awareness of C. difficile colitis in the clinical setting as a consequence of antibiotic therapy. This space can also provide a focal point as the instructor communicates the concept/significance of bacterial “strains” (genetic variants) throughout the course.

Educational Context (III) Tier III is designed to be a component of a Bio. majors course. These students will have sufficient background in genetics and cell biol., to identify problems, formulate questions, investigate literature, etc. (scientific method). Tier III activities would be a logical component of the laboratory of either an UD genetics or Mol. Bio course. Laboratory periods of 1.5 hr with computer stations available for all students would sufficiently support the investigation.

Goals of the Space Emphasize the connection between genomics and the pathogen’s clinical significance. (Re)Introduce students to various evolutionary concepts/relationships that play a role in disease and disease processes. Familiarize students with the variety and power of various BI tools.

Target Audience Tier I Allied health students with min./no genetics background, first year course Tier II** Allied health student with prior course content in genetics, second year course Tier III Biology/Mol Biol. major, third year course (prior preparation in genetics, cell biol., mol.biol.) **Stepwise progression through tiers

Tier I (tool use, and analysis) Problem Space- Using a provided data set—examine C. difficile strain variation over time. Q. 1: Can you correlate strain variation over time with emerging clinical impact/significance of the strain? (Comparative analysis of Clostridium difficile clinical isolates belonging to different genetic lineages and time periods. Spigaglia, P. Mastrantonio, P., 2006)

Tier I cont. Q.2 Look at evolution/change of/in 10 different C. difficile genes over time. Identify relationships between strains. -Does any one gene stand out as being more responsible for diversity of strains (correlate one gene with diversity)? Q.3 Does the emergence of antibiotic resistant strains correlate with increased strain variation?

Data Sets for Tier I Questions Variation in Cdif Strains (Sequences) Data Set#Sequences Cwp66 (surface protein)31 Cwp84 (cysteine protease)57 Fbp68 (fibronectin binding protein)30 FliC (flagellar subunit)44 FliD (flagellar subunit)33 GroEL (cytoplasmic chaperone)33 SlpA (surface S-layer protein)97 TcdA (toxin production)25 TcdB (toxin production)30 TcdD (toxin production)20

Data Sets for Tier I Questions Variation in C. diff Strains (Clinical Info) Data Set (still gathering data) Antibiotic resistance profile Patient outcome

Tool Set for Tier I & II Questions Variation in Cdif Strains QuestionTool(s) How much variation among strains? Covariation of gene change? Does variation cluster by year or geographic location? Why should I care about variation in this or that gene? ClustalW & tree-building algorithms PubMed & other literature search engines

Tier II (data acquisition, tool use, analysis) Q. Look at the geographical distribution of C. difficile strains. Correlate geo. distribution to virulence over time (need strain name, year, and geo. source) Data sets: PubMed paper with sequence detail –Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile. Lemee, L, Bourgeois I, Ruffin, E, Collignon, A., Lemeland, JF., Pons, JL Tools: BLAST, ClustalW, (alignment, matrices?, trees)

Data Sets for Tier II Questions Variation in Cdif Strains (Added Clinical Info) Data Set (still gathering data) Year of strain isolation Geographic location of strain isolation

Tier III (problem posing, data acq., tool use, analysis) Identify Problem –Spore formation in Clostridium species (induction/steps) –Toxin production (role of transposons/phage) in C. difficile –Antibiotic resistance mechanisms Students – formulate questions/hypothesis –Acquire data sets-- literature search –Analyze data and revisit hypothesis

Tool Set for Tier III Questions Variation in Cdif Strains QuestionTool(s) What is different between C. diff & nonpathogenic Clostridium species? Origin & evolution of antibiotic resistance genes in C. diff? Origin & evolution of toxin production genes in C. diff? What do we know about spore formation in Clostridium? PGraph Mummer, Protein v. Protein (TIGR CMR database) BLAST ClustalW & tree-building algorithms Gene Neighborhood (JGI IMG database) PubMed & other literature search engines