Transcript Using Bioinformatics to Investigate Evolution, Phylogeny

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 SpaceUsing 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
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Cwp66 (surface protein)
Cwp84 (cysteine protease)
Fbp68 (fibronectin binding protein)
FliC (flagellar subunit)
FliD (flagellar subunit)
GroEL (cytoplasmic chaperone)
SlpA (surface S-layer protein)
TcdA (toxin production)
TcdB (toxin production)
TcdD (toxin production)
#Sequences
31
57
30
44
33
33
97
25
30
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
Question
• 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?
Tool(s)
• 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. 2005.
• 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
Question
Tool(s)
• What is different between
C. diff & nonpathogenic
Clostridium species?
• PGraph
• Mummer, Protein v. Protein
(TIGR CMR database)
• 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?
• BLAST
• ClustalW & tree-building
algorithms
• Gene Neighborhood (JGI
IMG database)
• PubMed & other literature
search engines