Using Bioinformatics to Investigate Evolution, Phylogeny, and ...

underlingbuddhaBiotechnology

Oct 2, 2013 (3 years and 11 months ago)

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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


Question





Tool(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. 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?



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