Dr. Rita Tamayo is an associate professor at The University
of North Carolina at Chapel Hill. She received her Ph.D in Microbiology and
Immunology from UTSA’s Health Science Center in 2004.
Clostridium difficile is a pathogen
located in the human intestine that causes antibiotic associated pseudomembranous
colitis. It one of three bacterial groups listed by the Center for Disease
Control as a top threat because of its highly resistant vegetative spores that
secrete toxins resistant to antibiotics. Because not much is known about this
species, Dr. Tamayo investigated the mechanism of colonization in the gut and
how the genes responsible for virulence are regulated.
two key players involved in answering these questions which are cytotoxins TcdA
and TcdB, and the genes encoding the cell’s flagella. The cytotoxins disrupt
the integrity of the epithelium by breaking down tight junctions. However,
these toxins are linked to the expression of the flagella, which helps the cell
adhere to the gut. The gene responsible for encoding the flagella, flgB, has a
switch located upstream of the operon which controls both the production of the
flagella and the toxin through phase variation. This variation increases the
heterogeneity in the population by allowing there to be 2 different sequences
on the riboswitch (so that in adverse environmental conditions a small subset
of the bacteria can still survive to reproduce).
there were 8 highly conserved flagellar genes, a recombinase was suspected to
be responsible for the inversion. Each sequence was tested to see if the gene
could be inverted, using E. coli as
the host. This hypothesis was confirmed by using a system of 2 plasmids with
one allowing inducible expression of the tested recombinase sequence downstream
of the promoter, and the other containing the sequence for the promoter of the
flagellar switch. Using the polymerase chain reaction (PCR), 2 versions of the
plasmid having the on and off orientation sequence were tested to see if
inversion of the sequence occurred. Of the 8 sequences tested, a recombinase
named RecV was found to be sufficient for the inversion of the flagellar switch
from “phase ON” to “phase OFF” and vice versa. This ‘published,’ or “phase ON”
sequence codes for flagellated cells and the toxins through the cwpV switch,
while the sequence in the reverse direction showed no flagella or production of
toxins, meaning RecV was necessary for phase variation of these two genes.
These results occurred because the sequence encoding “phase OFF” is regulated
post-transcriptionally by a Rho factor encoded by the reverse sequence cwpV
switch which terminates its expression.
Tamayo’s research has increased understanding of the genetics of Clostridium difficile, her results show
applicability to an alternate way of diagnostically testing for the bacterium.
Traditionally, clinical labs use PCR to look for toxin genes, but from the
results of this experiment it is now known that the presence of a toxin gene
does not necessarily mean there will be expression. Testing can now look for
the orientation of the flagellar sequence in addition to toxin genes and
possibly provide a more accurate means of diagnosing pseudomembranous colitis.
of Dr. Tamayo’s research was interesting to me on a personal level because of
its direct applicability to medicine. Her presentation was well organized in
terms of experimental flow and results, but I found it extremely difficult to
understand because of the technical terms and nature of the experimental
methods. I feel overall this presentation was very informative for someone with
a Ph.D level understanding of molecular biology, but I found myself having to
do a lot of outside research on what a concrete definition of “phase variation”
was which was the main topic of the presentation. However, I overall enjoyed
the seminar and would attend another one given by her, now knowing I would need
to be prepared to do some outside learning to better understand her research.