In the life cycles of facultative pathogens, the ability to transition between the external environment and the host is key to survival. These transitions are accompanied by alterations in transcriptional and proteomic profiles (Merrell et al., 2002; Schild et al., 2007; LaRocque et al., 2008). Numerous studies have investigated the changes in gene expression that occur during these environmental shifts and have identified factors that are important for survival and growth in the two disparate settings. Less is known about the changes in gene expression that result as a pathogen transitions between microenvironments within the host or even the number of such transitions that occur. A single organ or tissue may in fact constitute a wide array of strikingly different environmental conditions for an invading pathogen

Mudrak, Benjamin

Tamayo, Rita

English

PubMed

Benjamin Mudrak

Rita Tamayo

Frontiers - Publisher Connector

www.frontiersin.org March 2011 | Volume 2 | Article 42 | 1
OpiniOn Article
published: 07 March 2011
doi: 10.3389/fmicb.2011.00042
consistent with the role of TCP in micro-
colony formation in V. cholerae (Kirn et al., 
2000). In vitro experiments demonstrated 
that these subpopulations were bistable and 
that the tcp expression phenotype could be 
induced with bicarbonate, a compound nat-
urally released by the intestine to neutral-
ize its contents (Kiela and Ghishan, 2009). 
More importantly, the bifurcation of TCP 
expression could be detected in vivo, after 
infection of rabbit ileal loops.
It has been shown previously that V. 
cholerae passaged through a host or grown 
in a biofilm in vitro becomes transiently 
hyperinfectious compared to isogenic bac-
teria grown in shaking culture (Merrell 
et al., 2002; Tamayo et al., 2010). Another 
study has demonstrated that clumps of V. 
cholerae are shed after host passage and 
that these “in vivo-induced biofilms” dis-
play greater hyperinfectivity than free-living 
bacteria that exit the human host (Faruque 
et al., 2006). Thus, biofilms or aggregates 
formed in vitro and in vivo are both poten-
tially primed for colonization of the host 
gastrointestinal tract. As noted by Nielsen 
et al. (2010) it is tempting to speculate that 
the TCP-positive aggregates observed in 
their study may be related to the hyperinfec-
tious aggregates shed by infected patients. 
Prior expression of TCP by host-passaged 
bacteria may facilitate attachment or micro-
colony formation in the small intestine.
Certainly, much work remains to be done 
to ascertain the signals influencing V. cholerae 
in different microenvironments within and 
outside the host. However, in their recent 
work and in an earlier report detailing the 
role of RpoS in the V. cholerae mucosal escape 
response (Nielsen et al., 2006), the Schoolnik 
lab has highlighted some added complica-
tions to the study of bacterial infection. 
Infections were once thought of in terms of 
early and late timepoints and location within 
specific organs (such as the small intestine for 
V. cholerae). It is now apparent that even cells 
Identifying new variables during infection: proximity to the 
host epithelium and epigenetic programs alter the expression 
of virulence factors in Vibrio cholerae
Benjamin Mudrak and Rita Tamayo*
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
*Correspondence: rtamayo@med.unc.edu
In the life cycles of facultative pathogens, 
the ability to transition between the exter-
nal environment and the host is key to sur-
vival. These transitions are accompanied by 
alterations in transcriptional and proteomic 
profiles (Merrell et al., 2002; Schild et al., 
2007; LaRocque et al., 2008). Numerous 
studies have investigated the changes in 
gene expression that occur during these 
environmental shifts and have identified 
factors that are important for survival and 
growth in the two disparate settings. Less is 
known about the changes in gene expres-
sion that result as a pathogen transitions 
between microenvironments within the 
host or even the number of such transitions 
that occur. A single organ or tissue may in 
fact constitute a wide array of strikingly 
different environmental conditions for an 
invading pathogen.
Many studies have also assumed a homo-
geneous response on the part of the popu-
lation of pathogenic cells, but increasing 
evidence suggests that bacterial cells can 
specialize, producing two stable phenotypes 
within a single isogenic population using 
an epigenetic mechanism called bistability 
(Veening et al., 2008). Recently, the pres-
ence of bistable subgroups has been noted 
in Bacillus subtilis biofilms (Chai et al., 2008) 
and in cultures of Pseudomonas aeruginosa 
(Turner et al., 2009) and Porphyromonas 
gingivalis (Suwannakul et al., 2010). In B. 
subtilis, key sporulation genes are differen-
tially expressed within isogenic subpopula-
tions in a biofilm. In P. aeruginosa and P. 
gingivalis, certain virulence factors exhibit 
bistable regulation. One hallmark of bist-
ability is the transience of the phenotype; 
cells in either subgroup give rise to cells of 
both types after further passaging. The exist-
ence of two populations has been likened 
to “bet-hedging,” with bacteria in bistable 
populations ready to thrive under more 
conditions than a homogeneous population 
(Veening et al., 2008).
Vibrio cholerae is the Gram-negative 
bacterium responsible for outbreaks of the 
deadly diarrheal disease cholera (Kaper 
et al., 1995). These outbreaks occur across 
the globe, and mortality rates can be con-
siderable when proper therapy, usually oral 
rehydration, is not available. The primary 
virulence factors produced by V. cholerae are 
cholera toxin (CT), which is responsible for 
generating the massive efflux of fluid and 
electrolytes that culminates in diarrhea, and 
the toxin-coregulated pilus (TCP), a bun-
dle-forming pilus required for colonization 
of the host intestine. Although the complex 
regulatory mechanisms controlling CT and 
TCP production have been defined in great 
detail, uncovering the dynamics of virulence 
factor expression during infection would 
improve our understanding of V. cholerae 
pathogenesis and transmission.
In a recent study, Nielsen et al. (2010) 
have provided strong evidence that two 
populations of V. cholerae cells arise during 
infection. Using the rabbit ileal loop model, 
the authors showed that the transcriptional 
profile of V. cholerae cells changes signifi-
cantly based on the proximity of the cells to 
the intestinal epithelium. Taking advantage 
of a clever reporter system, transient expres-
sion of the tcp locus, which encodes the sys-
tem required for TCP biogenesis, could be 
tracked by measuring bacterial fluorescence. 
Bacteria localized to the intestinal surface 
demonstrated much higher fluorescence, 
and thus higher tcp expression, than bac-
teria in the lumen. Similarly, expression of 
rrnB, a locus regulated by growth rate, was 
found to be highest in surface-associated 
bacteria, indicating that the bacteria near 
the host epithelium replicate more rapidly. 
Further studies showed that there were two 
populations of bacteria at late stages of 
infection: one group of cells continuing to 
express tcp and another group not express-
ing tcp. Interestingly, the tcp-expressing 
cells were frequently found in aggregates, 
Mudrak and Tamayo Identifying new variables during infection
Frontiers in Microbiology | Cellular and Infection Microbiology  March 2011 | Volume 2 | Article 42 | 2
infection increase fitness of Vibrio cholerae after 
release into the environment. Cell Host Microbe 2, 
264–277.
Suwannakul, S., Stafford, G. P., Whawell, S. A., and 
Douglas, C. W. (2010). Identification of bistable 
populations of Porphyromonas gingivalis that dif-
fer in epithelial cell invasion. Microbiology 156, 
3052–3064.
Tamayo, R., Patimalla, B., and Camilli, A. (2010). Growth 
in a biofilm induces a hyperinfectious phenotype in 
Vibrio cholerae. Infect. Immun. 78, 3560–3569.
Turner, K. H., Vallet-Gely, I., and Dove, S. L. (2009). 
Epigenetic control of virulence gene expression in 
Pseudomonas aeruginosa by a LysR-type transcrip-
tion regulator. PLoS Genet. 5, e1000779. doi: 10.1371/
journal.pgen.1000779
Veening, J. W., Smits, W. K., and Kuipers, O. P. (2008). 
Bistability, epigenetics, and bet-hedging in bacteria. 
Annu. Rev. Microbiol. 62, 193–210.
Received: 22 February 2011; accepted: 23 February 2011; 
published online: 07 March 2011.
Citation: Mudrak B and Tamayo R (2011) Identifying 
new variables during infection: proximity to the host epi-
thelium and epigenetic programs alter the expression of 
virulence factors in Vibrio cholerae. Front. Microbio. 2:42. 
doi: 10.3389/fmicb.2011.00042
This article was submitted to Frontiers in Cellular and 
Infection Microbiology, a specialty of Frontiers in Microbiology.
Copyright © 2011 Mudrak and Tamayo. This is an open-
access article subject to an exclusive license agreement 
between the authors and Frontiers Media SA, which 
permits unrestricted use, distribution, and reproduction 
in any medium, provided the original authors and source 
are credited.
Kaper, J. B., Morris, J. G. Jr., and Levine, M. M. (1995). 
Cholera. Clin. Microbiol. Rev. 8, 48–86.
Kiela, P. R., and Ghishan, F. K. (2009). Ion transport in 
the intestine. Curr. Opin. Gastroenterol. 25, 87–91.
Kirn, T. J., Lafferty, M. J., Sandoe, C. M., and Taylor, R. 
K. (2000). Delineation of pilin domains required for 
bacterial association into microcolonies and intesti-
nal colonization by Vibrio cholerae. Mol. Microbiol. 
35, 896–910.
Kroger, C., Srikumar, S., Ellwart, J., and Fuchs, T. M. 
(2011). Bistability in myo-inositol utilization 
by Salmonella enterica serovar Typhimurium. J. 
Bacteriol. 193, 1427–1435
LaRocque, R. C., Krastins, B., Harris, J. B., Lebrun, L. 
M., Parker, K. C., Chase, M., Ryan, E. T., Qadri, 
F., Sarracino, D., and Calderwood, S. B. (2008). 
Proteomic analysis of Vibrio cholerae in human stool. 
Infect. Immun. 76, 4145–4151.
Merrell, D. S., Butler, S. M., Qadri, F., Dolganov, N. A., 
Alam, A., Cohen, M. B., Calderwood, S. B., Schoolnik, 
G. K., and Camilli, A. (2002). Host-induced epi-
demic spread of the cholera bacterium. Nature 
417, 642–645.
Nielsen, A. T., Dolganov, N. A., Otto, G., Miller, M. C., 
Wu, C. Y., and Schoolnik, G. K. (2006). RpoS controls 
the Vibrio cholerae mucosal escape response. PLoS 
Pathog. 2, e109. doi: 10.1371/journal.ppat.0020109
Nielsen, A. T., Dolganov, N. A., Rasmussen, T., Otto, G., 
Miller, M. C., Felt, S. A., Toreilles, S., and Schoolnik, 
G. K. (2010). A bistable switch and anatomical site 
control Vibrio cholerae virulence gene expression in 
the intestine. PLoS Pathog. 6, e1001102. doi: 10.1371/
journal.ppat.1001102
Schild, S., Tamayo, R., Nelson, E. J., Qadri, F., Calderwood, 
S. B., and Camilli, A. (2007). Genes induced late in 
found in the same time and place (such as V. 
cholerae shed by a host) may represent mul-
tiple, distinct subpopulations, one or both 
of which may be responsible for previously 
observed phenotypes.
Looking beyond V. cholerae pathogen-
esis, epigenetic control of virulence fac-
tors may underlie other cases of transient 
infectivity. Indeed, a recent report detailing 
a bistable epigenetic response to bicarbo-
nate in Salmonella enterica indicates that 
such programs may be widespread, at least 
among enteric pathogens (Kroger et al., 
2011). Although it poses significant chal-
lenges, it is clearly important to consider 
variables specific to different microenviron-
ments (e.g., the lumen or epithelial surface) 
and different subpopulations of bacteria 
within those same spatiotemporal domains.
RefeRences
Chai, Y., Chu, F., Kolter, R., and Losick, R. (2008). 
Bistability and biofilm formation in Bacillus subtilis. 
Mol. Microbiol. 67, 254–263.
Faruque, S. M., Biswas, K., Udden, S. M., Ahmad, Q. S., 
Sack, D. A., Nair, G. B., and Mekalanos, J. J. (2006). 
Transmissibility of cholera: in vivo-formed biofilms 
and their relationship to infectivity and persistence 
in the environment. Proc. Natl. Acad. Sci. U.S.A. 103, 
6350–6355.


Identifying New Variables During Infection: Proximity to the Host Epithelium and Epigenetic Programs Alter the Expression of Virulence Factors in Vibrio Cholerae

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Frontiers in Microbiology

Benjamin eMudrak

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Identifying new variables during infection: proximity to the host epithelium and epigenetic programs alter the expression of virulence factors in Vibrio cholerae

Mudrak, B.

Tamayo, R.

Carolina Digital Repository

www.frontiersin.org March 2011 | Volume 2 | Article 42 | 1OpiniOn Articlepublished: 07 March 2011doi: 10.3389/fmicb.2011.00042consistent with the role of TCP in micro-colony formation in V. cholerae (Kirn et al., 2000). In vitro experiments demonstrated that these subpopulations were bistable and that the tcp expression phenotype could be induced with bicarbonate, a compound nat-urally released by the intestine to neutral-ize its contents (Kiela and Ghishan, 2009). More importantly, the bifurcation of TCP expression could be detected in vivo, after infection of rabbit ileal loops.It has been shown previously that V. cholerae passaged through a host or grown in a biofilm in vitro becomes transiently hyperinfectious compared to isogenic bac-teria grown in shaking culture (Merrell et al., 2002; Tamayo et al., 2010). Another study has demonstrated that clumps of V. cholerae are shed after host passage and that these “in vivo-induced biofilms” dis-play greater hyperinfectivity than free-living bacteria that exit the human host (Faruque et al., 2006). Thus, biofilms or aggregates formed in vitro and in vivo are both poten-tially primed for colonization of the host gastrointestinal tract. As noted by Nielsen et al. (2010) it is tempting to speculate that the TCP-positive aggregates observed in their study may be related to the hyperinfec-tious aggregates shed by infected patients. Prior expression of TCP by host-passaged bacteria may facilitate attachment or micro-colony formation in the small intestine.Certainly, much work remains to be done to ascertain the signals influencing V. cholerae in different microenvironments within and outside the host. However, in their recent work and in an earlier report detailing the role of RpoS in the V. cholerae mucosal escape response (Nielsen et al., 2006), the Schoolnik lab has highlighted some added complica-tions to the study of bacterial infection. Infections were once thought of in terms of early and late timepoints and location within specific organs (such as the small intestine for V. cholerae). It is now apparent that even cells Identifying new variables during infection: proximity to the host epithelium and epigenetic programs alter the expression of virulence factors in Vibrio choleraeBenjamin Mudrak and Rita Tamayo*Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA*Correspondence: rtamayo@med.unc.eduIn the life cycles of facultative pathogens, the ability to transition between the exter-nal environment and the host is key to sur-vival. These transitions are accompanied by alterations in transcriptional and proteomic profiles (Merrell et al., 2002; Schild et al., 2007; LaRocque et al., 2008). Numerous studies have investigated the changes in gene expression that occur during these environmental shifts and have identified factors that are important for survival and growth in the two disparate settings. Less is known about the changes in gene expres-sion that result as a pathogen transitions between microenvironments within the host or even the number of such transitions that occur. A single organ or tissue may in fact constitute a wide array of strikingly different environmental conditions for an invading pathogen.Many studies have also assumed a homo-geneous response on the part of the popu-lation of pathogenic cells, but increasing evidence suggests that bacterial cells can specialize, producing two stable phenotypes within a single isogenic population using an epigenetic mechanism called bistability (Veening et al., 2008). Recently, the pres-ence of bistable subgroups has been noted in Bacillus subtilis biofilms (Chai et al., 2008) and in cultures of Pseudomonas aeruginosa (Turner et al., 2009) and Porphyromonas gingivalis (Suwannakul et al., 2010). In B. subtilis, key sporulation genes are differen-tially expressed within isogenic subpopula-tions in a biofilm. In P. aeruginosa and P. gingivalis, certain virulence factors exhibit bistable regulation. One hallmark of bist-ability is the transience of the phenotype; cells in either subgroup give rise to cells of both types after further passaging. The exist-ence of two populations has been likened to “bet-hedging,” with bacteria in bistable populations ready to thrive under more conditions than a homogeneous population (Veening et al., 2008).Vibrio cholerae is the Gram-negative bacterium responsible for outbreaks of the deadly diarrheal disease cholera (Kaper et al., 1995). These outbreaks occur across the globe, and mortality rates can be con-siderable when proper therapy, usually oral rehydration, is not available. The primary virulence factors produced by V. cholerae are cholera toxin (CT), which is responsible for generating the massive efflux of fluid and electrolytes that culminates in diarrhea, and the toxin-coregulated pilus (TCP), a bun-dle-forming pilus required for colonization of the host intestine. Although the complex regulatory mechanisms controlling CT and TCP production have been defined in great detail, uncovering the dynamics of virulence factor expression during infection would improve our understanding of V. cholerae pathogenesis and transmission.In a recent study, Nielsen et al. (2010) have provided strong evidence that two populations of V. cholerae cells arise during infection. Using the rabbit ileal loop model, the authors showed that the transcriptional profile of V. cholerae cells changes signifi-cantly based on the proximity of the cells to the intestinal epithelium. Taking advantage of a clever reporter system, transient expres-sion of the tcp locus, which encodes the sys-tem required for TCP biogenesis, could be tracked by measuring bacterial fluorescence. Bacteria localized to the intestinal surface demonstrated much higher fluorescence, and thus higher tcp expression, than bac-teria in the lumen. Similarly, expression of rrnB, a locus regulated by growth rate, was found to be highest in surface-associated bacteria, indicating that the bacteria near the host epithelium replicate more rapidly. Further studies showed that there were two populations of bacteria at late stages of infection: one group of cells continuing to express tcp and another group not express-ing tcp. Interestingly, the tcp-expressing cells were frequently found in aggregates, Mudrak and Tamayo Identifying new variables during infectionFrontiers in Microbiology | Cellular and Infection Microbiology  March 2011 | Volume 2 | Article 42 | 2infection increase fitness of Vibrio cholerae after release into the environment. Cell Host Microbe 2, 264–277.Suwannakul, S., Stafford, G. P., Whawell, S. A., and Douglas, C. W. (2010). Identification of bistable populations of Porphyromonas gingivalis that dif-fer in epithelial cell invasion. Microbiology 156, 3052–3064.Tamayo, R., Patimalla, B., and Camilli, A. (2010). Growth in a biofilm induces a hyperinfectious phenotype in Vibrio cholerae. Infect. Immun. 78, 3560–3569.Turner, K. H., Vallet-Gely, I., and Dove, S. L. (2009). Epigenetic control of virulence gene expression in Pseudomonas aeruginosa by a LysR-type transcrip-tion regulator. PLoS Genet. 5, e1000779. doi: 10.1371/journal.pgen.1000779Veening, J. W., Smits, W. K., and Kuipers, O. P. (2008). Bistability, epigenetics, and bet-hedging in bacteria. Annu. Rev. Microbiol. 62, 193–210.Received: 22 February 2011; accepted: 23 February 2011; published online: 07 March 2011.Citation: Mudrak B and Tamayo R (2011) Identifying new variables during infection: proximity to the host epi-thelium and epigenetic programs alter the expression of virulence factors in Vibrio cholerae. Front. Microbio. 2:42. doi: 10.3389/fmicb.2011.00042This article was submitted to Frontiers in Cellular and Infection Microbiology, a specialty of Frontiers in Microbiology.Copyright © 2011 Mudrak and Tamayo. This is an open-access article subject to an exclusive license agreement between the authors and Frontiers Media SA, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.Kaper, J. B., Morris, J. G. Jr., and Levine, M. M. (1995). Cholera. Clin. Microbiol. Rev. 8, 48–86.Kiela, P. R., and Ghishan, F. K. (2009). Ion transport in the intestine. Curr. Opin. Gastroenterol. 25, 87–91.Kirn, T. J., Lafferty, M. J., Sandoe, C. M., and Taylor, R. K. (2000). Delineation of pilin domains required for bacterial association into microcolonies and intesti-nal colonization by Vibrio cholerae. Mol. Microbiol. 35, 896–910.Kroger, C., Srikumar, S., Ellwart, J., and Fuchs, T. M. (2011). Bistability in myo-inositol utilization by Salmonella enterica serovar Typhimurium. J. Bacteriol. 193, 1427–1435LaRocque, R. C., Krastins, B., Harris, J. B., Lebrun, L. M., Parker, K. C., Chase, M., Ryan, E. T., Qadri, F., Sarracino, D., and Calderwood, S. B. (2008). Proteomic analysis of Vibrio cholerae in human stool. Infect. Immun. 76, 4145–4151.Merrell, D. S., Butler, S. M., Qadri, F., Dolganov, N. A., Alam, A., Cohen, M. B., Calderwood, S. B., Schoolnik, G. K., and Camilli, A. (2002). Host-induced epi-demic spread of the cholera bacterium. Nature 417, 642–645.Nielsen, A. T., Dolganov, N. A., Otto, G., Miller, M. C., Wu, C. Y., and Schoolnik, G. K. (2006). RpoS controls the Vibrio cholerae mucosal escape response. PLoS Pathog. 2, e109. doi: 10.1371/journal.ppat.0020109Nielsen, A. T., Dolganov, N. A., Rasmussen, T., Otto, G., Miller, M. C., Felt, S. A., Toreilles, S., and Schoolnik, G. K. (2010). A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine. PLoS Pathog. 6, e1001102. doi: 10.1371/journal.ppat.1001102Schild, S., Tamayo, R., Nelson, E. J., Qadri, F., Calderwood, S. B., and Camilli, A. (2007). Genes induced late in found in the same time and place (such as V. cholerae shed by a host) may represent mul-tiple, distinct subpopulations, one or both of which may be responsible for previously observed phenotypes.Looking beyond V. cholerae pathogen-esis, epigenetic control of virulence fac-tors may underlie other cases of transient infectivity. Indeed, a recent report detailing a bistable epigenetic response to bicarbo-nate in Salmonella enterica indicates that such programs may be widespread, at least among enteric pathogens (Kroger et al., 2011). Although it poses significant chal-lenges, it is clearly important to consider variables specific to different microenviron-ments (e.g., the lumen or epithelial surface) and different subpopulations of bacteria within those same spatiotemporal domains.RefeRencesChai, Y., Chu, F., Kolter, R., and Losick, R. (2008). Bistability and biofilm formation in Bacillus subtilis. Mol. Microbiol. 67, 254–263.Faruque, S. M., Biswas, K., Udden, S. M., Ahmad, Q. S., Sack, D. A., Nair, G. B., and Mekalanos, J. J. (2006). Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proc. Natl. Acad. Sci. U.S.A. 103, 6350–6355.

Identifying new variables during infection: Proximity to the host epithelium and epigenetic programs alter the expression of virulence factors in Vibrio cholerae

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Identifying New Variables During Infection: Proximity to the Host Epithelium and Epigenetic Programs Alter the Expression of Virulence Factors in Vibrio Cholerae

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