INFECTION AND IMMUNITY, Aug. 2005, p. 4488–4493
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 8
Transcriptional Profiling of Vibrio cholerae Recovered Directly from
Patient Specimens during Early and Late Stages of
Regina C. LaRocque,1,2* Jason B. Harris,1Michelle Dziejman,3Xiaoman Li,4Ashraful I. Khan,5
Abu S. G. Faruque,5Shah M. Faruque,5G. B. Nair,5Edward T. Ryan,1,2,6Firdausi Qadri,5
John J. Mekalanos,3and Stephen B. Calderwood1,2,3
Division of Infectious Diseases, Massachusetts General Hospital,1Department of Medicine2and Department of Microbiology
and Molecular Genetics,3Harvard Medical School, and Department of Immunology and Infectious Diseases,
Harvard School of Public Health,6Boston, and Department of Biostatistics, Harvard University,
Cambridge,4Massachusetts, and International Centre for Diarrhoeal Disease Research,
Received 17 November 2004/Returned for modification 16 January 2005/Accepted 15 March 2005
Understanding gene expression by bacteria during the actual course of human infection may provide
important insights into microbial pathogenesis. In this study, we evaluated the transcriptional profile of Vibrio
cholerae, the causative agent of cholera, in clinical specimens from cholera patients. We collected samples of
human stool and vomitus that were positive by dark-field microscopy for abundant vibrios and used a
microarray to compare gene expression in organisms recovered directly from specimens collected during the
early and late stages of human infection. Our results reveal that V. cholerae gene expression within the human
host environment differs from patterns defined in in vitro models of pathogenesis. tcpA, the major subunit of
the essential V. cholerae colonization factor, was significantly more highly expressed in early than in late stages
of infection; however, the genes encoding cholera toxin were not highly expressed in either phase of human
infection. Furthermore, expression of the virulence regulators toxRS and tcpPH was uncoupled. Interestingly,
the pattern of gene expression indicates that the human upper intestine may be a uniquely suitable environ-
ment for the transfer of genetic elements that are important in the evolution of pathogenic strains of V. cholerae.
These findings provide a more detailed assessment of the transcriptome of V. cholerae in the human host than
previous studies of organisms in stool alone and have implications for cholera control and the design of
Bacterial behavior in the host is influenced by nutrient avail-
ability and by environmental substrates that change as infec-
tion progresses and tissue breakdown and inflammation occur.
These factors influence bacterial growth rate and population
dynamics and affect the production of virulence determinants.
To date, study of bacterial physiology during the actual course
of human infection has been technically difficult. However, the
development of highly sensitive microarray-based techniques
for evaluating global microbial gene expression has made such
an approach feasible. In this study, we compared the gene
expression profiles of Vibrio cholerae bacteria recovered di-
rectly from patient specimens during early and late stages of
human infection, using transcriptional profiling by microarray.
Our goal was to define virulence factors expressed in the hu-
man host and to identify differences with existing models of
cholera pathogenesis based on in vitro studies.
V. cholerae, the etiologic agent of cholera, has been exten-
sively studied using in vitro systems. This work indicates that
the coordinate expression of a network of pathogenicity genes
enables the organism to colonize the small intestine and pro-
duce cholera toxin (CTX), which leads to secretory diarrhea
(14). In addition to CTX, a second major virulence factor of V.
cholerae is the toxin-coregulated pilus (TCP), a type IV pilus
that is required for intestinal colonization (12, 23). TCP also
serves as the receptor for the entry of CTX?, the filamentous
bacteriophage that encodes cholera toxin (24). In vitro, two
transmembrane transcription complexes, ToxRS and TcpPH,
have been shown to sense environmental conditions and act
through a common downstream regulator, ToxT, to coordinate
the simultaneous expression of the genes encoding TCP and
CTX (4, 5, 9, 23).
Interestingly, analyses of V. cholerae in recently shed human
stool specimens have not identified high-level expression of
genes encoding CTX or TCP or of genes involved in virulence
regulation (3, 19). Rather, compared to in vitro-grown organ-
isms, V. cholerae in stool specimens appears to be in a physi-
ologic state of preparation for dissemination into the environ-
ment. These findings suggest that transcriptional profiling of
the organism in stool specimens may not identify virulence
genes essential in the early phases of colonization and patho-
genesis in the human. Furthermore, interpretation of the tran-
scriptional profile of V. cholerae recovered from stool has been
complicated by the lack of a biologically relevant comparator
Here we directly compare the transcriptional profile of V.
* Corresponding author. Mailing address: Division of Infectious
Diseases, Gray-Jackson 504, Massachusetts General Hospital, 55 Fruit
Street, Boston, MA 02114. Phone: (617) 726-3812. Fax: (617) 726-
7416. E-mail: firstname.lastname@example.org.
cholerae in the early phase of infection of the human upper
intestine, represented by organisms in vomitus, with that of V.
cholerae in stool, representing the late phase of human infec-
tion. Our results characterize the dynamic physiologic state of
V. cholerae during the course of human infection and identify
key differences from patterns of virulence gene expression
identified in vitro.
MATERIALS AND METHODS
Collection of clinical samples. At least 50 ml of stool or vomitus was collected
immediately upon passage from patients presenting to the International Centre
for Diarrhoeal Disease Research in Bangladesh (ICDDR,B) with V. cholerae O1
or O139 infection, prior to the receipt of antibiotics. As previously noted (3, 19),
such specimens contain high numbers of the infecting serogroup of V. cholerae
and are largely free of other organisms. Specimens that were positive by dark-
field microscopy for the darting movement of vibrios were plated onto tauro-
cholate-tellurite-gelatin agar for overnight culture and placed directly into Trizol
(Life Technologies) for subsequent extraction of total RNA. Two specimens of
vomitus and five specimens of stool that were positive by culture for V. cholerae
O1 or O139 were included in this analysis. Quantitative culture was performed
on samples of both stool and vomitus and yielded at least 108CFU/milliliter.
Human patients’ approval was obtained from the Massachusetts General Hos-
pital and the ICDDR,B.
RNA and genomic DNA extraction. Total RNA was isolated from the clinical
samples using Trizol (Life Technologies). RNA samples were treated with
DNase to remove contaminating DNA on an RNeasy column (QIAGEN).
Quantities of RNA were determined by spectrophotometry, and visualization on
a 1% agarose gel was used to verify the integrity of the RNA. Genomic DNA
from the sequenced V. cholerae O1 El Tor strain N16961 (10) was prepared using
the Easy-DNA kit (Invitrogen) according to the manufacturer’s instructions.
Microarrays and hybridization. The V. cholerae microarray consists of 3,890
full-length PCR products representing the annotated open reading frames from
the initial release of the V. cholerae N16961 genome (10). The construction of the
array, fluorescent cDNA and genomic DNA labeling, hybridization, and data
collection were carried out as previously described (3, 6). Each labeling and
hybridization was performed in duplicate. Genomic DNA was used as a universal
internal control for the quality of the microarray and also allowed for the
comparison of results across multiple experiments (22). Genes with insufficient
genomic DNA hybridization to the microarray were excluded from the analysis.
Statistical analysis. Data were normalized using locally weighted regression to
obtain the relative abundance of each transcript as an intensity ratio with respect
to that of genomic DNA (26). High correlation coefficients were observed be-
tween technical replicates (Pearson’s correlation coefficient [r] ? 0.80) and
between results for separate clinical specimens of vomitus (r ? 0.77) and of stool
(r ? 0.80). Hence, the results from the two clinical vomitus specimens and the
five clinical stool specimens were pooled, and Welch’s t test was used to assess
the statistical significance of differences in median V. cholerae gene expression
between the two phases of human infection. Adjustment for multiple compari-
sons was made using the false discovery rate control (P ? 0.05) (2). Fold changes
for the relative expression of a given gene between the two types of clinical
specimens were calculated by dividing the normalized median intensity ratios
with respect to genomic DNA. The full data sets are available as supplementary
material via the Gene Expression Omnibus website (http://www.ncbi.nlm.nih
Regional clusters of genes were identified by an iterative assessment of all
genes evaluated with the microarray; clusters were defined as regional groups in
which ?70% of genes showed similar expression patterns. The statistical signif-
icance of the clustering was calculated according to the hypergeometric distri-
Quantitative RT-PCR. Quantitative reverse transcription-PCR (RT-PCR) was
performed to verify the microarray results for tcpA on clinical samples with
sufficient remaining material (one clinical vomitus sample and three clinical stool
samples). RNA isolated as described above was reverse transcribed using the
Reverse Transcription System (Promega). Primers and probes for tcpA and V.
cholerae 16S RNA were designed using Primer Express (Applied Biosystems).
Quantitative RT-PCRs were done using the TaqMan system (Applied Biosys-
tems) and an Opticon 2 continuous fluorescence detector (MJ Research). Real-
time PCRs were performed in a final volume of 25 ?l containing 1? TaqMan
Universal Master Mix (Applied Biosystems), 900 nM forward and reverse Taq-
man primers, and 250 nM Taqman probe. Primers and probes were purchased
from Applied Biosystems, and reactions were performed in MicroAmp Optical
96-well plates (Applied Biosystems). Validation experiments were performed for
all TaqMan probe and primer sets, and these showed a linear relationship
between the cycle threshold (CT) and the logarithm of the template amount
(genomic DNA), as expected. To control for genomic DNA contamination,
reactions without reverse transcriptase were performed. Relative expression
levels in the different samples were calculated by using the comparative CT
method with 16S RNA as the internal reference for normalization.
Of the 3,882 individual genes evaluated, 42 (1%) were sig-
nificantly differentially expressed between the two phases of
human infection (Table 1). Most of the differentially expressed
genes were more highly expressed in early than in late human
infection, and many of these genes are involved in DNA rep-
lication, energy production, and protein synthesis. These re-
sults indicate that early human infection is a period of active
replication and metabolic activity for V. cholerae.
A number of virulence factors were significantly more highly
expressed in the earliest stage of cholera infection (Table 1).
The gene with the single most significant difference in expres-
sion (P ? 7 ? 10?7) was tcpA, which was ?6-fold more highly
expressed in early than in late human infection. By quantitative
RT-PCR, tcpA transcript abundance was ?150-fold higher in
vomitus than in stool. Although previous studies have demon-
strated that TCP is an essential colonization factor of V. chol-
erae (12, 23), this is the first direct evidence of its expression
during early human infection.
Two putative hemolysins were also among the virulence
factors that were differentially expressed during early human
infection (Table 1). V. cholerae hemolysins are structurally
similar to pore-forming toxins of other bacteria and may con-
tribute to the enterotoxic activity of the organism (13, 25).
Notably, 11 hypothetical proteins were identified in our anal-
ysis; further study of the role of these proteins in cholera
pathogenesis, as well as that of the two putative hemolysins, is
As with many other pathogenic bacteria, the major virulence
genes of V. cholerae are clustered in several chromosomal
regions; these pathogenicity islands appear to have been ac-
quired in horizontal gene transfer events that have been im-
portant in the evolution of pathogenic strains (7, 20). Because
of this, we looked for clusters of contiguous genes that were
similarly regulated in early or late V. cholerae infection. We
performed an iterative assessment of all genes evaluated by our
microarray; significant clusters were identified as regional
groups in which ?70% of genes showed similar expression
Our analysis identified four highly significant clusters of
genes (P ? 10?7), all of which were upregulated during early
infection (Fig. 1). One of these clusters, VC2568 to VC2597,
comprises ribosomal proteins and likely reflects the particu-
larly active replicative state of V. cholerae during early human
infection. The second regional cluster of genes is located on
the V. cholerae small chromosome from VCA0560 to
VCA0570 (P ? 4.9 ? 10?10). This cluster includes a number of
hypothetical proteins and a transcriptional regulator, raising
the possibility that these genes may represent an operon ex-
pressed in response to an environmental signal in human in-
fection. The most significant cluster of genes spans the V.
VOL. 73, 2005VIBRIO CHOLERAE TRANSCRIPTOME IN HUMAN INFECTION4489
TABLE 1. Individual Vibrio cholerae genes with statistically significant differential expression between early and late human infectiona
TIGR designation Protein characterization
More highly expressed in early human infection
Biosynthesis of cofactors: VCA0558 Gamma-glutamyltranspeptidase, putative3.94 1.1 ? 10?6
Cellular processes (pathogenesis)
Toxin coregulated pilin TcpA
1.2 ? 10?4
7.6 ? 10?7
6.6 ? 10?5
Cellular processes (chemotaxis and motility): VC2601Sodium-type flagellar protein MotX1.829.2 ? 10?5
Excinuclease ABC, subunit A
Holliday junction DNA helicase RuvB
Holliday junction DNA helicase RuvA
Site-specific DNA methyltransferase, putative
4.6 ? 10?4
2.0 ? 10?4
9.7 ? 10?6
5.0 ? 10?4
1.1 ? 10?5
Pyruvate dehydrogenase, E2 component, dihydrolipoamide
3.5 ? 10?4
2.8 ? 10?4
1.1 ? 10?5
4.9 ? 10?5
3.2 ? 10?4
Hypothetical proteins (conserved)
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
1.2 ? 10?4
4.0 ? 10?4
3.4 ? 10?4
3.3 ? 10?5
1.4 ? 10?4
2.1 ? 10?4
2.2 ? 10?4
1.8 ? 10?4
9.8 ? 10?6
Ribosomal protein L23
Ribosomal protein L4
1.1 ? 10?4
2.0 ? 10?4
Purines, pyrimidines, nucleosides, and nucleotides
4.2 ? 10?4
8.5 ? 10?5
Pyruvate dehydrogenase complex repressor
Sigma-E factor regulatory protein RseC
2.6 ? 10?4
2.9 ? 10?5
Transcription: VC0006 RNase P protein component1.803.3 ? 10?4
Enterobactin synthetase component F-related protein
DedA family protein, authentic frameshift
GGDEF family protein
1.6 ? 10?6
5.8 ? 10?5
1.2 ? 10?4
3.8 ? 10?7
More highly expressed in late human infection
4.6 ? 10?5
5.0 ? 10?4
2.3 ? 10?4
1.1 ? 10?4
Hypothetical protein (conserved): VC0688Conserved hypothetical protein0.701.8 ? 10?4
Protein fate: VC2675Protease HsIVU, subunit HsIV0.402.2 ? 10?4
Protein synthesis: VC1047Fatty oxidation complex, alpha subunit 0.50 4.0 ? 10?4
aAfter adjustment for multiple comparisons with the false discovery rate control.
4490LAROCQUE ET AL.INFECT. IMMUN.
cholerae large chromosome from VC1832 to VC1853 (P ? 1.5
? 10?17). Many of these genes are transcribed in the same
direction, suggesting that they may be under common control.
Included within this region are the contiguous genes tolQRA,
which together encode a membrane complex that is required
for CTX? entry into the microbe (11). Two genes contained in
this region, ruvA and ruvB, encode proteins that are essential
for homologous recombination (21). recA, although not part of
this cluster, was also significantly more highly expressed in
early than in late human infection (fold change, 3.94 [P ? 2.0
? 10?4]). With high levels of expression of tcpA, tolQRA, and
genes involved in homologous recombination, the human up-
per intestine may therefore be a particularly well suited envi-
ronment for horizontal gene transfer events that are important
in the evolution of pathogenic V. cholerae strains.
The final cluster identified in our analysis was the group of
genes encoding TCP (P ? 1.35 ? 10?9) (Fig. 2). Included in
this cluster is tcpA, which was among the 42 individual genes
identified as significantly differentially expressed between the
two phases of human infection (Table 1). TCP is part of a
previously described 40-kb pathogenicity island that may have
been acquired in a horizontal gene transfer event (15, 16). In
our microarray studies, 29 of the 31 genes on the TCP patho-
genicity island were upregulated in early compared with late
infection, including the cytoplasmic transcriptional factor toxT
(fold change, 12.12 [P ? 0.01]), although the upregulation of
genes other than tcpA did not reach individual statistical sig-
nificance with our small sample size. This suggests that the
ToxT-regulated expression of the entire set of genes involved
in the assembly of TCP may be one of the first steps in colo-
nization of the human intestine.
Notably, despite high levels of tcpA expression in early hu-
man infection, we did not observe high levels of expression of
genes encoded by CTX?, including ctxAB, in either early or
late human infection. This is consistent with other published
results (3) and points to important differences between the
regulation of virulence gene expression in the human intestine
and that in in vitro models, where tcpA and ctxAB are coordi-
nately expressed. Indeed, during the course of human infection
we additionally observed an uncoupling in expression of the
two upstream regulators of tcpA and ctxAB. In particular, toxR
expression did not differ between the two phases of human
infection (fold change, 1.08 [P ? 0.57]), nor did that of its
accessory transmembrane protein toxS (fold change, 0.97 [P ?
0.61]). In contrast, tcpP (fold change, 4.17 [P ? 0.06]) and its
accessory transmembrane protein tcpH (fold change, 12.02 [P
? 0.06]), both encoded on the TCP island, were each more
highly expressed in early than in late human infection, al-
FIG. 1. Regional clusters of genes on the V. cholerae large and small chromosomes that display similar patterns of expression during human
infection (P ? 10?7). All genes evaluated with the microarray are shown. For each gene, the log10-fold change in expression in early infection
(vomit) compared with late infection (stool) is represented graphically. Genes that are more highly expressed in early human infection are
represented in red, and genes that are more highly expressed in late human infection are represented in green. Significant clusters of genes are
represented in yellow.
VOL. 73, 2005VIBRIO CHOLERAE TRANSCRIPTOME IN HUMAN INFECTION4491
though the fold changes did not achieve statistical significance
(Fig. 1). These findings should be confirmed with additional
human samples; they suggest that tcpPH may play an earlier
role in the activation of V. cholerae virulence gene expression
in vivo than toxRS.
Here we have used a microarray-based approach to directly
study the gene expression pattern of V. cholerae during two
phases of human infection. We observed that the expression of
a key virulence factor, tcpA, is much more prominent in the
early than the late phase of human infection. On the other
hand, high levels of ctxAB expression were not observed in
vibrios recovered from either human vomit or stool. This could
indicate that natural infection requires only a basal level of
expression of ctxAB. Alternatively, CTX production may take
place in a unique intestinal microenvironment that is not rep-
resented by our samples, such as in the more distal small
intestine or in a subset of organisms that have attached to the
intestinal epithelium. Animal studies support the latter hy-
pothesis. In particular, studies using recombinase-based in vivo
expression technology with the infant mouse model of cholera
indicate that the production of cholera toxin is spatially sepa-
rate from and temporally dependent on the prior expression of
tcpA (18). In our study, toxRS and tcpPH, the two regulatory
complexes that have been shown in vitro to together control
the expression of V. cholerae virulence genes, also were uncou-
pled during early human infection. Together, these findings
illustrate the complexity of the environmental signals experi-
enced by V. cholerae during its passage through the human host
and underscore the difficulty of fully capturing these dynamic
interactions with laboratory-based models.
Our results also have implications for the development of
improved therapeutics and vaccine strategies for cholera. The
V. cholerae colonization factor TCP is very highly expressed
during the earliest stage of human infection, along with a
number of novel virulence genes. Studies with North American
volunteers and with cholera patients from Indonesia had pre-
viously suggested that TCP was not strongly immunogenic dur-
ing natural cholera infection (8). However, recent work in
Bangladesh using recombinant V. cholerae O1 El Tor TcpA has
shown that cholera patients in fact mount substantial mucosal
and systemic immune responses to the major subunit of TCP
(1). Overall, 93% of patients studied showed a TcpA-specific
mucosal or systemic response. High-level expression of tcpA in
the human upper intestine, combined with its potent immuno-
genicity, suggests that research on the role of immunity to
TcpA in protection from cholera is warranted. Further studies
of the two putative hemolysins and the hypothetical proteins
identified in our analysis may also identify novel therapeutic
Our evaluation of the gene expression pattern of V. cholerae
observed directly in clinical specimens also highlights an im-
portant evolutionary relationship between this microbe and the
human host. V. cholerae is unique among the major diarrheal
pathogens because it is part of the free-living bacterial flora of
aquatic environments. Through a series of incompletely under-
stood events, strains of V. cholerae emerge from estuarine
waters to cause widespread human disease. Our findings indi-
cate that the human upper intestine is a particularly suitable
niche for replication of V. cholerae outside the aquatic envi-
FIG. 2. Differential expression of genes in the TCP island during early compared with late human infection, represented as log10fold change.
Expression of the transposase sequence (tnp) of the TCP island is shown at the left, and that of the integrase gene (int) is at the right. Transcripts
in the TCP island that are more highly expressed in early human infection are shown in red, and transcripts that are more highly expressed in late
human infection are shown in green. Genes flanking the TCP island are represented in grey. The differential expression of the individual gene tcpA
achieved statistical significance after adjustment with the false discovery rate control.
4492LAROCQUE ET AL.INFECT. IMMUN.
ronment. This may in itself represent an evolutionary strategy
for dissemination, since the organism is shed in prodigious
quantities from an infected person (?108CFU/milliliter of
stool), and such organisms appear to exist in a hyperinfectious
state for the next host (19). Additionally, the human upper
intestine may be a particularly well suited environment for the
acquisition of foreign genetic material that is important in the
evolution of pathogenic V. cholerae strains. Studies with the
suckling mouse model of cholera have demonstrated the trans-
fer of CTX? between bacterial strains in vivo (17, 24). Our
transcriptional data suggest that optimal conditions for CTX?
transduction of V. cholerae exist during early infection of the
human host, the only known reservoir for the organism outside
of estuarine environments. Infection in the human intestine
may thus foster the development of pathogenic V. cholerae
strains, as well as enriching for their multiplication and subse-
In this study, we have taken advantage of the large quantities
of vibrios present in clinical samples in order to study an
important human pathogen within the host environment. With
the refinement of genome-based techniques, similar studies of
other microbial pathogens within specific human environments
will become increasingly feasible and may lead to new insights
into bacterial virulence.
Financial support was received from ICDDR,B and from NIH
grants TW07144 (R.C.L.), AI40725 (to E.T.R.), GM068851 (to J.J.M.
and S.M.F.), and U01-AI58935 (to S.B.C.). Jason Harris is an NICHD
fellow of the Pediatric Scientist Development Program (K12-
HD00850). Regina LaRocque was supported by a Burroughs Well-
come Fund Postdoctoral Fellowship in Tropical Infectious Diseases
from the American Society of Tropical Medicine and Hygiene.
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