fcgrt, egr1, and cebpd) and regenerating islet-derived protein 3 gamma and beta (reg3g and
reg3b), which were among the most highly induced genes (by factors of 16.8 and 13.1, respectively) (Fig. 2D and table S3). Thus, wild-type
V. cholerae triggered a greater innate immune
transcriptional response than did the T6SS mutant. We wondered whether the innate immune
responses observed in these experiments were
driven by release of microbe-associated molecular patterns (MAMPs), bacterial molecules recognized by the host innate immune system (11).
Consistent with this hypothesis, we found that
V. cholerae T6SS-dependent killing of E. coli releases MAMPs in vitro (fig. S3, A and B).
To test whether disease symptoms might be
modulated by the T6SS-mediated killing of commensal species, we gavaged mice with 108 CFU of
wild-type V. cholerae (C6706), its vipA T6SS mutant, or buffer and tested for fluid accumulation
(FA ratio, indicative of diarrhea) after 22 hours.
The FA ratio was highest for the wild type–
challenged group (0.0875 ± 0.0025) and was
significantly lower (0.0749 ± 0.0026) for the
T6SS-mutant group (Fig. 3A, P < 0.01). We also
monitored the bacterial load in the small intestine and colon of the infected mice. In both
organs, the wild-type CFU exceeded that of the
T6SS mutant by a factor of 3 to 9 (Fig. 3B, P < 0.01).
Furthermore, elimination of host gut commensals by antibiotic treatment abolished the observed T6SS-dependent enhancement of the FA
ratio and bacterial load for animals infected with
wild-type V. cholerae relative to the T6SS mutant
(Fig. 3, C and D).
The V. cholerae Tox T regulatory cascade is
known to enhance diarrheal responses and bacterial loads in vivo through its control of the ctx
and tcp virulence operons (12–15). Thus, we asked
whether T6SS-mediated killing of commensal
organisms could release a commensal-derived
signal that activates the Tox T regulatory cascade.
We found that tcpA and ctxA transcript levels in
bacteria recovered from infected mice were much
higher in the wild type than in its T6SS mutant
during the early stages of infection (fig. S4).
This difference significantly dropped when mice
where pretreated with streptomycin (Fig. 3, E
and F), indicating that T6SS-dependent killing
of streptomycin-sensitive commensal bacteria
can activate V. cholerae virulence gene expression
during early stages of infection. Because control
experiments showed that T6SS-mediated killing
of E. coli on laboratory media did activate tcpA or
ctxA transcription in vitro (fig. S5), we conclude
that the host is driving these T6SS-dependent
changes in virulence gene expression.
Genome analysis has recently revealed that
variant strains of the seventh pandemic El Tor
V. cholerae clade are now responsible for the
vast majority of cholera cases in South Asia,
Africa, and Haiti (12). Because these variant
strains cause more severe diarrhea in cholera
victims, we questioned whether they showed
higher levels of T6SS expression. Transcriptome
analysis revealed that the variant strains H1
(isolated early in the Haitian cholera epidemic)
vipA VCA0109VCA0111VCA0113 vasH vasF VCA0119 VCA0121 vgrG-3 VCA0105
vipB vasA VCA0112VCA0114clpV VCA0118 vasK VCA0122 VCA0124 VCA0106
VC1415 VC1416 VC1417 VC1418 VC1419 VC1420 VC1421 VCA0017 VCA0018 VCA0019 VCA0020 VCA0021 VCA0022 VCA0284 VCA0285 VCA0286
hcp-1 VC1417 VC1419 VC1421
vgrG-1 VC1418 VC1420
VCA0286 vgrG-2 VCA0020
hcp-2 VCA0019 VCA0021
Fig. 4. Constitutive transcriptional up-regulation of T6SS genes in recent El Tor variant strains.
The transcriptomes of past seventh pandemic El Tor strains N16961 (1971) and C6706 (1991) are compared to those of recent variant strains MDC126 (2008) and H1 (2010) within T6SS-related operons in
both chromosomes I and II. The relative expression levels of T6SS core/accessory, hcp, vgrG, and effector/
immunity genes are normalized as RPKM (reads per kilobase of transcript per million mapped reads) units.
T6SS- WT T6SS-
ctrl WT 6SS-
Ctrl WT 6SS-
Fig. 3. V. cholerae T6SS enhances the development of diarrhea disease symptoms. (A and B)
Fluid accumulation (FA) ratio (A) and V. cholerae CFU count per SI (B) of wild type–treated and
T6SS mutant–treated mice. (C and D) FA ratio (C) and V. cholerae CFU (D) measured in mice
pretreated with streptomycin. (E and F) tcpA and ctxA transcript levels were measured in mice not
treated with streptomycin (No Sm) (E) or Sm-treated (F) at 6 hours after infection. **P < 0.01,
***P < 0.005, ****P < 0.001 (unpaired t test); ns, not significant.