Keywords: Vibrio cholerae; ToxR Regulon; Accessory colonization factor; Chemotaxis
We have previously shown that two Vibrio Pathogenicity Island (VPI) genes (acfB, tcpI) under the control of the ToxR-ToxT regulatory cascade encode members of the methyl-accepting chemotaxis family of proteins [17,18]. V. cholerae strains with mutations in acfB were initially identified in a transposon library as being slightly defective in intestinal colonization [19]. TcpI was identified as a negative regulator of pilus synthesis [5,18]. In this report we find that V. cholerae strains bearing mutations in acfB were defective for chemotaxis towards mucus whereas tcpI mutants displayed wild type levels of chemotaxis towards mucus.
Strains |
Genotype |
Source/reference |
Vibrio cholerae |
|
|
0395 |
01 classical, Strr |
(19) |
0395 ΔacfB |
ΔacfB, Strr |
This study |
0395 ΔacfB/pACFB |
ΔacfB/pacfB, Strr, Ampr |
This study |
0395 ΔtoxR |
ΔtoxR, Strr |
(19) |
0395 ΔtcpI |
ΔtcpI, Strr |
This study |
0395 ΔcheW1 |
ΔcheW1, Strr |
This study |
0395 ΔcheW2 |
ΔcheW2, Strr |
This study |
0395 ΔcheW3 |
ΔcheW3, Strr |
This Study |
Escherichia coli |
|
|
Top10 |
F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1deoR araD139 Δ(ara-leu)7697 galUgalKrpsL (Strr) endA1 nupG |
Invitrogen |
DH5α (λpir) |
supE4 DlacU169 (Φ80 lacZDM15) hsdR17 recA1 endA1 gyrA96thi-1 relA1 λpir |
Invitrogen |
Sm10 (λpir) |
thi-1 thrleutonAlacYsupErecA::RP4-2Tc::Mu λpir R6K |
(37) |
Sy327(λpir) |
Δ(lac-pro) arE(Am) rifnalA recA56 λpir |
(37) |
Plasmids |
|
|
pBAD |
colE1 ori; araBAD promoter; Amp r |
Invitrogen |
pACFB |
acfB in pBAD; Amp r |
This study |
pCVD442 |
R6K ori;, mobRP4, bla, sacB |
(38) |
pGP704 |
R6K ori; Amp r |
(37) |
Strain |
Serine |
Mucus |
Galactose-6-sulfate |
0395 |
18 |
12 |
12 |
0395 ΔtoxR |
18 |
3 |
4 |
0395 acfB::CmR |
17 |
2 |
4 |
0395 acfB::CmR/pACFB |
17 |
11 |
11 |
0395 ΔtcpI |
17 |
12 |
11 |
0395 ΔcheW1 |
2 |
3 |
3 |
0395 ΔcheW2 |
18 |
11 |
13 |
0395 ΔcheW3 |
18 |
12 |
12 |
We and others have used computer algorithms to predict that AcfB is structurally related to methyl accepting chemotaxis proteins [17,29]. Over expression of acfB in both V. cholerae and E. coli alters the swarm plate response of these strains. This suggests that AcfB is capable of interacting with the general chemotaxis machinery of both organisms. In this report we demonstrate for the first time that AcfB functions in V. cholerae chemotaxis. V. cholerae bearing mutations in acfB fail to respond to a gradient of mucus and also fail to recognize galactose-6-sulfate as a chemoattractant. Wild type V.cholerae respond with a positive chemotactic response to both of these substances. As with acfB, over expression of tcpI in V. cholerae alters the swarm plate response of these organisms in LB soft agar plates suggesting that tcpI is able to interact with the general chemotaxis machinery. Unlike AcfB, however; TcpI does not appear to participate in the chemotactic response of V. cholerae to mucus/galactose-6-sulfate. TcpI is a pH dependent, negative regulator of TCP biogenesis. TcpI permits maximum synthesis of TCP in response to the pH of the culture medium. These findings and the relatedness of TcpI to MCPs, suggest that TcpI may "sense" pH. Capillary tube chemotaxis experiments in which V. cholerae 0395 and V. cholerae 0395ΔtcpI were exposed to KRT pH 6.5, KRT pH 7.4 and KRT pH 8.5 failed to demonstrate directed vibrio motility in response to pH (data not shown). Although regulation of TCP synthesis by TcpI appears to involve the ability of this inner membrane sensor protein to recognize pH, the mechanism by which it affects this regulation and any possible role for Che proteins in this regulation remains obscure. Chemotaxis in bacteria is accomplished via a complex signal transduction system that permits sensory adaptation and relates the input signal to the flagellar motor [20-22, 30,31]. In many bacteria the signal transduction apparatus contains multiple sets of the proteins required for signal transduction [32]. The genome V. cholerae is predicted to encode 22 open reading frames that are homologous to che genes, most of the V. cholerae che genes are clustered in three regions on both chromosomes. The precise role of multiple sets of che genes in V. cholerae and other bacteria is not known [15]. Several che genes have been shown to affect cellular functions not related to chemotaxis. HlyB and TcpI were shown to be involved in hemolysin secretion and pilus biogenesis respectively [17,33]. Previous work examining the role of V. cholerae che paralogues in chemotaxis have shown that genes located in the che cluster II are responsible for V. cholerae chemotaxis [15,16]. The role of the genes encoded in clusters I and III have not been elucidated. In order to examine the role of the three che gene clusters in the V. cholerae chemotactic response to mucus we generated vibrio strains with mutations in cheW1, cheW2 and cheW3. Only mutations within cheW1 abolished vibrio chemotaxis towards mucus and galactose-6-sulfate.
The V. cholerae acfC gene is predicted to encode a periplasmic sulfate binding protein and is part of a polycistronic operon downstream of acfB. Given its role in intestinal colonization, it seems likely that AcfC is involved in chemotaxis. Other periplasmic solute binding proteins such as the maltose binding protein and ribose binding protein have been shown to play a role in E. coli/Salmonella chemotaxis via interactions with methyl-accepting chemotaxis proteins [34]. Mucus is rich in sulfated molecules [35,36] and thus we hypothesized that the ACF proteins may represent a sulfate "sensing" mechanism whereby V. cholerae could sense intestinal mucus and promote penetration of the mucus layer by directing chemotaxis toward sulfated sugars. Galactose is a common sulfated sugar found in mucus [36] and thus we tested galactose-6-sulfate for its ability to act as a chemoattractant. Parental V. cholera 0395 was capable of chemotaxis towards this sulfated sugar whereas 0395ΔacfB and 0395ΔcheW1 were non-chemotactic towards galactose-6- sulfate. These findings support out the hypothesis regarding the role of AcfB and AcfC in intestinal colonization such that AcfC binds galactose-6-sulfate followed by interaction with AcfB which in turn activates the chemotaxis signal transduction cascade mediated by Che proteins encoded by the che II gene cluster.
Recent efforts aimed at generating live-attenuated Vc vaccine strains suggest that that motility may play a role in the residual virulence of Vc strains lacking cholera toxin genes [39,40]. These studies emphasize the importance of understanding the role of vibrio motility and chemotaxis proteins in intestinal colonization, as this might have a practical impact on the development of efficacious vaccines for the prevention of cholera and may also give new direction to vaccine research for other enteric pathogens. The results presented here, shed light on a novel aspect of Vc pathogenesis and promote a clearer understanding of the contribution of the Vc chemotaxis signaling proteins in the intestinal colonization process.
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