Article Figures & Data
Figures
- FIG. 1.
Surface polysaccharides expressed by LPS and CPS mutants. (A) Western blot analysis with O139-specific antiserum (see Materials and Methods). Reactive bands are indicated as follows: the upper band represents the capsular form of O139 side chain, and the lower band corresponds to the O side chain/core OS/lipid A portion. (B) Silver-stained polyacrylamide gel after SDS-PAGE. Stained bands are indicated as follows: the upper bands represent the O side chain/core OS/lipid A, and the lower bands are the core OS/lipid A portion. Lanes: wt, MO10 wild type; 1, MO10 ΔwaaL; 2, MO10 ΔwaaL pBADwaaL; 3, MO10 ΔgalEK; 4, MO10 ΔgalEK pACYCgalE; 5, MO10 wbfF; 6, MO10 wavB; 7, MO10 wavB pSSkanwavB2; 8, MO10 waaF; 9, MO10 waaF pACYCwaaF; 10, MO10 ΔwaaL ΔwbfF.
- FIG. 2.
Colonization deficiency of LPS and CPS mutants. Each point represents the value from a single mouse competition assay. The competitive index is defined as the output ratio of mutant to wild-type bacteria divided by the input ratio of mutant to wild-type bacteria. Strains with the wild-type ability to colonize have a competitive index of approximately 1.0. Data sets: 1, MO10 wbfF; 2, MO10 ΔgalEK; 3, MO10 ΔgalEK pACYCgalE; 4, MO10 ΔwaaL; 5, MO10 ΔwaaL pBADwaaL; 6, MO10 wavB; 7, MO10 wavB, pSSkanwavB2; 8, MO10 waaF; 9, MO10 waaF, pACYCwaaF; 10, MO10 ΔwaaL ΔwbfF. The defect in colonization of all mutant strains was statistically significant (P < 0.01) compared with the wild-type strain MO10 by Student's two-tailed t test.
- FIG. 3.
Expression of TcpA and cholera toxin in LPS and CPS mutants. Strains were grown under inducing conditions (Materials and Methods). (A) Whole-cell lysates were subjected to Western blot with TcpA antiserum. (B) Cell-free supernatants were measured for cholera toxin (CT) by ELISA and given in per optical density unit at 600 nm. Lanes: 1, MO10 (noninduced); 2, MO10 (induced); 3, MO10 wbfF (induced); 4 MO10 ΔwaaL (induced); 5, MO10 ΔgalEK (induced); 6, MO10 wavB (induced); 7, MO10 waaF (induced); 8, MO10 waaF pACYCwaaF (induced).
- FIG. 4.
Growth phenotypes of LPS and core OS mutants on TCBS plates. (A) TCBS plus ampicillin (100 μg/ml) containing 0.8% bile. (B) TCS plus ampicillin (100 μg/ml) without bile. (C) TCBS containing 0.8% bile. Sets: 1, MO10 pBAD28; 2, MO10 waaF pACYCwaaF; 3, MO10 waaF; 4, MO10 wavB pSSkanwavB2; 5, MO10 wavB; 6, MO10 ΔgalEK; 7, MO10 ΔwaaL; 8, MO10 wbfF.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant genotypea Reference or source Strains MO10 Clinical isolate of V. cholerae O139 42 PW96 MO10 lacZ::MCS 43 MO10 ΔwaaL MO10 ΔwaaL This study MO10 ΔwaaLwbfF MO10 ΔwaaL wbfF::pGP704 This study MO10 ΔgalEK ΔgalE::cm ΔgalK This study MO10 wbfF wbfF::pGP704 This study MO10 waaF waaF::pGP704 This study MO10 wavB wavB::pGP704 This study Plasmids pTrcAkan Cloning vector Kmr 24 pBAD28 Cloning vector Apr Kanr 8 pGP704 OriR6K mobRP4 Apr 22 pUC4K Apr Kmr 39 pGPwaaF ‘waaF’b Apr 25 pGPwavB ‘wavB’ Apr 25 pKEK229 OriR6K mobRP4 sacB Apr 6 pKEKΔgalE::cm ΔgalE::cm Apr 26 pBADwaaL waaL + Kmr 25 pACYCgalE galE + Kmr 26 pACYCwaaF waaF + Kmr 25 pAKwavB wavB tetR + Cmr 25 pGPwbfF ‘wbfF’ in pGP704 Apr This study pKEKΔgalK ΔgalK in pKEK229 Apr This study pKEKΔwaaL ΔwaaL in pKEK229 Apr This study pSSkanwavB1 pAKwavB tetR+ Cmr Kmr This study pSSkanwavB2 pSSkanwavB1 ΔtetR Cmr Kmr This study - TABLE 2.
Resistance to antimicrobial peptides and the complement system
Strain MBC of protamine (μg/ml) Serum resistancea (% survival) MO10 150 96.75 ± 6.50 MO10 ΔwaaL 150 99.15 ± 1.69 MO10 wbfF 150 3.68 ± 5.58 MO10 ΔwaaL ΔwbfF 150 0.03 ± 0.03 MO10 ΔgalEK 150 0.05 ± 0.06 MO10 ΔgalEK pACYCgalE ND 90.25 ± 11.84 MO10 waaFb 75 100 ± 0 MO10 waaF pACYCwaaF 150 ND MO10 wavB 150 92.48 ± 8.68 ↵ a Serum resistance was calculated as the ratio of the number of CFU found after cells were incubated for 45 min in 20% normal human serum divided by the number of CFU found after cells were incubated for 45 min in 20% heat-inactivated normal human serum. Indicated is the mean ± standard deviation. All assays were performed at least four times. ND, not determined.
↵ b This mutant was also tested in the presence of control plasmid pTrcAkan.
