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The O-antigen negative ∆wbaV mutant of Salmonella enterica serovar Enteritidis shows adaptive resistance to antimicrobial peptides and elicits colitis in streptomycin pretreated mouse model.

Jaiswal S, Pati NB, Dubey M, Padhi C, Sahoo PK, Ray S, Arunima A, Mohakud NK, Suar M - Gut Pathog (2015)

Bottom Line: Deletion of the above three genes resulted in the production of OAg-negative LPS.In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt.In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant.

View Article: PubMed Central - PubMed

Affiliation: KIIT School of Biotechnology, KIIT University, Bhubaneswar, Odisha 751024 India.

ABSTRACT

Background: Salmonella enterica serovar Enteritidis, the most common cause of human gastroenteritis, employs several virulence factors including lipopolysaccharide (LPS) for infection and establishment of disease inside the host. The LPS of S. enterica serovar Enteritidis consists of lipid A, core oligosaccharide and O-antigen (OAg). The OAg consists of repeating units containing different sugars. The sugars of OAg are synthesized and assembled by a set of enzymes encoded by genes organized into clusters. Present study focuses on the effect of deletion of genes involved in biosynthesis of OAg repeating units on resistance to antimicrobial peptides and virulence in mice.

Methods: In the present study, the OAg biosynthesis was impaired by deleting tyv, prt and wbaV genes involved in tyvelose biosynthesis and its transfer to OAg. The virulence phenotype of resulting mutants was evaluated by assessing resistance to antimicrobial peptides, serum complement, adhesion, invasion and in vivo colonization.

Results: Deletion of the above three genes resulted in the production of OAg-negative LPS. All the OAg-negative mutants showed phenotype reported for rough strains. Interestingly, ΔwbaV mutant showed increased resistance against antimicrobial peptides and normal human serum. In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt. In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant.

Conclusion: OAg-negative mutants are known to be avirulent; however, this study demonstrates that certain OAg negative mutants e.g. ∆wbaV may also show resistance to antimicrobial peptides and cause colitis in Streptomyces pretreated mouse model.

No MeSH data available.


Related in: MedlinePlus

Assessment of motility and sensitivity to antimicrobial peptides and serum. a Wild-type Salmonella and its isogenic mutants were placed at the center of the agar plate containing 0.3 % agar and incubated for 8 h. Diameter of growth zone was measured in centimeter (cm). Experiments were performed in triplicates at three independent occasions. b For AMP sensitivity assay, approximately 1 × 107 CFU/ml bacteria in PBS were incubated with or without antimicrobial peptides (1 µg/ml polymyxin B, 5 µg/ml protamine, 10 µg/ml LL37, and 2 µg/ml cecropin) for 1 h at 37 °C. The number of surviving bacteria was determined by plating serial dilutions on LB agar plates and survival percentage was calculated. Statistical analysis was performed using two-way ANOVA. c For analysis of serum resistance 1 × 107 CFU/ml of bacteria was incubated with or without 50 % of normal human serum and serial dilutions were plated to enumerate the number of surviving bacteria. Statistical analysis was performed using student t-test. The survival of Δprt and Δtyv mutants was compared with that of ΔwbaV. Level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant. Error bar indicate the standard deviation of three independent experiments
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Fig2: Assessment of motility and sensitivity to antimicrobial peptides and serum. a Wild-type Salmonella and its isogenic mutants were placed at the center of the agar plate containing 0.3 % agar and incubated for 8 h. Diameter of growth zone was measured in centimeter (cm). Experiments were performed in triplicates at three independent occasions. b For AMP sensitivity assay, approximately 1 × 107 CFU/ml bacteria in PBS were incubated with or without antimicrobial peptides (1 µg/ml polymyxin B, 5 µg/ml protamine, 10 µg/ml LL37, and 2 µg/ml cecropin) for 1 h at 37 °C. The number of surviving bacteria was determined by plating serial dilutions on LB agar plates and survival percentage was calculated. Statistical analysis was performed using two-way ANOVA. c For analysis of serum resistance 1 × 107 CFU/ml of bacteria was incubated with or without 50 % of normal human serum and serial dilutions were plated to enumerate the number of surviving bacteria. Statistical analysis was performed using student t-test. The survival of Δprt and Δtyv mutants was compared with that of ΔwbaV. Level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant. Error bar indicate the standard deviation of three independent experiments

Mentions: Bacterial strains lacking OAg are known to be less motile as compared to those with long OAg repeating units. To confirm if the OAg-negative mutants used in this study were also less motile, the motilities of the mutants were analyzed by soft agar assay. All the OAg-negative mutants were found to be less motile as compared to the wild-type and showed almost two fold reduction in their motility (Fig. 2a).Fig. 2


The O-antigen negative ∆wbaV mutant of Salmonella enterica serovar Enteritidis shows adaptive resistance to antimicrobial peptides and elicits colitis in streptomycin pretreated mouse model.

Jaiswal S, Pati NB, Dubey M, Padhi C, Sahoo PK, Ray S, Arunima A, Mohakud NK, Suar M - Gut Pathog (2015)

Assessment of motility and sensitivity to antimicrobial peptides and serum. a Wild-type Salmonella and its isogenic mutants were placed at the center of the agar plate containing 0.3 % agar and incubated for 8 h. Diameter of growth zone was measured in centimeter (cm). Experiments were performed in triplicates at three independent occasions. b For AMP sensitivity assay, approximately 1 × 107 CFU/ml bacteria in PBS were incubated with or without antimicrobial peptides (1 µg/ml polymyxin B, 5 µg/ml protamine, 10 µg/ml LL37, and 2 µg/ml cecropin) for 1 h at 37 °C. The number of surviving bacteria was determined by plating serial dilutions on LB agar plates and survival percentage was calculated. Statistical analysis was performed using two-way ANOVA. c For analysis of serum resistance 1 × 107 CFU/ml of bacteria was incubated with or without 50 % of normal human serum and serial dilutions were plated to enumerate the number of surviving bacteria. Statistical analysis was performed using student t-test. The survival of Δprt and Δtyv mutants was compared with that of ΔwbaV. Level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant. Error bar indicate the standard deviation of three independent experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559907&req=5

Fig2: Assessment of motility and sensitivity to antimicrobial peptides and serum. a Wild-type Salmonella and its isogenic mutants were placed at the center of the agar plate containing 0.3 % agar and incubated for 8 h. Diameter of growth zone was measured in centimeter (cm). Experiments were performed in triplicates at three independent occasions. b For AMP sensitivity assay, approximately 1 × 107 CFU/ml bacteria in PBS were incubated with or without antimicrobial peptides (1 µg/ml polymyxin B, 5 µg/ml protamine, 10 µg/ml LL37, and 2 µg/ml cecropin) for 1 h at 37 °C. The number of surviving bacteria was determined by plating serial dilutions on LB agar plates and survival percentage was calculated. Statistical analysis was performed using two-way ANOVA. c For analysis of serum resistance 1 × 107 CFU/ml of bacteria was incubated with or without 50 % of normal human serum and serial dilutions were plated to enumerate the number of surviving bacteria. Statistical analysis was performed using student t-test. The survival of Δprt and Δtyv mutants was compared with that of ΔwbaV. Level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant. Error bar indicate the standard deviation of three independent experiments
Mentions: Bacterial strains lacking OAg are known to be less motile as compared to those with long OAg repeating units. To confirm if the OAg-negative mutants used in this study were also less motile, the motilities of the mutants were analyzed by soft agar assay. All the OAg-negative mutants were found to be less motile as compared to the wild-type and showed almost two fold reduction in their motility (Fig. 2a).Fig. 2

Bottom Line: Deletion of the above three genes resulted in the production of OAg-negative LPS.In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt.In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant.

View Article: PubMed Central - PubMed

Affiliation: KIIT School of Biotechnology, KIIT University, Bhubaneswar, Odisha 751024 India.

ABSTRACT

Background: Salmonella enterica serovar Enteritidis, the most common cause of human gastroenteritis, employs several virulence factors including lipopolysaccharide (LPS) for infection and establishment of disease inside the host. The LPS of S. enterica serovar Enteritidis consists of lipid A, core oligosaccharide and O-antigen (OAg). The OAg consists of repeating units containing different sugars. The sugars of OAg are synthesized and assembled by a set of enzymes encoded by genes organized into clusters. Present study focuses on the effect of deletion of genes involved in biosynthesis of OAg repeating units on resistance to antimicrobial peptides and virulence in mice.

Methods: In the present study, the OAg biosynthesis was impaired by deleting tyv, prt and wbaV genes involved in tyvelose biosynthesis and its transfer to OAg. The virulence phenotype of resulting mutants was evaluated by assessing resistance to antimicrobial peptides, serum complement, adhesion, invasion and in vivo colonization.

Results: Deletion of the above three genes resulted in the production of OAg-negative LPS. All the OAg-negative mutants showed phenotype reported for rough strains. Interestingly, ΔwbaV mutant showed increased resistance against antimicrobial peptides and normal human serum. In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt. In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant.

Conclusion: OAg-negative mutants are known to be avirulent; however, this study demonstrates that certain OAg negative mutants e.g. ∆wbaV may also show resistance to antimicrobial peptides and cause colitis in Streptomyces pretreated mouse model.

No MeSH data available.


Related in: MedlinePlus