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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

Attachment and invasion of S. Enteritidis wild-type and its isogenic mutants to HCT116 cell line. a HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Infection was performed on ice followed by incubation at 4 °C for 30 min. Non adherent bacteria were removed by washing and number of adhering bacteria was enumerated by plating serial dilution. b For invasion, HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Number of invading bacteria was determined through standard gentamicin protection assay. SPI1 negative strain ΔinvC served as an experimental control. Experiments were performed in triplicates. Percentage of adhesion, invasion and uptake of mutant was compared to wild-type (normalized to 100). Error bar indicate the standard deviation of three independent experiments. Statistical analysis was performed using t test. The percentage of adhesion and invasion replication of Δprt and Δtyv was compared with that of ΔwbaV and level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant
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Fig3: Attachment and invasion of S. Enteritidis wild-type and its isogenic mutants to HCT116 cell line. a HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Infection was performed on ice followed by incubation at 4 °C for 30 min. Non adherent bacteria were removed by washing and number of adhering bacteria was enumerated by plating serial dilution. b For invasion, HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Number of invading bacteria was determined through standard gentamicin protection assay. SPI1 negative strain ΔinvC served as an experimental control. Experiments were performed in triplicates. Percentage of adhesion, invasion and uptake of mutant was compared to wild-type (normalized to 100). Error bar indicate the standard deviation of three independent experiments. Statistical analysis was performed using t test. The percentage of adhesion and invasion replication of Δprt and Δtyv was compared with that of ΔwbaV and level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant

Mentions: To study the effect of deletion of above genes in adhesion and invasion, the ability of different OAg mutants to adhere and invade HCT-116 cells was compared. OAg- negative mutants attached and invaded HCT-116 cells more efficiently as compared to the parental strain. Among all the three mutants, ΔwbaV mutant showed the highest rate of adhesion (~2.5-fold) and invasion (~4.5-fold) when compared with the wild-type (Fig. 3a, b) and the complemented strain showed adhesion and invasion similar to wild type. However, variations in the efficiency of adhesion and invasion among mutants were observed. The ΔwbaV mutant showed significantly increased adhesion and invasion when compared with other two OAg- negative mutants (P < 0.05–0.001).Fig. 3


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)

Attachment and invasion of S. Enteritidis wild-type and its isogenic mutants to HCT116 cell line. a HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Infection was performed on ice followed by incubation at 4 °C for 30 min. Non adherent bacteria were removed by washing and number of adhering bacteria was enumerated by plating serial dilution. b For invasion, HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Number of invading bacteria was determined through standard gentamicin protection assay. SPI1 negative strain ΔinvC served as an experimental control. Experiments were performed in triplicates. Percentage of adhesion, invasion and uptake of mutant was compared to wild-type (normalized to 100). Error bar indicate the standard deviation of three independent experiments. Statistical analysis was performed using t test. The percentage of adhesion and invasion replication of Δprt and Δtyv was compared with that of ΔwbaV and level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Attachment and invasion of S. Enteritidis wild-type and its isogenic mutants to HCT116 cell line. a HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Infection was performed on ice followed by incubation at 4 °C for 30 min. Non adherent bacteria were removed by washing and number of adhering bacteria was enumerated by plating serial dilution. b For invasion, HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Number of invading bacteria was determined through standard gentamicin protection assay. SPI1 negative strain ΔinvC served as an experimental control. Experiments were performed in triplicates. Percentage of adhesion, invasion and uptake of mutant was compared to wild-type (normalized to 100). Error bar indicate the standard deviation of three independent experiments. Statistical analysis was performed using t test. The percentage of adhesion and invasion replication of Δprt and Δtyv was compared with that of ΔwbaV and level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant
Mentions: To study the effect of deletion of above genes in adhesion and invasion, the ability of different OAg mutants to adhere and invade HCT-116 cells was compared. OAg- negative mutants attached and invaded HCT-116 cells more efficiently as compared to the parental strain. Among all the three mutants, ΔwbaV mutant showed the highest rate of adhesion (~2.5-fold) and invasion (~4.5-fold) when compared with the wild-type (Fig. 3a, b) and the complemented strain showed adhesion and invasion similar to wild type. However, variations in the efficiency of adhesion and invasion among mutants were observed. The ΔwbaV mutant showed significantly increased adhesion and invasion when compared with other two OAg- negative mutants (P < 0.05–0.001).Fig. 3

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