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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

Colonization of cecum, mLN, spleen and liver of C57BL/6 mouse by S. Enteritidis and OAg-negative mutants: Streptomycin pretreated mice were infected with respective bacterial strains through oral route of administration. Mice were sacrificed on 3 days p. i. and cecum (a), mLN (b), spleen (c) and liver (d) were collected and CFU in each organ were determined. Statistical significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant
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Fig4: Colonization of cecum, mLN, spleen and liver of C57BL/6 mouse by S. Enteritidis and OAg-negative mutants: Streptomycin pretreated mice were infected with respective bacterial strains through oral route of administration. Mice were sacrificed on 3 days p. i. and cecum (a), mLN (b), spleen (c) and liver (d) were collected and CFU in each organ were determined. Statistical significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant

Mentions: To understand the effect of deletion of above genes on virulence of ΔwbaV mutant in vivo, C57BL/6 mice were infected with wild-type, ΔwbaV and Δprt mutants. In a 3 days p. i. experiment, the cecal colonization of ΔwbaV mutant was comparable to wild-type, whereas the Δprt mutant colonized less efficiently than ΔwbaV mutant and the wild-type (Fig. 4a), however, the differences were not statistically significant. On the contrary, the pathogen load in the mesenteric lymph node (mLN) varied between both the mutants. The CFU of ΔwbaV mutant recovered from mLN was significantly (P = 0.0085) higher than Δprt mutant (Fig. 4b). Similarly, the bacterial load in spleen and liver was also found to be significantly higher in case of ΔwbaV mutant as compared to Δprt mutant (P < 0.05) (Fig. 4c, d). Notably, the ΔwbaV mutant showed reduced colonization in mLN, spleen and liver as compared to the wild-type. Further, histopathological evaluation was carried out to compare the cecal inflammation between the mutants and wild-type. The ΔwbaV mutant was found to induce moderate cecal inflammation whereas Δprt mutant was unable to induce inflammation in the C57BL/6 mice (Fig. 5a). The cecal pathology was assessed by a semi-quantitative scoring method as described in materials and methods. The day 3 p. i. experiment showed a significant difference in the cecal pathology of the mice infected with Δprt mutant (pathoscore: 8) and ΔwbaV mutant (pathoscore: 2) as compared to the wild-type (P < 0.01) (Fig. 5b). The phenotype of complemented ΔwbaV strain was similar to wild type.Fig. 4


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)

Colonization of cecum, mLN, spleen and liver of C57BL/6 mouse by S. Enteritidis and OAg-negative mutants: Streptomycin pretreated mice were infected with respective bacterial strains through oral route of administration. Mice were sacrificed on 3 days p. i. and cecum (a), mLN (b), spleen (c) and liver (d) were collected and CFU in each organ were determined. Statistical 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

Fig4: Colonization of cecum, mLN, spleen and liver of C57BL/6 mouse by S. Enteritidis and OAg-negative mutants: Streptomycin pretreated mice were infected with respective bacterial strains through oral route of administration. Mice were sacrificed on 3 days p. i. and cecum (a), mLN (b), spleen (c) and liver (d) were collected and CFU in each organ were determined. Statistical significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant
Mentions: To understand the effect of deletion of above genes on virulence of ΔwbaV mutant in vivo, C57BL/6 mice were infected with wild-type, ΔwbaV and Δprt mutants. In a 3 days p. i. experiment, the cecal colonization of ΔwbaV mutant was comparable to wild-type, whereas the Δprt mutant colonized less efficiently than ΔwbaV mutant and the wild-type (Fig. 4a), however, the differences were not statistically significant. On the contrary, the pathogen load in the mesenteric lymph node (mLN) varied between both the mutants. The CFU of ΔwbaV mutant recovered from mLN was significantly (P = 0.0085) higher than Δprt mutant (Fig. 4b). Similarly, the bacterial load in spleen and liver was also found to be significantly higher in case of ΔwbaV mutant as compared to Δprt mutant (P < 0.05) (Fig. 4c, d). Notably, the ΔwbaV mutant showed reduced colonization in mLN, spleen and liver as compared to the wild-type. Further, histopathological evaluation was carried out to compare the cecal inflammation between the mutants and wild-type. The ΔwbaV mutant was found to induce moderate cecal inflammation whereas Δprt mutant was unable to induce inflammation in the C57BL/6 mice (Fig. 5a). The cecal pathology was assessed by a semi-quantitative scoring method as described in materials and methods. The day 3 p. i. experiment showed a significant difference in the cecal pathology of the mice infected with Δprt mutant (pathoscore: 8) and ΔwbaV mutant (pathoscore: 2) as compared to the wild-type (P < 0.01) (Fig. 5b). The phenotype of complemented ΔwbaV strain was similar to wild type.Fig. 4

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