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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

Schematic presentation of final steps of dideoxy sugar biosynthesis, location of genes on chromosome and LPS profile of wild-type S. Enteritidis and its isogenic mutants. a CDP-paratose is synthesized by CDP-4-keto-3,6-dideoxy-d-glucose by the action of prt which is epimerized by Tyv to tyvelose. After synthesis, tyvelose is transferred to OAg backbone of mannose (Man), rhamnose (Rha) and galactose (Gal) by WbaV. CDP-abequose is also synthesized from same CDP sugar intermediate as CDP-paratose. b Location of genes on chromosome. The regions enclosed by the black bar were deleted with the help of lambda red recombinase system. White arrow indicate that genes are located on negative strand. c LPS from wild-type Salmonella as well as the mutants were isolated using the protocol described in materials and methods and separated on polyacrylamide gel electrophoresis (PAGE) gel using tricine-SDS buffer system. LPS were visualized by silver staining. c∆ indicates the mutants are complemented with the corresponding genes
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Fig1: Schematic presentation of final steps of dideoxy sugar biosynthesis, location of genes on chromosome and LPS profile of wild-type S. Enteritidis and its isogenic mutants. a CDP-paratose is synthesized by CDP-4-keto-3,6-dideoxy-d-glucose by the action of prt which is epimerized by Tyv to tyvelose. After synthesis, tyvelose is transferred to OAg backbone of mannose (Man), rhamnose (Rha) and galactose (Gal) by WbaV. CDP-abequose is also synthesized from same CDP sugar intermediate as CDP-paratose. b Location of genes on chromosome. The regions enclosed by the black bar were deleted with the help of lambda red recombinase system. White arrow indicate that genes are located on negative strand. c LPS from wild-type Salmonella as well as the mutants were isolated using the protocol described in materials and methods and separated on polyacrylamide gel electrophoresis (PAGE) gel using tricine-SDS buffer system. LPS were visualized by silver staining. c∆ indicates the mutants are complemented with the corresponding genes

Mentions: Salmonella are Gram-negative, intracellular bacteria that cause diseases ranging from acute gastroenteritis to typhoid fever, posing a significant threat to public health globally. Infections with non typhoidal serovars of Salmonella enterica, predominantly S. enterica serovar Enteritidis (S. Enteritidis) and S. enterica serovar Typhimurium (S. Typhimurium) are more frequent and occur in both developing and industrialized nations [1]. Salmonella employs a number of virulence factors to successfully colonize and replicate inside the host. The most critical virulence determinants of Salmonella infections are Salmonella pathogenicity islands (SPI), of which SPI-1 and SPI-2 play crucial role in invasion and intracellular replication respectively. Apart from these, numerous additional virulence factors such as pilli or fimbria [2], flagella [3], lipopolysaccharide (LPS) [4] etc. are required for a successful infection. Among these factors, role of LPS in Salmonella virulence has been established by several studies [5–7]. LPS is a major structural component of the outer membrane of all the Gram-negative bacteria and plays a critical role in the bacterial pathogenesis. It consists of three distinct domains: lipid A, core oligosaccharide (OS) and O-antigen polysaccharide (OAg). The different components of LPS interact with different parts of host cells and contribute to bacterial pathogenesis. For example, different sugars of outer core interact with epithelial cells, whereas, lipid A interacts with the TLR4, a surface receptor of immune cells. While lipid A and core OS structures are fairly conserved, the OAg is highly variable, leading to serological specificity among Gram-negative bacteria. OAg is a modular assembly of oligosaccharide units that varies with respect to the sugar composition and number of their modal repeats [8]. Typically, in all the Gram-negative bacteria, this region comprises of 16 to more than 100 repeats of oligosaccharide units containing 4–6 monosaccharides each. Bacteria lacking OAg are called rough. The repeating unit of OAg of Salmonella mainly consists of three hexose sugars namely mannose, rhamnose, galactose and one dideoxy hexose as the fourth component [9]. Mannose, rhamnose and galactose form the backbone of the OAg and are conserved across different serovars of Salmonella. The dideoxy sugar linked (α-1, 3) to mannose residue varies among different serovars. Based on the agglutination by antibodies against different O-antigens, Salmonella have been grouped into six serogroups namely A, B, C1, C2, D and E. The group A (e.g. S. Typhi) contain paratose (3,6-dideoxy-d-ribo-hexose), group B (S. Typhimurium) has abequose, whereas group D (e.g. S. Enteritidis) contain tyvelose (3,6-dideoxy-d-arabino-hexose) as fourth component of OAg repeating unit [10]. A schematic representation of the biosynthetic pathway of these sugars is shown in Fig. 1a. In S. Enteritidis, enzyme paratose synthase (Prt, formerly known as RfbS), synthesizes paratose, which is further epimerized by the enzyme CDP-tyvelose-2-epimerase (Tyv, formerly known as RfbE), to tyvelose which is then transferred to OAg repeating unit by tyvelosyl transferase (WbaV, formerly known as rfbV) (Fig. 1a). The genes responsible for OAg biosynthesis are generally found on the chromosome as an OAg gene cluster and genetic variation in this cluster reflects the structural variations of OAg across Gram-negative bacteria. This gene cluster encodes proteins that can be further categorized into three groups [11]. First group involve proteins that synthesize nucleotide sugar precursors. Proteins of the second group are mainly glycosyl transferases (GTase) that build sequentially, the OAg-repeating unit on the carrier lipid, undecaprenyl phosphate (UndP). The third group of enzyme is mainly OAg processing enzyme involved in the polymerization and translocation of OAg across the membrane. The enzymes, Tyv and Prt are involved in biosynthesis of CDP-sugars whereas WbaV is a GTase. The location of the above genes on chromosome has been depicted in Fig. 1b.Fig. 1


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)

Schematic presentation of final steps of dideoxy sugar biosynthesis, location of genes on chromosome and LPS profile of wild-type S. Enteritidis and its isogenic mutants. a CDP-paratose is synthesized by CDP-4-keto-3,6-dideoxy-d-glucose by the action of prt which is epimerized by Tyv to tyvelose. After synthesis, tyvelose is transferred to OAg backbone of mannose (Man), rhamnose (Rha) and galactose (Gal) by WbaV. CDP-abequose is also synthesized from same CDP sugar intermediate as CDP-paratose. b Location of genes on chromosome. The regions enclosed by the black bar were deleted with the help of lambda red recombinase system. White arrow indicate that genes are located on negative strand. c LPS from wild-type Salmonella as well as the mutants were isolated using the protocol described in materials and methods and separated on polyacrylamide gel electrophoresis (PAGE) gel using tricine-SDS buffer system. LPS were visualized by silver staining. c∆ indicates the mutants are complemented with the corresponding genes
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schematic presentation of final steps of dideoxy sugar biosynthesis, location of genes on chromosome and LPS profile of wild-type S. Enteritidis and its isogenic mutants. a CDP-paratose is synthesized by CDP-4-keto-3,6-dideoxy-d-glucose by the action of prt which is epimerized by Tyv to tyvelose. After synthesis, tyvelose is transferred to OAg backbone of mannose (Man), rhamnose (Rha) and galactose (Gal) by WbaV. CDP-abequose is also synthesized from same CDP sugar intermediate as CDP-paratose. b Location of genes on chromosome. The regions enclosed by the black bar were deleted with the help of lambda red recombinase system. White arrow indicate that genes are located on negative strand. c LPS from wild-type Salmonella as well as the mutants were isolated using the protocol described in materials and methods and separated on polyacrylamide gel electrophoresis (PAGE) gel using tricine-SDS buffer system. LPS were visualized by silver staining. c∆ indicates the mutants are complemented with the corresponding genes
Mentions: Salmonella are Gram-negative, intracellular bacteria that cause diseases ranging from acute gastroenteritis to typhoid fever, posing a significant threat to public health globally. Infections with non typhoidal serovars of Salmonella enterica, predominantly S. enterica serovar Enteritidis (S. Enteritidis) and S. enterica serovar Typhimurium (S. Typhimurium) are more frequent and occur in both developing and industrialized nations [1]. Salmonella employs a number of virulence factors to successfully colonize and replicate inside the host. The most critical virulence determinants of Salmonella infections are Salmonella pathogenicity islands (SPI), of which SPI-1 and SPI-2 play crucial role in invasion and intracellular replication respectively. Apart from these, numerous additional virulence factors such as pilli or fimbria [2], flagella [3], lipopolysaccharide (LPS) [4] etc. are required for a successful infection. Among these factors, role of LPS in Salmonella virulence has been established by several studies [5–7]. LPS is a major structural component of the outer membrane of all the Gram-negative bacteria and plays a critical role in the bacterial pathogenesis. It consists of three distinct domains: lipid A, core oligosaccharide (OS) and O-antigen polysaccharide (OAg). The different components of LPS interact with different parts of host cells and contribute to bacterial pathogenesis. For example, different sugars of outer core interact with epithelial cells, whereas, lipid A interacts with the TLR4, a surface receptor of immune cells. While lipid A and core OS structures are fairly conserved, the OAg is highly variable, leading to serological specificity among Gram-negative bacteria. OAg is a modular assembly of oligosaccharide units that varies with respect to the sugar composition and number of their modal repeats [8]. Typically, in all the Gram-negative bacteria, this region comprises of 16 to more than 100 repeats of oligosaccharide units containing 4–6 monosaccharides each. Bacteria lacking OAg are called rough. The repeating unit of OAg of Salmonella mainly consists of three hexose sugars namely mannose, rhamnose, galactose and one dideoxy hexose as the fourth component [9]. Mannose, rhamnose and galactose form the backbone of the OAg and are conserved across different serovars of Salmonella. The dideoxy sugar linked (α-1, 3) to mannose residue varies among different serovars. Based on the agglutination by antibodies against different O-antigens, Salmonella have been grouped into six serogroups namely A, B, C1, C2, D and E. The group A (e.g. S. Typhi) contain paratose (3,6-dideoxy-d-ribo-hexose), group B (S. Typhimurium) has abequose, whereas group D (e.g. S. Enteritidis) contain tyvelose (3,6-dideoxy-d-arabino-hexose) as fourth component of OAg repeating unit [10]. A schematic representation of the biosynthetic pathway of these sugars is shown in Fig. 1a. In S. Enteritidis, enzyme paratose synthase (Prt, formerly known as RfbS), synthesizes paratose, which is further epimerized by the enzyme CDP-tyvelose-2-epimerase (Tyv, formerly known as RfbE), to tyvelose which is then transferred to OAg repeating unit by tyvelosyl transferase (WbaV, formerly known as rfbV) (Fig. 1a). The genes responsible for OAg biosynthesis are generally found on the chromosome as an OAg gene cluster and genetic variation in this cluster reflects the structural variations of OAg across Gram-negative bacteria. This gene cluster encodes proteins that can be further categorized into three groups [11]. First group involve proteins that synthesize nucleotide sugar precursors. Proteins of the second group are mainly glycosyl transferases (GTase) that build sequentially, the OAg-repeating unit on the carrier lipid, undecaprenyl phosphate (UndP). The third group of enzyme is mainly OAg processing enzyme involved in the polymerization and translocation of OAg across the membrane. The enzymes, Tyv and Prt are involved in biosynthesis of CDP-sugars whereas WbaV is a GTase. The location of the above genes on chromosome has been depicted in Fig. 1b.Fig. 1

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