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Genetic and Functional Diversity of Pseudomonas aeruginosa Lipopolysaccharide.

Lam JS, Taylor VL, Islam ST, Hao Y, Kocíncová D - Front Microbiol (2011)

Bottom Line: Most P. aeruginosa strains produce two distinct forms of O-Ag, one a homopolymer of D-rhamnose that is a common polysaccharide antigen (CPA, formerly termed A band), and the other a heteropolymer of three to five distinct (and often unique dideoxy) sugars in its repeat units, known as O-specific antigen (OSA, formerly termed B band).Compositional differences in the O units among the OSA from different strains form the basis of the International Antigenic Typing Scheme for classification via serotyping of different strains of P. aeruginosa.The focus of this review is to provide state-of-the-art knowledge on the genetic and resultant functional diversity of LPS produced by P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph Guelph, ON, Canada.

ABSTRACT
Lipopolysccharide (LPS) is an integral component of the Pseudomonas aeruginosa cell envelope, occupying the outer leaflet of the outer membrane in this Gram-negative opportunistic pathogen. It is important for bacterium-host interactions and has been shown to be a major virulence factor for this organism. Structurally, P. aeruginosa LPS is composed of three domains, namely, lipid A, core oligosaccharide, and the distal O antigen (O-Ag). Most P. aeruginosa strains produce two distinct forms of O-Ag, one a homopolymer of D-rhamnose that is a common polysaccharide antigen (CPA, formerly termed A band), and the other a heteropolymer of three to five distinct (and often unique dideoxy) sugars in its repeat units, known as O-specific antigen (OSA, formerly termed B band). Compositional differences in the O units among the OSA from different strains form the basis of the International Antigenic Typing Scheme for classification via serotyping of different strains of P. aeruginosa. The focus of this review is to provide state-of-the-art knowledge on the genetic and resultant functional diversity of LPS produced by P. aeruginosa. The underlying factors contributing to this diversity will be thoroughly discussed and presented in the context of its contributions to host-pathogen interactions and the control/prevention of infection.

No MeSH data available.


Related in: MedlinePlus

Organization of the genes within OSA biosynthesis clusters. (A) OSA biosynthesis cluster of serotype O5 adapted from Burrows et al. (1996). The gene cluster is located on the complementary strand; genes which match the PFAM designation are colored accordingly. Genes not involved in OSA biosynthesis are depicted in gray including a large insertion sequence (IS). (B) Adapted from Raymond et al. (2002), the OSA biosynthesis gene clusters were organized into 11 groups based on sequence conservation. Genes were designated using the PFAM database; specific protein families which occur a minimum of three times throughout all 20 OSA biosynthesis clusters are represented by a specific color. A red outline depicts an ORF with potential transmembrane-spanning domains. Previously identified genes are labeled above the respected cluster if present within the serotype. Insertion sequences (IS) present within genes are depicted by a secondary gray box.
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Figure 2: Organization of the genes within OSA biosynthesis clusters. (A) OSA biosynthesis cluster of serotype O5 adapted from Burrows et al. (1996). The gene cluster is located on the complementary strand; genes which match the PFAM designation are colored accordingly. Genes not involved in OSA biosynthesis are depicted in gray including a large insertion sequence (IS). (B) Adapted from Raymond et al. (2002), the OSA biosynthesis gene clusters were organized into 11 groups based on sequence conservation. Genes were designated using the PFAM database; specific protein families which occur a minimum of three times throughout all 20 OSA biosynthesis clusters are represented by a specific color. A red outline depicts an ORF with potential transmembrane-spanning domains. Previously identified genes are labeled above the respected cluster if present within the serotype. Insertion sequences (IS) present within genes are depicted by a secondary gray box.

Mentions: The importance of the OSA toward virulence and its use in serotyping has made it an attractive target for genetic studies to help understand both the plasticity of this region of the LPS and the complex steps of OSA biosynthesis. OSA biosynthesis follows the Wzy-dependent pathway model, originally proposed by Whitfield (1995); it involves the sequential activities of a series of integral inner membrane (IM) proteins, for which we have recently obtained comprehensive topological data helping to explain their respective functions (Islam et al., 2010). In this model, OSA sugar repeats are sequentially built on the lipid carrier undecaprenyl pyrophosphate (UndPP) on the cytoplasmic face of the IM. The UndPP-linked OSA repeats are then translocated to the periplasmic face of the IM by the flippase Wzx (Burrows and Lam, 1999), where they are polymerized by Wzy (De Kievit et al., 1995) through a putative catch-and-release mechanism (Islam et al., 2011), to modal lengths regulated by Wzz1 (Burrows et al., 1997) and Wzz2 (Daniels et al., 2002). The modal length of OSA imparted by Wzz2 (40–50 repeat length) is longer than that imparted by Wzz1 (12–16 repeats and 22–30 repeats; Daniels et al., 2002). However, Wzz1 is apparently more important for virulence than Wzz2 (Kintz et al., 2008). Finally, the complete OSA chain is ligated to lipid A-core by the O-Ag ligase WaaL (Abeyrathne et al., 2005; Abeyrathne and Lam, 2007). Preliminary investigations mapped the OSA cluster to 37 min of the P. aeruginosa PAO1 (serotype O5) genome (Lightfoot and Lam, 1993), corresponding to pa3141 to pa3160 in the annotated genome of strain PAO1 (Stover et al., 2000). This first reported LPS OSA cluster was isolated from a cosmid-based genomic library. Clone pFV100 from the library was able to complement mutant ge6, a Tn5–751 insertional mutant of PAO1, defective in B band (OSA) biosynthesis (Lightfoot and Lam, 1993). Subsequently, Burrows et al. (1996) obtained the sequence of the entire OSA cluster. To characterize the function of the genes encoded in this cluster, knockout mutant constructs were generated for each of the genes and the mutants were examined for their effect on LPS production in P. aeruginosa. The list of OSA biosynthesis genes in this serotype O5 cluster and their functions were determined based on genetic studies as well as biochemical and chemical evidence (Figure 2A; Table 2, Burrows et al., 1996). Following the success in characterizing the O5 OSA biosynthesis locus, the sequences of the O6 (Belanger et al., 1999) and O11 (Dean et al., 1999) OSA loci were also determined. Comparisons among the newly sequenced loci revealed that all three OSA loci were flanked by himD/ihfB (pa3161) on the 5′ end and terminated with wbpM (pa3141) on the 3′ end. These observations are essential for establishing the conserved chromosomal locus for the OSA cluster, though the genes within the locus are the most varied in the P. aeruginosa genomes regardless of serotype. This information eventually allowed Raymond et al. (2002) to clone and sequence the OSA loci from all 20 IATS serotypes. Based on their sequencing data, the general genetic structures of the OSA loci of all the serotypes could be divided into 11 distinct groups based on the protein families that the genes in these loci encode, as well as the presence of insertion sequences (IS) and deletions (Figure 2B; Raymond et al., 2002). This group has also presented the sequences of a set of primers for PCR amplification of each of the IATS serotypes, meaning that a PCR-based approach can be used to correctly type clinical isolates that have previously been evaluated as NT by any typing antisera. However, thus far, there has been no systematic study conducted by any group to test the capability of using these primers for consistently typing clinical strains of P. aeruginosa even though the potential to do so exists. These initial genetic investigations helped to reveal differences among the OSA clusters; worth noting is the anomaly discovered regarding the entire loss of the OSA cluster in the O15 serotype. Strains that belong to this serotype were previously identified using both polyclonal antibody typing kits and mAb-based serotyping (Lam et al., 1987b). It was proposed by Raymond et al. (2002) that in serotype O15, the genes involved in its OSA biosynthesis may not necessarily be residing in the usual OSA locus as in other serotypes. Additionally, in serotype O6, the wzy gene does not reside in the OSA cluster (Belanger et al., 1999). Further, in serotype O5, the transcriptional start site for wzx exists within the wzy gene, and there is a large IS at the 3′ end of the O5 cluster upstream of wbpM (Burrows et al., 1996). This variation helps to explain the diversity of the LPS in P. aeruginosa as a property of the genetic differences among the IATS serotypes. Other factors that influence the OSA diversity are outlined in the following sections.


Genetic and Functional Diversity of Pseudomonas aeruginosa Lipopolysaccharide.

Lam JS, Taylor VL, Islam ST, Hao Y, Kocíncová D - Front Microbiol (2011)

Organization of the genes within OSA biosynthesis clusters. (A) OSA biosynthesis cluster of serotype O5 adapted from Burrows et al. (1996). The gene cluster is located on the complementary strand; genes which match the PFAM designation are colored accordingly. Genes not involved in OSA biosynthesis are depicted in gray including a large insertion sequence (IS). (B) Adapted from Raymond et al. (2002), the OSA biosynthesis gene clusters were organized into 11 groups based on sequence conservation. Genes were designated using the PFAM database; specific protein families which occur a minimum of three times throughout all 20 OSA biosynthesis clusters are represented by a specific color. A red outline depicts an ORF with potential transmembrane-spanning domains. Previously identified genes are labeled above the respected cluster if present within the serotype. Insertion sequences (IS) present within genes are depicted by a secondary gray box.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108286&req=5

Figure 2: Organization of the genes within OSA biosynthesis clusters. (A) OSA biosynthesis cluster of serotype O5 adapted from Burrows et al. (1996). The gene cluster is located on the complementary strand; genes which match the PFAM designation are colored accordingly. Genes not involved in OSA biosynthesis are depicted in gray including a large insertion sequence (IS). (B) Adapted from Raymond et al. (2002), the OSA biosynthesis gene clusters were organized into 11 groups based on sequence conservation. Genes were designated using the PFAM database; specific protein families which occur a minimum of three times throughout all 20 OSA biosynthesis clusters are represented by a specific color. A red outline depicts an ORF with potential transmembrane-spanning domains. Previously identified genes are labeled above the respected cluster if present within the serotype. Insertion sequences (IS) present within genes are depicted by a secondary gray box.
Mentions: The importance of the OSA toward virulence and its use in serotyping has made it an attractive target for genetic studies to help understand both the plasticity of this region of the LPS and the complex steps of OSA biosynthesis. OSA biosynthesis follows the Wzy-dependent pathway model, originally proposed by Whitfield (1995); it involves the sequential activities of a series of integral inner membrane (IM) proteins, for which we have recently obtained comprehensive topological data helping to explain their respective functions (Islam et al., 2010). In this model, OSA sugar repeats are sequentially built on the lipid carrier undecaprenyl pyrophosphate (UndPP) on the cytoplasmic face of the IM. The UndPP-linked OSA repeats are then translocated to the periplasmic face of the IM by the flippase Wzx (Burrows and Lam, 1999), where they are polymerized by Wzy (De Kievit et al., 1995) through a putative catch-and-release mechanism (Islam et al., 2011), to modal lengths regulated by Wzz1 (Burrows et al., 1997) and Wzz2 (Daniels et al., 2002). The modal length of OSA imparted by Wzz2 (40–50 repeat length) is longer than that imparted by Wzz1 (12–16 repeats and 22–30 repeats; Daniels et al., 2002). However, Wzz1 is apparently more important for virulence than Wzz2 (Kintz et al., 2008). Finally, the complete OSA chain is ligated to lipid A-core by the O-Ag ligase WaaL (Abeyrathne et al., 2005; Abeyrathne and Lam, 2007). Preliminary investigations mapped the OSA cluster to 37 min of the P. aeruginosa PAO1 (serotype O5) genome (Lightfoot and Lam, 1993), corresponding to pa3141 to pa3160 in the annotated genome of strain PAO1 (Stover et al., 2000). This first reported LPS OSA cluster was isolated from a cosmid-based genomic library. Clone pFV100 from the library was able to complement mutant ge6, a Tn5–751 insertional mutant of PAO1, defective in B band (OSA) biosynthesis (Lightfoot and Lam, 1993). Subsequently, Burrows et al. (1996) obtained the sequence of the entire OSA cluster. To characterize the function of the genes encoded in this cluster, knockout mutant constructs were generated for each of the genes and the mutants were examined for their effect on LPS production in P. aeruginosa. The list of OSA biosynthesis genes in this serotype O5 cluster and their functions were determined based on genetic studies as well as biochemical and chemical evidence (Figure 2A; Table 2, Burrows et al., 1996). Following the success in characterizing the O5 OSA biosynthesis locus, the sequences of the O6 (Belanger et al., 1999) and O11 (Dean et al., 1999) OSA loci were also determined. Comparisons among the newly sequenced loci revealed that all three OSA loci were flanked by himD/ihfB (pa3161) on the 5′ end and terminated with wbpM (pa3141) on the 3′ end. These observations are essential for establishing the conserved chromosomal locus for the OSA cluster, though the genes within the locus are the most varied in the P. aeruginosa genomes regardless of serotype. This information eventually allowed Raymond et al. (2002) to clone and sequence the OSA loci from all 20 IATS serotypes. Based on their sequencing data, the general genetic structures of the OSA loci of all the serotypes could be divided into 11 distinct groups based on the protein families that the genes in these loci encode, as well as the presence of insertion sequences (IS) and deletions (Figure 2B; Raymond et al., 2002). This group has also presented the sequences of a set of primers for PCR amplification of each of the IATS serotypes, meaning that a PCR-based approach can be used to correctly type clinical isolates that have previously been evaluated as NT by any typing antisera. However, thus far, there has been no systematic study conducted by any group to test the capability of using these primers for consistently typing clinical strains of P. aeruginosa even though the potential to do so exists. These initial genetic investigations helped to reveal differences among the OSA clusters; worth noting is the anomaly discovered regarding the entire loss of the OSA cluster in the O15 serotype. Strains that belong to this serotype were previously identified using both polyclonal antibody typing kits and mAb-based serotyping (Lam et al., 1987b). It was proposed by Raymond et al. (2002) that in serotype O15, the genes involved in its OSA biosynthesis may not necessarily be residing in the usual OSA locus as in other serotypes. Additionally, in serotype O6, the wzy gene does not reside in the OSA cluster (Belanger et al., 1999). Further, in serotype O5, the transcriptional start site for wzx exists within the wzy gene, and there is a large IS at the 3′ end of the O5 cluster upstream of wbpM (Burrows et al., 1996). This variation helps to explain the diversity of the LPS in P. aeruginosa as a property of the genetic differences among the IATS serotypes. Other factors that influence the OSA diversity are outlined in the following sections.

Bottom Line: Most P. aeruginosa strains produce two distinct forms of O-Ag, one a homopolymer of D-rhamnose that is a common polysaccharide antigen (CPA, formerly termed A band), and the other a heteropolymer of three to five distinct (and often unique dideoxy) sugars in its repeat units, known as O-specific antigen (OSA, formerly termed B band).Compositional differences in the O units among the OSA from different strains form the basis of the International Antigenic Typing Scheme for classification via serotyping of different strains of P. aeruginosa.The focus of this review is to provide state-of-the-art knowledge on the genetic and resultant functional diversity of LPS produced by P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph Guelph, ON, Canada.

ABSTRACT
Lipopolysccharide (LPS) is an integral component of the Pseudomonas aeruginosa cell envelope, occupying the outer leaflet of the outer membrane in this Gram-negative opportunistic pathogen. It is important for bacterium-host interactions and has been shown to be a major virulence factor for this organism. Structurally, P. aeruginosa LPS is composed of three domains, namely, lipid A, core oligosaccharide, and the distal O antigen (O-Ag). Most P. aeruginosa strains produce two distinct forms of O-Ag, one a homopolymer of D-rhamnose that is a common polysaccharide antigen (CPA, formerly termed A band), and the other a heteropolymer of three to five distinct (and often unique dideoxy) sugars in its repeat units, known as O-specific antigen (OSA, formerly termed B band). Compositional differences in the O units among the OSA from different strains form the basis of the International Antigenic Typing Scheme for classification via serotyping of different strains of P. aeruginosa. The focus of this review is to provide state-of-the-art knowledge on the genetic and resultant functional diversity of LPS produced by P. aeruginosa. The underlying factors contributing to this diversity will be thoroughly discussed and presented in the context of its contributions to host-pathogen interactions and the control/prevention of infection.

No MeSH data available.


Related in: MedlinePlus