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![Representation of the Wzx/Wzy-Dependent Pathway for Biosynthesis of CPS 9A Pictured is a hypothetical model for capsule biosynthesis in S. pneumoniae based on a mixture of experimental evidence and speculation. For a recent review, see Yother [15]. (1) Non-housekeeping nucleotide sugar biosynthesis. (2) The initial transferase (WchA in this case) links the initial sugar as a sugar phosphate (Glc-P) to a membrane-associated lipid carrier (widely assumed to be undecaprenyl phosphate). (3) Glycosyl transferases sequentially link further sugars to generate repeat unit. (4) Wzx flippase transports the repeat unit across the cytoplasmic membrane. (5) Wzy polymerase links individual repeat units to form lipid-linked CPS. (6) Wzd/Wze complex translocates mature CPS to the cell surface and may be responsible for the attachment to peptidoglycan. The complex of WchA, Wzy, Wzx, Wzd, and Wze shown in the membrane is based on that in Figure 2 of Whitfield and Paiment [47] for the related Escherichia coli Type 1 capsule. Found at DOI: 10.1371/journal.pgen.0020031.g001](profile/Angeliki-Mavroidi-2/publication/7246568/figure/fig1/AS:281213664284675@1444057970589/Representation-of-the-Wzx-Wzy-Dependent-Pathway-for-Biosynthesis-of-CPS-9A-Pictured-is-a.png)
Representation of the Wzx/Wzy-Dependent Pathway for Biosynthesis of CPS 9A Pictured is a hypothetical model for capsule biosynthesis in S. pneumoniae based on a mixture of experimental evidence and speculation. For a recent review, see Yother [15]. (1) Non-housekeeping nucleotide sugar biosynthesis. (2) The initial transferase (WchA in this case) links the initial sugar as a sugar phosphate (Glc-P) to a membrane-associated lipid carrier (widely assumed to be undecaprenyl phosphate). (3) Glycosyl transferases sequentially link further sugars to generate repeat unit. (4) Wzx flippase transports the repeat unit across the cytoplasmic membrane. (5) Wzy polymerase links individual repeat units to form lipid-linked CPS. (6) Wzd/Wze complex translocates mature CPS to the cell surface and may be responsible for the attachment to peptidoglycan. The complex of WchA, Wzy, Wzx, Wzd, and Wze shown in the membrane is based on that in Figure 2 of Whitfield and Paiment [47] for the related Escherichia coli Type 1 capsule. Found at DOI: 10.1371/journal.pgen.0020031.g001
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Synopsis
Several major bacterial pathogens are coated by a polysaccharide capsule that is important for virulence. Each strain of Streptococcus pneumoniae (the pneumococcus) produces one of 90 different capsular polysaccharides, which are distinguished by using a set of antisera that recognise the chemical differences in the capsules. The capsule i...
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... pneumoniae (the pneumococcus) is a major cause of morbidity and mortality worldwide, causing diseases that range in severity from meningitis, septicaemia, and pneumonia to sinusitis and acute otitis media [1,2]. Factor (typing) sera are used to divide pneumococci into serotypes and serogroups, which include immunologically related serotypes. These sera have been developed by a process of multiple cross-absorptions, which render them specific for the immunochemical differences between the pneumococcal capsular polysaccharides (CPSs) [3]. At present, 90 individual serotypes are recognised by their patterns of reactivity with the factor sera [4], and serotypes vary in the extent to which they are carried in the nasopharynx and the degree to which they are recovered from different disease states [5,6]. Expression of a capsule is important for survival in the blood and is strongly associated with the ability of pneumococci to cause invasive disease. The capsule is surface exposed, and antibodies against CPS provide protection against pneumococcal disease. Consequently, polyvalent polysaccharide vaccines have been developed in which CPS from the serotypes most commonly associated with invasive disease in children are linked to a protein carrier, and a seven-valent conjugated polysaccharide vaccine has been introduced and shown to be highly effective [7,8]. A 23-valent polysaccharide vaccine is also available for use in adults [9]. With the exception of types 3 and 37, which are synthesised by the synthase pathway [10–14], pneumococcal CPSs are generally synthesised by the Wzx/Wzy-dependent pathway ( Figure 1). The genes for the latter pathway are located at the same chromosomal locus (cps), between dexB and aliA [15–17]. CPSs are synthesised by transfer of an initial monosaccharide phosphate from a nucleotide diphosphate sugar to a membrane-associated lipid carrier, followed by the sequential transfer of further monosaccharides to produce the lipid- linked repeat unit. This is transferred to the outer face of the cytoplasmic membrane by the repeat-unit transporter or flippase, polymerised to form the mature CPS, and then attached to the peptidoglycan [18]. The cps locus therefore typically encodes the enzymes to build the repeat unit, including an initial glycosyl phosphate transferase, and additional transferases responsible for the formation of the linkages, and to allow for the addition of sugars (or other moieties), or to otherwise modify the repeat unit, as well as a repeat-unit flippase and polymerase [15]. The substantial diversity of pneumococcal CPSs is believed to have arisen as a consequence of selection for antigenic diversity imposed by the human immune system [6]. The evolutionary timescales and the genetic events by which novel serogroups and serotypes arise are unclear. Comparisons of the available cps loci indicate a variety of genetic mechanisms and show that the central genes responsible for the synthesis and polymerisation of the repeat unit are highly variable and often non-homologous between serotypes. These genes have a low percentage G C content, and new serotypes may frequently have been generated by the introduction of novel cps genes into pneumococci by lateral gene transfer from other species. A much better understanding of the complex mechanisms by which antigenic diversity arises could be obtained by using the sequences of the complete set of pneumococcal cps loci. We therefore obtained sequences of the cps locus for all 90 serotypes and used these data, together with the available polysaccharide structures and the patterns of serological reactions with typing sera, to explore the genetics of capsular diversity in this major pathogen. Here we present highlights of our analysis to date, and a more exhaustive analysis will be reported elsewhere. PCR products were generated from genomic DNA using primers specific for the dexB and aliA genes and ranged in size from 10,337 bp (serotype 3) to 30,298 bp (serotype 38) with an average of 20,714 bp. The synthase gene (wchE) of serotype 3 is located within the cps locus, but the type 37 cps locus, which was very similar to that of serotype 33F, is defective and serotype is determined by the type 37 synthase gene (tts) located elsewhere on the chromosome [10]. Annotation and analysis of the cps sequences revealed the generality of several previously observed characteristics. Genes for the generation of CPSs are always orientated in the same direction as the dexB and aliA genes (Figures 2 and S1). The regulatory and processing genes wzg, wzh, wzd, and wze (also known as cpsABCD ) are conserved with high sequence identity in all cases and are almost always in this gene order at the 5 9 end of the cps locus. In most cps clusters, the fifth gene encodes the initial glucose phosphate transferase, WchA (also known as CpsE), responsible for linkage of an activated glucose phosphate to the lipid carrier (see below). The polysaccharide polymerase (wzy) and flippase (wzx) genes are always present downstream together with a varying set of genes for glycosyl transferases, acetyl transferases, nucleotide diphosphate sugar biosynthesis, and modifying enzymes. In every case, there is a region of low percentage G þ C content within the cps locus. The first four genes and the non- housekeeping sugar biosynthesis genes have typical percentage G þ C content for S. pneumoniae, while the ‘‘ serotype- specific ’’ genes, particularly wzy and wzx, tend to have more AT-rich sequences. In the regions between the cps genes and the flanking dexB and aliA genes, there is almost always evidence of mobile genetic elements. This is largely man- ifested as intact or disrupted genes for insertion-sequence (IS) transposases [19,20], although in four cases we identified group-II introns [21] (serotypes 19F, 25F, 25A, and 38). We could assign a functional designation to the products of all but 26 of the 1,999 predicted coding sequences in the 90 cps regions, with most of the remainder showing weak similarities to products of genes in bacterial polysaccharide gene clusters. Unsurprisingly, many coding sequences fall into the broad functional categories of glycosyl transferase (351), acetyl transferase (74), and sugar phosphate transferase (71). To make more specific assignments within such categories, we used the TribeMCL program to assemble all the annotated proteins into homology groups (HGs). With from two to 90 members in each, 91% of the proteins assembled into 175 HGs, with the remainder forming 74 single-member HGs (Table S1). The products of wzg, wzh, wzd, and wze each fall into a single HG covering every serotype. Ignoring IS element transposases, the next largest HG comprises 65 WchA initial transferases (HG5). At the other extreme, the serotype-specific gene products are diverse, with 87 HGs for non-initial sugar transferases and 40 and 13 groups of Wzy repeat-unit polymerases and Wzx flippases, respectively. Of the 18 sugars and related compounds found in S. pneumoniae capsules, seven are available from housekeeping metabolic pathways and nine from known dedicated pathways encoded within the cps cluster (Figure S2). This includes 4- keto- N -acetyl-D-quinovosamine (UDP-KDQNAc), which is the intermediate in the two step reaction catalysed by FnlA [22]. We found a perfect correlation between the presence of a non- housekeeping sugar in the CPS and the presence of the appropriate biosynthetic genes in the cps locus. Two of the three remaining compounds are the sugar alcohol phosphates arabinitol-1-P and mannitol-6-P. The precursors for these have not been identified, but nucleotide-diphosphate-linked precursors can be easily derived from D-xylulose-5-phosphate or D-fructose-6-phosphate, respectively, by two-step pathways parallel to that for CDP-ribitol formation from ribulose-5- phosphate [23]. D-xylulose-5-phosphate and D-fructose-6- phosphate are central to major pathways, and there are appropriate genes for their conversion in the associated cps loci. The precursor for ribofuranose has also not been identified, but a proposed pathway for its biosynthesis by the product of a gene within CPS 19F (cps19R) [24] is supported by our observation that an orthologous gene (renamed rbsF ) is present for all CPS that contain ribofuranose. Choline-1-phosphate, glycerol-1-phosphate, and glycerol-2- phosphate are also found in some of the structures. CDP- choline is known to be produced by S. pneumoniae as a precursor for teichoic acid biosynthesis [25]. For glycerol-1- phosphate, we find an intact gct gene for CDP-glycerol synthesis [26] in the cps where expected, and there are four genes associated with presence of glycerol-2-phosphate, ...
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... which are thought to encode a CDP-2-glycerol pathway [27], while wchX encodes the glycerol phosphotransferase. The situation is illustrated in Figure 1 for cps 9A, which has pathway genes for N -acetylmannosamine pyranose and glucuronic acid, but not for glucopyranose (Glc p ) or galactopyranose (Gal p ) as these are available in S. pneumoniae from central metabolism. Initial transferase WchA adds glucose-1-phosphate to undecaprenol phosphate [28] to create Und-PP-Glc (Figure 1), and we assume it performs that function in all 65 serotypes where it is present. For the known structures, there is a perfect correlation between the presence/absence of wchA and the presence/absence of glucose in the repeating unit. Where wchA is absent, the products of the fifth cps gene fall into three HGs (WciI, WcjG, and WcjH) all with the same Pfam [29] domain and similar hydrophobicity profiles to the carboxy-terminal region of WchA. We suggest that they function as the initial sugar transferases, as it is known that for the Salmonella enterica wchA homologue, wbaP, the 3 9 end of the gene is sufficient for transferase activity [30]. By correlation with CPS constituents, we predict the transferred initial sugars as N -acetylgalactosamine pyranose (Gal p NAc) or N -acetylglucosamine pyranose (Glc p NAc) for WciI, Gal p or galactofuranose for WcjG and Gal p for WcjH. Serotype 1 is an exception as no gene product with similarity to an initial sugar transferase has yet been identified. The initial sugar of the repeat unit is also the donor sugar in the polymerisation of the repeat units (Figure 1), and the specificity of the Wzy polymerase determines the other component of this linkage, which in the case of CPS 9A is a beta (1–4) linkage to the terminal glucose of the next repeat unit. For the known structures [31], identification of the initial sugar allowed us to determine the polymerase linkage as both donor and acceptor sugar, and the linkages were defined once the initial sugar had been identified (see Figures 2 and S1). Where there is ambiguity due to two residues of the initial sugar in the repeat unit, the polymerase linkage can be provisionally identified by considering the linkage catalysed by other members of the same Wzy HG. The predictions for initial sugars, and subsequent repeat-unit polymerisation linkage, correlate well with the polymerase HGs (Table S2). There are 32 polymerase HGs associated with WchA, five with WciI, four with WcjG and one with WcjH. These associations are mostly exclusive, with only five polymerase HGs associated with two initial transferases. In such cases, the linkages involve the same acceptor sugar anomerism ( a or b isomer) and the same or a closely related donor sugar. This adds strong support to the inferences drawn for the specificity of the initial transferases. The availability of all of the annotated cps sequences allowed us to look for correlations between genes, known CPS structures, and serology (gene clusters, CPS structures, and antigenic formulae are summarised in Figure S1 and Table S3). In this way, we can attempt both to infer gene function and, by comparing related cps loci, to account for differences in CPS structure and serology. Variations between cps loci range from two base substitutions for 18B and 18C to wholesale differences in gene complement. Within this range, the variations likely to have a phenotypic effect include gene inactivation due to single base substitutions generating a premature stop codon, single base insertion/deletions leading to translational frameshifts, change of sequence leading to change of enzyme specificity, recombination or IS element insertion leading to gene truncation, and insertion/deletion/replacement of single and multiple genes. Within serogroups, the genetic differences were often subtle but were also sometimes surprisingly prominent. Comparisons also revealed some strong common- ality between the cps of different serogroups and serotypes. Illustrative examples that demonstrate how structure, genetics, and serology were combined to analyse the cps loci are shown in Figure 2 and are discussed below. Previously described CPS structures [31] for all four serotypes of serogroup 9 show only subtle differences and provide an example of multiple serotypes arising by divergence from a single cps locus. Their cps genes fall into two pairs, with 9A highly similar to 9V [32], and 9L highly similar to 9N, but with the two pairs differing significantly in sequence ( Figure 2), suggesting an initial divergence to form two ancestral serotypes; this split correlates with a difference at residue 5 of the repeat unit, where 9L and 9N CPSs have Glc p NAc, whereas 9A and 9V have Glc p . Factor sera 9d reacts with 9A and 9V but not with 9L and 9N, suggesting that it is interacting with Glc p but not with Glc p NAc. Both are housekeeping sugars, and their differential incorporation is likely to be due to divergent forms of glycosyl transferase WcjC. Subsequently, one of these ancestral serotypes diverged to form 9L and 9N, the latter becoming unique in the group in having Glc p rather than Gal p as residue 3 in the repeat unit. Their dexB–aliA loci have the same gene complement, and within the cps genes there are only 79 nucleotide differences. The highest number of amino acid substitutions (13) is within glycosyl transferase WcjA; ten are unique to 9N and presumably result in its altered specificity for Glc p ...
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... which are thought to encode a CDP-2-glycerol pathway [27], while wchX encodes the glycerol phosphotransferase. The situation is illustrated in Figure 1 for cps 9A, which has pathway genes for N -acetylmannosamine pyranose and glucuronic acid, but not for glucopyranose (Glc p ) or galactopyranose (Gal p ) as these are available in S. pneumoniae from central metabolism. Initial transferase WchA adds glucose-1-phosphate to undecaprenol phosphate [28] to create Und-PP-Glc (Figure 1), and we assume it performs that function in all 65 serotypes where it is present. For the known structures, there is a perfect correlation between the presence/absence of wchA and the presence/absence of glucose in the repeating unit. Where wchA is absent, the products of the fifth cps gene fall into three HGs (WciI, WcjG, and WcjH) all with the same Pfam [29] domain and similar hydrophobicity profiles to the carboxy-terminal region of WchA. We suggest that they function as the initial sugar transferases, as it is known that for the Salmonella enterica wchA homologue, wbaP, the 3 9 end of the gene is sufficient for transferase activity [30]. By correlation with CPS constituents, we predict the transferred initial sugars as N -acetylgalactosamine pyranose (Gal p NAc) or N -acetylglucosamine pyranose (Glc p NAc) for WciI, Gal p or galactofuranose for WcjG and Gal p for WcjH. Serotype 1 is an exception as no gene product with similarity to an initial sugar transferase has yet been identified. The initial sugar of the repeat unit is also the donor sugar in the polymerisation of the repeat units (Figure 1), and the specificity of the Wzy polymerase determines the other component of this linkage, which in the case of CPS 9A is a beta (1–4) linkage to the terminal glucose of the next repeat unit. For the known structures [31], identification of the initial sugar allowed us to determine the polymerase linkage as both donor and acceptor sugar, and the linkages were defined once the initial sugar had been identified (see Figures 2 and S1). Where there is ambiguity due to two residues of the initial sugar in the repeat unit, the polymerase linkage can be provisionally identified by considering the linkage catalysed by other members of the same Wzy HG. The predictions for initial sugars, and subsequent repeat-unit polymerisation linkage, correlate well with the polymerase HGs (Table S2). There are 32 polymerase HGs associated with WchA, five with WciI, four with WcjG and one with WcjH. These associations are mostly exclusive, with only five polymerase HGs associated with two initial transferases. In such cases, the linkages involve the same acceptor sugar anomerism ( a or b isomer) and the same or a closely related donor sugar. This adds strong support to the inferences drawn for the specificity of the initial transferases. The availability of all of the annotated cps sequences allowed us to look for correlations between genes, known CPS structures, and serology (gene clusters, CPS structures, and antigenic formulae are summarised in Figure S1 and Table S3). In this way, we can attempt both to infer gene function and, by comparing related cps loci, to account for differences in CPS structure and serology. Variations between cps loci range from two base substitutions for 18B and 18C to wholesale differences in gene complement. Within this range, the variations likely to have a phenotypic effect include gene inactivation due to single base substitutions generating a premature stop codon, single base insertion/deletions leading to translational frameshifts, change of sequence leading to change of enzyme specificity, recombination or IS element insertion leading to gene truncation, and insertion/deletion/replacement of single and multiple genes. Within serogroups, the genetic differences were often subtle but were also sometimes surprisingly prominent. Comparisons also revealed some strong common- ality between the cps of different serogroups and serotypes. Illustrative examples that demonstrate how structure, genetics, and serology were combined to analyse the cps loci are shown in Figure 2 and are discussed below. Previously described CPS structures [31] for all four serotypes of serogroup 9 show only subtle differences and provide an example of multiple serotypes arising by divergence from a single cps locus. Their cps genes fall into two pairs, with 9A highly similar to 9V [32], and 9L highly similar to 9N, but with the two pairs differing significantly in sequence ( Figure 2), suggesting an initial divergence to form two ancestral serotypes; this split correlates with a difference at residue 5 of the repeat unit, where 9L and 9N CPSs have Glc p NAc, whereas 9A and 9V have Glc p . Factor sera 9d reacts with 9A and 9V but not with 9L and 9N, suggesting that it is interacting with Glc p but not with Glc p NAc. Both are housekeeping sugars, and their differential incorporation is likely to be due to divergent forms of glycosyl transferase WcjC. Subsequently, one of these ancestral serotypes diverged to form 9L and 9N, the latter becoming unique in the group in having Glc p rather ...
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... which are thought to encode a CDP-2-glycerol pathway [27], while wchX encodes the glycerol phosphotransferase. The situation is illustrated in Figure 1 for cps 9A, which has pathway genes for N -acetylmannosamine pyranose and glucuronic acid, but not for glucopyranose (Glc p ) or galactopyranose (Gal p ) as these are available in S. pneumoniae from central metabolism. Initial transferase WchA adds glucose-1-phosphate to undecaprenol phosphate [28] to create Und-PP-Glc (Figure 1), and we assume it performs that function in all 65 serotypes where it is present. For the known structures, there is a perfect correlation between the presence/absence of wchA and the presence/absence of glucose in the repeating unit. Where wchA is absent, the products of the fifth cps gene fall into three HGs (WciI, WcjG, and WcjH) all with the same Pfam [29] domain and similar hydrophobicity profiles to the carboxy-terminal region of WchA. We suggest that they function as the initial sugar transferases, as it is known that for the Salmonella enterica wchA homologue, wbaP, the 3 9 end of the gene is sufficient for transferase activity [30]. By correlation with CPS constituents, we predict the transferred initial sugars as N -acetylgalactosamine pyranose (Gal p NAc) or N -acetylglucosamine pyranose (Glc p NAc) for WciI, Gal p or galactofuranose for WcjG and Gal p for WcjH. Serotype 1 is an exception as no gene product with similarity to an initial sugar transferase has yet been identified. The initial sugar of the repeat unit is also the donor sugar in the polymerisation of the repeat units (Figure 1), and the specificity of the Wzy polymerase determines the other component of this linkage, which in the case of CPS 9A is a beta (1–4) linkage to the terminal glucose of the next repeat unit. For the known structures [31], identification of the initial sugar allowed us to determine the polymerase linkage as both donor and acceptor sugar, and the linkages were defined once the initial sugar had been identified (see Figures 2 and S1). Where there is ambiguity due to two residues of the initial sugar in the repeat unit, the polymerase linkage can be provisionally identified by considering the linkage catalysed by other members of the same Wzy HG. The predictions for initial sugars, and subsequent repeat-unit polymerisation linkage, correlate well with the polymerase HGs (Table S2). There are 32 polymerase HGs associated with WchA, five with WciI, four with WcjG and one with WcjH. These associations are mostly exclusive, with only five polymerase HGs associated with two initial transferases. In such cases, the linkages involve the same acceptor sugar anomerism ( a or b isomer) and the same or a closely related donor sugar. This adds strong support to the inferences drawn for the specificity of the initial transferases. The availability of all of the annotated cps sequences allowed us to look for correlations between genes, known CPS structures, and serology (gene clusters, CPS structures, and antigenic formulae are summarised in Figure S1 and Table S3). In this way, we can attempt both to infer gene function and, by comparing related cps loci, to account for differences in CPS structure and serology. Variations between cps loci range from two base substitutions for 18B and 18C to wholesale differences in gene complement. Within this range, the variations likely to have a phenotypic effect include gene inactivation due to single base substitutions generating a premature stop codon, single base insertion/deletions leading to translational frameshifts, change of sequence leading to change of enzyme specificity, recombination or IS element insertion leading to gene truncation, and insertion/deletion/replacement of single and multiple genes. Within serogroups, the genetic differences were often subtle but were also sometimes surprisingly prominent. Comparisons also revealed some strong common- ality between the cps of different serogroups and serotypes. Illustrative examples that demonstrate how structure, genetics, and serology were combined to analyse the cps loci are shown in Figure 2 and are discussed below. Previously described CPS structures [31] for all four serotypes of serogroup 9 show only subtle differences and provide an example of multiple serotypes arising by divergence from a single cps locus. Their cps genes fall into two pairs, with 9A highly similar to 9V [32], and 9L highly similar to 9N, but with the two pairs differing significantly in sequence ( Figure 2), suggesting an initial divergence to form two ancestral serotypes; this split correlates with a difference at residue 5 of the repeat unit, where 9L and 9N CPSs have Glc p NAc, whereas 9A and 9V have Glc p . Factor sera 9d reacts with 9A and 9V but not with 9L and 9N, suggesting that it is interacting with Glc p but not with Glc p NAc. Both are housekeeping sugars, and their differential incorporation is likely to be due to divergent forms of glycosyl transferase WcjC. Subsequently, one of these ancestral serotypes diverged to form 9L and 9N, the latter becoming unique in the group in having Glc p rather than Gal p as residue 3 in the repeat unit. Their dexB–aliA loci have the same gene complement, and within the cps genes there are only 79 nucleotide differences. The highest number of amino acid substitutions (13) is within glycosyl transferase WcjA; ten are unique to 9N and presumably result in its altered specificity for Glc p rather than for Gal p . The other ancestral serotype gave rise to 9V and 9A, which differ from each other only in their CPS acetylation; the former CPS has an O -acetylation pattern unique in the serogroup. This is likely due to the O -acetyl transferase– encoding wcjE gene, which is intact and apparently functional in 9V, disrupted by a frameshift mutation in 9A (deletion of guanine, nucleotide 726), and truncated in 9L and 9N by the insertion of an IS element. Interestingly, factor sera 9g reacts only with serotype 9V and may recognise an acetyl-based epitope determined by wcjE . Serogroup 9 cps loci also differ by the insertion, in 9A and 9V relative to 9L and 9N, of an O -acetyl transferase gene (wcjD) and an adjacent IS element. This correlates with recent nuclear magnetic resonance data (I. C. Skovsted, unpublished data), indicating that 9A CPS is partially acetylated. The cps gene clusters of serogroup 12 and serotypes 44 and 46 are almost identical, differing only in IS transposase genes, and provide an example of common ancestry that is not apparent from serology. Structures have been determined ...
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... communication systems. Capsule biosynthesis is influenced by galactose utilization as some of the precursors of capsule biosynthesis are generated through galactose catabolism 22 . Furthermore, our previous work showed that the Rgg/SHP144 and Rgg/SHP1518 cell-cell communication systems are responsive to galactose [23][24][25][26] To understand how the catabolic pathways contributed to pneumococcal virulence, we evaluated the expression of selected genes in ΔgalK, ΔlacD, and ΔgalKΔlacD recovered 4 h post-infection from the lungs (Fig. 6a). ...
Efficient utilization of nutrients is crucial for microbial survival and virulence. The same nutrient may be utilized by multiple catabolic pathways, indicating that the physical and chemical environments for induction as well as their functional roles may differ. Here, we study the tagatose and Leloir pathways for galactose catabolism of the human pathogen Streptococcus pneumoniae. We show that galactose utilization potentiates pneumococcal virulence, the induction of galactose catabolic pathways is influenced differentially by the concentration of galactose and temperature, and sialic acid downregulates galactose catabolism. Furthermore, the genetic regulation and in vivo induction of each pathway differ, and both galactose catabolic pathways can be turned off with a galactose analogue in a substrate-specific manner, indicating that galactose catabolic pathways can be potential drug targets.
... Pneumococcus possesses high genetic diversity due to mutation events, frequent horizontal gene transfer, and selection pressure from diverse human hosts [14][15][16]. The polysaccharide capsule that surrounds the bacterial cell is essential for evading the human immune system and is the basis for the serotyping classification scheme [17,18]. The pneumococcus comprises more than 100 antigenically distinct serotypes, a subset of which are the target of current vaccines [8- 10,19]. ...
Understanding how pathogens spread across geographical space is fundamental for control measures such as vaccination. Streptococcus pneumoniae (the pneumococcus) is a respiratory bacterium responsible for a large proportion of infectious disease morbidity and mortality globally. Even in the post-vaccination era, the rates of invasive pneumococcal disease (IPD) remain stable in most countries, including Israel. To understand the geographical spread of the pneumococcus in Israel, we analysed 1174 pneumococcal genomes from patients with IPD across multiple regions. We included the evolutionary distance between pairs of isolates inferred using whole-genome data within a relative risk (RR) ratio framework to capture the geographical structure of S. pneumoniae . While we could not find geographical structure at the overall lineage level, the extra granularity provided by whole-genome sequence data showed that it takes approximately 5 years for invasive pneumococcal isolates to become fully mixed across the country.
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... The outer capsule polysaccharide (cps) is a major pneumococcal virulence factor, protecting the pneumococcus against desiccation, complement-mediated opsonophagocytosis and other host antimicrobial pathways [5][6][7]. The cps biosynthesis genes are found on a single locus controlled by a single promoter region for most serotypes [8]. Cps consists of diverse sugar structures that vary among isolates, serving as the basis for classifying S. pneumoniae serotypes, with more than 100 immunologically-distinct serotypes identified to-date [9]. ...
... We used parsnp v1.7.4 (https://github.com/marbl/parsnp) to extract and identify single nucleotide polymorphisms (SNPs) within the Blantyre pneumococcal cps locus by aligning them against reference serotype specific cps locus described by Bentley et al. [8]. These SNPs were then annotated to distinguish synonymous from non-synonymous mutations using the vcf-annotator tool v0.5 (https://github.com/rpetit3/vcf-annotator). ...
... Additionally, we identified differences between lineages in DNA binding site sequences for SpxR and CpsR in the 37-CE, which are known to suppress capsule expression [8] (Fig. 3b). Notably, GPSC14 and GPSC455 showed a higher similarity (92 %, SpxR1 and CpsR region) to the reference strain (D39 Serotype 4) in the SpxR1 and CpsR binding sites described by Glanvielle et al., compared to GPSC20, 228, 22, 857, 882, 10, and 116 (62 % similarity, SpxR1 and CpsR region). ...
Since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in Malawi in 2011, there has been persistent carriage of vaccine serotype (VT) Streptococcus pneumoniae , despite high vaccine coverage. To determine if there has been a genetic change within the VT capsule polysaccharide (cps) loci since the vaccine’s introduction, we compared 1022 whole-genome-sequenced VT isolates from 1998 to 2019. We identified the clonal expansion of a multidrug-resistant, penicillin non-susceptible serotype 23F GPSC14-ST2059 lineage, a serotype 14 GPSC9-ST782 lineage and a novel serotype 14 sequence type GPSC9-ST18728 lineage. Serotype 23F GPSC14-ST2059 had an I253T mutation within the capsule oligosaccharide repeat unit polymerase Wzy protein, which is predicted in silico to alter the protein pocket cavity. Moreover, serotype 23F GPSC14-ST2059 had SNPs in the DNA binding sites for the cps transcriptional repressors CspR and SpxR. Serotype 14 GPSC9-ST782 harbours a non-truncated version of the large repetitive protein (Lrp), containing a Cna protein B-type domain which is also present in proteins associated with infection and colonisation. These emergent lineages also harboured genes associated with antibiotic resistance, and the promotion of colonisation and infection which were absent in other lineages of the same serotype. Together these data suggest that in addition to serotype replacement, modifications of the capsule locus associated with changes in virulence factor expression and antibiotic resistance may promote vaccine escape. In summary, the study highlights that the persistence of vaccine serotype carriage despite high vaccine coverage in Malawi may be partly caused by expansion of VT lineages post-PCV13 rollout.
... In general, pneumococcus CPS are synthesized via a Wzx/ Wzy-dependent pathway, except for serotypes 3 and 37 which use synthase-dependent pathway [11]. The capsule polysaccharide (cps) locus contains the majority of the genes for S. pneumoniae capsular biosynthesis. ...
... However, it can only be performed on viable isolates and relatively expensive. Bentley et al. [11] in 2006 found that the cps locus of S. pneumoniae associated to biochemical structure and immunological patterns and made a breakthrough for molecular serotyping assays. Afterwards, molecular typing methods continue to performed, including multiplex polymerase chain reaction (PCR), whole-genome sequencing, and microarray [13]. ...
... S. pneumoniae CPS have a major influence in pneumococcal pathogenesis [11,24]. Most CPS serotypes has positive charge to aid colonization by preventing these bacteria from mucus trap [25]. ...
This narrative review describes genomic characteristic, serotyping, immunogenicity, and vaccine development of Streptococcus pneumoniae capsular polysaccharide (CPS). CPS is a primary virulence factor of S. pneumoniae. The genomic characteristics of S. pneumoniae CPS, including the role of biosynthetic gene and genetic variation within cps (capsule polysaccharide) locus which may lead to serotype replacement are still being investigated. One hundred unique serotypes of S. pneumoniae have been identified through various methods of serotyping using phenotypic and genotypic approach. The advantages and limitations of each method are various, emphasizing the need for accurate and comprehensive serotyping for effective disease surveillance and vaccine targeting. In addition, we elaborate the critical role of CPS in vaccine development by providing an overview of immunogenicity, ongoing research of pneumococcal vaccines, and the impact on disease burden.
... In the 68 serotypes where synthesis initiates with Glc(glucose),thissequence encodes a CpsE homolog(WchA homology group; poly isoprenyl phosphate hexose-1-P family). The remaining serotypes lack Glc, and initiation with another sugar is catalyzed by a glycosyltransferase of the WciI, WcjG, or WcjH homology group (11). Among all serotypes, the cpsA sequences are highly conserved, whereas the cpsBCD sequences (and cpsE, where present) can be divided into two clusters The downstream regions of the loci do not exhibit the sequence conservation or clustering seen in the upstream regions, and they are considered to be serotype specific. ...
Estimation of effects of coumarin,7-ethyl-4-methyl coumarin, 4,7dimethyl-6-nitro coumarin and7-hydroxy-4-methyl coumarin on the expression fold of eps Ggene with different concentration (100 ,200, 300 and 400)µg/ml. The results shown that the Coumarin and its derivatives at the concentration 200 µg/ml was more effect in expression fold epsG gene compared with control. The gene expression of epsG gene responsible of production of glucosyl transferase affected by coumarin and its derivatives at different ratio.
... The CPS locus is known to be physically located between aliA and dexB genes in the pneumococcus [24]. The prototypical serotype 3 CPS reference locus [24] has the following genes, dexB, aliB, tnp, wzg, wzh, wzd, wze, tnp, ugd, wchE, galU, and pgm, and our analysis is based on the gene content comparisons at the physical locations with the serotype 3 reference CPS locus. ...
... The CPS locus is known to be physically located between aliA and dexB genes in the pneumococcus [24]. The prototypical serotype 3 CPS reference locus [24] has the following genes, dexB, aliB, tnp, wzg, wzh, wzd, wze, tnp, ugd, wchE, galU, and pgm, and our analysis is based on the gene content comparisons at the physical locations with the serotype 3 reference CPS locus. Although only the core CPS genes (wzg, wzh, wzd, and wze) have known capsule biosynthesis functions [24,25], others have unknown or potential auxiliary functions. ...
... The prototypical serotype 3 CPS reference locus [24] has the following genes, dexB, aliB, tnp, wzg, wzh, wzd, wze, tnp, ugd, wchE, galU, and pgm, and our analysis is based on the gene content comparisons at the physical locations with the serotype 3 reference CPS locus. Although only the core CPS genes (wzg, wzh, wzd, and wze) have known capsule biosynthesis functions [24,25], others have unknown or potential auxiliary functions. Therefore, to avoid confusion, we refer to any genes located in the entire region known to harbor genes involved in capsule biosynthesis for serotype 3 as CPS genes. ...
Background
Streptococcus pneumoniae serotype 3 remains a problem globally. Malawi introduced 13-valent pneumococcal conjugate vaccine (PCV13) in 2011, but there has been no direct protection against serotype 3 carriage. We explored whether vaccine escape by serotype 3 is due to clonal expansion of a lineage with a competitive advantage.
Methods
The distribution of serotype 3 Global Pneumococcal Sequence Clusters (GPSCs) and sequence types (STs) globally was assessed using sequences from the Global Pneumococcal Sequencing Project. Whole-genome sequences of 135 serotype 3 carriage isolates from Blantyre, Malawi (2015–2019) were analyzed. Comparative analysis of the capsule locus, entire genomes, antimicrobial resistance, and phylogenetic reconstructions were undertaken. Opsonophagocytosis was evaluated using serum samples from vaccinated adults and children.
Results
Serotype 3 GPSC10-ST700 isolates were most prominent in Malawi. Compared with the prototypical serotype 3 capsular polysaccharide locus sequence, 6 genes are absent, with retention of capsule polysaccharide biosynthesis. This lineage is characterized by increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing.
Conclusions
A serotype 3 variant in Malawi has genotypic and phenotypic characteristics that could enhance vaccine escape and clonal expansion after post-PCV13 introduction. Genomic surveillance among high-burden populations is essential to improve the effectiveness of next-generation pneumococcal vaccines.
... The capsular polysaccharide of the pneumococcus defines its serotype and is the main virulence determinant as well as the target for pneumococcal conjugate vaccines (PCVs) [2]. As of 2020, over one hundred serotypes have been identified [3,4]. The carriage prevalence of S. pneumoniae in the nasopharynx varies with age, environment, and the presence of upper respiratory infections and, when assessed by culture methods, typically ranges from 5-10 % among adults without children and 20-60 % in school-age children [2]. ...
Background. Despite use of highly effective conjugate vaccines, invasive pneumococcal disease (IPD) remains a leading cause of morbidity and mortality and disproportionately affects Indigenous populations. Although included in the 13-valent pneumococcal conjugate vaccine (PCV13), which was introduced in 2010, serotype 3 continues to cause disease among Indigenous communities in the Southwest USA. In the Navajo Nation, serotype 3 IPD incidence increased among adults (3.8/100 000 in 2001–2009 and 6.2/100 000 in 2011–2019); in children the disease persisted although the rates dropped from 5.8/100 000 to 2.3/100 000.
Methods. We analysed the genomic epidemiology of serotype 3 isolates collected from 129 adults and 63 children with pneumococcal carriage ( n =61) or IPD ( n =131) from 2001 to 2018 of the Navajo Nation. Using whole-genome sequencing data, we determined clade membership and assessed changes in serotype 3 population structure over time.
Results. The serotype 3 population structure was characterized by three dominant subpopulations: clade II ( n =90, 46.9 %) and clade Iα ( n =59, 30.7 %), which fall into Clonal Complex (CC) 180, and a non-CC180 clade ( n =43, 22.4 %). The proportion of clade II -associated IPD cases increased significantly from 2001 to 2010 to 2011–2018 among adults (23.1–71.8 %; P <0.001) but not in children (27.3–33.3 %; P =0.84). Over the same period, the proportion of clade II- associated carriage increased; this was statistically significant among children (23.3–52.6 %; P =0.04) but not adults (0–50.0 %, P =0.08).
Conclusions. In this setting with persistent serotype 3 IPD and carriage, clade II has increased since 2010. Genomic changes may be contributing to the observed trends in serotype 3 carriage and disease over time.
... In S. pneumoniae, two distinct CPS synthesis pathways are documented: the prevalent Wzy-dependent pathway and the rarer synthase-dependent pathway (confined to two serotypes, type 3 and type 37). The Wzy-dependent loci are distinguished by two characteristic proteins: Wzy, an oligosaccharide repeat unit polymerase (CpsI), and Wzx, an oligosaccharide transporter(CpsJ) (7). The expansive CPS operon consists of four invariant regulatory proteins succeeded by a plethora of variable glycosyltransferases. Insightful studies on CPS loci from other pathogenic streptococci like Streptococcus agalactiae (8,9), Streptococcus suis (10), and Streptococcus iniae (11) have revealed their crucial role in bacterial virulence. ...
Capsular polysaccharides (CPS) in Streptococcus pneumoniae are pivotal for bacterial virulence and present extensive diversity. While oral streptococci show pronounced antigenicity toward pneumococcal capsule-specific sera, insights into evolution of capsule diversity remain limited. This study reports a pneumococcal CPS-like genetic locus in Streptococcus parasanguinis, a predominant oral Streptococcus. The discovered locus comprises 15 genes, mirroring high similarity to those from the Wzy-dependent CPS locus of S. pneumoniae. Notably, S. parasanguinis elicited a reaction with pneumococcal 19B antiserum. Through nuclear magnetic resonance analysis, we ascertained that its CPS structure matches the chemical composition of the pneumococcal 19B capsule. By introducing the glucosyltransferase gene cps19cS from a pneumococcal serotype 19C, we successfully transformed S. parasanguinis antigenicity from 19B to 19C. Furthermore, substituting serotype-specific genes, cpsI and cpsJ, with their counterparts from pneumococcal serotype 19A and 19F enabled S. parasanguinis to generate 19A- and 19F-specific CPS, respectively. These findings underscore that S. parasanguinis harbors a versatile 19B-like CPS adaptable to other serotypes. Remarkably, after deleting the locus’s initial gene, cpsE, responsible for sugar transfer, we noted halted CPS production, elongated bacterial chains, and diminished biofilm formation. A similar phenotype emerged with the removal of the distinct gene cpsZ, which encodes a putative autolysin. These data highlight the importance of S. parasanguinis CPS for biofilm formation and propose a potential shared ancestry of its CPS locus with S. pneumoniae.
IMPORTANCE
Diverse capsules from Streptococcus pneumoniae are vital for bacterial virulence and pathogenesis. Oral streptococci show strong responses to a wide range of pneumococcal capsule-specific sera. Yet, the evolution of this capsule diversity in relation to microbe-host interactions remains underexplored. Our research delves into the connection between commensal oral streptococcal and pneumococcal capsules, highlighting the potential for gene transfer and evolution of various capsule types. Understanding the genetic and evolutionary factors that drive capsule diversity in S. pneumoniae and its related oral species is essential for the development of effective pneumococcal vaccines. The present findings provide fresh perspectives on the cross-reactivity between commensal streptococci and S. pneumoniae, its influence on bacteria-host interactions, and the development of new strategies to manage and prevent pneumococcal illnesses by targeting and modulating commensal streptococci.
... Glycoconjugates are complex heterogeneous biopolymers found on the cell surface and secreted from all cells. In prokaryotes, (Tytgat and Lebeer, 2014) glycoconjugates play important roles in survival, (Chen et al., 2004) antibiotic resistance, (Campos et al., 2004) immune evasion and immunogenicity (Tan et al., 2020;Bentley et al., 2006), and biofilm formation (Vu et al., 2009) Genetic disruption or inhibition of the enzymes involved in glycoconjugate assembly can result in deleterious effects in many microorganisms and have been reported to attenuate virulence and pathogenicity (Chua et al., 2021;Hong and Reeves, 2016;Yethon et al., 2000) Despite the overwhelming compositional diversity of prokaryotic glycoconjugates, the underlying logic for their biosynthesis is often conserved and most commonly localized to cell membranes (Whitfield et al., 2020a). Thus, structural and mechanistic studies of the machinery for glycoconjugate assembly are of considerable interest although the membrane localization of glycoconjugate assembly enzymes poses a significant hurdle for in-depth characterization. ...
Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.
... When applied to individual colonies, such methods have a low sensitivity for detecting co-carriage, although this can be improved using latex agglutination of plate sweeps (15). Nevertheless, these methods cannot identify novel serotypes; these may be discovered through whole genome sequencing (WGS) approaches, which detect specific sequence variants of the Capsular Biosynthetic Locus (CBL) (18)(19)(20), the operon which defines pneumococcal serotype (21). Yet WGS of individual colonies is difficult to deploy at scale in resource-limited settings as it is expensive and time-consuming, requiring specific expertise and access to specialist laboratory equipment (22). ...
... To improve enrichment by NAS, we specifically enriched for the pneumococcal CBL, which is generally absent from streptococci other than S. pneumoniae (21). We sequenced the same library as described in Figure 1 Figure 3: Difference in normalised coverage per locus between NAS and control channels across the Spn23F genome when targeting whole genome (blue) or CBL (red). ...
... 2.3 NAS can simultaneously enrich for multiple pneumococcal CBL in the same mixture NAS is therefore capable of distinguishing encapsulated S. pneumoniae from other streptococci, but effective serotype surveillance requires the identification of multiple serotypes in cases of co-carriage. CBL are highly structurally diverse (21), potentially allowing differentiation of multiple CBL in co-carriage by phasing contiguous structural variants using long reads (43). To determine whether NAS can differentiate and enrich for multiple CBL sequences, we generated a set of mock communities where Spn23F was mixed in 50:50 proportions with other S. pneumoniae strains with different genotypes and serotypes (Figure 5a). ...
Serotype surveillance of Streptococcus pneumoniae (the pneumococcus) is critical for understanding the effectiveness of current vaccination strategies. However, existing methods for serotyping are limited in their ability to identify co-carriage of multiple pneumococci and detect novel serotypes. To develop a scalable and portable serotyping method that overcomes these challenges, we employed Nanopore Adaptive Sampling (NAS), an on-sequencer enrichment method which selects for target DNA in real-time, for direct detection of S. pneumoniae in complex samples. Whereas NAS targeting the whole S. pneumoniae genome was ineffective in the presence of non-pathogenic streptococci, the method was both specific and sensitive when targeting the capsular biosynthetic locus (CBL), the operon that determines S. pneumoniae serotype. NAS significantly improved coverage and yield of the CBL relative to sequencing without NAS, and accurately quantified the relative prevalence of serotypes in samples representing co-carriage. To maximise the sensitivity of NAS to detect novel serotypes, we developed and benchmarked a new pangenome-graph algorithm, named GNASTy. We show that GNASTy outperforms the current NAS implementation, which is based on linear genome alignment, when a sample contains a serotype absent from the database of targeted sequences. The methods developed in this work provide an improved approach for novel serotype discovery and routine S. pneumoniae surveillance that is fast, accurate and feasible in low resource settings. GNASTy therefore has the potential to increase the density and coverage of global pneumococcal surveillance.
One sentence summary
Pangenome graph-based Nanopore Adaptive Sampling, presented in our tool GNASTy, is a sensitive, portable and cost-effective method for Streptococcus pneumoniae surveillance.
