Judy K. Morona

Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae

Serotype 19F variants of the major Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae have been proposed to have arisen by recombinational exchanges at the capsular biosynthetic locus. Members of the Spanish multiresistant serotype 23F clone and the serotype 19F variants were confirmed to be essentially identical in overall genotype, as they were indistinguishable by REP‐PCR, and had identical sequences at three polymorphic housekeeping genes. Eight serotype 19F variants were studied and all had large recombinational replacements at the capsular biosynthetic locus. In all cases, one of the recombinational cross‐over points appeared to be upstream of dexBwhich flanks one end of the capsular locus, and in six of the variants the other cross‐over point was downstream of aliA, which flanks the other end of the locus. In two strains a recombinational cross‐over point between the introduced serotype 19F capsular region and that of the Spanish serotype 23F clone could be clearly identified, within cpsN in one strain and within cpsM in the other. The differences in the recombinational junctions and sequence polymorphisms within the introduced capsular genes, suggested that the eight serotype 19F variants emerged on at least four separate occasions. Changes in capsular type by recombination may therefore be relatively frequent in pneumococci and this has implications for the long‐term efficacy of conjugate pneumococcal vaccines that will protect against only a limited number of serotypes.

Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae

In Streptococcus pneumoniae, the first four genes of the capsule locus (cpsA to cpsD) are common to most serotypes. By analysis of various in‐frame deletion and site‐directed mutants, the function of their gene products in capsular polysaccharide (CPS) biosynthesis was investigated. We found that while CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and CpsD have similarity to the amino‐terminal and carboxy‐terminal regions, respectively, of the autophosphorylating protein‐tyrosine kinase Wzc from Escherichia coli. Alignment of CpsD with Wzc and other related proteins identified conserved Walker A and B sequence motifs and a tyrosine rich domain close to the carboxy‐terminus. We have shown that CpsD is also an autophosphorylating protein‐tyrosine kinase and that point mutations in cpsD affecting either the ATP‐binding domain (Walker A motif) or the carboxy‐terminal [YGX]4 repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysaccharide production and show that they affect CPS production differently. Whereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX]4 repeat domain resulted in an apparent increase in encapsulation and a mucoid phenotype. These data suggest that autophosphorylation of CpsD at tyrosine attenuates its activity and reduces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephosphorylation of CpsD. These results are consistent with CpsD tyrosine phosphorylation acting to negatively regulate CPS production. This has implications for the function of CpsC/CpsD homologues in both Gram‐positive and Gram‐negative bacteria and provides a mechanism to explain regulation of CPS production during pathogenesis.

Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway

We have previously reported the nucleotide sequence of the first six genes of the Streptococcuspneumoniae type 19F capsular polysaccharide biosynthesis locus (cps19f). In this study we used plasmid insertion/rescue and inverse polymerase chain reaction (PCR) to clone an additional 10 kb downstream region containing the remainder of the cps19f locus, which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion‐duplication mutants in seven out of the nine new ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (non‐encapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. T7 expression studies confirmed that cps19fH, cps19fK, cps19fL, cps19fM and cps19fN directed the production of polypeptides of the expected size in Escherichia coli. The function of the cps19fK product was confirmed by its ability to complement a mutation in nfrC (rffE ) in E. coli, as judged by restoration of sensitivity to bacteriophage N4. Interestingly, the last four genes of the locus (cps19fL–O ) exhibit very strong homology (up to 70% amino acid identity) to a portion of the Shigella flexneri rfb gene cluster encoding biosynthesis of dTDP‐rhamnose. When expressed in E. coli, cps19fL–O were capable of complementing a mutation deleting the respective Shigella flexneri homologues. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and, within this, cps19fI was unique to type 19F.

Streptococcus pneumoniae Capsule Biosynthesis Protein CpsB Is a Novel Manganese-Dependent Phosphotyrosine-Protein Phosphatase

The first four genes of the capsule locus (cps) of Streptococcus pneumoniae (cpsA to cpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that an S. pneumoniae strain with point mutations in cpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which the cpsB gene had been deleted.

Mutational Analysis of the Carboxy-Terminal (YGX)4 Repeat Domain of CpsD, an Autophosphorylating Tyrosine Kinase Required for Capsule Biosynthesis in Streptococcus pneumoniae

In Streptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions of cpsB, cpsC, or cpsD exhibit rough colony morphologies. CpsD is an autophosphorylating protein-tyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regulates CPS production. In this study, we further investigated the role of the carboxy-terminal (YGX)(4) repeat domain of CpsD in encapsulation. A CpsD truncation mutant in which the entire (YGX)(4) repeat domain was removed was indistinguishable from a strain in which the entire cpsD gene had been deleted, indicating that the carboxy-terminal (YGX)(4) tail is required for CpsD activity in capsular polysaccharide production. Double mutants having a single tyrosine residue at position 2, 3, or 4 in the (YGX)(4) repeat domain and lacking CpsB exhibited a rough colony morphology, indicating that in the absence of an active protein-tyrosine phosphatase, phosphorylation of just one of the tyrosine residues in the (YGX)(4) repeat was sufficient to inactivate CpsD. When various mutants in which CpsD had either one or combinations of two or three tyrosine residues in the (YGX)(4) repeat domain were examined, only those with three tyrosine residues in the (YGX)(4) repeat domain were indistinguishable from the wild-type strain. The mutants with either one or two tyrosine residues exhibited mucoid colony morphologies. Further analysis of the mucoid strains indicated that the mucoid phenotype was not due to overproduction of capsular polysaccharide, as these strains actually produced less capsular polysaccharide than the wild-type strain. Thus, the tyrosine residues in the (YGX)(4) repeat domain are essential for normal functioning of CpsD.

Comparison of the clinical effectiveness of different off‐loading devices for the treatment of neuropathic foot ulcers in patients with diabetes: a systematic review and meta‐analysis

Effective off‐loading is considered to be an important part of the successful clinical management of diabetic foot ulcers. The aim of this systematic review is to investigate the safety and effectiveness of different off‐loading devices for the treatment of diabetic foot ulcers. The medical literature was extensively searched from January 1966 to May 2012. Systematic reviews and controlled studies that compared the use of different off‐loading devices formed the evidence base. Studies were critically appraised to determine their risk of methodological bias, and data were extracted. Results were pooled using random effects meta‐analysis and tested for heterogeneity. When compared with removable devices, non‐removable off‐loading devices were found, on average, to be more effective at promoting the healing of diabetic foot ulcers (RRp = 1.43; 95% CI 1.11, 1.84; I2 = 66.9%; p = 0.001; k = 10). Analysis, stratified by type of removable device, did not detect a statistically significant difference between non‐removable off‐loading devices and removable cast walkers; however, on average non‐removable off‐loading devices performed better than therapeutic shoes at promoting the healing of diabetic foot ulcers (RRp = 1.68; 95% CI 1.09, 2.58; I2 = 71.5%; p = 0.004; k = 6). The two types of non‐removable off‐loading devices i.e. total contact casts and instant total contact casts (removable cast walker rendered irremovable by securing with bandage or lace), were found to be equally effective (RRp = 1.06; 95% CI 0.88, 1.27; I2 = 3.3%; p = 0.31; k = 2). In conclusion, non‐removable off‐loading devices regardless of type, are more likely to result in ulcer healing than removable off‐loading devices, presumably because patient compliance with off‐loading is facilitated. Copyright

Construction of plasmid vectors with a non-antibiotic selection system based on the Escherichia coli thyA+ gene: application to cholera vaccine development

The construction of live oral carriers based on attenuated Salmonella strains as vectors offers a new approach to vaccine development. We have constructed a set of plasmid vectors which have the thyA gene of Escherichia coli (encoding thymidylate synthetase) as the marker for selection and maintenance of plasmid clones. The thyA system offers an alternative to antibiotic-resistance selection markers. It can be easily adapted to a particular host-vector combination since thyA chromosomal mutations can be readily introduced by trimethoprim selection. We also describe the construction of thyA-based plasmids with the Vibrio cholerae rfb genes (encoding O-antigen biosynthesis of the Inaba serotype). These have been found to be useful in the construction of candidate bivalent cholera-typhoid vaccines.

Construction of K88- and K99-expressing clones of Salmonella typhimurium G30: immunogenicity following oral administration to pigs

Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99. Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed. These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively. When administered orally to adult pigs, a dose of 10(11) vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens. Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms. Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system.

Surface co-expression of Vibrio cholerae and Salmonella typhi O-antigens on Ty21a clone EX210

In an attempt to construct a bivalent, live, oral cholera-typhoid vaccine, genes specifying the biosynthesis of Vibrio cholerae O-antigen have been transferred into a modified version of the attenuated, oral typhoid vaccine strain Salmonella typhi Ty21a. The present report investigates the production of V. cholerae and S. typhi O-antigens by one such clone, EX210. When cultured without galactose supplementation EX210 produces surface O-antigen of V. cholerae type, as detected by haemagglutination-inhibition and bactericidal assays, and by immuno-electron microscopy. However, the protective efficacy of Ty21a depends upon growth in the presence of exogenous galactose and under these conditions only S. typhi O-antigen is detectable on the surface of EX210. Subsequent experiments revealed that the proportion of polysaccharide of S. typhi type is dependent upon the level of galactose supplementation, and identified a limiting sugar concentration which results in surface co-expression of both O-antigens. Visualization of the two polysaccharides on silver-stained polyacrylamide gels indicates that S. typhi O-antigen subunits are polymerized into longer sidechains, suggesting that at higher galactose concentrations their predominance results in a masking of the shorter V. cholerae O-antigen.

Identification of Streptococcus pneumoniae Cps2C Residues That Affect Capsular Polysaccharide Polymerization, Cell Wall Ligation, and Cps2D Phosphorylation

A number of single amino acid substitutions throughout Streptococcus pneumoniae Cps2C were found to affect its function and confer either a mucoid or a small colony phenotype. These mutants exhibit significant changes in capsular polysaccharide (CPS) profile relative to that of wild-type pneumococci. The introduced mutations affect either polymerization or ligation of CPS to the cell wall and/or Cps2D phosphorylation.