Insertion-duplication mutagenesis of neisseria: use in characterization of DNA transfer genes in the gonococcal genetic island.

We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of traG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.

A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates.

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation-like secretion system. Consistent with this hypothesis, a wild-type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.

Pneumococcal diversity: considerations for new vaccine strategies with emphasis on pneumococcal surface protein A (PspA).

Streptococcus pneumoniae is a problematic infectious agent, whose seriousness to human health has been underscored by the recent rise in the frequency of isolation of multidrug-resistant strains. Pneumococcal pneumonia in the elderly is common and often fatal. Young children in the developing world are at significant risk for fatal pneumococcal respiratory disease, while in the developed world otitis media in children results in substantial economic costs. Immunocompromised patients are extremely susceptible to pneumococcal infection. With 90 different capsular types thus far described, the diversity of pneumococci contributes to the challenges of preventing and treating S. pneumoniae infections. The current capsular polysaccharide vaccine is not recommended for use in children younger than 2 years and is not fully effective in the elderly. Therefore, innovative vaccine strategies to protect against this agent are needed. Given the immunogenic nature of S. pneumoniae proteins, these molecules are being investigated as potential vaccine candidates. Pneumococcal surface protein A (PspA) has been evaluated for its ability to elicit protection against S. pneumoniae infection in mouse models of systemic and local disease. This review focuses on immune system responsiveness to PspA and the ability of PspA to elicit cross-protection against heterologous strains. These parameters will be critical to the design of broadly protective pneumococcal vaccines.

The pathogenic neisseriae contain an inactive rpoN gene and do not utilize the pilE sigma54 promoter.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae.

We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae. The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild-type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non-permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild-type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16h in stationary phase, both strains underwent an apparent replication event, after which the wild-type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild-type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild-type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase.

Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae.

The capsular polysaccharide is the major virulence factor of Streptococcus pneumoniae. Previously, we identified and cloned a region from the S. pneumoniae chromosome specific for the production of type 3 capsular polysaccharide. Now, by sequencing the region and characterizing mutations genetically and in an in vitro capsule synthesis assay, we have assigned putative functions to the products of the type-specific genes. Using DNA from the right end of the region in mapping studies, we have obtained further evidence indicating that the capsule genes of each serotype are contained in a gene cassette located adjacent to this region. We have cloned the region flanking the left end of the cassette from the type 3 chromosome and have found that it is repeated in the S. pneumoniae chromosome. The DNA sequence and hybridization data suggest a model for recombination of the capsule gene cassettes that not only describes the replacement of capsule genes, but also suggests an explanation for binary capsule type formation, and the creation of novel capsule types.

Genetic and molecular characterization of capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 3.

To achieve a better understanding of the genetics of capsular polysaccharide synthesis in Streptococcus pneumoniae, we have identified and characterized mutants deficient in type 3 capsule production. We identified a clone that restored encapsulation in one of our mutants and in a mutant deficient in UDP-glucose dehydrogenase. By hybridization, we developed a chromosomal map of the type 3-specific region and identified a flanking region containing DNA common to all capsule types examined. Insertion mutations were used to identify chromosomal loci required for capsule synthesis, and to map transcription within the region. Using non-destructive insertions linked to type-specific genes of type 2, 3, or 5, we were able to select for the transformation of all necessary genes specific for capsule type. Our data provide molecular evidence to show that all the type-specific genes are linked in a cassette and can be transferred as a unit during transformation.

Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae.

To assess the role of capsular serotypes in the virulence of Streptococcus pneumoniae, we have constructed isogenic derivatives differing only in the type of capsule expressed. Strains of types 2, 5, and 6B were converted to type 3 by transformation and selection for an erythromycin resistance marker linked to the type 3 capsule locus. Characterization studies revealed that these type 3 derivatives were indistinguishable from the type 2, type 5, and type 6B parental strains in terms of restriction enzyme fragment pattern and expression of pneumococcal surface protein A (PspA). Expression of the type 3 capsule did not alter the mouse virulence of the similarly virulent type 2 strain. However, alteration of capsule type had a profound effect on the virulence of the type 5 and type 6B derivatives. The highly virulent type 5 strain was essentially avirulent when expressing the type 3 capsule. Conversely, the 50% lethal dose of the relatively avirulent type 6B strain was reduced > 100-fold when the type 3 capsule was expressed. Thus, the serotype of capsule expressed has a major effect on virulence, and this effect is dependent upon the genetic background of the recipient strain.

Analysis of Streptococcus pneumoniae sequences cloned into Escherichia coli: effect of promoter strength and transcription terminators.

Difficulties encountered in the cloning of DNA from Streptococcus pneumoniae and other AT-rich organisms into ColE1-type Escherichia coli vectors have been proposed to be due to the presence of a large number of strong promoter-acting sequences in the donor DNA. The use of transcription terminators has been advocated as a means of reducing instability resulting from disruption of plasmid replication caused by strong promoters. However, neither the existence of promoter-acting sequences of sufficient strength and number to explain the reported cloning difficulties nor their role as a source of instability has been proven. As a direct test of the "strong promoter" hypothesis, we cloned random fragments from S. pneumoniae into an E. coli vector containing transcription terminators, identified strong promoter-acting sequences, and subsequently removed the transcription terminators. We observed that terminator removal resulted in reduced copy numbers for the strongest promoter-acting sequences but not in reduced promoter strengths or altered plasmid stabilities. Our results indicate that promoters strong enough to require transcription terminators for plasmid stability are probably rare in S. pneumoniae DNA.

Rapid progression of peripheral vascular disease after diagnostic angiography.

The authors retrospectively reviewed records of percutaneous transluminal angioplasties performed at three institutions. Seven stenotic lesions in peripheral vessels were identified that had progressed to total occlusion in the interval between the time that diagnostic run-off angiography and attempted angioplasty were performed. The post-angiographic occlusions were 3-16 cm long (mean, 9 cm), and the interval between diagnostic angiography and discovery of the occlusions ranged from 1 hour to 91 days (median, 2 days). Treatment of the occlusions was riskier and more difficult than simple angioplasty of the original stenoses. The authors conclude that some aspect of the angiographic procedure probably precipitated the transition from stenosis to occlusion. This complication can be prevented by preparation for angioplasty immediately after diagnostic run-off angiography or perhaps by use of heparin during the diagnostic study.