Daniel Llull

Characteristic Signatures of the lytA Gene Provide a Basis for Rapid and Reliable Diagnosis of Streptococcus pneumoniae Infections

ABSTRACT The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.

Current trends in capsular polysaccharide biosynthesis of Streptococcus pneumoniae

The capsular gene cluster (cap/cps) of 13 out of the 90 known pneumococcal types has been sequenced. The cap/cps operon, located between dexB and aliA in the Streptococcus pneumoniae chromosome, contains some of the genes responsible for the synthesis of the type-specific polysaccharide flanked by four conserved open reading frames. The biochemical function of only a few capsular genes has been established, whereas the role of the flanking regions is controversial. Remarkably, only one gene (tts) located outside the cap locus is required for the synthesis of type 37 capsule. Moreover, other genes not linked to the cap gene cluster are also needed for capsule synthesis in pneumococcus.

Genetic bases and medical relevance of capsular polysaccharide biosynthesis in pathogenic streptococci.

Many streptococci are human and/or animal pathogens and the frequent cause of life-threatening diseases. Among various streptococcal virulence factors, capsular polysaccharides (CPs) are recognized as essential to prevent phagocytosis by macrophages and neutrophils. In the last decade, an impressive advance on the knowledge of the genetic bases underlying capsule formation has been achieved. The capsular gene cluster driving the formation of the CP of Streptococcus pyogenes and other hyaluronate-producing streptococci, represents one of the simplest cases of gene organization to synthesize a capsule. A more complex situation has been found in Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, and other streptococci. On the whole, there exists a direct relationship between the structural and chemical complexity of the repeating unit of the polysaccharide and the number of genes found in the corresponding capsular locus. Streptococcal vaccines, either polysaccharide or conjugate, are currently being tested in clinical trials to overcome the rise of worldwide antibiotic resistance, although, for different reasons, none of these vaccines are expected to provide the required full coverage in a near future. This concern has prompted to explore alternative possibilities with an improved therapeutic potential against streptococcal diseases.

Molecular structure of the gene cluster responsible for the synthesis of the polysaccharide capsule of Streptococcus pneumoniae type 33F

The organization and nucleotide sequence of the capsular gene cluster involved in the biosynthesis of the type 33F capsular polysaccharide of Streptococcus pneumoniae have been determined. The complete type 33F operon (cap33f) is composed of 14 potential open reading frames where the last ten genes are group-specific. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. A functional promoter located immediately upstream from the first gene of the cap33f gene cluster has been demonstrated. A 20 kb DNA fragment containing the cap33f genes and the operon promoter was sufficient to transform a S. pneumoniae type 3 unencapsulated mutant to the type 33F capsule.

Molecular Characterization of Disease-Associated Streptococci of the Mitis Group That Are Optochin Susceptible

ABSTRACT Eight optochin-susceptible (Opts) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae suggested that the Opts isolates corresponded to streptococci of the mitis group. Besides, the Opts streptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytA and ply) but also with ant, a gene not present in most S. pneumoniae strains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytA gene revealed that the Opts streptococci carried lytA alleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atp operon encoding the FoF1 H+-ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae by horizontal gene transfer.

In Vitro Bactericidal Activity of the Antiprotozoal Drug Miltefosine against Streptococcus pneumoniae and Other Pathogenic Streptococci

ABSTRACT Miltefosine (hexadecylphosphocholine), the first oral drug against visceral leishmaniasis, triggered pneumococcal autolysis at concentrations higher than 2.5 μM. Bactericidal activity was also observed in cultures of other streptococci, although these failed to undergo lysis. The autolysis elicited by miltefosine can be attributed to triggering of the pneumococcal autolysin LytA.

Skl, a novel choline-binding N-acetylmuramoyl-l-alanine amidase of Streptococcus mitis SK137 containing a CHAP domain

The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N‐acetylmuramoyl‐l‐alanine amidase (EC 3.5.1.28), showing optimum activity at 30 °C and at a pH of 6.5. Skl is a unique member of the choline‐binding family of proteins since it contains a cysteine, histidine‐dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP‐containing choline‐binding proteins.