Genetic and Epidemiological Characterization of Streptococcus pneumoniae disease determinants.
Melchiorre, Sara (2011) Genetic and Epidemiological Characterization of Streptococcus pneumoniae disease determinants. [Tesi di dottorato] (Inedito)
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The Gram-positive pathogen Streptococcus pneumoniae is a major cause of community acquired pneumonia as well as of upper respiratory tract infections such as acute otitis media and sinusitis, and invasive diseases like meningitis, bacteremia, and endocarditis. However, S. pneumoniae is also a commensal of the upper respiratory tract, especially of young children, which represent the reservoir for pneumococcal transmission within the community. A major virulence factor of Streptococcus pneumoniae is the polysaccharide capsule. The pneumococcal capsule displays an extremely high variability (it exists in fact in at least ninety different types, known as serotypes) and provides an effective barrier against host-cell mediated phagocytosis allowing bacterial persistence in the blood. In addition to the capsular determinant, many other secreted or surface exposed factors have been described to be of importance for virulence through in vivo animal model studies and in vitro experiments (i.e. choline-binding protein A (CbpA), the pneumococcal toxin pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pilus components; however, their direct contribution to and essentiality for disease development in humans have still to be determined. Streptococcus pneumoniae pilus islet-1 (PI-1)-encoded pilus enhances in vitro adhesion to the respiratory epithelium and may contribute to pneumococcal nasopharyngeal colonization and transmission. The pilus subunits are regarded as potential protein vaccine candidates. In the first part of this study, we sought to determine PI-1 prevalence in carried pneumococcal isolates and explore its relationship with transmissibility or carriage duration. We studied 896 pneumococcal isolates collected during a longitudinal carriage study that included monthly nasopharyngeal swabbing of 234 infants and their mothers between the ages of 1 and 24 months. These were cultured according to the WHO pneumococcal carriage detection protocol. PI-1 PCR and genotyping by multilocus sequence typing were performed on isolates chosen according to specific carriage and transmission definitions. Overall, 35.2% of the isolates were PI-1-positive, but PI-1 presence was restricted to ten of the 34 serotypes studied and was most frequently associated with serotypes 19F and 23F; 47.5% of transmitted and 43.3% of non-transmitted isolates were PI-1-positive (OR 1.2; 95% CI 0.8–1.7; p 0.4). The duration of first-ever infant pneumococcal carriage was significantly longer with PI-1-positive organisms, but this difference was not significant at the individual serotype level. In conclusion, PI-1 is commonly found in pneumococcal carriage isolates, but does not appear to be associated with pneumococcal transmissibility or carriage duration. In the second part of this work, we focused on non-typeable Streptococcus pneumoniae (NTPn). NTPns are typically isolated from nasopharyngeal carriage or from conjunctivitis. Since the isolation of NTPn from invasive disease is rare, we characterized the genetic basis for non-typeability of two isolates obtained in Italy from two cases of bacteremic pneumonia. Multi Locus Sequence Typing (MLST) revealed that both NTPn belonged to ST191, which, according to the MLST database, is associated with serotype 7F. Sequencing of the capsular locus (cps) confirmed the presence of a 7F cps in both strains and revealed the existence of distinct single point mutations in the wchA gene (a glycosyltransferase), both leading to the translation of proteins truncated at the C-terminus. To verify if these mutations were responsible for non-typeability of the isolates, a functional 7F WchA was over-expressed in both NTPn. The two NTPn along with their WchA over-expressing derivatives were analyzed by Transmission Electron Microscopy and by high-resolution magic angle spinning NMR spectroscopy. Both NTPn were devoid of a polysaccharide capsule and WchA over-expression was sufficient to restore the assembly of a serotype 7F capsule on the surface of the two NTPn. In conclusion, we identified two new naturally-occurring point-mutations leading to the non typeability in pneumococcus and demonstrated that WchA is essential for the biosynthesis of the serotype 7F capsule.
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