Genetic approaches towards understanding penumococcal virulence and biology

Sammanfattning: Streptococcus pneumoniae, the pneumococcus, is a major human pathogen giving rise to death and illness worldwide every year. It causes a wide variety of diseases, from normally harmless infections such as otitis media to potentially life-threatening systemic diseases such as pneumonia, meningitis and sepsis. However, it is commonly carried in the nasopharynx of healthy children. Pneumococcal infections are in general treated with antibiotics such as penicillin, but over the last decades antibiotic resistance has become an increasing problem. The aim of this thesis was to learn more about pneumococcal virulence as well as the mechanisms leading to resistance. First, we studied the lytic behavior of pneumococcal clinical isolates, and found a link between the degree of lysis and the capsular serotype. Lytic antibiotics such as penicillin and vancomycin kill pneumococci partly by activating the pneumococcal autolysin LytA. We showed that strains of serotypes 1, 4, 6B and 23F were generally less lytic towards penicillin than other isolates. In addition, the isolates belonging to serotype 9V were the only ones showing reduced lysis towards vancomycin. Nonencapsulated strains were also more lytic than encapsulated ones, both upon addition of lytic antibiotics and in the stationary phase. In the second study we identified a novel pneumococcal virulence factor, the pilus. This molecule is encoded by the rlrA pathogenicity islet, containing three structural genes (rrgA-C), three sortases (srtB-D) and a positive regulator (rlrA). With electron microscopy and immunogold staining we could show that pili are mainly built up by RrgB, with RrgA located at the base and RrgC present on the tip of the structure. Mutants lacking pili were less virulent and caused less inflammation in an animal model. The importance of pili was further established in the third study, where we looked at the distribution of pneumococci nonsusceptible to penicillin (PNSP) in Sweden. Between 1999 and 2003 the frequency of serotype 14 increased from 12% to 26% of all PNSP. By investigating a large number of PNSP isolates of types 9V, 14 and 19F with multilocus sequence typing and pulsed-field gel electrophoresis we concluded that a serotype switch had occurred with a 9V clone obtaining the type 14 capsule. The rise seen in PNSP of serotype 14 could be attributed to this clone, known as Spain9V-3 of ST156. We also showed that about 50% of all PNSP in 2003 belonged to this clonal cluster and that its success may be explained by the fact that it carries the rlrA islet and expresses pili. Two type 19F isolates of ST156 were studied in a mouse model of colonization. These isolates were identical as shown by microarray, except that one of them was piliated. The piliated strain outcompeted the nonpiliated one, again showing the advantage of piliation in colonization. In the final study we correlated in vitro fitness to in vivo virulence. The fitness of a panel of clinical isolates with known virulence in mice was monitored. Two isolates each of types 1, 6B, 7F, 14 and 19F were included, chosen to be as genetically different as possible. Only one of the isolates, a type 14 isolate, had a growth rate much slower than the rest. Interestingly, the type 14 strains differed the most in virulence, when comparing the strains within each serotype pair to each other. The slow-growing strain was less virulent than the other type 14 isolate in a mouse model of infection. To further investigate the effect of fitness defects on virulence, five mutants were constructed and analyzed. These were mutated in five well-conserved housekeeping genes, resulting in growth defects for three of them. These three mutants were, to different degrees, attenuated in virulence in an intranasal mouse model. We conclude that pneumococcal fitness is of great importance for in vivo virulence.