Pneumococcal Pili and Other Cell Surface Properties Affect the Infection Biology of Streptococcus Pneumoniae

The pneumococcus, Streptococcus pneumoniae, is a major respiratory tract disease causing pathogen with an estimated mortality of 1-2 million per year worldwide. It is the most common bacterial cause of sinusitis, otitis media and community acquired pneumonia and also a major cause of invasive diseases such as septicaemia and meningitis. Rarely bacteraemia is associated with cardiac complications, such as endocarditis and pericarditis. Pneumococci are also frequent colonizers of the nasopharynx of healthy children attending day-care centers and may be considered as mucosal commensals of the nasopharyngeal microflora. The aim of this thesis was to investigate pneumococcal adhesion factors and other cell wall properties involved in the interaction with the human host in regard to colonization, virulence, and treatment. Clinical isolates, from patients hospitalised with a pneumococcal bacteraemia associated with a pneumococcal endocarditis or pericarditis, were collected and analysed. An associated condition such as pneumonia, sinusitis, meningitis or sepsis, appeared to be a prerequisite for disease, and was present in all cases. In the study we found that streptococci seem to require pre-existing endothelial lesions in order to adhere to cardiac valves and cause endocarditis. We could not find any specific virulence attributes, such as capsular types or adhesive properties in the examined isolates. A novel pilus-like structure was found on the surface of pneumococci, encoded by the rlrA pathogenicity islet. The rlrA islet contains the genes rlrA, encoding the positive regulator, rrgA, rrgB, rrgC, encoding three structural pili subunits and srtB, srtC, srtD, encoding three sortases involved in the linkage and assembly of the pilus structure. We found that the pilus shaft consists of a polymer of RrgB subunits with RrgC as the tip protein. RrgA is distributed in intervals along the shaft and monomers are also associated with the pneumococcal cell wall. The pilus is one of the major, but not the only, adherence factor to lung epithelial cells. We show that RrgA is the major adhesion among the pilus proteins. However, the pilus as such does no seem to be essential for in vitro adherence as long as RrgA is present. Our observations also show, that RrgA is the major protein involved in virulence and pathogenicity, inducing earlier death and high levels of TNF and IL-6 in murine infection models. Lysis is a characteristic feature of pneumococci. We examined contributions of cell wall charge and pH alterations on LytA-dependent stationary phase and antibiotic induced lysis. An exogenous pH below 6 or fermentation of glucose and subsequent low pH levels in the growth medium effectively abolished lysis but not growth or death of the bacteria. Pneumococcal cell wall pH levels were detected by FITC emission. We could not find any significant pH changes in the cell wall of bacteria in liquid cultures. However, a biofilm model in which non-encapsulated T4R and T4R∆lytA attached as monolayers, showed that treatment with penicillin G, vancomycin or valinomycin (a proton motive force dissipator), but not the non-lytic erythromycin, led to an instant pH shift. This pH shift was dependent upon endogenous H202 production as it required an intact pyruvate oxidase gene, spxB, and could be prevented with exogenous addition of catalase. LIST OF PUBLICATIONS I. Kan B, Ries J, Normark B.H., Chang FY, Feldman C, Ko WC, Rello J, Snydman DR, Yu VL, Ortqvist A. 2006. Endocarditis and pericarditis complicating pneumococcal bacteraemia, with special reference to the adhesive abilities of pneumococci: results from a prospective study. Clin Microbiol Infect. 12(4):338-44. II. Barocchi MA, Ries J, Zogaj X, Hemsley C, Albiger B, Kanth A, Dahlberg S, Fernebro J, Moschioni M, Masignani V, Hultenby K, Taddei AR, Beiter K, Wartha F, von Euler A, Covacci A, Holden DW, Normark S, Rappuoli R, Henriques-Normark B. 2006. A pneumococcal pilus influences virulence and host inflammatory responses. Proc Natl Acad Sci U S A. 103(8):2857-62. III. Nelson, AL, Ries J, Bagnoli F, Dahlberg S, Rounioja S, Tschöp J, Morfeldt E, Holden D, Rappuoli R, Normark S, Barocchi MA and Henriques-Normark B. 2007. RrgA is a pilus-associated adhesin and virulence factor in S. pneumoniae. Manuscript. IV. Ries, J, Kjäll P, Beiter K, Wartha F, Syk A, Richter-Dahlfors A, Morfeldt E, Normark S and Henriques-Normark B. 2007. Lytic antibiotics affect cell wall pH of Streptococcus pneumoniae dependent upon endogenous H202 production. Submitted manuscript. * Authors contributed equally.