Characterization of host and microbe interactions affecting adherence, clearance and systemic spread of S. pneumoniae

Sammanfattning: Streptococcus pneumoniae (the pneumococcus) mainly gives rise to diseases of the respiratory tract, such as pneumonia, acute otitis media and sinusitis, but it also causes severe invasive diseases, such as meningitis and sepsis. The pneumococcus accounts for approximately two million deaths world-wide every year, the majority of which are children in developing countries. However, the most common form of pneumococcushost interaction is asymptomatic colonization of the nasopharynx. The factors determining why the pneumococcus is sometimes a harmless colonizer, while other times a detrimental pathogen are incompletely understood, and both bacterial and host factors are likely to contribute. The aim of the work presented in this thesis was, thus, to study the interplay between the pneumococcus and the host in adherence, clearance, and systemic spread of pneumococci. Several host receptors, such as Toll-like receptors (TLRs), complement receptors (CRs) and scavenger receptors, are involved in recognition and clearance of microorganisms. The TLRs recognize danger-associated molecular patterns, and TLR9 signaling is induced by CpG motifs in bacterial DNA. Many of the TLRs signal through the adaptor protein MyD88, which is crucial for the innate immune defense against pneumococci. CR3 is another receptor expressed on phagocytes, such as macrophages and neutrophils. It has been shown to mediate phagocytosis of several different bacterial species. The recently discovered pneumococcal pilus is encoded by the rlrA pathogenicity islet. RrgA, RrgB and RrgC are the subunits building up the pilus fiber. RrgB constitutes the backbone while the other two are ancillary subunits decorating the pilus shaft. We identified RrgA as the main pilus adhesin, and found that this protein was able to mediate adherence to epithelial cells in vitro, even in the absence of a pilus polymer. In a mouse model of colonization, RrgA expression led to higher numbers of bacteria in the nasopharynx. Additionally, we identified RrgA as the main player in the interaction with murine primary macrophages. Here, expression of RrgA enhanced both phagocytosis of bacteria and motility of macrophages. The increased uptake of RrgA-positive bacteria also resulted in longer intracellular survival in macrophages. CR3 was identified as the macrophage receptor interacting with RrgA. In vivo, murine expression of CR3 and bacterial expression of RrgA led to faster appearance of bacteria in the bloodstream and a more rapid onset of disease. Taken together, these data led us to hypothesize that a CR3-RrgA interaction promotes translocation of bacteria from the peritoneal cavity to the bloodstream, possibly by employing phagocytes. The role of TLRs in the innate immune defense against pneumococci was also investigated. We found that TLR9, but not TLR1, TLR2, TLR4, TLR6 or IL-1R/IL-18R, played a non-redundant role in defense against pneumococcal infection. TLR9-deficient mice showed an impaired clearance of the bacteria in the lungs within the first hours of infection. In vitro, murine primary macrophages were also impaired in their ability to phagocytose pneumococci, and the intracellular killing was delayed in the absence of TLR9. Therefore, we hypothesize that TLR9 mediates efficient clearance of pneumococci in the lungs by upregulating phagocytic receptors on alveolar macrophages. The work presented in this thesis provides deeper knowledge of certain factors that affect the transition from colonization to disease. The pilus-associated proteins are putative vaccine candidates, and it is therefore important to characterize their contribution to pneumococcal pathogenesis. By further increasing the understanding of effector mechanisms involved in the immune defense, treatment regimens may be improved.