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Streptococcus pneumoniae (the pneumococcus) is an opportunistic human pathogen that causes life-threatening diseases including pneumonia, sepsis, meningitis but also non-invasive local infections such as otitis media. Pneumococci have evolved versatile strategies to colonize the upper respiratory tract (URT) of humans. Binding to epithelial surfaces is thereby mediated through direct interactions with host cell receptors or indirectly via binding to components of the extracellular matrix (ECM). However, successful colonization and subsequent infection require S. pneumoniae to cross tissue barriers protected by the immune system of the host. Pneumococci have therefore evolved a wide range of mechanisms to circumvent the antibacterial activity of the immune system such as the acquisition or expression of serine protease activity. Serine protease enzymes have emerged during evolution as one of the most abundant and functionally diverse groups of proteins in eukaryotic and prokaryotic organisms. However, the epithelial barriers, integrins, and other cell surface receptors are often initially inaccessible for pneumococci colonizing the nasopharyngeal cavity. Therefore, pneumococci recruit host-derived extracellular serine proteases such as plasmin(ogen) for extracellular matrix and mucus degradation, which results in enhanced binding to epithelial and endothelial cells. S. pneumoniae expresses four surface-anchored or surface-associated serine proteases depending on the serotype: HtrA, SFP, PrtA, and CbpG. These enzymes belong to the category of trypsin-like or subtilisin-like family proteins, which are characterized by the presence of three-conserved amino acid residues, Ser-His-Asp. The catalytic triads are critical for the cleavage of peptide bonds. Studies focusing on the deletion of single pneumococcal serine proteases are difficult to interpret due to the compensatory effects of the other serine proteases.
Initially, a comprehensive in silico analysis of the distribution and genes organization of pneumococcal serine proteases was carried out in this study. Interestingly, the genes encoding PrtA, HtrA, and CbpG were highly conserved among the 11 analyzed strains. Surprisingly, the gene encoding the subtilisin-like protein SFP was not present in some of the strains and seems to be strain-dependent. Therefore, pneumococci have at least three serine proteases as shown e.g., for serotype 19F_EF3030 strain. Computer-assisted analyses of the structure of pneumococcal serine proteases showed high similarities in the catalytic domains between HtrA and CbpG or between PrtA and SFP in 3D structural models.
The focus of this study lies on the impact of single extracellular pneumococcal serine proteases on pneumococcal pathogenesis during adherence, colonization, virulence and biofilm formation. Therefore, double and triple deletion mutants were generated in the colonizing S. pneumoniae serotype 19F strain EF3030 and the more invasive serotype 4 strain TIGR4, respectively. In adherence studies with human Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F_EF3030 mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. In a mouse colonization model, the inactivation of serine proteases in strain 19F_EF3030 strongly reduced nasopharyngeal colonization in CD-1 mice. The bacterial load in the nasopharynx was thereby monitored for a period of 14 days. Mutant strains showed significantly lower bacterial numbers in the nasopharynx on days 2, 3, 7, and 14 post-inoculations.
Following up on pneumococcal pathogenesis, an in vivo acute pneumonia mouse infection model and in vitro phagocytosis was used to analyze the impact of single serine proteases during infection and phagocytosis. Mice were intranasally infected with the bioluminescent TIGR4lux wild-type or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. The acute lung infection was monitored in real-time by using an IVIS®-Spectrum in vivo imaging system. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. Phagocytosis assays were conducted using murine J77A.1 macrophages. The number of triple serine protease mutants internalized by macrophages were significantly reduced in comparison to the isogenic wild-type.
Finally, two different experimental biofilm models were used to study the influence of serine proteases on biofilm formation grown on an abiotic surface (glass) and a biological surface. Biofilm development on living epithelial cells was stronger after 48 and 72h than on the glass surface. On epithelial substratum, the serine protease mutant with only CbpG+ showed higher and denser biofilm development after 48h and 72h of incubation compared to the parental strains and other serine protease mutants. Moreover, the bacterial dispersal from biofilms was significantly more in the mutant strains lacking serine proteases than in the wild type.
In conclusion, nasopharyngeal colonization is a prerequisite for invasive diseases and transmission. Pneumococcal serine proteases are indispensable for nasopharyngeal colonization and facilitate access to eukaryotic cell-surface receptors by the cleavage of ECM proteins. Thus, serine proteases could be promising candidates for developing antimicrobials to reduce pneumococcal colonization and transmission.