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Lipoproteins of Staphylococcus aureus represent a major class of surface proteins, which are anchored to the outer leaflet of the cell membrane. Although they play a key role in the immune response and virulence, the majority of lipoproteins in this organism is still of unknown function. The aim of our study was to investigate the function of so far poorly or uncharacterized lipoproteins in S. aureus strain Newman. To this end, an integrated bioinformatical approach was applied to define the pan-lipoproteome of 123 completely sequenced S. aureus strains. In total, this analysis predicted 192 different potential lipoproteins, with a core lipoproteome of 39 and a variable lipoproteome of 153 lipoproteins. Out of those 192 lipoproteins, 141 are so far functionally uncharacterized. Primarily focusing on members of the core-lipoproteome with unknown or poorly characterized function, 24 lipoproteins or co-encoded neighbor proteins were selected for further characterization. Of those 24 proteins, 20 S. aureus markerless deletion mutants were constructed (S. aureus delta l01 - delta l20) and screened for an altered growth behavior under various conditions. Here, three mutants showed a temperature-sensitive phenotype, two mutants formed aggregates in the TSB of the manufacturer Merck (TSBMerck), and four mutants showed reduced growth under osmotic stress with 8% NaCl. An altered aggregation behavior was observed for four mutants in the presence of Triton X-100 and for eleven mutants in the presence of SDS. Furthermore, ten mutants revealed an impaired biofilm formation capacity as well as reduced hemolytic activity. Interestingly, S. aureus deletion mutants delta l14 (delta NWMN_1435) and delta l16 (delta NWMN_0646) showed an altered phenotype under nearly all tested growth and stress conditions. Most strikingly, both deletion mutants demonstrated dramatic defects in cell morphology and cell division during the transient growth phase in TSBMerck and were therefore selected for further detailed characterization. Electron microscopy imaging of the two mutants revealed an irregular cell shape, increased cell size, multiple displaced division septa, and incomplete separation of daughter cells resulting in the formation of cell aggregates in TSBMerck. Complementarily, microarray-based transcriptome analysis and whole-genome sequencing of S. aureus delta l14 and delta l16 suppressor mutants strongly point to a functional association of both lipoproteins with cell envelope- or cell division-related processes. Specifically, multiple hints suggest a functional connection of both lipoproteins with lipo- or wall teichoic acids. Of note, the phenotypes of S. aureus delta l14 and delta l16 are conditional and appear under some, but not all growth conditions. Thus, it is conceivable that the function of L14 and L16 is modulated by metabolic processes, or that the proteins might be part of a “backup system” becoming important only under certain conditions. Collectively, we propose that L14 and L16 fulfill a basic role in cell envelope- or cell division-related processes under specific growth conditions. Particularly, the activity of L14 and L16 might be necessary for the function or localization of lipo- or wall teichoic acids, and thus, might be linked to the regulation of autolysins. In conclusion, this study reveals important insights into the function of two so far uncharacterized but highly conserved lipoproteins in S. aureus.
Streptococcus pneumoniae (pneumococci) are lancet-shaped, Gram-positive, alpha-hemolytic, facultative anaerobic human specific commensals of the upper and lower respiratory tract. Pneumococci may convert to pathogenic bacteria and spread to the lungs and blood. In different population groups, such as children, the elderly and immunocompromised individuals, pneumococci can cause local infections such as bronchitis, rhinitis, acute sinusitis, and otitis media as well as life-threatening invasive diseases such as community-acquired pneumonia, sepsis and meningitis. Pneumococci are surrounded by a rigid and complex exoskeleton, the peptidoglycan, also referred to as murein sacculus. The peptidoglycan (PNG) protects the cells from rupture by osmotic pressure and maintains their characteristic shape. The PNG is a heteropolymer made up of glycan strands that are cross-linked by short peptides and during growth the existing murein is continuously hydrolyzed by specific lytic enzymes to enable the insertion of new peptidoglycan. Bacterial cell-wall hydrolases are essential for peptidoglycan turnover and crucial to preserve cell shape. The D,D-carboxypeptidase DacA and L,D-carboxypeptidase DacB of Streptococcus pneumoniae function in a sequential manner. This study determined the crystal structure of the surface-exposed lipoprotein DacB, which differs considerably from the DacA structure. DacB contains a Zn2+ ion in its catalytic center located in the middle of a fully exposed, large groove. Two different conformations with differently arranged active site topology were identified. In addition the critical residues for catalysis and substrate specificity were identified. Deficiency in DacA or DacB resulted in a modified peptidoglycan peptide composition and led to an altered cell shape of the dac-mutants. In contrast, lgt-mutant lacking lipoprotein diacylglyceryl transferase activity required for proper lipoprotein maturation retained L,D-carboxypeptidase activity and showed an intact murein sacculus. Furthermore, this study demonstrated the pathophysiological effects of disordered DacA or DacB activities. Real-time bioimaging of intranasally infected mice indicated a substantially attenuated virulence of dacB- and dacAdacB-mutants pneumococci, while loss of function of DacA had no significant effect. In addition, uptake of these mutants by professional phagocytes was enhanced, while their adherence to lung epithelial cells was decreased. The second part of this study focused on the functional and structure determination of the soluble dimeric pneumococcal lipoprotein PccL. Because of its calycin fold and structural homology with the lipocalin YxeF from Bacillus subtilis, PccL was introduced as the first member of the lipocalin protein family in pneumococci and named “PccL” (Pneumococcal calycin fold containing Lipoprotein). Similar to other lipocalins, the distinct beta-barrel, which is open at one end, is significantly conserved in PccL. Moreover, the application of the in vivo acute pneumonia mouse infection model and the in vitro phagocytosis as well as adherence invasion studies revealed considerable differences in colonization and invasive infection between the wild-type D39 and the pccL-mutant. In conclusion, this study characterized the crucial role of pneumococcal carboxypeptidases DacA and DacB for PGN architecture, bacterial shape and pathogenesis. By applying in vivo and in vitro approaches, a close relationship between PGN metabolism and pathophysiological effects was discovered. In addition, the high resolution structure of DacB has been solved and analyzed and a structure model with a resolution of 2.0 Å is provided. Furthermore, analysis of the PGN composition was applied to indicate the impact of an impaired lipoprotein biogenesis pathway on localization and activity of DacB. The major impact of carboxypeptidases on cell shape and virulence proposes DacB as a promising target for the development of novel drugs or due to its surface exposition also as a promising vaccine candidate. PccL is the first pneumococcal lipocalin-like protein and this study indicated its contribution to pneumococcal virulence. However, the mechanism and the mode of action of PccL are still unknown and have to be deciphered in further studies.