| Abstract: | The Gram-positive pathogen Staphylococcus aureus secretes various proteins into its extracellular milieu. Bioinformatics analyses have indicated that most of these proteins are directed to the canonical Sec pathway, which consists of the translocation motor SecA and a membrane-embedded channel composed of the SecY, SecE, and SecG proteins. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins. Here, we have addressed the roles of the nonessential channel components SecG and SecY2 in the biogenesis of the extracellular proteome of S. aureus. The results show that SecG is of major importance for protein secretion by S. aureus. Specifically, the extracellular accumulation of nine abundant exoproteins and seven cell wall-bound proteins was significantly affected in an secG mutant. No secretion defects were detected for strains with a secY2 single mutation. However, deletion of secY2 exacerbated the secretion defects of secG mutants, affecting the extracellular accumulation of one additional exoprotein and one cell wall protein. Furthermore, an secG secY2 double mutant displayed a synthetic growth defect. This might relate to a slightly elevated expression of sraP, encoding the only known substrate for the Sec2 pathway, in cells lacking SecG. Additionally, the results suggest that SecY2 can interact with the Sec1 channel, which would be consistent with the presence of a single set of secE and secG genes in S. aureus.Staphylococcus aureus is a well-represented component of the human microbiota as nasal carriage of this Gram-positive bacterium has been shown for 30 to 40% of the population (32). This organism can, however, turn into a dangerous pathogen that is able to infect almost every tissue in the human body. S. aureus has become particularly notorious for its high potential to develop resistance against commonly used antibiotics (20, 49). Accordingly, the S. aureus genome encodes an arsenal of virulence factors that can be expressed when needed at different stages of growth. These include surface proteins and invasins that are necessary for colonization of host tissues, surface-exposed factors for evasion of the immune system, exotoxins for the subversion of protective host barriers, and resistance proteins for protection against antimicrobial agents (37, 57).Most proteinaceous virulence factors of S. aureus are synthesized as precursors with an N-terminal signal peptide to direct their transport from the cytoplasm across the membrane to an extracytoplasmic location, such as the cell wall or the extracellular milieu (38, 45). As shown for various Gram-positive bacteria, the signal peptides of S. aureus are generally longer and more hydrophobic than those of Gram-negative bacteria (38, 54). On the basis of signal peptide predictions using a variety of algorithms, it is believed that most exoproteins of S. aureus are exported to extracytoplasmic locations via the general secretory (Sec) pathway (38). This seems to involve precursor targeting to the Sec machinery via the signal recognition particle instead of the well-characterized proteobacterial chaperone SecB, which is absent from Gram-positive bacteria (16, 19, 53). The preproteins are then bound by the translocation motor protein SecA (38, 45). Through repeated cycles of ATP binding and hydrolysis, SecA pushes the protein in an unfolded state through the membrane-embedded SecYEG translocation channel (12, 30, 33, 52). Upon initiation of the translocation process, the proton motive force is thought to accelerate preprotein translocation through the Sec channel (26). Recently, the structure of the SecA/SecYEG complex from the Gram-negative bacterium Thermotoga maritima was solved at 4.5 Å resolution (58). In this structure, one SecA molecule is bound to one set of SecYEG channel proteins. The core of the Sec translocon consists of the SecA, SecY, and SecE proteins, which are essential for growth and viability of bacteria, such as Escherichia coli and Bacillus subtilis (6, 9, 22). In contrast, the channel component SecG is dispensable for growth, cell viability, and protein translocation (26, 48). Nevertheless, SecG does enhance the efficiency of preprotein translocation through the SecYE channel (26, 48). This is of particular relevance at low temperatures and in the absence of a proton motive force (17). Several studies suggest that E. coli SecG undergoes topology inversion during preprotein translocation (25, 27, 43). Even so, van der Sluis et al. reported that SecG cross-linked to SecY is fully functional despite its fixed topology (46). During or shortly after membrane translocation of a preprotein through the Sec channel, the signal peptide is removed by signal peptidase. This is a prerequisite for the release of the translocated protein from the membrane (1, 47).Several pathogens, including Streptococcus gordonii, Streptococcus pneumoniae, Bacillus anthracis, Bacillus cereus, and S. aureus, contain a second set of chromosomal secA and secY genes named secA2 and secY2, respectively (39). Comparison of the amino acid sequences of the SecY1 and SecY2 proteins shows that their similarity is low (about 20% identity) and that the conserved regions are mainly restricted to the membrane-spanning domains. It has been shown for S. gordonii that the transport of at least one protein is dependent on the presence of SecA2 and SecY2. This protein, GspB, is a large cell surface glycoprotein that is involved in platelet binding (4). The protein contains an unusually long N-terminal signal peptide of 90 amino acids, large serine-rich repeats, and a C-terminal LPXTG motif for covalent cell wall binding. The gspB gene is located in a gene cluster with the secA2 and secY2 genes. Two other genes in this cluster encode the glycosylation proteins GftA and GftB, which seem to be necessary for stabilization of pre-GspB. Furthermore, the asp4 and asp5 genes in the secA2 secY2 gene cluster show similarity to secE and secG, and they are important for GspB export by S. gordonii (44). Despite this similarity, SecE and SecG cannot complement for the absence of Asp4 and Asp5, respectively. The secA2-secY2 gene cluster is also present in S. aureus, but homologues of the asp4 and asp5 genes are lacking. This seems to suggest that SecA2 and SecY2 of S. aureus share the SecE and SecG proteins with SecA1 and SecY1. The sraP gene in the secA2-secY2 gene cluster of S. aureus encodes a protein with features similar to those described for GspB. Siboo and colleagues (41) have shown that SraP is glycosylated and capable of binding to platelets. Importantly, the disruption of sraP resulted in a decreased ability to initiate infective endocarditis in a rabbit model. Consistent with the findings in S. gordonii, SraP export was shown to depend on SecA2/SecY2 (40). However, it has remained unclear whether other S. aureus proteins are also translocated across the membrane in an SecA2/SecY2-dependent manner.The present studies were aimed at defining the roles of two Sec channel components, SecG and SecY2, in the biogenesis of the S. aureus exoproteome. The results show that secG and secY2 are not essential for growth and viability of S. aureus. While the absence of SecY2 by itself had no detectable effect, the absence of SecG had a profound impact on the composition of the exoproteome of S. aureus. Various extracellular proteins were present in decreased amounts in the growth medium of secG mutant strains, which is consistent with impaired Sec channel function. However, a few proteins were present in increased amounts. Furthermore, the absence of secG caused a serious decrease in the amounts of the cell wall-bound Sbi protein. Most notable, a secG secY2 double mutant strain displayed synthetic growth and secretion defects. |
