Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus

Abstract

SummaryExcretion of cytoplasmic proteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG-binding protein, which is secreted via the Secpathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan-binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non-excreted Ndh2, a soluble NAD-H:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.

Non-classical Protein Excretion Is Boosted by PSMα-Induced Cell Leakage

“…There are arguments for both possibilities. For example, cells in the stationary growth phase or mutants with increased autolysis activity or altered cell wall structure release more CPs to the supernatant (Ebner et al, 2015a; Nega et al, 2015). On the other hand there are also evidences for a defined mechanism in ECP.…”

SagB Glucosaminidase Is a Determinant of Staphylococcus aureus Glycan Chain Length, Antibiotic Susceptibility, and Protein Secretion

“…Boiling staphylococci in suspension with ionic detergent does not lead to cell lysis or leakage of cytoplasmic protein, suggesting that staphylococcal murein sacculi are impenetrable for proteins (32). Nevertheless, during exponential growth, S. aureus secretes at least 59 proteins processed from signal peptide-bearing precursors into the culture medium in addition to excreting 53 polypeptides that apparently do not travel via the Sec pathway (33)(34)(35). S. aureus genes for cell wall synthesis and peptidoglycan processing were heretofore not reported to contribute to protein secretion.…”

Excreted Cytoplasmic Proteins Contribute to Pathogenicity in Staphylococcus aureus

“…We studied the excretion of these proteins in various staphylococcal strains to see whether the amount of excreted proteins correlates with the virulence of the strains. Since ECP occurs mainly during bacterial growth (19), samples were collected after 4 h (mid-exponential phase), and the relative amounts of FbaA and GAPDH in the cytoplasm (control) and culture supernatant were determined by Western blotting. The largest amounts of secreted FbaA and GAPDH were found in the Newman, HG001, HG003, and JE2 strains.…”

“…Subsequently cells were pelleted by centrifugation, and supernatant was applied to SDS-PAGE gels. Preparation of cytoplasmic proteins, enrichment of extracellular proteins, and Western blot analyses were performed as described by Ebner et al (19).…”

“…One of the main arguments is that only certain CPs were excreted, while others, although highly abundant in the cytoplasm, were not found extracellularly, suggesting that there is a selection principle at work (18). Studying the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase (FbaA) and enolase (Eno), showed that they are excreted mainly during the exponential growth phase in S. aureus and that the exit site is the septal cleft of dividing cells (19). Another typical excreted CP is the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is, like FbaA, an essential enzyme in glycolysis of S. aureus (18,20,21).…”