Septal secretion of protein A in Staphylococcus aureus requires SecA and lipoteichoic acid synthesis
- University of Chicago, United States
Figures
Figure 1
Mutagenesis of the signal peptide of staphylococcal protein A (SpA).
(a) Schematic illustrating the primary structure of SpA and of SpAED with the immunoglobulin binding domains (IgBDs, designated E, A, B, C and D), region X (Xr), LysM domain and LPXTG sorting …
Figure 2
SpA signal peptide variants defective in precursor processing and septal secretion.
(a) S. aureus cultures were fractionated into cytoplasm (C), membrane (M), cell wall (W) and culture supernatant (S) and analyzed by immunoblotting with αSpA to reveal the subcellular location of …
Figure 3
Proteolytic cleavage of the SpA signal peptide.
(a) Wild-type SpAED or SpAED/ΔIA, SpAED/S18L, and SpAED/R10A variant precursors and cleavage products were purified from detergent-solubilized staphylococcal membranes using affinity chromatography …
Figure 4 with 1 supplement
Crosslinking of staphylococcal proteins to SpAED/S18L or SpAED/SP-SasF.
(a) Bacteria from S. aureus WY110 (pSpAED/S18L) and S. aureus WY110 (pSpAED/ SP-SasF) cultures were crosslinked with 0.9% formaldehyde, membrane proteins detergent-solubilized and SpAED/S18L as well …
Figure 4—figure supplement 1
SpA septal secretion analysis in S. aureus mutants with knockout or conditional mutations in genes whose products were crosslinked to SpAED/S18L.
(a) Localization of SpA secretion in ΔezrA, ΔrseP, ΔclpC and PBP2-depleted S. aureus. To detect intracellular localization of SpA in mutants with known cell division or peptidoglycan synthesis …
Figure 5
Depletion of SecA in S. aureus.
(a) Diagram of the secA gene locus in S. aureus RN4220 (wild-type parent, WT) and its Pspac-secA variant. (b) Bacteria from overnight cultures of wild-type S. aureus and S. aureus Pspac-secA grown …
Figure 6
SecA depletion diminishes septal secretion of SpA in S. aureus.
(a) SpA precursor processing of [35S]methionine pulse-labeled S. aureus RN4220 (WT) or S. aureus Pspac-secA grown in the presence or absence of 1 mM IPTG. Bacteria were pulse-labeled for 60 s with …
Figure 7
SecA localization in staphylococci.
(a) Diagram of the secA gene locus and of the pCL55-mediated att insertion site for secA-sfGFP in the staphylococcal genome. (b) Bacteria from overnight cultures of S. aureus RN4220 (WT), S. aureus …
Figure 8
Intracellular trafficking of SpA in the presence and absence of SecA.
(a) S. aureus Pspac-secA cells were grown for 3 hr in the presence (+SecA) or absence of 1 mM IPTG (-SecA) and, alongside S. aureus ∆spa control cells, were trypsin treated to remove extracellular …
Figure 9
SecDF contributes to SpA secretion at septal membranes.
(a) S. aureus cultures were centrifuged to sediment the bacteria into the pellet (P) fraction and separate them from the extracellular medium (S, supernatant). Following lysostaphin digestion of …
Figure 10
Localization of SpA secretion in LtaS-depleted S. aureus.
(a) Schematic to illustrate the ltaS locus in S. aureus RN4220 and ANG499. (b) Fluorescence microscopy with BODIPY-FL-vancomycin (green) and αSpA (red) stained samples 20 min after trypsin removal …
Tables
Table 1
MALDI-TOF-MS ion signals of purified SpAED species and their variants
https://doi.org/10.7554/eLife.34092.005| Protein | Observed m/z | Calculated m/z* | ∆obs.- calc. |
|---|---|---|---|
| SpAED-1 | 16776.47 | 16777.7 | 1.23 |
| SpAED-2 | 15273.92 | 15273.78 | 0.14 |
| SpAED-3 | 14418.92 | 14418.78 | 0.14 |
| SpAED-4 | 13152.40 | 13152.32 | 0.08 |
| SpAED/S18L-1 | 16803.02 | 16803.78 | 0.76 |
| SpAED/S18L-2 | 15298.89 | 15299.86 | 0.97 |
| SpAED/S18L-3 | 14417.89 | 14418.78 | 0.89 |
| SpAED/S18L-4 | 13152.22 | 13152.32 | 0.10 |
| SpAED/∆IA-1 | 16592.95 | 16593.46 | 0.51 |
| SpAED/∆IA-2 | 15088.71 | 15089.54 | 0.83 |
| SpAED/∆IA-3 | 14417.86 | 14418.78 | 0.92 |
| SpAED/∆IA-4 | 13151.55 | 13152.32 | 0.77 |
| SpAED/R10A-1 | 16692.99 | 16692.59 | 0.40 |
| SpAED/R10A-2 | 15584.97 | 15586.2 | 1.23 |
| SpAED/R10A-3 | 14418.46 | 14418.78 | 0.32 |
| SpAED/R10A-4 | 13152.07 | 13152.32 | 0.25 |
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*Based on average mass calculated with the online ExPASy tool.
Additional files
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Supplementary file 1
List of ESI-MS identified tryptic peptides crosslinked to SpAED/S18L.
- https://doi.org/10.7554/eLife.34092.014
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Supplementary file 2
Strains and plasmids used in this study.
- https://doi.org/10.7554/eLife.34092.015
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Supplementary file 3
Oligonucleotide primers used in this study.
- https://doi.org/10.7554/eLife.34092.016
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Transparent reporting form
- https://doi.org/10.7554/eLife.34092.017
