Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors
- Mechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore
- Biochemistry and Molecular Biology, University of Texas Medical Branch, United States
Figures
Figure 1
Scheme for site-specific fluorescent labeling of SPI-2 effectors.
(A) Chemical structure of 4-azidophenylalanine (AzF), 7-(Hydroxy-coumarin-4-yl) ethylglycine (Hco), trans-Cyclooct-2-en – L - Lysine (TCO*Lys), BDP-DBCO, and SiR-tetrazine. (B) Incorporation of a …
Figure 2 with 5 supplements
AzF is site-specifically incorporated into SifA-E52TAG in Salmonella.
(A) Left: Coomassie stained SDS-PAGE; right: SDS-PAGE fluorescence imaging confirms selective labeling of SifA-E52AzF with BDP-DBCO in the cell lysate. (B) Expression of SifA-E52AzF in Salmonella …
Figure 2—figure supplement 1
SsaP is required for SPI-2 secretion.
In the wild-type background, the secreted translocon protein SseB appears in both whole cell and secreted fractions (left panel, lane 1). In the ssaP null strain, SseB is produced, but not secreted. …
Figure 2—figure supplement 2
Selection of ncAA incorporation sites.
Residue conservation scores calculated from multiple sequence alignments were mapped onto the crystal structure of SifA (PDB ID: 3CXB) using the ConSurf web server (http://consurf.tau.ac.il/). The …
Figure 2—figure supplement 3
Western blot of SseB (translocon) and SsaP-HA secretion.
Whole cell and secreted fractions were harvested after growth in SPI-2-inducing conditions. The SseB translocon was secreted in the wild-type strain (far left), but not in the ssaP null strain. …
Figure 2—figure supplement 4
AzF and subsequent Alexa555 sDIBO Alkyne incorporation is highly specific.
Cells were transformed with pEVOL and psifA52TAG-GFP or psifA-WT-GFP were grown in the presence of 1 mM AzF and 0.2% L-arabinose in MgM pH 5.6, 37°C. Cells were harvested at OD600 = 0.4. After …
Figure 2—figure supplement 5
Average fraction of cells that display polarly localized SsaP (ntotal = 112 cells).
Error bars are standard deviation across 18 ROIs pooled from three independent experiments.
-
Figure 2—figure supplement 5—source data 1
Source data for Figure 2—figure supplement 5.
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig2-figsupp5-data1-v2.xlsx
Figure 3 with 5 supplements
Functional complementation of SIF formation by SifA-E52AzF.
(A) HeLa cells were infected with a sifA null strain of Salmonella expressing SifA-E52AzF and incubated for 16 hr. Infected cells were fixed with 4% PFA and immuno-stained for Salmonella (yellow) …
Figure 3—figure supplement 1
The amber suppression system (pEVOL) does not affect Salmonella pathogenesis.
(A) Growth curves of wild-type Salmonella and its isogenic sifA mutant in LB media in the presence of pEVOL. The error bars represent the mean ± standard deviation (n = 3). (B) Survival assay of …
-
Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig3-figsupp1-data1-v2.xlsx
-
Figure 3—figure supplement 1—source data 2
Source data for Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig3-figsupp1-data2-v2.xlsx
Figure 3—figure supplement 2
A sifA null strain lacks SIFs.
HeLa cells were infected with Salmonella for 16 hr, fixed and immuno-stained for LAMP1 (red), LPS (yellow), and DAPI (blue). As expected, the formation of SIFs was observed in cells infected with WT …
Figure 3—figure supplement 3
No off-target labeling of secreted effectors is observed within host cells.
HeLa cells were infected with WT-Salmonella harboring SseJ-HA and pEVOL in the presence of AzF. At 10 hr post infection, excess AzF was washed out and replaced with fresh growth media without AzF …
Figure 3—figure supplement 4
The average fraction of infected cells that contain LAMP1 positive SIFs and SIFs containing labeled SifA.
The total number of LAMP1 positive cells was 85 for WT, 91 for sifA (psifA52TAG + pEVOL), and 70 for sifA (psifA52TAG + pEVOL) labeled with the click reaction. The error bars indicate the standard …
-
Figure 3—figure supplement 4—source data 1
Source data for Figure 3—figure supplement 4.
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig3-figsupp4-data1-v2.xlsx
Figure 3—figure supplement 5
The proportion of tubules with labeled SifA and its colocalization with LAMP1 (n = 27); n is the total number of cells analyzed.
Error bars represent the standard deviation across three data sets pooled from separate infections.
-
Figure 3—figure supplement 5—source data 1
Source data for Figure 3—figure supplement 5.
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig3-figsupp5-data1-v2.xlsx
Figure 4 with 4 supplements
Super-resolution imaging of GCE-labeled SifA.
(Ai) SifA colocalizes with SseJ. Spinning-disk SIM image of an infected HeLa cell, 16 hr after invasion, fixed with PFA and stained for SifA (green) and SseJ-HA (red). (Aii) Magnified view of SifA …
-
Figure 4—source data 1
Source data for Figure 4(iii-iv)..
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig4-data1-v2.xls
-
Figure 4—source data 2
Source data for Figure 4(iii-iv).
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig4-data2-v2.xls
Figure 4—figure supplement 1
The proportion of tubules with labeled SifA and its colocalization with SseJ (n = 33) and kinesin (n = 35); n is the total number of cells analyzed.
Error bars represent the standard deviation across three data sets pooled from separate infections.
-
Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/67789/elife-67789-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
GA fixation preserves microtubules.
(A) HeLa cells were infected with sifA mutants harboring pSifA52TAG and pEVOL-AzF. At 10 hr post infection, excess AzF was washed out and replaced with fresh growth media without AzF for 2 hr. At 14 …
Figure 4—figure supplement 3
Functional complementation of SIFs by expression of fluorescent SifA-E52Hco.
(A) HeLa cells were infected with the sifA null strain of Salmonella expressing SifA-E52Hco and SseJ-HA for 16 hr, fixed and immuno-stained for LAMP1 (green), SseJ (red). The SifA-E52Hco strain was …
Figure 4—video 1
Live cell imaging of secreted SifA labelled with BDP-DBCO dye during HeLa cell infection.
Figure 5 with 5 supplements
SsaP is secreted during HeLa cell infection.
(A) SPIEDAC labeling of secreted SifA-Y65TCO with fluorogenic SiR-Tz. HeLa cells were infected with the ssaP null mutant of Salmonella expressing SsaP-Y65TCO in the presence of TCO*Lys for 12 hr. …
Figure 5—figure supplement 1
An ssaP null mutant is defective in SPI-2 secretion.
(A) HeLa cells were infected with the ssaP null mutant for 16 hr, fixed and immuno-stained for LPS (green), SseJ (red) and DAPI (blue). In the absence of ssaP, SseJ is not secreted. (B) …
Figure 5—figure supplement 2
Survival of S. Typhimurium SsaP-55HA mutant in HeLa cells.
Strains of Salmonella wild-type, ssaP null, ssaP/λssaP (complemented with ssaP integrated a λatt), and ssaP/ λssaP-55HA (ssaP with an HA tag at amino acid 55) were grown in LB to late exponential …
Figure 5—figure supplement 3
SsaP is secreted.
Strains of Salmonella containing ssaP/λssaP-55HA (left) ssaP/λssaP-110HA (middle and right) were grown in LB to late exponential phase and added to HeLa cells at an MOI of 100:1 for 30 min. The …
Figure 5—figure supplement 4
SsaP was associated with bacteria and mostly localized to the poles during early infection times.
(A) HeLa cells were infected with an ssaP null mutant of Salmonella expressing SsaP-YTCO*Lys in the presence of TCO*Lys. After 6 hr post infection, HeLa cells were incubated with 1.5 μM SiR-Tz in …
Figure 5—figure supplement 5
Schematic of endosomal tubule (SIF) formation.
Upon entry into the host, Salmonella resides within an acidic vacuole. The histidine kinase EnvZ senses the acid pH of the Salmonella cytoplasm (Park et al., 2017) and drives OmpR-dependent …
