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. 2008 May 14;3(5):e2178.
doi: 10.1371/journal.pone.0002178.

Hierarchical effector protein transport by the Salmonella Typhimurium SPI-1 type III secretion system

Brit Winnen  1 , Markus C Schlumberger, Alexander Sturm, Kaspar Schüpbach, Stefan Siebenmann, Patrick Jenny, Wolf-Dietrich Hardt
Affiliations

Hierarchical effector protein transport by the Salmonella Typhimurium SPI-1 type III secretion system

Brit Winnen et al. PLoS One. .

Abstract

Background: Type III secretion systems (TTSS) are employed by numerous pathogenic and symbiotic bacteria to inject a cocktail of different "effector proteins" into host cells. These effectors subvert host cell signaling to establish symbiosis or disease.

Methodology/principal findings: We have studied the injection of SipA and SptP, two effector proteins of the invasion-associated Salmonella type III secretion system (TTSS-1). SipA and SptP trigger different host cell responses. SipA contributes to triggering actin rearrangements and invasion while SptP reverses the actin rearrangements after the invasion has been completed. Nevertheless, SipA and SptP were both pre-formed and stored in the bacterial cytosol before host cell encounter. By time lapse microscopy, we observed that SipA was injected earlier than SptP. Computer modeling revealed that two assumptions were sufficient to explain this injection hierarchy: a large number of SipA and SptP molecules compete for transport via a limiting number of TTSS; and the TTSS recognize SipA more efficiently than SptP.

Conclusions/significance: This novel mechanism of hierarchical effector protein injection may serve to avoid functional interference between SipA and SptP. An injection hierarchy of this type may be of general importance, allowing bacteria to precisely time the host cell manipulation by type III effectors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SipA and SptP are co-expressed in S. Typhimurium.
(A) SipA and SptP expression in wt S. Typhimurium. M1269 was grown under TTSS-1 inducing conditions, immobilized on gelatine-coated coverslips, fixed, permeabilized and SptP and SipA pools in the bacterial cytosol were immuno-strained (Materials and Methods). DNA was stained with DAPI. (B) Fraction of bacteria harboring SipA (open bars), SptP (black bars) and fraction of the SipA+ bacteria also harboring SptP (grey bars); Data was obtained from experiments as described in (A) and (C); n>400 bacteria, two independent experiments; (C) SipA and SptP expression in hilA over-expressing S. Typhimurium. M1269 (pHilA) was grown and analyzed as described in (A). (D) FACS-analysis of pHilA-induced sipA-expression. M2001 harbors the transcriptional reporter tsr-venus integrated downstream of the chromosomal sipA gene. FACS analysis of Tsr-Venus fluorescence is shown for M2001 and M2001 (pHilA). Wt S. Typhimurium (w/o tsr-venus reporter) served as a negative control. The gate is indicated as a box harboring 1.1% of wt control bacteria, 19.6% of M2001 and 90.8% of M2001(pHilA).
Figure 2
Figure 2. Hierarchical injection of SipA and SptP.
(A) Phases of the SipA- and SptP injection process; (B) Typical time lapse movie of the infection process. The bacteria harbored a pGFP plasmid to facilitate detection (GFP fluorescence) in the presence of membrane ruffling (phase contrast). (C) Representative images of M1269 and M1223 (pHilA, pGFP) in the early/intermediate or late phase of SipA and SptP injection into COS7 cells. Cells were fixed, permeabilized with lysozyme, and immunostained for LPS (blue), SipA (red) and SptP (green). (D) Time course of SipA and SptP depletion from hilA-overexpressing bacteria during the infection of COS7 cells. Infection was monitored as in (B) and intrabacterial SipA- and SptP pools were stained as in (C). For each bacterium, the graph shows the time between docking and fixation, the presence/absence of SipA (red) and SptP (green) in the bacterial cytosol. Gray lines connect SipA and SptP data from the same bacterium. Circles represent data obtained from M1223 (pHilApGFP; sipAHAsptPM45) and triangles data from M1269 (pHilApGFP; sipAM45sptPHA). The data was fitted using a rolling average algorithm (red and green lines, see Materials and Methods) to determine when injection was completed with 50% probability (t50%). tu = time units.
Figure 3
Figure 3. Computer simulation exploring hierarchical SipA and SptP injection by hilA-over-expressing bacteria (M1223 or M1269 (pHilA)).
(A) Model for TTSS injection of SipA and SptP. Key parameters for SipA binding to the chaperone InvB2 and interaction of the InvB2-SipA complex to the TTSS are shown. The same types of parameters describe SptP secretion (not shown). Brackets: numbers of particles. (B) Simulation of SipA and SptP secretion assuming identical parameters for both effector proteins. (C) Simulation of SipA and SptP secretion assuming that InvB2-SipA has a 10-fold higher affinity for the TTSS than SicP2-SptP. All other steps of SipA- and SptP-secretion had identical parameters. In this case, SipA is secreted before SptP. For details of the simulation and additional simulations see Supporting Materials.
Figure 4
Figure 4. Model for the timing of host cell manipulation by TTSS-1.
The bacterium harbors a pre-formed effector protein pool and the TTSS is triggered upon docking to the host cell (0s p.i.). SipA and SopE are injected during the first phase (first 30–150s p.i.) and trigger ruffling and invasion. Afterwards, SptP is injected and begins to reverse Rac1 and Cdc42 activation (150–500s p.i.). Finally, SopE (and presumably SipA) are degraded while SptP persists considerably longer in the host cell cytosol. As a result, the host cell actin cytoskeleton returns back to its normal shape.
See this image and copyright information in PMC

References

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