Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Mar 8.
Published in final edited form as: Chem Biol. 2008 Jun;15(6):619–628. doi: 10.1016/j.chembiol.2008.04.014

Quantification of Real-Time Salmonella Effector Type-III Secretion Kinetics Reveals Differential Secretion Rates for SopE2 and SptP

Schuyler B VanEngelenburg 1, Amy E Palmer 1
PMCID: PMC3297674  NIHMSID: NIHMS59595  PMID: 18559272

Summary

Gram negative pathogenic bacteria such as Salmonella utilize the Type-III Secretion System to inject bacterial effector proteins into a host cell. Upon entry, these effectors bind mammalian cell proteins, hijack cellular signaling pathways, and re-direct cellular function, thus enabling bacterial infection. In this study we use the FlAsH/tetracysteine labeling system to fluorescently-tag specific effectors in Salmonella to observe real-time secretion of these proteins into a mammalian host cell. The tetracysteine tag is genomically incorporated, thus preserving endogenous control of bacterial effectors. Here we demonstrate for the first time that two effectors, SopE2 and SptP, exhibit different secretion kinetics, as well as different rates of degradation within the host cell. These proteins respectively activate and suppress GTPase Cdc42, suggesting that there is a temporal hierarchy for effector delivery and persistence within the cell that is directly related to effector function.

Introduction

Salmonella sub-species are intracellular bacterial pathogens and the etiological agents associated with food poisoning and typhoid fever. Salmonella have evolved a sophisticated macromolecular protein complex called the Type-III Secretion System (TTSS) to translocate bacterial effector proteins into the host cytosol. These bacterial effector proteins then coordinate invasion by modulating eukaryotic host signaling pathways such as actin cytoskeleton dynamics, vesicular transport, and nuclear responses associated with inflammation (Hardt, W. D., 1998;Zhou, D., 2001a). Effectors exhibit both antagonistic and synergistic functions, e.g. SopE2 and SptP activate and repress, respectively, GTPase Cdc42 (Fu, Y., 1999;Stender, S., 2000), while SipA, SopB, and SopE act in concert to trigger actin polymerization at sites of bacterial invasion (Fu, Y., 1999;Zhou, D., 1999;Zhou, D., 2001b). This carefully concerted action of the bacterial effector repertoire is critical for bacterial persistence within the host (Hapfelmeier, S., 2004;Zhou, D., 2001a). These actions are likely to be regulated both spatially and temporally in order to preserve the hierarchy of events necessary for invasion. Indeed, work by Kubori et al. has shown that SopE, an effector highly homologous to SopE2, is rapidly degraded upon host cell entry leaving SptP to deactivate bacterial-mediated actin polymerization (Kubori, T., 2003). However, no temporal regulatory mechanisms have been reported for effector protein delivery, perhaps because of the dearth of available tools to monitor the spatio-temporal nature of effector secretion.

Translocation of bacterial effector proteins into host cells has traditionally been investigated by Western blotting or immunofluorescence (Abrahams, G. L., 2006;Kubori, T., 2003), which have limited temporal resolution, or by enzymatic assays (Schlumberger, M. C., 2005;Sory, M. P., 1994), which are indirect and have low spatial resolution. Direct labeling of effectors with bulky fluorescent proteins perturbs TTSS processes (Akeda, Y., 2005), thus preventing fluorescent proteins from being used to monitor direct protein translocation. Schlumberger et al. developed the first assay to image accumulation of effector proteins within a host cell by recruitment of a fluorescently-labeled chaperone within the host (Schlumberger, M. C., 2005). This assay is effective at monitoring the appearance of effectors within the host, however because the fluorescence signal relies on both effector accumulation and chaperone recruitment, the assay does not directly monitor the kinetics of secretion. The fluorescein-based biarsenical small molecule FlAsH offers a convenient alternative for direct labeling of tagged proteins within living cells. FlAsH was designed to bind a 12 amino acid tetracysteine (TC) motif with high affinity and specificity (Adams, S. R., 2002;Martin, B. R., 2005). Previously, Enninga et al. demonstrated that the FlAsH/TC system can be used to fluorescently label effector proteins expressed on an exogenous plasmid (Enninga, J., 2005). Here we extend this work and develop a system that is sufficiently sensitive to detect secretion of effectors under endogenous control. By monitoring disappearance of the labeled effector from the bacteria, we can directly measure the kinetics of secretion. Moreover, by incorporating the non-secreted mCherry fluorescent protein as a bacterial marker we can normalize for Z-plane movement and detect accumulation of effectors in real-time within mammalian cells.

In this work we focus on two Salmonella effectors, SptP and SopE2, because their demonstrated antagonistic activities make them likely candidates for effectors who are regulated temporally, either in their delivery or in their host cell mediated degradation (Fu, Y., 1999;Hardt, W. D., 1998). We utilize the FlAsH/TC labeling system combined with live cell microscopy to monitor effector secretion throughout the invasion process. We identify an unprecedented difference in secretion rates between SopE2 and SptP, as well as a difference in the rate of host cell-mediated degradation, which correlates with the known function of these effectors in manipulating CDC42 signaling.

Results

In situ fluorescence labeling of bacterial effectors and visualization of Type-III secretion by fluorescence microscopy is depicted in Figure 1a. To preserve endogenous control of TTSS effector expression, the tetracysteine (TC) tag was genomically incorporated into the Salmonella effector genes SptP and SopE2 using the lambda red recombinase system developed by Datsenko et al. (Datsenko, K. A., 2000). As illustrated in Supplementary Figure 8, the expression level of TC-tagged effectors is identical to non-tagged effectors. Therefore, this strategy ensures that effector proteins are expressed at wild type levels from their endogenous promoters, thus minimally perturbing the delicate balance of bacterial invasion. Because we anticipated low levels of protein expression, we compared FlAsH labeling of the single TC with a 3-fold repeat (3XTC; [GSFLNCCPGCCMEP]3).

Figure 1.

Figure 1

Open in a new tab

Visualizing fluorescently-labeled effectors in Salmonella. (a) Schematic illustration of the FlAsH labeling strategy for Salmonella strains expressing 3XTC effectors. (b) Average background-corrected FlAsH fluorescence intensity of different Salmonella strains. A significant increase (P < 0.001) in the average FlAsH signal intensity was observed for strains expressing TC tagged effectors, with the 3XTC tag exhibiting a small but significant increase in intensity as compared to the 1XTC. Treating bacteria with the TTSS inhibitor 1 resulted in an increase in the FlAsH signal intensity for both SptP- and SopE2-3XTC. Error bars: S.E.M., n > 100 individual bacteria were quantified for each group. Bacteria were selected and fluorescence was quantified as described in Experimental Procedures. Comparison of TTSS leak rates of individual bacteria in the absence of host cell contact for FlAsH labeled (c) SopE2-3XTC and (d) SptP-3XTC. The slow decay of FlAsH fluorescence in the absence of mammalian cell contact is a combination of the documented leak of effectors and FlAsH photobleaching. There is no significant difference (P = 0.8526) in the leak rate (kleakage) between the two labeled effector proteins (n = 8 for each). Statistical comparisons were made using a two-tailed t-test with post F-test to confirm the assumption of equal variances (P = 0.4649). Data represents mean ± SEM.

Salmonella strains encoding either SptP- or SopE2-3XTC were grown under invasion conditions and labeled with FlAsH. Salmonella expressing SptP- or SopE2-3XTC demonstrated a significant increase in FlAsH fluorescence intensity compared with wild-type (SL1344) bacteria (Figure 1b), indicating that FlAsH labeling is specific for bacteria expressing TC-tagged proteins. On average ~25% of bacteria were labeled with FlAsH, suggesting that only ~ 25% of bacteria express the effectors SopE2 and SptP when grown under invasion conditions. This is consistent with a previous report that only ~26% of bacteria express SipA, another invasion associated effector, when grown under similar conditions (Schlumberger, M. C., 2005). We observe 20% higher FlAsH intensity for SptP compared to SopE2. Because the fluorescence intensity of FlAsH can vary in different environments (Enninga, J., 2005;Martin, B. R., 2005), we determined the FlAsH labeling efficiency (defined as FlAsH intensity per protein) for SopE2 vs. SptP. We observed a 2-fold increase in the FlAsH fluorescence/protein ratio for SopE2-3XTC compared with SptP-3XTC indicating that labeling of SopE2 yields a higher fluorescence signal (Supplementary Figure 9). Thus, the higher FlAsH signal for SptP vs. SopE2-3XTC indicates that SptP is expressed at higher levels than SptP. Salmonella strains expressing 3XTC-tagged SptP yielded small, but significant increases in fluorescence intensity compared with 1XTC-SptP, prompting us to utilize the 3XTC tag for all subsequent experiments. Given the marginal increase in FlAsH intensity (~15%), we suspect that fewer than three FlAsH molecules bind per 3XTC. We are currently comparing the fluorescence intensities for 1X, 2X, and 3X-TC tags and optimizing the signal enhancement.

Treatment of bacteria with a recently identified small molecule (3,5-dichlorosalicylaldehyde benzoylhydrazone; 1), which inhibits Type-III secretion of effectors (Hudson, D. L., 2007), resulted in an increase (~ 30–40 %) in FlAsH signal intensity over non-treated bacteria. The observed increase indicates that a subset of the labeled effector pool is lost prior to infection, consistent with reports that the Salmonella TTSS possesses a slow intrinsic leak when grown under TTSS inducing conditions (Collazo, C. M., 1995;Schlumberger, M. C., 2006). To characterize the kinetics of this leak, we labeled both SptP and SopE2 with FlAsH and monitored the fluorescence decay in the absence of mammalian cells. A slow decrease in FlAsH fluorescence in the absence of host cell contact was observed (kleak/bleach(SopE2-3XTC) = 3.2×10−4 ± 8.5×10−5 s−1, n = 8; kleak/bleach(SptP-3XTC) = 3.0×10−4 ± 8.2×10−5 s−1, n = 8). This decrease is likely due to a combination of effector leakage and photobleaching (Figure 1c–d). Importantly, there is no difference between the fluorescence decay of SptP and SopE2 (P = 0.8526).

To address whether addition of the 3XTC tag and/or treatment with FlAsH perturbed TTSS function and the coordinated action of effectors, we performed a quantitative bacterial invasiveness assay. We reasoned that if incorporation of the 3XTC tag onto effectors or treatment with FlAsH compromised either bacterial viability or the ability of bacteria to invade HeLa cells, we would see a reduction in the number of internalized bacteria. As depicted in Figure 2a, we detected no difference in the invasiveness of the SopE2-3XTC and SptP-3XTC strains as compared to wild-type SL1344, suggesting that incorporation of the tag did not significantly perturb TTSS function. Additionally, we detected no difference in invasiveness due to FlAsH and BAL treatment. We also verified that FlAsH treatment did not perturb the overall viability of Salmonella using a growth assay (Supplementary Figure 10). We next wanted to address whether the activities of the effectors themselves were perturbed by incorporation of the 3XTC tag. SptP functions to restore membrane architecture after internalization of invading bacteria in addition to regulating the activity of Cdc42. To quantify SptP activity, internalized bacterial microcolonies were scored for their association with actin rich foci (Figure 2b). As expected, a bacterial strain deficient in SptP (ΔSptP) showed strong enrichment of actin, whereas the WT strain SL1344 showed a drastic reduction in actin association with microcolonies due to restoration of membrane architecture. We observed a comparable reduction in the number of bacterially associated actin foci in the SptP-3XTC strain compared with wild-type SL1344, indicating SptP function is not compromised by addition of the 3XTC tag. SopE2 activates Cdc42 which functions to induce a pro-inflammatory response leading to the production of Interleukin-8 (IL-8) (Huang, F. C., 2004;Patel, J. C., 2005). Therefore, to assess SopE2 activity we measured the production of IL-8 using an enzyme-linked immunosorbent assay (ELISA). As depicted in Figure 2c, ΔSopE2 showed a marked reduction in IL-8 production as compared to the wild type SL1344 strain. The SopE2-3XTC strain elicited a significant pro-inflammatory response, although there was a slight reduction compared to wild type SL1344. Collectively, these results suggest that the 3XTC tag and FlAsH labeling of the SopE2 and SptP effectors minimally perturbs effector function and the TTSS mediated invasion process. We also observed no significant perturbations in the levels of other effector proteins secreted by the TTSS in SptP-and SopE2-3XTC tagged strains compared to wild-type bacteria (Figure 2d).

Figure 2.

Figure 2

Open in a new tab

Incorporation of the 3XTC tag onto the effectors SopE2 and SptP minimally perturbs invasiveness and protein function. (a) Salmonella strains in the presence and absence of the FlAsH and BAL were scored for the number of internalized bacteria per HeLa cell using DIC and fluorescence microscopy. No statistical difference between the number of Salmonella per HeLa cell for WT SL1344 and 3XTC epitope tagged strains +/− FlAsH treatment was observed (n ≥ 40 for each experiment; 3 independent experiments for a total n > 120). (b) ΔSptP resulted in a marked increase in actin-associated foci whereas both wild type SL1344 and SptP-3XTC exhibited much lower levels, indicating membrane restoration in these strains. Salmonella strains expressing the mCherry bacterial marker were allowed to infect HeLa cells for 90 minutes and then fixed. Cells were stained with phalloidin-FITC and Hoechst 33258 to visualize the host cell cytoskeleton and nuclei respectively. (c) HeLa cells were infected with Salmonella strains and 5 hours after infection the growth medium was harvested and secreted IL-8 was quantified using an ELISA. Similar to wild type SL1344, SopE2-3XTC induces the production of IL-8, although the extent of IL-8 production is slightly reduced (P < 0.01). Conversely, a bacterial strain carrying a null mutation in SopE2 (ΔSopE2) is significantly attenuated for IL-8 production (P < 0.05 compared to SopE2-3XTC). Experiments were performed in triplicate. Data represents mean ± SD. (d) The secreted proteome (invasion inducing conditions) was TCA precipitated from the culture supernatant and resolved using SDS-PAGE. No change in secreted protein levels is observed for the strains SptP- and SopE2-3xTC compared to wild-type.

Having established that 3XTC tagged effectors expressed under endogenous control resulted in detectable FlAsH labeling and validated that effector function is not significantly perturbed by this labeling technology, we then assessed whether we could monitor translocation of FlAsH labeled effector proteins into HeLa epithelial cells. Initially, we observed large focal plane deviations due to bacterial cell engulfment. In order to circumvent this problem and enable the use of wide-field fluorescence microscopy for monitoring type-III secretion processes, we engineered Salmonella strains to express a non-secreted fluorescent protein, mCherry (Shaner, N. C., 2004). This method yields a ratiometric readout (FlAsH/mCherry), where decreases in the ratio correspond with disappearance of the FlAsH signal (i.e. secretion) regardless of the focal position of bacteria. We observed heterogeneity in the initial FlAsH/mCherry fluorescence ratios due to small differences in FlAsH labeling and mCherry expression between the two strains used in this study (Figure 11a; Supplemental Information). Although we observe some heterogeneity in the initial fluorescence ratios between the two effectors, the secretion rate constant does not depend on the starting ratio or ratiometric span of individual invading bacteria (see Figure 12a–f, Supplemental Information). A representative time-lapse experiment of FlAsH labeled SptP-3XTC is presented in Figure 3 and Supplementary Video 1. Over the course of 15–20 minutes we observed the hallmark invasion phenotype of membrane ruffling, bacterial engulfment, and membrane architecture restoration. This latter phenotype results from the action of SptP demonstrating that fusion of the 3XTC motif to SptP does not inhibit its function (Fu, Y., 1999). As seen in Figure 3a, upon mammalian cell contact the ratio of FlAsH/mCherry fluorescence decreases over time due to secretion of SptP into the host cell. Analogously, SopE2 secretion was readily detected upon mammalian cell contact (Figure 13a, Supplemental Information).

Figure 3.

Figure 3

Open in a new tab

Representative time-course for invasion of HeLa cells by individual bacteria. (a) Images of FlAsH labeled SptP-3XTC Salmonella infecting a HeLa cell. Presented are overlaid images of the FlAsH channel (in green) and DIC channel. For clarity, the mCherry fluorescence channel is not depicted. Salmonella triggers membrane ruffling (6 and 7.5 min), bacterial internalization (7.5 and 15 min), and restoration of the membrane architecture (21 min). The mCherry fluorescence channel was used to normalize for Z-plane movement during bacterial internalization. A decrease in the intrabacterial FlAsH fluorescence is observed over the course of invasion. (b) Representative HeLa infection using SptP-3XTC Salmonella pre-treated (3 hrs) with the TTSS inhibitor 1. The box highlights quantified Salmonella in contact with a HeLa cell over a relevant infection period. No TTSS mediated membrane ruffling events, actin foci, or (FlAsH/mCherry) decreases were observed. Scale bar represents 10 μm.

Treatment of SptP-3XTC with TTSS inhibitor 1 prevented cellular invasion and mammalian cell contact-mediated decreases in fluorescence, as illustrated in Figure 3b. For Salmonella treated with inhibitor 1 we did not detect any membrane ruffling events during the course of an invasion indicating that invasion-associated effector proteins were not released. Likewise, treatment of SopE2-3XTC with inhibitor 1 abolished contact-mediated decreases in fluorescence (Figure 13b Supplemental Information). As a control we measured the rate of decay for FlAsH-treated wild type SL1344 bacteria. These bacteria do not contain a tetracysteine tag, but on average do exhibit a small amount of non-specific staining (Figure 1b). As demonstrated in Figure 14 (Supplemental Information), there is a slow decay in fluorescence over time. This decay appears more linear than exponential, but we fit the data to a single exponential to compare the rate constants. The average rate of decay was 6.2 ± 3.4 × 10−4 s−1 for internalized bacteria and 5.4 ± 8.8 × 10−4 s−1 for bacteria in the absence of mammalian cell contact. These decay rates are comparable to the leak/bleach rate for fluorescence decay of non-secreted effectors (3.0 × 10−4; Figure 1c and d), suggesting that photobleaching dominates this process.

We chose to examine SptP and SopE2 because their demonstrated antagonistic activities make them likely candidates for effectors which are regulated temporally, either in their delivery or degradation (Fu, Y., 1999;Hardt, W. D., 1998). The secretion kinetics of representative individual bacteria are presented in Figure 4, demonstrating that SopE2 is secreted faster than SptP. The fit parameters for all quantified bacteria are presented in Figure 9e of Supplementary Information. Type-III secretion was modeled as an irreversible first-order decay process as effectors are translocated in an ATP dependent manner (Akeda, Y., 2005). Quantification revealed that SopE2-3XTC was secreted into HeLa (epithelial) cells at a rate roughly 2-fold that of SptP-3XTC (ksecretion(SopE2) = 4.0 × 10−3 ± 0.4 × 10−3 s−1 vs. ksecretion(SptP) = 2.1 × 10−3 ± 0.4 × 10−3 s−1) (Figure 4b). Bacteria which did not contact mammalian cells displayed only minimal changes in the FlAsH/mCherry ratio over the duration of the experiment. The average rate of fluorescence decay for bacteria not contacting mammalian cells was 1.4 × 10−4 ± 8.6 × 10−5 s−1, and was the same for both SptP-3XTC and SopE2-3XTC. These rates are consistent with the rate we observed for the combination of photobleaching and the slow effector leak in the absence of mammalian cells (Figure 1c and d).

Figure 4.

Figure 4

Open in a new tab

Salmonella effector SopE2 is secreted faster than effector SptP. (a) Representative kinetic traces and corresponding fits for SopE2 (circles, R2 = 0.9196), SptP (squares, R2 = 0.9169), and a control bacterium expressing FlAsH labeled SptP but which did not contact HeLa cells during the invasion (triangles, R2 = 0.5143). Figure 10 of Supporting Information presents the fitting parameters for the remainder of the bacteria. (b) Average first-order rate constants for SptP-3XTC (ksecretion = 2.1 × 10−3 ± 0.4 × 10−3 s−1; S.E.M., n = 11) and SopE2-3XTC (ksecretion = 4.0 × 10−3 ± 0.4 × 10−3 s−1; S.E.M., n = 14). SopE2-3XTC shows a significant increase in the rate of type-III secretion compared with SptP-3XTC (α = 0.05; P = 0.0029). Data represents mean ± SEM.

To further show that the secretion rates we observed are not due to the cell type used, we chose to quantify secretion of the effectors SopE2 and SptP in a marcrophage-like cell line. The secretion rates in RAW 264.7 macrophage-like cells were comparable to those in HeLa cells, with SopE2-3XTC yielding an average secretion rate of 4.5 × 10−3 ± 8.8 × 10−4 s−1 (n = 4) and SptP-3XTC yielding an average secretion rate of 2.2 × 10−3 ± 4.8 × 10−4 s−1 (n = 4) (Figure 5a and b). To ensure that contact dependent decreases in fluorescence were not due to interaction with membrane ruffles or bacterial internalization, TTSS inhibitor 1 treated bacteria were opsonized and phagocytosed by RAW 264.7 macrophage-like cells. Inhibitor treated bacteria internalized by macrophage in a TTSS independent manner exhibited fluorescence decays similar to those observed for bleach/leak kinetics (kbleach(SopE2) = 4.6 × 10−4 ± 1.5 × 10−4 s−1 (n = 3) and kbleach(SptP) = 2.4 × 10−4 ± 3.7 × 10−4 s−1 (n= 3); Figure 5c and d). This control demonstrates that the fluorescence decay for SopE2-3XTC and SptP-3XTC represent specific secretion through the TTSS. Furthermore, as this decay rate is similar to what we observed for the bleach/leak rate of decay for bacteria not contacting mammalian cells we conclude that the rate of photobleaching dominates that of effector leakage. These results provide the first direct evidence that temporal regulation of effectors can occur by modulating the kinetics of effector delivery, and suggests that delivery of antagonistic effectors may be pre-organized to ensure that activators are secreted more rapidly than repressors. We observed invasion initiation throughout a time-course experiment, highlighting the importance of monitoring secretion at the single bacterium level, as bulk assays average the kinetics of multiple invasions that occur at different times.

Figure 5.

Figure 5

Open in a new tab

Quantification of SopE2- and SptP-3xTC secretion with mouse macrophage-like (RAW 264.7) cells. FlAsH labeled effector strains SopE2-3xTC (a) and SptP-3XTC (b) were quantified for effector secretion after infecting RAW264.7 cells. SopE2-3XTC (c) and SptP-3XTC (d) strains were pre-treated with TTSS inhibitor (1) prior to FlAsH staining and opsonization. RAW 264.7 cells were allowed to engulf opsonized Salmonella strains and individual bacteria were quantified using the FlAsH/mCherry fluorescence ratio.

To build a complete picture of the regulation of SopE2 vs. SptP, we examined the rate of degradation within the host cell. Previously, Kubori et al. demonstrated that the antagonistic SopE and SptP activities are regulated through differential proteasomal degradation rates, with SopE being turned-over at a ~ 6.5× elevated rate (t1/2 ≈ 7.5 min vs. 45 min) (Kubori, T., 2003). Using the method of Kubori et. al. (Kubori, T., 2003), we quantified translocated effector proteins by Western blotting. As shown in Figure 6a, SopE2-3XFLAG was degraded with a t1/2 = 40 min, as compared with SptP-3XFLAG t1/2 = 110 min, yielding a ~ 2.8× difference in mammalian cell-mediated degradation kinetics. We also observed similar degradation rates by inhibiting de novo effector protein synthesis post-invasion, indicating that the persistence of the effectors SptP- and SopE2-3XFLAG is not the result of re-synthesis after bacterial internalization (Figure 6b). The observed 2.5× difference in t1/2 of SptP (Kubori's vs. this work) may result from the different cell types used in the respective experiments or the addition of the 3XFLAG epitope to the effectors. Even so, it is clear that SopE2 is degraded faster than SptP and that the relative difference in rates (SopE2 vs. SptP) is less than that observed for SopE vs. SptP. Therefore, in addition to enhanced secretion rates, SopE2 is also degraded ~2.8× faster than the repressor SptP.

Figure 6.

Figure 6

Open in a new tab

Intracellular degradation time course for translocated SopE2- and SptP-3XFLAG effectors. (a) SopE2-3XFLAG SL1344 infection time course illustrating gradual degradation kinetics. (b) SptP-3XFLAG SL1344 infection time course. (c) SopE2-3XFLAG SL1344 infection time course showing similar degradation rates with inhibition of de novo protein synthesis using chloramphenicol. (d) SptP-3XFLAG SL1344 infection time course performed in the presence of chloramphenicol. Intact bacteria were separated by selective lysis of mammalian cells to ensure selective measurement of translocated effector protein. SopE2 and SptP are both present in host cells post-invasion (~15 minutes). The degradation rate for SopE2-3XFLAG is approximately 2× that of SptP-3XFLAG. Anti-β-actin antibody was used as loading control. Positive control lane (+) represents 20% of input Salmonella for each time course (total bacterial lysate). (*) denotes anti-FLAG M2 cross-reactive bacterial lysate protein in positive control lane.

Discussion

To coordinate invasion, Salmonella need to turn on and turn off mammalian cell signaling pathways at explicit times. Consequently, Salmonella have evolved a repertoire of effector proteins, some of which act in concert and some of which act in direct opposition to one another. In order to maintain the hierarchy of events necessary for invasion, these effectors must be regulated both spatially and temporally. In this work we demonstrate for the first time that two effectors which exhibit antagonistic functions are secreted at different rates. The difference in temporal regulation of SopE2 vs. SptP and SopE vs. SptP correlates well with their relative known functions. SopE2 possesses guanine nucleotide exchange factor (GEF) activity towards Cdc42 (Stender, S., 2000), where Cdc42 activation leads to a nuclear pro-inflammatory response (Patel, J. C., 2005). In contrast, SopE activates Rac1, contributing to actin polymerization events during bacterial invasion (Patel, J. C., 2005;Rudolph, M. G., 1999). Post invasion, SptP serves to deactivate both Cdc42 and Rac1 leading to repression of nuclear responses and membrane architecture restoration respectively (Fu, Y., 1999). The enhanced rate of secretion of SopE2 would enable activation of Cdc42 before SptP is delivered. Although Kubori et al. did not detect differential secretion of SopE and SptP this may result from the limited temporal resolution of their assay. Upon bacterial invasion, the entire pool of SopE is degraded within 15 minutes which corresponds with the time required for invasion (Kubori, T., 2003). Conversely, SopE2 persists within the host cell (≥ 120 minutes) far longer than SopE, perhaps to provide a more graded nuclear response.

In the present work we modified an experimental approach employed previously by Enninga et. al. (Enninga, J., 2005). We have made three improvements. First we demonstrate that the TC-tag can be genomically incorporated, thus allowing us to study effectors expressed under control of their endogenous promoter. This is important as it ensures that the system is minimally perturbed. Second we discovered that the 3XTC allows for higher fluorescent labeling than the 1XTC. We believe that the current 3XTC tag is not fully optimized and therefore we are in the process of optimizing this tag to enhance the fluorescence signal, using a strategy similar to that reported for enhancing 1XTC sensitivity (Martin, B. R., 2005). Lastly, by incorporating the mCherry fluorescent protein as a non-secreted bacterial marker, we were able to monitor secretion on a standard wide field fluorescent microscope. Moreover, the mCherry bacterial marker enables us to identify accumulated effectors within the host cell paving the way for studying real-time effector localization and trafficking. As depicted in Figure 7, the decrease in bacterial FlAsH fluorescence correlates with an increase in mammalian fluorescence, where the bacteria are readily marked by the presence of mCherry. Our current signal:noise is low, requiring high MOIs to observe effector accumulation, however we are hopeful that this signal can be increased upon optimization of the 3XTC. Based on these results we believe this experimental approach shows promise for monitoring the kinetics of labeled effector trafficking and localization within living host cells in real time.

Figure 7.

Figure 7

Open in a new tab

Real-time accumulation of SopE2-3XTC/FlAsH fluorescence in a mammalian cell. Presented are overlaid images of the mCherry channel (red) and FlAsH channel (green), where yellow denotes direct overlap between the two channels. At -1 min there is very little cellular fluorescence in either channel. At 1 min post invasion, bacteria are clearly yellow/green indicating the presence of SopE2. At 35 min post-invasion, the bacteria are red because they have secreted all their SopE2 but retain the mCherry bacterial marker. Conversely, at 35 min the mammalian cell fluorescence in the FlAsH channel increases due to accumulated effector protein. An increase in mammalian cell fluorescence occurs only for the cell that is readily invaded (arrow) with no signal occurring in the cells that were not invaded (*). Scale bar represents μm.

The FlAsH/TC system displays a number of advantages over other approaches to studying Type-III Secretion. The primary methods for monitoring effector secretion are Western blotting and immunofluoresence. While these methods can provide critical snapshots of effector concentration and localization, the first time point is typically acquired at 15 min. As is evident from our data presented in Figure 4, the effector protein from an individual bacterium is > 75 % depleted at this 15 min time point. Therefore Western blotting and immunofluorescence techniques are incapable of resolving fast secretion events. Additionally, we found that bacteria initiate invasion throughout the time course, highlighting the importance of monitoring secretion from individual bacteria. Therefore, bulk assays assessing effector translocation, such as western blotting, temporally average multiple bacterial invasions. Schlumberger et. al. developed an innovative assay to monitor accumulation of effector proteins within a host cell by recruitment of a fluorescently-labeled chaperone expressed within the host cell (Schlumberger, M. C., 2005). While this assay is effective at monitoring localization of effector proteins, it does not permit direct assessment of effector secretion kinetics. Perhaps for this reason, the kinetics of recruitment upon SipA accumulation were found to be heterogeneous. Moreover, this method is limited to studying effectors for which the chaperone has been identified. Tetracysteine tagging and FlAsH labeling of endogenously expressed effectors enables us to single out a specific effector, without perturbing the remaining effectors and provides a system for monitoring effector translocation into live mammalian cells.

Significance

We have demonstrated a powerful new technique for quantitatively assaying Type-III secretion kinetics of endogenous Salmonella effector proteins. Importantly, because of the time resolution of this method, we were able to discern a difference in the rate at which effectors are delivered into the host. We propose that the combined effect of faster secretion and faster degradation for SopE2 compared with SptP creates a temporal hierarchy based on effector function. This study highlights the power of using real-time methods to study Type-III secretion processes. As pathogens are hard pressed to rapidly establish a replicative niche within the host, it is likely many more sophisticated spatiotemporal regulatory mechanisms will be uncovered as our understanding of host pathogen interactions matures.

Experimental Procedures

Media and Reagents

Oligonucleotides were obtained from IDT (Coralville, IA). PCR amplifications of the TC-tag and resistance cassette DNA was carried out using Phusion DNA polymerase (Finnzymes/New England Biolabs) to ensure the fidelity of chromosomal insertions. Taq DNA polymerase was used for all colony PCR reactions to ensure the presence of the TC-tag tagged genes. L-arabinose (Sigma) was used at a concentration of 50 mM for induction of bacterial cultures. LB was purchased from Invitrogen (Carlsbad, CA). SOB media was prepared as described (Hanahan, D., 1983).

Plasmids

Plasmids pKD46, pKD3, pKD4 were obtained from B. Wanner (Purdue University, West Lafayette, IN) by means of C. Detweiler (University of Colorado, Boulder, CO). Plasmid pKD46 carries the bacteriophage λ red genes (γ, β, and exo) which catalyze chromosomal recombination using double stranded linear DNA in bacteria (Datsenko, K. A., 2000). The plasmid pKD4 is a π-dependent suicide vector carrying the kanamycin-resistance gene (KnR) (Datsenko, K. A., 2000).

Construction of the template plasmids pTetCys, p3XTetCys, and p3XFLAG

Oligonucleotide primers encoding the high affinity tetracysteine coding sequence (GSFLNCCPGCCMEP) (Martin, B. R., 2005) and attB recombination sequences were used for amplification of the KnR resistance gene from plasmid pKD4. The resultant amplicon was cloned into the vector pDONR221 by recombination using the Gateway system (Invitrogen), yielding the vector pTetCys. Oligonucleotide primers encoding a 2× tetracysteine coding sequence and attB sites were used to amplify a 3× tetracysteine encoding amplicon from the vector pTetCys. This amplicon was recombined into the pDONR221 vector using the Gateway system (Invitrogen). The resulting vector p3XTetCys encodes the three copies of the tetracysteine epitope followed by the Knr resistance gene. The vector p3XFLAG was created using an identical procedure.

Construction of plasmid pAMCh

The bacterial marker plasmid pAMCh was constructed from the parent plasmid pACYC177 (New England Biolabs). Oligonucleotide primers complimentary to the fluorescent protein mCherry were used as primers for PCR amplification from the parent vector pRSET-B mCherry courtesy of R.Y. Tsien. The resulting amplicon was cloned into the pACYC177 vector using XhoI and HindIII restriction sites. The ribosomal protein promoter, P-rpsM, was cloned from the Salmonella enterica serovar LT2 genome using complimentary oligonucleotides. The P-rpsM amplicon was cloned upstream of the mCherry coding sequence using the XhoI restriction site, yielding a vector capable of constitutive mCherry fluorescent protein expression.

Bacteria

Salmonella enterica serovar Typhimurium derived from the strain LT2 which harbors the plasmid pKD46 was used for all recombination exchanges described (λ red). Strain SL1290 was used for creation of P22 bacteriophage stocks. The S. Typhimurium wild-type strain SL1344 (StrR) was used for P22 transduction of the antibiotic resistance gene and the coupled epitope fusion. Where indicated, strains carrying null mutations in the TTSS effectors SopE2 and SptP were created using recombination exchanges with the kanamycin resistance gene as previously described (Datsenko, K. A., 2000). Eschericia coli strains DH5α were used for all cloning experiments (Invitrogen).

FlAsH Fluorescence Intensity Quantification

Average fluorescent intensity analysis was performed on individual Salmonella (Figure 1b). Briefly, bacteria were stained with FlAsH (5 μM), washed 3× with 0.25 mM BAL (1,2-dimercaptoethanol; Sigma) in Hank's Balanced Salt Solution (HBSS), and tethered to imaging dishes using poly(L)-lysine (100 μg × mL−1; Sigma). Random fields of view were chosen and images were acquired for the FlAsH and mCherry channels. These image files were then imported into ImageJ, background subtracted, thresholded to the average intensity of background staining (WT SL1344, no TC tag), and regions of interest (ROI) were auto-assigned by the software. These ROIs were then used as a masking template for the original background subtracted image (no thresholding) and the average fluorescence intensity for each ROI was acquired. Greater than 100 individual bacteria (n > 100) were quantified from each group.

Invasiveness assay

Invasion efficiency was quantified as previously described (Drecktrah, D., 2006). HeLa cells were seeded at a density of 3.0 × 105 cells per 3.5 cm2 dish 18 hours prior to infection. Salmonella cultures were grown under invasion associated TTSS inducing conditions (0.3 M NaCl, low O2, LB medium) and HeLa cells were infected with a multiplicity of infection (MOI) of 50. The infection was allowed to proceed for 15 minutes before the addition of 100 μg × mL−1 gentamycin (Sigma) to kill extracellular bacteria. The infection was allowed to proceed for an additional 75 minutes. The invasion process was quenched by the addition of 4% formaldehyde. Cells were then stained with 2 μg × mL−1 Hoechst 33258 dye for 20 minutes to visualize mammalian nuclei. This protocol allowed for visualization and differentiation of extracellular bacteria while leaving intracellular bacteria unlabeled. All strains of Salmonella used in these experiments express the fluorescent protein mCherry to mark individual bacteria for enumeration. Invasion efficiency was scored by DIC and fluorescence microscopy blind to the Salmonella strain under investigation. Random viewing fields were selected for enumerating the number of bacteria infecting each HeLa cell. Experiments for each genotype were performed in triplicate (n ≥ 40 HeLa cells for each replicate). Where indicated, Salmonella strains were treated with 5 μM FlAsH-EDT2 for 1 hour at 37 °C and washed 3× with 0.25 mM BAL in HBSS prior to infection.

SptP activity assay

The actin foci/membrane ruffling assay was performed as described previously (Kubori, T., 2003). HeLa cells were plated and infected with Salmonella as described above. Infections were quenched as described above. Cells were permeabilized with .1% Triton X-100 (Sigma) and stained with Hoechst 33258 and phalloidin-FITC (Sigma) to visualize nuclei and actin respectively. Experiments were performed using fluorescence microscopy blind to the Salmonella strain under investigation. Random fields of infected HeLa cells were scored for the association of bacterial microcolonies with actin rich foci. All experiments were performed in triplicate (n ≥ 40 bacterial microcolonies for each replicate).

SopE2 activity assay

Quantification of SopE2-mediated upregulation of interleukin-8 (IL-8) was performed as previously described (Huang, F. C., 2004). Salmonella cultures were grown under SPI-1 conditions as described above. HeLa cells were seeded at a density of 4.0 × 104 cells per well in standard 6-well plates 48 hrs prior to infection (2% FCS DMEM medium). TTSS induced bacteria were added at an MOI of 50. Infections were allowed to proceed for 1 hour before the addition of 100 μg × mL−1 gentamycin. Six hours post-infection the culture medium was collected and clarified by centrifugation at 14K rpm for 5 minutes. Quantification of IL-8 production was performed using an enzyme-linked immunosorbent assay according to the manufacturers instructions (BioLegend). ELISA reactions were performed in triplicate for each genotype.

TCA Precipitation of the Secreted Proteome

Salmonella strains were grown overnight under invasion associated conditions to induce SPI-1 effector secretion into the culture supernatants. Culture supernatants were collected by centrifugation at 14 K× g for 30 minutes followed by 0.22 μm filtration. The supernatants were then precipitated with 10% TCA as described above. Precipitated proteins were washed in acetone and redissolved in SDS-loading buffer and resolved using 10–20% SDS-PAGE. Secreted proteins were visualized with Coomassie Brilliant Blue R-250 (Sigma).

Time-lapse Microscopy

Fluorescence imaging of bacterial effector secretion was performed using an Axiovert 200M wide-field microscope (Zeiss), equipped with Lambda 10–3 filter changer (Sutter Instruments). This system is configured to allow rapid acquisition of fluorescence images in order to facilitate live cell imaging. Images were acquired using METAFLUOR software (Universal Imaging) and a Cascade 512B CCD camera (Roper Scientific) with an EM Gain of 2500 and a 5 MHz transfer speed. All images were obtained using a 1.4 NA 100× PlanAPO objective (Zeiss), and the following filter combinations: FlAsH: 495/10 (excitation), 542/50 (emission), 515 (dichroic filter cube); mCherry: 577/20 (excitation), 630/60 (emission), 595 (dichroic filter cube).

Kinetic Secretion Assay

SL1344 strains carrying 3XTC fusions to the effector proteins SopE2 and SptP were grown for ~16 hrs. at 37°C under invasion associated conditions conditions (LB 0.3M NaCl, low O2, OD600 ~0.4–0.5) (Collazo, C. M., 1996). HeLa cells were seeded onto 3.5 cm2 imaging dishes at ~70–90% confluence. Macrophage-like RAW264.7 cell infections were treated identically to HeLa infections. Approximately 500 μL of bacterial culture was treated with 5 μM FlAsH-EDT2 (Invitrogen) for 1 hour at 37°C before incubation (~10 minutes) with 10 μg × mL−1 poly(L)-lysine. Bacteria were then washed 3× with Hank's HEPES Balanced Salt Solution (HHBSS) containing 0.25 mM BAL (2,3-dimercaptoethanol). HeLa cells were infected at an MOI of ~10 at 25°C. Image acquisition was initiated before infection and frames for each channel were acquired every 15 s with an exposure time of 500 ms and 900 ms for the FlAsH and mCherry channels, respectively. DIC images were acquired at 15 s intervals with an exposure time of 1 s. Only those bacteria for which initial mammalian cell contact was observed were quantified. A total of 11 individual bacteria for the SptP-3XTC and 16 bacteria containing the SopE2-3XTC obtained from independent experiments on different days were used for analysis of Type-III secretion kinetics. The fluorescence intensity of individual bacteria was quantified by summing their intensity in a region-of-interest (roughly the size of the bacterium) for both the FlAsH and reference mCherry channels. The fluorescence intensities for both channels were background subtracted, where a region of interest of identical size was placed in a region of the dish containing no cells and no bacteria. Background corrected FlAsH and mCherry signals were then processed as a ratiometric (FlAsH/mCherry) time-course. Bleaching corrections were not performed on any data sets as independent experiments demonstrated that rate of photobleaching was not significant compared to the rate of decay for our experimental conditions (see Figure 2). Kinetic traces were individually fit to single exponential equations and the rate constant (ksecretion) and error in this fit were extracted. Only those bacteria which remained isolated for the majority of an experiment were included in the analysis. In the case of clear interference by other bacteria, as assessed by DIC and the mCherry channel, time course data points were excluded from exponential fits.

3,5-dichlorosalicylaldehyde benzoyl hydrazone (1)

The type-III secretion system inhibitor 1 was synthesized as previously described from commercially available starting materials (Sigma) (Johnson, D. K., 1982). Compound 1 was recrystallized from ethanol and confirmed to be >95% pure as assess by NMR: 1H NMR (600 MHz, D6-DMSO) δ 12.54 (s, 2H), 8.58 (s, 1H) 7.96 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 2.5 Hz, 1H), 7.63 (t, J = 7.3, 1H), 7.62 (d, J = 2.5 Hz, 1H), 7.56 (t, J = 7.8 Hz, 2H); 13C NMR (600 MHz, D6-DMSO) δ 163.07, 152.30, 147.10, 132.39, 132.20, 130.30, 128.67, 128.47, 127.81, 122.95, 121.49, 120.79. Inhibition of type-III secretion was performed by pre-treating invasion induced Salmonella with 100 μM of compound 1 (DMSO as carrier) for 3 hours prior to the kinetic secretion assay. To enhance bacterial uptake with macrophage-like RAW264.7 infections, inhibitor treated bacteria were opsonized with 14% fetal calf serum for 30 minutes prior to mammalian cell addition.

Proteasomal Degradation Assay

Host cell mediated degradation rates of the effectors SopE2 and SptP were assessed using the method of Kubori et al. (Kubori, T., 2003) with slight modifications. Breifly, SL1344 strains carrying genomic 3XFLAG epitope fusions to the effector proteins SopE2 and SptP were grown under invasion conditions for ~16 hrs at 37°C. HeLa cells (50–80% confluence) were infected at an MOI of ~100 at 37°C and 5% CO2. After 15 minutes of infection, cells were washed 2× with warm HHBSS and overlaid with 10 mL of HHBSS containing 0.1 mg/mL of gentamycin (Sigma). For de novo protein synthesis inhibition experiments, 100 μg × mL−1 chloramphenicol was added to all time points 15 minutes post-invasion. Effector proteins were harvested at the appropriate time points by washing the cultures 2× with cold HHBSS containing the proteasome inhibitor Mg132 (10 μM; Sigma) followed by lysis in 1.0 mL lysis buffer (HHBSS, 0.1% Triton X-100, 1 mM PMSF, and 10 μM Mg132). HeLa cell lysates were clarified by centrifugation at 14 Krpm for 30 minutes (4°C). The supernatant fraction was filtered through a 0.2 μm filter, TCA precipitated, the proteins resolved by SDS-PAGE (10–20% gradient), and transferred to PVDF membrane for western blotting. The membranes were simultaneously probed with a mouse anti-FLAG M2 antibody (1:500), mouse anti β-actin antibody (1:7,500), and rabbit anti-mouse HRP conjugate (1:5,000) as secondary antibody. Membranes were stripped and re-probed with a mouse monoclonal anti-DnaK antibody (1:1,000) to confirm the absence of bacterial cytoplasmic contaminants in the translocated effector fractions. SopE2 and SptP half-lives (t1/2) were estimated by comparing the area × intensity normalized to the β-actin loading control.

Statistical Analysis

All statistical tests were performed using GraphPad Prism software (GraphPad Software, Inc.). Statistical significance for FlAsH staining intensities was obtained using the Kruskal-Walis test and Dunn's multiple comparison post test. This nonparametric test was used because each group did not conform to normality as the mean fluorescence intensities were just above the detection limit of the system. For kinetic comparisons of the effectors SopE2- and SptP-3XTC, n values for secretion assays were obtained from individual experiments on separate days. A two-tailed unpaired t-test was used for statistical analysis (α = 0.05; P = 0.0029) and the assumption of equal variances was substantiated using an F test (α = 0.05; P = 0.4173).

Supplementary Material

01
02
Download video file (76.5MB, avi)

Acknowledgements

We would like to thank Dr. Corrella Detweiler for providing bacterial strains, plasmids, and advice, Dr. Richard Shoemaker for help with NMR samples and structure determination, and Dr. Jorge E. Galán for providing the monoclonal anti-SptP antibody. We would like to acknowledge the Creative Training in Molecular biology Grant (NIH 5 T32 GM07135-33) and the University of Colorado for financial support.

Abbreviations

TTSS

Type-Three Secretion System

SPI-1

Salmonella Pathogenicity Island 1

FlAsH

fluorescein-based biarsenical hairpin binder

TC

tetracysteine

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Competing Financial Interests We declare no competing financial interests.

References

  1. Abrahams GL, Muller P, Hensel M. Functional Dissection of SseF, a Type III Effector Protein Involved in Positioning the Salmonella-Containing Vacuole. Traffic. 2006;7:950–965. doi: 10.1111/j.1600-0854.2006.00454.x. [DOI] [PubMed] [Google Scholar]
  2. Adams SR, Campbell RE, Gross LA, Martin BR, Walkup GK, Yao Y, Llopis J, Tsien RY. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am.Chem.Soc. 2002;124:6063–6076. doi: 10.1021/ja017687n. [DOI] [PubMed] [Google Scholar]
  3. Akeda Y, Galan JE. Chaperone release and unfolding of substrates in type III secretion. Nature. 2005;437:911–915. doi: 10.1038/nature03992. [DOI] [PubMed] [Google Scholar]
  4. Collazo CM, Galan JE. Requirement for exported proteins in secretion through the invasion-associated type III system of Salmonella typhimurium. Infect.Immun. 1996;64:3524–3531. doi: 10.1128/iai.64.9.3524-3531.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collazo CM, Zierler MK, Galan JE. Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus. Mol.Microbiol. 1995;15:25–38. doi: 10.1111/j.1365-2958.1995.tb02218.x. [DOI] [PubMed] [Google Scholar]
  6. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc.Natl.Acad.Sci.U.S.A. 2000;97:6640–6645. doi: 10.1073/pnas.120163297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drecktrah D, Knodler LA, Ireland R, Steele-Mortimer O. The Mechanism of Salmonella Entry Determines the Vacuolar Environment and Intracellular Gene Expression. Traffic. 2006;7:39–51. doi: 10.1111/j.1600-0854.2005.00360.x. [DOI] [PubMed] [Google Scholar]
  8. Enninga J, Mounier J, Sansonetti P, Tran VN. Secretion of type III effectors into host cells in real time. Nat Methods. 2005;2:959–965. doi: 10.1038/nmeth804. [DOI] [PubMed] [Google Scholar]
  9. Fu Y, Galan JE. A salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion. Nature. 1999;401:293–297. doi: 10.1038/45829. [DOI] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol.Biol. 1983;166:557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. Hapfelmeier S, Ehrbar K, Stecher B, Barthel M, Kremer M, Hardt WD. Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice. Infect.Immun. 2004;72:795–809. doi: 10.1128/IAI.72.2.795-809.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hardt WD, Chen LM, Schuebel KE, Bustelo XR, Galan JE. S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell. 1998;93:815–826. doi: 10.1016/s0092-8674(00)81442-7. [DOI] [PubMed] [Google Scholar]
  13. Huang FC, Werne A, Li Q, Galyov EE, Walker WA, Cherayil BJ. Cooperative Interactions between Flagellin and SopE2 in the Epithelial Interleukin-8 Response to Salmonella enterica Serovar Typhimurium Infection. Infect.Immun. 2004;72:5052–5062. doi: 10.1128/IAI.72.9.5052-5062.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hudson DL, Layton AN, Field TR, Bowen AJ, Wolf-Watz H, Elofsson M, Stevens MP, Galyov EE. Inhibition of Salmonella type III secretion by small-molecule inhibitors. Antimicrob.Agents Chemother. 2007 doi: 10.1128/AAC.01492-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnson DK, Murphy TB, Rose NJ, Goodwin WH, Pickart L. Cytotoxic chelators and chelates 1. Inhibition of DNA synthesis in cultured rodent and human cells by aroylhydrazones and by a copper(II) complex of salicylaldehyde benzoyl hydrazone. Inorganica Chimica Acta. 1982;67:159–165. [Google Scholar]
  16. Kubori T, Galan JE. Temporal Regulation of Salmonella Virulence Effector Function by Proteasome-Dependent Protein Degradation. Cell. 2003;115:333–342. doi: 10.1016/s0092-8674(03)00849-3. [DOI] [PubMed] [Google Scholar]
  17. Martin BR, Giepmans BN, Adams SR, Tsien RY. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat Biotechnol. 2005;23:1308–1314. doi: 10.1038/nbt1136. [DOI] [PubMed] [Google Scholar]
  18. Patel JC, Rossanese OW, Galan JE. The functional interface between Salmonella and its host cell: opportunities for therapeutic intervention. Trends in Pharmacological Sciences. 2005;26:564–570. doi: 10.1016/j.tips.2005.09.005. [DOI] [PubMed] [Google Scholar]
  19. Rudolph MG, Weise C, Mirold S, Hillenbrand B, Bader B, Wittinghofer A, Hardt WD. Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases. J Biol.Chem. 1999;274:30501–30509. doi: 10.1074/jbc.274.43.30501. [DOI] [PubMed] [Google Scholar]
  20. Schlumberger MC, Hardt WD. Salmonella type III secretion effectors: pulling the host cell's strings. Curr.Opin.Microbiol. 2006;9:46–54. doi: 10.1016/j.mib.2005.12.006. [DOI] [PubMed] [Google Scholar]
  21. Schlumberger MC, Muller AJ, Ehrbar K, Winnen B, Duss I, Stecher B, Hardt WD. Real-time imaging of type III secretion: Salmonella SipA injection into host cells. Proc.Natl.Acad.Sci.U.S.A. 2005;102:12548–12553. doi: 10.1073/pnas.0503407102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004;22:1567–1572. doi: 10.1038/nbt1037. [DOI] [PubMed] [Google Scholar]
  23. Sory MP, Cornelis GR. Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol.Microbiol. 1994;14:583–594. doi: 10.1111/j.1365-2958.1994.tb02191.x. [DOI] [PubMed] [Google Scholar]
  24. Stender S, Friebel A, Linder S, Rohde M, Mirold S, Hardt WD. Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell. Mol.Microbiol. 2000;36:1206–1221. doi: 10.1046/j.1365-2958.2000.01933.x. [DOI] [PubMed] [Google Scholar]
  25. Zhou D, Galan J. Salmonella entry into host cells: the work in concert of type III secreted effector proteins. Microbes.Infect. 2001a;3:1293–1298. doi: 10.1016/s1286-4579(01)01489-7. [DOI] [PubMed] [Google Scholar]
  26. Zhou D, Mooseker MS, Galan JE. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science. 1999;283:2092–2095. doi: 10.1126/science.283.5410.2092. [DOI] [PubMed] [Google Scholar]
  27. Zhou D, Chen LM, Hernandez L, Shears SB, Galan JE. A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization. Mol.Microbiol. 2001b;39:248–259. doi: 10.1046/j.1365-2958.2001.02230.x. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS59595-supplement-01.pdf (571.7KB, pdf)
02
Download video file (76.5MB, avi)

RESOURCES