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Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named ‘inclusion’ and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion.
Chlamydia trachomatis constitutes the leading cause of bacterially induced sexually transmitted diseases worldwide (Beagley and Timms, 2000). Chlamydia is an obligate intracellular bacterium that accomplishes its entire life cycle inside host cells. A typical infection starts with the entry of elementary bodies (EBs) – the bacterial infectious form – into host cells. The EBs rapidly differentiate into reticulate bodies (RBs), the metabolically active but non-infectious form of chlamydial organisms. After numerous rounds of replication, the RBs differentiate back into EBs for spreading to adjacent cells (Abdelrahman and Belland, 2005). As soon as they are formed, Chlamydia-containing vesicles dissociate from the classical endocytic/phagocytic/lysosomal pathway and intercept components of the biosynthetic route (Fields and Hackstadt, 2002). These bacteria-containing vesicles traffic to the peri-Golgi region and fuse to form a single vacuole termed inclusion. The inclusion membrane serves as both a barrier for protecting the intravacuolar bacteria from the immune system, and a gate for C. trachomatis interactions with host cells to acquire nutrients (Saka and Valdivia, 2010). The mechanisms by which these bacteria generate their convenient intracellular niche have attracted intensive investigation.
Rab are host proteins in charge of the control of intracellular transport pathways (Hutagalung and Novick, 2011). Chlamydia manipulates host cell trafficking to its own benefit by means of the interaction with Rabs and Rab effectors. A subset of Rabs (Rab1, Rab4, Rab6, Rab11 and Rab14) is selectively recruited to C. trachomatis inclusions (Rzomp et al., 2003; Brumell and Scidmore, 2007; Moorhead et al., 2007). Recently, we and others have demonstrated that Rab11 and Rab14 are implicated in chlamydial inclusion development and promote bacterial replication by favouring the capture of sphingolipids-enriched vesicles (Rejman et al., 2009a; Capmany and Damiani, 2010). In order to go deeper into the participation of Rab11 and Rab14 in the biogenesis of intracellular chlamydial vacuoles, we focused our interest on a dual Rab-binding protein named Family of Interacting Protein 2 (FIP2) (Hales et al., 2001; Wallace et al., 2002). FIP2 has a Rab-binding domain (RBD) at its C-terminus, and at its N-terminus presents a C2 domain with high affinity to several phospholipids such as phosphatidic acid and phosphatidylinositol-(3,4,5)-trisphosphate (Prekeris, 2003; Lindsay and McCaffrey, 2004). FIP2 functions in recycling of molecules such as transferrin receptors (Lindsay and McCaffrey, 2002), in regulating transcytosis (Ducharme et al., 2007), and in the apical targeting of several proteins (Ducharme et al., 2011). Recently, it has been described that FIP2 specifically binds both, Rab11 and Rab14 (Kelly et al., 2010). We became interested in studying this dual Rab interacting protein, given the relevance of these Rabs for chlamydial infection outcome.
Our data show that FIP2 recruitment is specific, since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusion membrane. Furthermore, the FIP2's association depends on bacterial protein synthesis; whereas microtubule dynamics and Golgi integrity of host cells are not necessary. FIP2 associates to Rab11 at the chlamydial inclusion membrane through its RBD and promotes Rab14 recruitment. Finally, our results demonstrate that FIP2 is required for an efficient bacterial replication and infectivity.
FIP2, a member of the Rab11-Family of Interacting Proteins, is recruited to the chlamydial inclusions
Since Rab11 and Rab14, associate with C. trachomatis inclusions and are important for the bacterium's nutrition and development (Capmany et al., 2011), we analysed the role of FIP2, a recently described dual Rab interacting protein, in infected cells. FIP2, is predicted to form complexes with Rab11 such as (Rab11)2–(FIP2)2 or a heterotetramer Rab11–(FIP2)2–Rab14 (Kelly et al., 2010). In order to determine whether FIP2 localizes to chlamydial inclusions, we examined the intracellular distribution of this protein in infected cells. HeLa cells overexpressing GFP-FIP2 were infected with C. trachomatis serovar L2 and fixed at 18 h post infection (p.i.). Cells were viewed by laser-scanning confocal microscopy (LSCM) and the resulting images clearly showed GFP-FIP2 surrounding chlamydial inclusions (Fig. 1A, left panel). FIP2 decorated the periphery of the inclusion in a defined rim-like staining pattern in the totality of the infected cells. The association of FIP2 to the chlamydial inclusion was further confirmed by the analysis of the distribution of GFP-FIP2 (green) and Hoechst-labelled bacteria (blue) along a line traversing the chlamydial inclusion. Hoechst labels eukaryotic and bacterial DNA. The intensity profile showed Hoechst-labelled bacteria between two peaks of GFP-FIP2 (Fig. 1A, right panel). This observation was supported by the detection of the endogenous FIP2 surrounding the chlamydial inclusions, as shown in Fig. 1B. FIP2 recruitment was independent of the multiplicity of infection (moi) used, since we have observed FIP2 associated with the inclusions in cells infected at a moi of less than 1 or more than 50. The presence of FIP2 associated with chlamydial inclusions was further confirmed by the analysis of z-sections through the centre of the inclusion. The confocal planes revealed a fine punctuate pattern of GFP-FIP2 surrounding the inclusion with Hoechst-labelled Chlamydia inside (Supplementary Fig. S1A). Several sections along the y-axis from a three dimensional reconstruction of the z-optical planes are shown in Supplementary Fig. S1B. The white arrow points out a chlamydial inclusion (blue) decorated by a punctuated rim-like staining of FIP2 in a three dimensional reconstruction of infected cells (Supplementary Fig. S2).
The temporal expression pattern of C. trachomatis genes varies along their developmental cycle (Abdelrahman and Belland, 2005). As a consequence, a differential timing has been demonstrated in the association of Rab GTPases with the chlamydial inclusion. Rab11 is recruited earlier than Rab14 and the latter remains associated to the inclusion during the complete cycle of development (Capmany and Damiani, 2010). To analyse the recruitment of FIP2 to the inclusions along the developmental cycle, HeLa cells overexpressing GFP-FIP2 were infected with C. trachomatis and fixed at different post-infection times. At the initial stages of infection (2 h p.i.), we observed GFP-FIP2 surrounding the newly formed inclusions. The recruitment of GFP-FIP2 was more evident at 6 h to 10 h p.i. when the vacuoles arrived at their peri-nuclear localization. The amount of GFP-FIP2 associated to chlamydial inclusions peaked at mid-stage of development (18 h p.i.), showing a rim-shape staining pattern limiting the inclusions. Finally, at 24 h p.i., GFP-FIP2 association with the chlamydial inclusion was lost (Fig. 1C). Experiments over time demonstrated that the association of FIP2 to the chlamydial inclusions closely correlated with the recruitment of Rab11. In contrast, we have established that Rab14 is recruited later, when the inclusions arrive to the perinuclear region (10 h p.i.), and remains associated until the end of the bacterial developmental cycle.
FIP2 possesses a conserved RBD but shares little overall sequence homology with the other members of the Rab11-Family of Interacting Proteins (Hales et al., 2001; Wallace et al., 2002). To assess the specificity of FIP2 recruitment, HeLa cells were transiently transfected with either pGFP-RCP or pGFP-FIP2 (class I FIPs), or with pGFP-FIP3 (class II FIP) for 24 h and then, were infected with C. trachomatis. At 18 h post infection, cells were fixed and bacterial and eukaryotic DNA was stained using Hoechst and analysed by confocal microscopy. The images showed that only GFP-FIP2 localized to the chlamydial inclusions, as demonstrated by the distinct rim-like staining pattern surrounding the inclusions. On the other hand, the distribution of RCP and FIP3 remained unaltered in infected cells and did not localize at the inclusions, as shown by the lack of staining at the periphery of the inclusions (Fig. 2). These results demonstrate the specificity of the recruitment of FIP2 to chlamydial inclusions.
FIP2 recruitment is a bacteria-driven process and is independent of microtubules and Golgi integrity
To characterize the association with FIP2 to the chlamydial inclusions, HeLa cells overexpressing GFP-FIP2 infected for 24 h were maintained under different conditions, fixed and analysed by confocal microscopy. A batch of cells was treated for 22 h with chloramphenicol. The results showed that FIP2 recruitment requires active bacterial protein synthesis since no association was observed in chloramphenicol-treated cells (Fig. 3A, see inset). Similarly, other cells were infected for 18 h and cultivated at 32°C. At this temperature, IncA and probably other bacterial proteins are neither trafficked nor exposed at the chlamydial inclusion membrane (Fields et al., 2002). Confocal images showed that GFP-FIP2 did not localize to the inclusions of infected cells maintained at 32°C (Fig. 3B). Our observations suggest that a temperature-sensitive exported Inc protein located at the chlamydial inclusion membrane is necessary for FIP2 association. Thus, the recruitment of FIP2 requires a Chlamydia-driven modification of the inclusion membrane.
To understand the function of FIP2 in chlamydial pathogenesis, we further characterize its recruitment to the inclusion membrane. HeLa cells overexpressing FIP2 were infected with C. trachomatis for 18 h. Microtubule polymerization was inhibited by treatment with 20 μM nocodazole during the last 4 h of infection. Then, cells were fixed and DNA was labelled with Hoechst (blue). The disruption of cytoskeleton was confirmed by tubulin staining (data not shown). FIP2 was found associated to chlamydial inclusion membrane even under conditions where microtubule dynamic was abolished (Fig. 4A, lower panels). Our observations showed that FIP2 recruitment to C. trachomatis inclusions persists in the absence of an intact microtubule network.
Since the interference with microtubule polymerization disrupts the Golgi apparatus, our results suggest that FIP2 recruitment does not require Golgi integrity. To further address this observation, we added 1 μg ml−1 Brefeldin A (BFA), a fungal metabolite that collapses Golgi apparatus into the endoplasmic reticulum, during the last 4 h of infection. Then, cells were fixed while eukaryotic and bacterial DNA was stained with Hoechst. Labelling the Golgi apparatus with anti-GM130 antibodies confirmed its disruption (data not shown). Confocal images showed that FIP2 remained associated to the chlamydial inclusions in BFA-treated cells (Fig. 4A, middle panels). Our data demonstrated that FIP2 trafficking to chlamydial inclusions does not require an intact Golgi apparatus. Previously, it has been shown that microtubule polymerization and Golgi integrity are not required for the recruitment of several Rabs to C. trachomatis inclusions (Rzomp et al., 2003). Our observations are in agreement with these findings; however, we noted a slight but significant decrease in the amount of FIP2 associated with the chlamydial inclusion membrane in both, nocodazole- and BFA-treated cells (Fig. 4B, right panel). In addition, the size of the inclusions was slightly reduced in nocodazole-treated cells (Fig. 4B, left panel). These observations suggest that microtubule- and Golgi-dependent trafficking pathways might play minor roles, though are not essential, for the recruitment of FIP2 to chlamydial inclusions.
Recruitment of FIP2 to chlamydial inclusions is mediated by its RBD
To characterize which domain of FIP2 is involved in its association with the chlamydial inclusions, HeLa cells overexpressing GFP-FIP2 and several mutant forms were infected with C. trachomatis and fixed at 18 h p.i. Confocal images showed a clear association of GFP-FIP2 full length (green) with perinuclear chlamydial inclusions at mid-stage of development (Fig. 5A, upper panels). The binding of high amounts of FIP2 to the chlamydial inclusions interfered with the formation of a single vesicle, leading to multiple vacuoles of reduced size (Fig. 5B, white bars). Then, we analyse infected cells overexpressing GFP-FIP2 (129–512) designated as FIP2ΔC2. This mutant protein is devoid of its N-terminal C2 domain that has high affinity for acidic phospholipids. In the images, a well-defined ring of the mutant protein limiting the bacterial inclusions was evident (Fig. 5A, middle panels). These results indicate that the lipids are not involved in the binding of FIP2 to the chlamydial inclusion membrane. Next, we analysed the role of the RBD of FIP2. The RBD is an amphipathic α helical domain that interacts with Rab11 and is located at the C-terminus of FIP2 within a predicted coiled-coil region. For these assays, we infected cells overexpressing GFP-FIP2 (129–378) named GFP-FIP2ΔC2ΔRBD. Interestingly, the association with the chlamydial inclusions of the mutant protein, lacking both the C2 and the RBDs, was lost (Fig. 5A, lower panels). Confocal images showed that the mutant protein lacking the RBD displayed a diffuse cellular pattern without evident association with the inclusions. Our data suggest that the presence of the RBD is essential for the binding of FIP2 to the chlamydial inclusion membrane.
Other groups of infections were carried out (under the same conditions except fixation at 10 h p.i.) to prove the requirement of the RBD of FIP2 for its association with the chlamydial inclusions. According to the above observations, FIP2 and FIP2ΔC2 associated with chlamydial inclusions. In contrast, FIP2ΔC2ΔRBD was not recruited to chlamydial inclusions, confirming that the RBD is fundamental for the binding (Supplementary Fig. S3). The finding of smaller and more numerous inclusions labelled with FIP2 was more evident at early stages of infection, suggesting a delayed fusion of vesicles in the presence of an excess of this Rab interacting protein (Supplementary Fig. S3, see insets). Taken together, our results indicate that the RBD of FIP2 is necessary for its association with chlamydial inclusions.
Since FIP2 interacts with Rab11 through its RBD, we infected cells overexpressing both RFP-Rab11wt (red) and GFP-FIP2 or its mutants (green). Confocal images clearly showed the colocalization between Rab11wt and FIP2 or FIP2ΔC2, whereas the mutant lacking the RBD (FIP2ΔC2ΔRBD) did not colocalize with Rab11, neither at the chlamydial inclusion membrane nor at other intracellular membranes (Fig. 6). The right panels show the intensity profile scanned along the white line drawn on the merged images. The intensity profiles clearly showed the colocalization of RFP-Rab11wt (red) with GFP-FIP2 or GFP-FIP2ΔC2 (green) at the inclusion membrane level. The blue profile indicates the bacterial DNA and represents the bacteria within the inclusion. The absence of green peaks at the inclusion membrane level in cells overexpressing GFP-FIP2ΔC2ΔRBD unequivocally demonstrated that the mutant protein lacking the RBD is not recruited, despite of the finding of red peaks corresponding to RFP-Rab11wt flanking the inclusion. These observations show that the presence of Rab11 at the inclusion membrane is not sufficient for the binding of the FIP2 mutant protein that lacks the RBD.
Given that the GDP-bound mutant of Rab11 (Rab11S25N) binds to FIP2 but does not associate with chlamydial inclusions (Junutula et al., 2004; Wei et al., 2006), we infected cells overexpressing FIP2, Rab11S25N or both proteins. As expected, confocal images showed the association of FIP2 (green) with the chlamydial inclusion (Fig. 7, upper panels). Furthermore, we confirmed the lack of recruitment of the negative mutant of Rab11 (red) (Fig. 7, middle panels). Interestingly, the association of FIP2 with the chlamydial inclusions was abolished in the presence of the negative mutant of Rab11 (Fig. 7, lower panels). In order to analyse in more detail the degree of colocalization between FIP2 and Rab11 (wild type or its GDP-bound mutant), we performed an intensity correlation analysis. HeLa cells co-overexpressing FIP2 (green) and, Rab11WT or Rab11S25N (red), were infected with C. trachomatis for 18 h, fixed and analysed by confocal microscopy. The pixels in the images pseudo-coloured plotted the frequency of the red-green pixel combination in the original image, and thereby represent Manders' coefficients. The white dots correspond to the highest values whereas blue represent the lowest values of colocalization between both proteins. Representative images are shown in Supplementary Fig. S4, right panels. In agreement with the findings presented above, FIP2 and Rab11WT colocalized not only at the chlamydial inclusion membrane but also at vesicles throughout the cell (Supplementary Fig. S5, upper panels). On the other hand, neither FIP2 nor the GDP-bound mutant of Rab11 associated to the inclusion membrane. However, both proteins colocalized in tubulovesicular structures distributed along the cell (Supplementary Fig. S4, lower panels). These findings suggest that FIP2 might be sequestered by the negative mutant of Rab11 forming an inactive complex, and as a consequence, FIP2 association with chlamydial inclusions is interfered.
Rab11–FIP2–Rab14 complex at the chlamydial inclusion membrane
It has been recently shown that FIP2 interacts not only with Rab11 but also with Rab14 (Kelly et al., 2010). In order to analyse the colocalization of FIP2 with these two Rabs at the chlamydial inclusion membrane, we infected cells co-overexpressing RFP-Rab11 (red), GFP-FIP2 (green) and immunodetected endogenous Rab14 (blue). During the last 6 h of infection, cells were treated with BFA (1 μg ml−1), nocodazole (20 μm) or maintained without treatment (control cells). Then, cells were fixed and analysed by confocal microscopy. Representative images are shown in Supplementary Fig. S5.
To address more accurately the degree of colocalization between FIP2 and both Rabs, we applied tools of quantitative confocal microscopy. We calculated the Manders' and Pearson's coefficients that measure overlapping between proteins (Bolte and Cordelieres, 2006; Ramirez et al., 2010). We observed a high level of colocalization between both Rabs and FIP2 under all conditions (with the coefficients rounded at 1 meaning complete colocalization). We only noted a slight but significant decrease in the colocalization between Rab11 and FIP2 (M2 = 0.933) and between Rab14 and FIP2 (M2 = 0.799) when microtubule polymerization was impaired (Table 1). However, the almost complete overlapping among Rab11, Rab14 and FIP2 strongly suggests that these proteins coexist in a multimeric complex located at the chlamydial inclusion membrane.
Table 1. Pearson's and Manders' colocalization coefficients
HeLa cells co-overexpressing GFP-FIP2 (green) and RFP-Rab11wt (red) were infected with C. trachomatis L2 (moi 5) and treated with: 1 μg ml−1 Brefeldin A (6 h) or 20 μM nocodazole (6 h) prior fixation, or left untreated as control cells. At 18 h p.i., cells were fixed and processed for immunofluorescence. Endogenous Rab14 was detected by using specific rabbit anti-Rab14 antibodies followed by Alexa Fluor 633 goat anti-rabbit IgG. Confocal images of 50 cells were taken from three independent experiments. The inclusion was crop and pixel fluorescence was analysed at the different channels. Pearson's and Manders' coefficients were calculated using the Image J software. Data were statistically analysed by one-way anova and Tukey–Kramer post-test (P < 0.05; **P < 0.01).
The interplay between Rab11 and Rab14 with the dual Rab-binding protein FIP2 was analysed in cells in which the expression of FIP2 was reduced by using specific short interfering RNA (siRNA). Briefly, HeLa cells were transfected with chemically synthesized siRNA targeted to FIP2 or negative control siRNA. At 72 h post transfection, cells were infected with C. trachomatis for 18 h. Then, cells were fixed and processed for the detection of endogenous Rabs by immunofluorescence. Confocal images showed endogenous Rab11 surrounding the chlamydial inclusions despite of the silencing of FIP2 expression (Fig. 8, upper panels). On the other hand, the recruitment of endogenous Rab14 to the chlamydial inclusion membrane decreased in FIP2-silenced cells (Fig. 8, lower panels). It is important to note that FIP2-silencing neither affected Rab14 expression assessed by Western blot (Supplementary Fig. S6), nor Rab14 intracellular localization. Images show that endogenous Rab14 displayed a punctuated pattern that remained mostly unchanged after FIP2 depletion in non-infected cells observed by confocal microscopy (Supplementary Fig. S7).
A schematic illustration summarizes the connection between Rab11 and Rab14 with the dual Rab-binding protein FIP2 at the chlamydial inclusion membrane (Fig. 9). Our findings that: (i) FIP2 associates with growing inclusions in a similar time frame to Rab11 binding (2 h p.i.), (ii) FIP2 binds to the chlamydial inclusion through its RBD, and (iii) FIP2 binding is lost in the presence of the GDP-bound mutant of Rab11 which does not associate with the chlamydial inclusions and iv. Rab11 binding to the inclusions persists in FIP2-silenced cells; suggest that FIP2 is recruited to the chlamydial inclusions by binding to Rab11 through its RBD. In addition, our results showed that Rab14 is the latest component of this complex to be recruited, since its association with chlamydial inclusions starts later during inclusion development (10 h p.i.) and is interfered in FIP2-silenced cells.
The overall findings suggest a likely sequential recruitment of Rab11–FIP2–Rab14 to the chlamydial inclusion. Nevertheless, bacterial proteins could be playing additional roles in the recruitment of these host trafficking controllers.
FIP2 favours chlamydial multiplication
The role of host FIP2 on C. trachomatis infection outcomes was assessed in cells in which the endogenous protein expression was silenced by RNA interference. Briefly, HeLa cells were transfected with specific siRNA against FIP2 or negative control siRNA. At 72 h post transfection, cells were infected with C. trachomatis. Then, cells were lysed at 48 h p.i. and infectious particles were titrated on fresh HeLa cells by inclusions forming unit (IFU) assays. Silencing of FIP2 substantially reduced bacterial progeny in comparison with control cells (Fig. 10A). These results further confirmed the importance of this Rab-binding protein for bacterial replication. Knock-down of FIP2 (more than 90%) was confirmed by Western blot using rabbit polyclonal anti-FIP2 antibodies and goat anti-rabbit HRP-conjugated IgG (Fig. 10B). The detrimental impact on bacterial multiplication caused by FIP2 silencing was similar to that found in Rab14-silenced cells (data not shown), supporting that this Rab-binding protein could be relevant for Rab14 targeting to the inclusion. The recovery of FIP2 expression after gene silencing and its effect on chlamydial inclusion development is shown in Supplementary Fig. S8. Briefly, HeLa cells were transfected with specific siRNA against FIP2 or negative control siRNA. At 72 h post transfection, a batch of FIP2-silenced cell was transfected with the vector pGFP-FIP2. As a control of the efficiency of transfection, a batch of siRNA control-treated cells was also transfected with pGFP-FIP2. After 24h, the four groups of cells (siRNA control cells, siRNA control cells overexpressing GFP-FIP2, FIP2-silenced cells and FIP2-silenced cells overexpressing GFP-FIP2) were infected with C. trachomatis for 18 h. Then, cells were fixed and the size of the inclusions was measured by confocal microscopy. Images show that FIP2 silencing significantly reduced the development of chlamydial inclusions and there was a partial retrieval of the inclusion growth in those cells which were overexpressing GFP-FIP2 (Supplementary Fig. S8A). A quantification of chlamydial inclusion size from siRNA control cells and FIP2-depleted cells, overexpressing or not GFP-FIP2, is shown in Supplementary Fig. S8B. These results show that the rescue of FIP2 expression causes a favourable impact on chlamydial inclusion growth.
In summary, our data suggest that C. trachomatis actively recruits host FIP2 that promotes the development of chlamydial inclusions leading to an increased bacterial progeny and infectivity.
Intracellular pathogens establish complex host–pathogen interactions for the successful development of their niche (Brumell and Scidmore, 2007). An increasing body of data point out that these intruders co-opt intracellular transport not only for avoiding phagolysosome degradation but also for capturing host nutrients (Saka and Valdivia, 2010). Chlamydia actively modifies the inclusion's membrane through the exposition of bacterial proteins on its surface for the recruitment of selected host proteins (Valdivia, 2008; Dehoux et al., 2011). Several host trafficking controllers have been found associated to C. trachomatis inclusions, such as Rab1, Rab4, Rab6, Rab11 and Rab14 (Rzomp et al., 2003; Rejman et al., 2009b; Capmany and Damiani, 2010). Conveniently, pathogenic Chlamydia exploits several Rab-mediated transport steps for the re-direction of lipids towards the inclusion since those molecules are essential for chlamydial inclusion growth and bacterial replication (Carabeo et al., 2003; Cocchiaro et al., 2008; Moore et al., 2008). More than one Rab, among them Rab11 and Rab14, contributes to lipid transport to the inclusion, thereby affecting the intracellular development of these bacteria (Rejman et al., 2009a; Capmany and Damiani, 2010). However, little is known about the participation of Rab interacting proteins in the Chlamydia-driven usurpation of host trafficking. In this study, we characterize the recruitment to the chlamydial inclusions of a novel dual Rab interacting protein known as FIP2 that binds to both, Rab11 and to Rab14 through a well-conserved RBD.
In this report, we show the recruitment of the endogenous FIP2 protein to chlamydial inclusions. This recruitment is specific since other members of the Rab11-Family of Interacting Proteins (FIPs) do not associate with chlamydial inclusions. Furthermore, our results show that bacterial protein synthesis is required for host FIP2 recruitment. On the other hand, the association of FIP2 with the inclusions does not require an intact Golgi apparatus or microtubule polymerization. Coincidently, it has been reported that the recruitment of other Rabs is maintained in the absence of microtubule dynamics or when the Golgi apparatus is disrupted by BFA treatment (Rzomp et al., 2003; Moorhead et al., 2007; 2010).
The C-terminus of FIP2 adopts an α-helical structure with conserved residues forming a hydrophobic Rab-binding patch that is involved in the binding to Rabs (Jagoe et al., 2006). Interestingly, our results demonstrate that this RBD is essential for FIP2 recruitment to chlamydial inclusions, whereas the N-terminus lipid binding C2 domain of FIP2 is not required for this association. Furthermore, our findings indicate that FIP2 associates with the chlamydial inclusion membrane by linking to Rab11. In agreement, in cells overexpressing the Rab11-negative mutant (the GDP-bound protein that does not associate with chlamydial inclusions) the binding of FIP2 is lost. Taken together, our data indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its RBD.
Similarly, the oculocerebrorenal syndrome of Lowe protein I (OCRL1), a Golgi-associated phosphatidylinositol-5-phosphatase that interacts with Rab1, Rab5, Rab6 and Rab14, associates with chlamydial inclusions through a region that includes the RBD (Moorhead et al., 2010). On the contrary, the bicaudal effector of Rab6, BICD1, is recruited to chlamydial inclusions independently of Rab6 (Moorhead et al., 2007).
The presence of FIP2 surrounding chlamydial inclusions is evident already at 2 h p.i. and increases along the developmental cycle until its peak at 18 h p.i. At later stages of inclusion development, FIP2 recruitment is lost (24 h p.i. and so on). This time pattern of association resembles Rab11 recruitment, whereas Rab14 reaches the inclusion later, being evident at 10 h p.i. and remains associated to the inclusions throughout the entire cycle of development. Quantitative confocal microscopy analysis shows almost complete colocalization among Rab11, Rab14 and FIP2 at the chlamydial inclusion membrane, even upon disruption of Golgi integrity or microtubule dynamics. The silencing of FIP2 expression does not affect Rab11 association with chlamydial inclusions whereas it interferes the binding of Rab14 to chlamydial inclusions. Thus, our findings suggest the chance of a sequence of linkage Rab11–FIP2–Rab14. However, the participation of bacterial proteins in the targeting/binding of Rab11–FIP2–Rab14 complex to the chlamydial inclusion membrane should not be dismissed.
On the other hand, the IFU analysis clearly shows that the presence of FIP2 favours bacteria replication and infectivity since the amount of infectious particles released from FIP2-depleted cells is significantly lower than in control cells. The detrimental impact on bacterial multiplication observed after FIP2 silencing, together with the partial rescue after FIP2 expression recovery; suggest that this protein might be co-opted by Chlamydia to hijack trafficking pathways of host cells for its own benefit. The scaffold protein FIP2 might stabilize the Rab/FIP complex at the chlamydial inclusion membrane to promote the capture of Rab14-labelled vesicles rich in host cell lipids.
Chlamydia trachomatis manipulates functionally redundant intracellular trafficking pathways to ensure its survival and replication (Cocchiaro and Valdivia, 2009; Valdivia, 2008). Our results might contribute to better delineate the complex molecular machinery co-opted by Chlamydia for the capture of host nutrients and thus provide insights into bacterial manipulation of vesicular trafficking. This knowledge could be useful for the development of novel anti-chlamydial therapies. Nevertheless, further experimentation is required to reveal which chlamydial protein is involved in the recruitment of Rab11–FIP2–Rab14 complex to the inclusion and to elucidate at a deeper level the role of Rab/FIP platforms in the usurpation of host cell intracellular trafficking by pathogenic Chlamydia.
Cells and bacteria
HeLa 229 cells (ABAC, Bs.As., Argentina) were grown in infection medium (IM): D-MEM high glucose (Gibco-BRL, Bs.As., Argentina) supplemented with 10% fetal bovine serum (FBS) (Internegocios SA, Bs.As., Argentina), 0.3 mg ml−1l-glutamine (ICN Biomedicals, Ohaio, USA) and 1.55 mg ml−1 glucose (Biopack, Bs.As., Argentina). C. trachomatis serovar L2 (gently given and typified by Unidad de Estudios de Chlamydias, FFyB, UBA, Bs.As., Argentina) were used. For bacterial propagation, HeLa cells were infected at a moi of 20 and incubated at 37°C in an atmosphere of 5% CO2 and 95% humidified air for 48 h. Then, infected cells were lysed with glass beads and EBs were purified by centrifugation as previously described (Caldwell et al., 1981). The purified EBs were suspended in 0.2 M sucrose–5% FBS–0.02 M phosphate buffer (pH = 7.2) and titrated by determination of IFU.
Plasmids and antibodies
The plasmids pGFP-FIP3, pGFP-FIP2WT, pGFP-FIP2ΔC2 and pGFP-FIP2ΔC2ΔRBD were generously provided by Dr James R. Goldering (Vanderbilt University School of Medicine, Nashville, USA). pGFP-RCP was given by Dr Mary McCaffrey (Cork University, Duke, USA) and pRFP-Rab11 was provided by Dr María I. Colombo (IHEM, UNCuyo, Argentina). pcDNA3.1-Myc-Rab11S25N was given by Dr Rytis Prekeris (University of Colorado Denver, Aurora, USA) and pcDNA3.1-HA-Rab11S25N was a gift of Dr David Sabatini (New York University, New York, USA). pGFP-Rab14 was constructed in our lab. The antibodies used were: rabbit anti-FIP2, rabbit anti-Rab14 and mouse anti-tubulin (Abcam, Cambridge, USA); rabbit anti-Rab11 (Invitrogen, Argentina); mouse anti-actin (Sigma, Bs.As, Argentina); rabbit anti-Myc and rabbit anti-HA (Santa Cruz Biotechnology, CA, USA); rabbit polyclonal anti-IncG antibodies (generously provided by Dr Ted Hackstadt, Rocky Mountain Laboratories, NIH, Montana, USA), FITC-labelled anti-rabbit IgG, Texas Red-labelled anti-rabbit IgG and Alexa Fluor 633-labelled anti-rabbit IgG (Molecular Probes, USA); goat anti-rabbit HRP-conjugated IgG and goat anti-mouse HRP-conjugated IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA).
Cell transfection and infection
HeLa 229 cells were grown on 12-mm-diameter glass coverslips in 24-well plates (ETC Internacional, Bs.As, Argentina) until 80% confluence. Cells were washed once with serum-free D-MEM (Gibco-BRL Bs.As., Argentina) and transfected with Lipofectamine 2000 (Invitrogen, Bs.As., Argentina) using 1 μl per 2 μg of DNA per well according to the manufacturer's protocol. Eighteen hours post transfection, cells were infected with C. trachomatis serovar L2 at a moi of 1–50. HeLa cells with bacteria were centrifuged for 10 min at 30°C at 1100 r.p.m. and then maintained for two and a half hour at 37°C. After that, cells were washed two times with phosphate-buffered saline (PBS) to eliminate non-internalized bacteria, and finally, cells were incubated in the presence of infection medium (D-MEM without antibiotics) at 37°C in an atmosphere of 5% CO2 and 95% humidified air for the indicated times (post-infection period).
Reagents and treatments
20 μM nocodazole (Calbiochem, San Diego, CA), 200 μg ml−1 chloramphenicol (Rontag, Bs.As., Argentina) or 1 μg ml−1 BFA (Merck, Bs.As., Argentina) were added to infected cells at the indicated post-infection times (p.i.). For fixation, 3% p-formaldehyde (PFA) in PBS and NH4Cl (Calbiochem, USA) were used. As mounting medium was used 0.1 μg ml−1 Hoechst/Mowiol (Molecular Probes and Calbiochem, USA respectively).
Fluorescent labelling and confocal microscopy
For colocalization studies, HeLa cells cultured on coverslips in 24-well plates were transfected with the appropriate plasmids. After 24 h, cells were infected with C. trachomatis according to the assay. Then, cells were fixed in 3% PFA for 15 min, washed and incubated with 50 mM NH4Cl at room temperature for 15 min and finally, mounted in Hoechst/Mowiol. To detect endogenous proteins (FIP2, Rab11, Rab14), cells were fixed in 3% PFA, permeabilized for 20 min with 0.2% saponin/BSA in PBS and then, incubated for 60 min with the appropriated primary antibody followed by incubation with a fluorescent-labelled secondary antibody. Samples were mounted in Hoechst/Mowiol and observed by confocal fluorescence microscopy. Cells from the different experimental conditions were equally and simultaneously processed. Confocal images were captured using the same parameters setting: equal optical magnification (60×) and electronic zoom (2×), identical laser potency (5%), identical photodetector gain (HV 480 V), identical scanning speed (12 ms pixel−1). The fixation of the parameters described above determined exposure and acquisition times that were identical for all experimental groups. To quantify FIP2 association, fluorescence intensities of GFP-FIP2 (473 nm laser) were determined by defining regions of interest (ROI) coincident with chlamydial inclusions (405 nm laser) by confocal microscopy and were expressed as arbitrary units (a.u.). Images were acquired at 512 by 512 pixels. The size of the inclusions was determined as pixel area assessed by confocal microscopy. An Olympus FV-1000 spectral confocal unit mounted on an IX-25 Olympus inverted microscope was used. Confocal images were acquired and analysed with the FV10-ASW 1.7 Software (Olympus America, Melville, NY) and then processed using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA). Adobe Illustrator CS3, MacBiophotonic Image J and Windows Movie Maker were used to perform figures and videos.
Quantitative confocal microscopy
The degree of colocalization between the Rabs and FIP2 was quantified in infected cells without treatment (control cells) or after BFA or nocodazole treatment. Pearson's coefficient measures the relationship between two fluorophores calculated by linear regression, ranging from 1 to −1, with 1 standing for complete positive correlation and −1 for a negative correlation, with zero standing for no correlation. This coefficient is distorted when the intensity of both fluorophores is different. Manders' coefficient indicates the proportion of the green signal coincident with a signal in the red channel over its total intensity (M1) and vice versa (M2); and varies from 0 to 1, the former corresponding to non-overlapping images and the latter reflecting 100% colocalization between both images. This coefficient is useful to analyse images with different intensity of fluorochromes in the two channels. Confocal images were captured using the same parameters setting: equal optical magnification (60×) and electronic zoom (2×), identical laser potency (5%), identical photodetector gain (HV 480V), identical scanning speed (12 μs pixel−1). Both coefficients were calculated analysing 50 cells from each condition by MacBiophotonic Image J. The degree of colocalization between FIP2 and Rab11WT, or between FIP2 and Rab11S25N, was assessed by intensity correlation analysis. The images generated are a representation of the Manders' coefficients. The pixels in the pseudocoloured image represent the frequency of the red-green pixel combination in the original image (white colour corresponds to the highest values whereas blue represents the lowest values of colocalization, by convention). Analysis was performed using the appropriated plug-in of MacBiophotonic Image J software.
Transfected cells were infected with C. trachomatis L2 for 48 h, lysed with glass beads and titrated on fresh HeLa cells. First, cell lysate was centrifuged for 10 min at 500 g to remove debris and progressive dilutions were inoculated onto fresh HeLa cells seeded on a 96-well plate. After 24 h, the number of inclusions formed by chlamydial progeny was assessed by immunofluorescence and expressed as IFU per ml.
FIP2 silencing and Western blot
HeLa cells were seeded onto six well tissue culture plates and 24 h later were transfected with 1 nM All Stars-negative control siRNA or 1 nM of a mix of Predesigned siRNA directed against human FIP2 using HiPerFect transfection reagent (Qiagen, Berlin, Germany) and Opti-MEM (Invitrogen, Bs.As., Argentina) accordingly to siRNA transfection protocol suggested at the Qiagen web page. At 72 h post transfection, cells were infected with C. trachomatis. Two days later, the amount of infectious particles was measured by IFU. To confirm the decrease in FIP2 protein, cell lysates obtained at 72 h post siRNA transfection were resolved by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes and then detected using rabbit polyclonal anti-FIP2 (1:1000) followed by goat anti-rabbit HRP-conjugated antibodies (1:5000). Protein loading was controlled with mouse monoclonal anti-actin (1:1000) and goat anti-mouse HRP-labelled antibodies (1:5000). In another set of experiments, the expression of Rab14 was analysed with rabbit anti-Rab14 (1:300) followed by goat anti-rabbit HRP-conjugated antibodies (1:2000). Protein loading was assessed with mouse monoclonal anti-tubulin (1:7000) and goat anti-mouse HRP-labelled antibodies (1:5000). Amersham ECL PlusTM was used to evidence HRP activity (GE Healthcare Life Sciences, Bs.As., Argentina). The ratio between Rab14 and tubulin was analysed by using the LAS-400 EPUV luminometer and LAS image reader software (FUJI Life Science, Japan) and expressed as relative units.
For the experiments of the rescue of FIP2 expression, cells seeded in 24-well tissue culture plates were transfected with 1 nM control siRNA or 1 nM specific siRNA against FIP2. At 72 h post transfection, a batch of control siRNA-treated cells and a batch of FIP2-silenced cells were transfected with pGFP-FIP2 using the Lipofectamine protocol. Twenty-four hours later, all groups (control siRNA, control siRNA + pGFP-FIP2, FIP2 siRNA and FIP2 siRNA + pGFP-FIP2) were infected with C. trachomatis for 18 h. Then, cells were fixed, permeabilized and processed for immunofluorescence as described under the subtitle ‘Fluorescent labelling and confocal microscopy’. Anti-IncG was used as primary antibody to detect the bacterial protein IncG that delineates the chlamydial inclusions.
Data were analysed by one-way anova and Tukey–Kramer post-test.
We thank Alejandra Medero and Marcelo Furlán for technical assistance, and Milton Aguilera for his help with quantitative confocal microscopy. We are indebted with María Isabel Colombo for her constant interest and helpful suggestions during the course of this study. We thank Luis Mayorga for his critical comments on the manuscript. This work was supported by Fundación Bunge y Born, PIP-CONICET and Sepcyt-UNCuyo Grants to M.T.D.