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. Author manuscript; available in PMC: 2013 Jul 3.
Published in final edited form as: Cell Rep. 2013 May 30;3(6):1921–1931. doi: 10.1016/j.celrep.2013.04.027

Chlamydia trachomatis inclusion membrane protein CT228 recruits elements of the myosin phosphatase pathway to regulate release mechanisms

Erika I Lutter 1, Alexandra C Barger 1, Vinod Nair 2, Ted Hackstadt 1,*
PMCID: PMC3700685  NIHMSID: NIHMS474532  PMID: 23727243

Summary

Chlamydia trachomatis replicates within a membrane bound compartment termed an inclusion. The inclusion membrane is modified by the insertion of multiple proteins known as Incs. In a yeast two-hybrid screen, an interaction was found between the inclusion membrane protein CT228 and MYPT1, a subunit of myosin phosphatase. MYPT1 was recruited peripherally around the inclusion while the phosphorylated inactive form was localized to active Src-family kinase-rich microdomains. Phosphorylated myosin light chain 2 (MLC2), myosin light chain kinase (MLCK), and myosin IIA and IIB also colocalized with inactive MYPT1. The role of these proteins was examined in the context of host-cell exit mechanisms; cell lysis or extrusion of intact inclusions. Inhibition of myosin II or siRNA depletion of myosin IIA and IIB, MLC2, or MLCK reduced chlamydial extrusion thus favoring lytic events as the primary means of release. These studies provide insights into regulation of egress mechanisms by C. trachomatis.

Introduction

Chlamydiae are Gram-negative obligate intracellular bacteria that cause a variety of human and veterinary infections. Chlamydia trachomatis is comprised of multiple serological variants, or serovars that are responsible for different diseases. Among these are the causative agents of the most prevalent bacterial sexually transmitted disease in the United States (Schachter, 1999) as well as the etiological agents of trachoma, the leading cause of infectious blindness worldwide (Burton and Mabey, 2009). All chlamydiae share common infection strategies which include a biphasic developmental cycle that consists of metabolically inactive infectious elementary bodies (EBs) and metabolically active and noninfectious reticulate bodies (RBs) (Moulder, 1991). The chlamydial developmental cycle occurs within a unique parasitophorous vacuole termed the inclusion (Hackstadt et al., 1997). At the end of the developmental cycle, EBs are released to initiate new cycles of infection. Release may be either by lysis of the host cell or extrusion of intact inclusions by an active process requiring actin polymerization (Chin et al., 2012; Hybiske and Stephens, 2007; Todd and Caldwell, 1985).

The inclusion membrane is the interface for interaction between chlamydiae and the host cell. Early in infection the inclusion membrane is extensively modified by the insertion of bacterially synthesized, type III secreted proteins termed inclusion membrane proteins (Incs) which decorate the cytosolic face of the inclusion (Hackstadt et al., 1999; Rockey et al., 1997; Scidmore and Hackstadt, 2001). The number of Incs predicted varies between species with C. trachomatis predictions ranging between 36–59 Incs (Bannantine et al., 2000; Dehoux et al., 2011; Li et al., 2008; Lutter et al., 2012; Shaw et al., 2000; Toh et al., 2003). These Incs are comprised of a highly diverse set of proteins that are suspected of being involved in modulating host signaling pathways and cellular functions. Virtually all interactions of the chlamydial inclusion with the eukaryotic host cell require de novo chlamydial protein synthesis and, presumably, modification of the inclusion membrane. These functions include acquisition of host lipids (Carabeo et al., 2003; Hackstadt et al., 1996) and trafficking to the microtubule organizing center (MTOC) where the inclusion is typically observed in close proximity with the host cell nucleus and centrosomes (Clausen et al., 1997; Grieshaber et al., 2003; Grieshaber et al., 2006; Higashi, 1965; Mital and Hackstadt, 2011; Mital et al., 2010). There are relatively few confirmed interactions of host proteins with specific Incs. These include the interactions of CT229 with Rab4 (Rzomp et al., 2006), IncG (CT118) recruitment of 14-3-3β (Scidmore and Hackstadt, 2001), and IncD (CT115) interaction with CERT (Derre et al., 2011).

In this study, a yeast two-hybrid screen of C. trachomatis Inc CT228 identified an interaction with myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin phosphatase. MYPT1 was recruited peripherally around the chlamydial inclusion while the phosphorylated, inactive form of MYPT1 was enriched at Src-family kinase rich microdomains (Mital et al., 2010) on the inclusion membrane. Phosphorylated myosin light chain 2 (MLC2), myosin light chain kinase (MLCK), and myosin IIA and IIB were also identified in the inclusion microdomains. Depletion of myosin IIA and IIB, MLCK, and MLC2 by siRNA interference resulted in decreased extrusion formation by C. trachomatis suggesting that CT228 functions in regulation of release mechanisms at the end of the developmental cycle.

Results

Yeast two hybrid screen of CT228

The C-terminus of CT228 downstream of the bilobed hydrophobic domain (aa 86–196) (Figure 1A) was cloned into Gal4 DNA binding domain (BD) pGBKT7 (Clontech) (Figure 1B) and transformed into yeast strain AH109. To identify putative interacting partners, AH109 containing pGBKT7-CT228 was used as bait to screen yeast strain Y187 containing a random normalized HeLa cDNA library (Clontech). A low stringency screen was first employed to select for diploids and confirmed in higher stringency conditions on SD-Trp Leu His Ade + X-α Galactosidase plates. Prey plasmids were isolated and sequenced. One of the positive diploids identified after the bait dependency tests, Prey-43, contained the C-terminus of the PPP1R12A gene which corresponds to the leucine zipper domain of MYPT1. The interaction between CT228 and MYPT1 was verified in a targeted screen using inframe fusions of Gal4AD to full length MYPT1(Figure 1C).

Figure 1. Yeast two-hybrid screen and interaction of C. trachomatis CT228 with MYPT1.

Figure 1

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(A) Kyte and Doolittle plot (Kyte and Doolittle, 1982) of CT228 showing location of the bi-lobed hydrophobic domain. (B) Schematic of bait and prey yeast-two hybrid constructs. The bait construct was generated fusing the C-terminus of CT228 in frame with Gal4BD. The interacting prey identified in the screen consisted of the leucine zipper of MYPT1 fused to Gal4AD (Prey 43). (C) Interactions in the yeast two hybrid screen were verified by β-galactosidase assays of S. cerevisiae diploids generated from mating pairs of pGBKT7 (bait) and pGADT7 (prey) fusions. Liquid β-galactosidase assays confirmed interactions between the positive control p53 and T-antigen, CT228 (C-terminus) and Prey-43 (yeast two-hybrid prey containing the leucine zipper of MYPT1), and CT228 (C-terminus) and full length MYPT1. No interactions were detected with CT228 and MYPT1ΔLZ, pGBKT7 and MYPT1, pGBKT7 and MYPT1ΔLZ, or in the negative control of Lamin and T-antigen. β-galactosidase activity was determined in triplicate with error bars representing standard deviation. (D) Immunoblot of MYPT1 coimmunoprecipitated from infected L2 infected HeLa lysates with rabbit polyclonal antisera to CT228. No interaction was detected in uninfected HeLa lysates or the negative control antisera to R. rickettsii (anti-Rr). (E) Diagram of myosin phosphatase showing relevant regulatory phosphorylation sites on MYPT1, the small subunit (M20), and the catalytic subunit Protein Phosphatase 1 (PP1). Also shown are the location of the ankyrin repeats and the leucine zipper. (F) Interactions in the yeast-two hybrid were verified by α-galactosidase assays of S. cerevisiae diploids generated from mating pairs of GBKT7-CT228 and N-terminally truncated MYPT1 Gal4AD fusions (starting at amino acids 445, 632 or 845; either lacking or retaining the leucine zipper (a–f) (Ankyrin repeats (AR); Leucine zipper (LZ)). Diploids were grown in SD-Trp Leu and interactions were detected on high stringency SD-Trp Leu His Ade + α Gal. Interactions were detected between CT228 and MYPT1-Gal4AD fusions containing the leucine zipper (b, d, and f) as well as the positive control p53 + T-antigen (+). No interactions were detected with MYPT1-Gal4AD fusions lacking the leucine zipper (a, c, and e) and the negative control Lamin + T antigen (−).

Coimmunoprecipitation experiments confirmed the interaction between CT228 and MYPT1 in vivo. MYPT1 was coprecipitated by anti-CT228 in the C. trachomatis L2 infected lysates but not from uninfected lysates nor by an irrelevant antiserum (Figure 1D).

To verify the domain of MYPT1 (Figure 1E) interacting with the C-terminus of CT228, full length and N-terminal truncations of MYPT1 starting at amino acids 445, 632 and 845 either with (MYPT1) or without the leucine zipper (MYPT1ΔLZ) were fused to Gal4AD and screened for interaction with CT228 (Figure 1F). The interaction of MYPT1 with the C-terminus of CT228 was specific as MYPT1 did not interact with the empty vector, pGADT7. The interaction of CT228 with MYPT1 was limited to the constructs that contained the leucine zipper suggesting that the interaction was specific to that region.

CT228 and MYPT1 localization to the inclusion membrane

HeLa cell monolayers infected with C. trachomatis L2 for 18 hrs were stained with rabbit anti-CT228 and mouse anti-MOMP or mouse anti-Src pY419. CT228 was observed around the circumference of the inclusion membrane but enriched in discrete microdomains colocalizing with active Src-family kinases (Figure 2A).

Figure 2. CT228 and MYPT1 localization to the C. trachomatis inclusion membrane.

Figure 2

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(A) HeLa cell monolayers were infected with C. trachomatis L2 at an MOI ~1 for 18 hrs. Cells were fixed and labeled with anti-CT228 and anti-MOMP or anti-Src-pY419. Merged images show CT228 localizing to the periphery of the inclusion and enriched with active Src kinase. Bar = 5 µm. (B) HeLa cells were infected at an MOI of ~ 1 for 18 hrs, fixed and labeled with rabbit anti-MYPT1 and mouse anti-C. trachomatis MOMP or mouse anti-14-3-3β. (C) Expression of mCherry-MYPT1 showing recruitment to the C. trachomatis inclusion membrane at 18 hr post-infection. Bar = 5 µm in panels B and C. (D). Immunoelectron microscopy showing localization of MYPT1 with enrichment at specific sites in the inclusion membrane. Bar = 2 µm.

The distribution of MYPT1 in infected cells was also examined in C. trachomatis L2 infected HeLa cells. At 18 hrs post infection, cells were fixed and labeled with polyclonal rabbit anti-MYPT1 and mouse anti-MOMP (Figure 2B). MYPT1 was localized to the inclusion membrane and not the intracellular bacteria similar to the patterns as previously observed for IncA and IncG (Hackstadt et al., 1999; Scidmore-Carlson et al., 1999). Dual labeling with MYPT1 and 14-3-3β, a host cell protein previously shown to be recruited to the inclusion membrane through its interaction with IncG (Scidmore and Hackstadt, 2001), demonstrated localization of MYPT1 to the inclusion membrane (Figure 2B). The recruitment patterns of MYPT1 to both the periphery of the inclusion and microdomains matches the localization of CT228. To confirm the recruitment of MYPT1 to the inclusion membrane, an N-terminal mCherry fusion of full length MYPT1 was expressed in C. trachomatis L2 infected HeLa cells. In HeLa cells expressing mCherry-MYPT1, MYPT1 was recruited to the periphery of the inclusion membrane in the same pattern seen with anti-MYPT1 antibodies (Figure 2C). The recruitment of MYPT1 to the periphery of the inclusion membrane with enrichment at microdomains was corroborated by immunoelectron microscopy using anti-MYPT1 with immunoperoxidase labeling to show diaminobenzidine reaction product at the cytosolic face of the inclusion membrane (Figure 2D).

MYPT1 recruitment is species specific

The recruitment of MYPT1 to inclusions of other chlamydial serovars and chlamydiae species was examined. HeLa cells were infected with C. trachomatis serovars A, B, D, or L2, C. muridarum, C.caviae and C. pneumoniae and stained with anti- chlamydia LPS and anti-MYPT1 antibodies. MYPT1 localized to all C. trachomatis inclusions except serovar B/Jali20 (Figure 3), which encodes a truncated form of CT228 (Seth-Smith et al, 2009) and does not detectably express CT228. C. muridarum also recruited MYPT1 to the inclusion membrane but C. caviae and C. pneumoniae did not, suggesting that there are diverse requirements for MYPT1 among the different chlamydial species and C. trachomatis serovars. The absence of MYPT1 from serovar B/Jali20, which does not express CT228 confirms the role of this inclusion membrane protein in recruitment of MYPT1.

Figure 3. Species specific recruitment of MYPT1.

Figure 3

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HeLa cells were infected with C. trachomatis serovars A, B, D and L2, C. muridarum mouse pneumonitis (MoPn), C. caviae (GPIC) or C. pneumoniae AR-39 (Cpn). At specific times, cells were fixed (A = 30 hrs; B = 30 hrs; D =30 hrs; L2 = 18 hrs; MoPn =30 hrs; GPIC =30 hrs and Cpn =48 hrs), permeabilized, and labeled with anti- chlamydia LPS antibody and anti-MYPT1 antibody. Chlamydial serovars and species grow at different rates. Images were selected to show approximately the same size inclusions. MYPT1 recruitment was specific for C. trachomatis serovars A, D, and L2 and C. muridarum. Bar = 5 µm.

C. trachomatis infection induces phosphorylation changes in MYPT1 and selective recruitment of pT853-MYPT1 to chlamydial microdomains

CT228 is among the Incs transcribed early in infection (Belland et al., 2003; Shaw et al., 2000) . By 4 hr post-infection, C. trachomatis has initiated protein synthesis and modified the inclusion membrane. Recruitment of MYPT1 is observed by this time (Figure 4A) and is retained circumferentially around the inclusion membrane for the duration of the developmental cycle.

Figure 4. Infection by C. trachomatis L2 causes phosphorylation changes of MYPT1 and selective time dependent recruitment of pT853-MYPT1 in microdomains.

Figure 4

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(A) Indirect immunofluorescence of C. trachomatis L2 infected HeLa cells at 4, 12, 24, and 42 hrs post infection at an MOI of ~10 stained with anti-MYPT1 (green) and anti-L2 EB (red) antisera to show early recruitment and retention of MYPT1 to the inclusion membrane. By 4 hr post-infection, the inclusion membrane has been modified by insertion of chlamydial protein and trafficking of nascent to the MTOC is evident (Grieshaber et al, 2003). By 12 hr post-infection, replication of RBs is apparent but the inclusions have not yet fused into a single inclusion as is typical of C. trachomatis (Hackstadt et al, 1999). Bar = 10 µm. (B) Immunoblot analysis of cell lysates infected with C. trachomatis L2 at an MOI of ~5 for 0, 18, 30, and 42 hrs showing levels of Pan-MYPT1, pT853-MYPT1, and pT696-MYPT1. (C) Indirect immunofluorescence of C. trachomatis L2 infected HeLa cells at 18, 30, and 42 hrs post infection at an MOI of ~1 stained with anti-pT853-MYPT1 (green) and anti-Src-pY419 (red). The lumen of the inclusion (I) is indicated. Bar = 10 µm.

MYPT1 is known to be phosphorylated by a number of kinases, specifically by Rho-associated kinase (ROCK) at threonines 853 and 696 (Feng et al., 1999; Kawano et al., 1999). Phosphorylation of these sites is inhibitory to MYPT1 function due to tertiary folding changes to MYPT1 that no longer allow for the binding of the myosin phosphatase complex to MLC2. Dephosphorylation of MLC2 at threonine 18 and serine 19 by myosin phosphatase is inhibitory to myosin motor activity, thus phosphorylation of MYPT1 favors MLC2 activity. Because the phosphorylation state of MYPT1 is important for the activity, the phosphorylation of MYPT1 was investigated during C. trachomatis infection. Specific antibodies to MYPT1 phosphorylated at T853 (pT853) or T696 (pT696) as well as anti-MYPT1 were used to probe immunoblots of C. trachomatis infected HeLa cell lysates taken at intervals throughout the developmental cycle (Figure 4B). Total MYPT1 levels remained constant in infected and uninfected lysates, however, by 42 hrs post-infection, a dramatic reduction in phosphorylation of MYPT1 at both phosphorylation sites (T696 and T853) was seen in infected cell lysates.

To determine if there was differential recruitment of the phosphorylated forms of MYPT1, C. trachomatis L2-infected HeLa cells were labeled with anti-pT853-MYPT1 and anti-Src-pY419 and examined by immunofluorescence. Phosphorylated MYPT1 was recruited to the inclusion but was enriched in microdomains (Figure 4C) rather than circumferentially around the inclusion membrane as observed with the anti-MYPT1 antibody (Figure 2 and 4A). The pattern was similar to previously described microdomains that are laden with active Src-family kinases (Mital et al., 2010). At 18 hrs post infection, pT853-MYPT1 was rarely observed at the microdomains. However, as the infection progressed, the pT853 form of MYPT1 become more prominent in the microdomains suggesting a time dependent phosphorylation change or recruitment of pT853-MYPT1 late in the chlamydial developmental cycle. Collectively, the data suggest that C. trachomatis infection results in decreased phosphorylation of the inhibitory sites on MYPT1 but selective recruitment of the remaining pT853-MYPT1 to microdomains on the chlamydial inclusion late in development. Thus phosphorylation and activity of MLC2 is expected to be enhanced at these focal points on the inclusion membrane at the time of egress.

Active myosin light chain kinase, serine 19 phosphorylated myosin light chain 2, and myosin IIA and IIB are recruited to microdomains

When phosphorylated at T853, the phosphatase activity of MYPT1 is inhibited and is correlated with increased levels of S19 phosphorylated MLC2. S19 phosphorylated MLC2 interacts with heavy chain myosin II. Antagonistic to MYPT1 is myosin light chain kinase (MLCK), which actively phosphorylates MLC2. MLCK activity is also regulated by phosphorylation state. Phosphorylation of Y464 (pY464) and Y471 (pY471) promotes kinase activity (Birukov et al., 2001). Because the inactive, phosphorylated T853 form of MYPT1 is recruited to inclusion membrane microdomains known to be areas of active Src-family kinase activity, the potential for MLCK, pS19-MLC2, and myosin II recruitment was also examined. C. trachomatis L2-infected HeLa cell monolayers were fixed and labeled with anti-Src-pY419 and either anti-pS19-MLC2, anti-pY464-MLCK, anti-pY471-MLCK, anti-myosin IIA, or myosin IIB (Figure 5). Each of these were enriched at inclusion microdomains and colocalized with active Src-family kinases.

Figure 5. Active MLCK, S19-MLC2, myosin IIA, and myosin IIB are recruited to microdomains on the chlamydial inclusion.

Figure 5

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Recruitment of pY464- and pY471-phosphorylated MLCK, S19-phosphorylated MLC2, and myosin IIA and IIB were examined by indirect immunofluorescence for colocalization with Src-pY419 in C. trachomatis infected HeLa cells at 18 hrs post-infection with an MOI of ~1. Representative images are shown. Bar = 5 µm.

C. trachomatis release by extrusion requires MLCK, MLC2, and myosin II

Myosin II has been implicated in exit mechanisms of C. trachomatis at the end of the developmental cycle and is required for extrusion formation by C. trachomatis through the use of the inhibitor blebbistatin (Hybiske and Stephens, 2007), therefore, the role of the myosin phosphatase pathway in extrusion formation was investigated.

Extrusion formation by C. trachomatis was monitored over time to identify peak times of extrusion formation and release. A peak of extrusion formation was seen at 48 hrs post-infection (Suppl. Figure 1).

C. trachomatis infected cells were also treated with DMSO, blebbistatin (myosin II inhibitor) or jasplakinolide (actin depolymerization inhibitor) and monitored for extrusion formation and total IFUs at 48 hrs post-infection. As previously described (Hybiske and Stephens, 2007), blebbistatin significantly inhibited extrusion formation (p<0.0001) compared to the jasplakinolide or DMSO treated cells (Suppl. Fig. 1). The reduced extrusion formation was not due to growth inhibition since the total infectious PFUs were constant under all conditions.

The effects on release mechanisms of C. trachomatis by siRNA depletion of MLCK, MYPT1, MLC2, and myosin IIA and IIB were assessed. Efficacy of siRNA knockdowns was confirmed by QuantiGene analysis. All targeted messages were decreased by greater than 75%. Depletion of MYPT1 had no effect on extrusion formation (Figure 6A). This result was not surprising because it is the inactive, phosphorylated form of MYPT1 that is associated with activation of the myosin motor complex, thus absence of MYPT1 is indistinguishable from its inactivation. However, MLCK, MLC2, and myosin IIA and IIB depletion significantly reduced extrusion formation suggesting that each of these proteins was essential for C. trachomatis to produce extrusions. Dual knockdowns of MYPT1 and MLCK produced similar levels of extrusion events as the MLCK depletion alone. The extent of extrusion formation was not dependent on total progeny IFUs as C. trachomatis produced similar levels of infectious progeny in all siRNA treatments (Figure 6B). The effects of MLCK, MYPT1, MLC2, MLCK and MYPT1 combined, and myosin IIA and IIB depletion on dynein-dependent interactions with the early inclusion were assessed by two independent assays that had previously been used to analyze C. trachomatis inclusion trafficking (Mital and Hackstadt, 2011). None of the depletions altered early trafficking of nascent inclusions to the MTOC or distance of mature inclusions from centrosomes (not shown). The interactions of the C. trachomatis inclusion with the myosin phosphatase pathway and the actin cytoskeleton do not appear to disrupt interactions with dynein and microtubule dependent trafficking.

Figure 6. Depletion of MLCK, MLC2, and myosin IIA and IIB inhibit extrusion formation.

Figure 6

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(A) Effect on extrusion production by siRNA depletion of components of the myosin phosphatase and myosin kinases pathways. (B) Effect on total infectious progeny production by siRNA depletion of components of the myosin phosphatase and myosin kinases pathways. Experiments were performed in triplicate; error bars represent standard deviation. *p < 0.0001 See also Figure S1.

Discussion

In a yeast two-hybrid screen for Inc-interacting host proteins, an interaction between the inclusion membrane protein CT228 and a subunit of myosin phosphatase, MYPT1, was observed. The recruitment of MYPT1 to the C. trachomatis inclusion was confirmed and showed a unique pattern of circumferential labeling of the inclusion membrane with enrichment at inclusion membrane microdomains. The activity of MYPT1 is regulated by phosphorylation state (Amano et al., 1996). MYPT1 is phosphorylated by a number of kinases, including Rho-associated kinase (ROCK) at T696 and T853 (Feng et al., 1999; Kawano et al., 1999), protein kinase C (PKC) at the ankyrin repeats (Toth et al., 2000), integrin-linked kinase at T34 (Kiss et al., 2002), and Zip kinase at T695 (Haystead, 2005). Of these sites, T696 and T853 are inhibitory whereupon phosphorylation induces tertiary folding changes to MYPT1 that no longer allow for the binding of MLC2 and thereby prevent the dephosphorylation of MLC2. MLC2 thus remains in an active state to interact with myosin IIA and myosin IIB to constitute an active myosin motor complex. The recruitment of T853 phosphorylated MYPT1 to chlamydial inclusion microdomains suggests that myosin phosphatase would be in an inactive state at these specific sites on the inclusion membrane. Accordingly, the myosin phosphatase substrate, MLC2, was observed in the S19 phosphorylated, active state within these Src-family kinase rich microdomains. MLCK is the kinase which phosphorylates MLC2 at T18 and S19, although only recruitment of pS19-MLC2 was addressed in our study. MLCK is activated by phosphorylation at Y464 and Y471 (Birukov et al., 2001) and recruitment of active MLCK to the chlamydial inclusion was confirmed. The enrichment of active Src-family kinases at C. trachomatis inclusion microdomains (Mital et al., 2010) suggests that they play an important role in the phosphorylation and activation of MLCK thus contributing to the phosphorylation of MLC2 and the activation of the myosin motor complex at inclusion membrane microdomains. Recently, myosin has been implicated in one of the egress mechanisms of C. trachomatis; extrusion of intact inclusions from the host cell rather than a lytic event (Hybiske and Stephens, 2007). Myosin IIA and myosin IIB recruitment to inclusion membrane microdomains was confirmed here. A model depicting key elements of the myosin regulatory pathway associated with the inclusion membrane is shown in Figure 7. In this model, the antagonistic activities of myosin phosphatase and myosin kinase would be expected to shift the balance of lytic vs. extrusion egress mechanisms in response to external environmental stimuli and appropriate cellular signaling pathways.

Figure 7. Schematic representation of myosin phosphatase pathway in C trachomatis microdomains.

Figure 7

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The role of myosin phosphatase is to dephosphorylate MLC2. When phosphorylated at T853, MYPT1 is inhibited and can no longer dephosphorylate MLC2. Src-family kinases (SFK) are known to phosphorylate MLCK which in turn actively phosphorylates MLC2. Myosin IIA and IIB and phosphorylated MLC2 and MLCK are required for C. trachomatis extrusion. Dashed lines are used to present potential inhibitory upstream regulators of MYPT1 including Rho-associated protein kinase (ROCK), Protein Phosphatase 1 (PP1), C-kinase potentiated Protein phosphatase-1 Inhibitor (CPI-17), ROCK, Protein Kinase C isoforms (PKCs), diacylglycerol (DAG), and phospholipase C (PLC) that could contribute to myosin phosphatase inhibition and chlamydial extrusion.

All intracellular pathogens must eventually exit the host cell (Friedrich et al., 2012; Hybiske and Stephens, 2008). C. trachomatis has evolved at least two, and possibly three, distinct mechanisms of host-cell egress, cell lysis and extrusion (Hybiske and Stephens, 2007) and a non-lytic exocytosis-like mechanism (Todd and Caldwell, 1985). The extrusion mechanism is an actin dependent process that involves protrusion of the inclusion from the host cell followed by constriction and resealing of the plasma membrane and subsequent release of the intact membrane bound inclusion with survival of the host cell. The extrusion mechanism is believed to be dependent on actin polymerization, N-WASP, Rho GTPase, and myosin II through the use of specific inhibitors of each of the activities (Hybiske and Stephens, 2007). Recruitment of the actin coat to the inclusion prior to extrusion appears to be a sporadic and dynamic event relying on a combination of both bacterial and host factors (Chin et al., 2012).

C. trachomatis manipulates the myosin phosphatase pathway throughout the course of its developmental cycle. MYPT1 associates with CT228 at the inclusion membrane early and remains throughout the developmental cycle. MYPT1 in this state is not phosphorylated thus is an active MLC2 phosphatase expected to favor inactive myosin motor complexes at the inclusion membrane which may prevent premature release of inclusions from the cell. This is in contrast to the localized enrichment of phosphorylated MYPT1 that occurs at discrete microdomains late in infection. Comparison of HeLa cell lysates from uninfected and infected cells show an overall dephosphorylation of MYPT1 in the later stages of infection. Thus the majority of the phosphorylated, inactive MYPT1 is localized at the inclusion membrane where it appears to serve as focal point at which activation of MLC2 by MLCK is favored and the the myosin motor complex is activated to promote egress of the intact chlamydial inclusion. The manipulation of MYPT1 phosphorylation by C. trachomatis not only limits active myosin complexes in the host cell but also permits selective exploitation of active myosin motor complexes by Chlamydia for egress.

Recruitment of MYPT1 among the different chlamydial species and serovars can be at least partially accounted for by the diversity of CT228. C. trachomatis serovar B/Jali20 encodes a truncated CT228 (Seth-Smith et al., 2009). Specifically, a deletion at position 86 of CT228 results in a frameshift predicted to produce a truncated CT228 of 38 amino acids in length which lacks the C-terminus that interacts with the leucine zipper of MYPT1. We confirmed the lack of expression with specific antibodies. Accordingly, C. trachomatis B/Jali20 does not recruit MYPT1 to the inclusion membrane. Interestingly, C. trachomatis B/Jali20 did not form extrusions (not shown). The most closely related species to C. trachomatis that also recruited MYPT1, C. muridarum, contains a CT228 homologue that shares 50% identity at the amino acid level to CT228 of C. trachomatis L2. C. muridarum does not, however, exhibit Src-kinase rich microdomains (Mital et al., 2010). C. caviae and C. pneumoniae did not recruit MYPT1. C. caviae contains a distant homolog which may be too divergent to have retained the same function and C. pneumoniae lacks a CT228 homologue (Lutter et al., 2012). It is likely that additional chlamydial or host factors, potentially those involved in the initiation of actin recruitment around the late inclusion (Hybiske and Stephens, 2007; Kumar and Valdivia, 2008), are required for the commencement of the actual extrusion process. Interestingly, neither MYPT1 nor inclusion membrane microdomains (Mital et al., 2010) are observed in C. caviae although C. caviae has been described as utilizing the extrusion mechanism of release, albeit at an apparently reduced rate (Hybiske and Stephens, 2007). The recognition of Chlamydia variants lacking CT228 and/or Src-kinase rich microdomains should be of value in deciphering the many potential regulatory pathways that control C. trachomatis egress from infected cells.

Modulation of MLC2 and MLCK phosphorylation states during the infection process of other pathogens has been described. MYPT1 had not been previously reported to be recruited to the site of infection of other intracellular pathogens although changes in phosphorylation of MYPT1 were identified during Trypanosoma cruzi infection (Mott et al., 2009). Evidence of MLC2 phosphorylation during infection of enteropathogenic E. coli (EPEC) was observed in cell fractions associated with the cytoskeleton (Manjarrez-Hernandez et al., 1996). Alterations in intestinal epithelial cell permeability induced by either EPEC or enterohemorrhagic E. coli involve the phosphorylation of MLCK (Philpott et al., 1998; Yuhan et al., 1997). Myosin is a known target for other intracellular pathogens including Listeria monocytogenes which usurps myosin VIIA for uptake into host cells (Sousa et al., 2004) and Shigella flexneri which requires myosin II (Rathman et al., 2000) for dissemination and myosin-X for filopodia formation (Bishai et al., 2012). Salmonella enterica serovar Typhimurium also co-opts myosin II via SopB to regulate salmonella-containing vacuole dynamics such as positioning and stability (Wasylnka et al., 2008). Several viruses (Santangelo and Bao, 2007; van Leeuwen et al., 2002) also utilize myosin in egress from host cells although these are not within a membrane bound compartment.

C. trachomatis inclusion membrane microdomains were first identified as areas of focal recruitment of activated Src-family tyrosine kinases and the microtubular cytoskeleton (Mital et al., 2010). It now appears that microdomains are also focal points for interactions with the actin cytoskeleton and involvement in egress at the end of the developmental cycle. Inclusion membrane microdomains thus appear to be platforms for interactions with both the actin and microtubular cytoskeleton. Whether the two pathways might interact cooperatively or antagonistically remains to be determined. The implication of Src-family tyrosine kinases and early expressed Incs in regulation of both early trafficking of the inclusion and egress suggests the potential for antagonistic activities that may hold egress mechanisms in check until an appropriate signal is received at the end of the developmental cycle. Tyrosine phosphorylation seems to play multiple, species specific roles in early vs. late interactions with the cytoskeleton throughout the chlamydial developmental cycle (Mital and Hackstadt, 2011) and may be a key regulator of egress mechanisms.

It is interesting that chlamydiae would have two independent means of egress. It is suspected that the underlying purpose for chlamydial extrusion may lie in evasion of innate or acquired immune responses (Hybiske and Stephens, 2007). The release of infectious EBs encapsulated within an intact inclusion membrane may allow the chlamydiae to evade local inflammatory mediators for subsequent release and infection of susceptible cells at more distal sites. Interestingly, two potential upstream regulators of the myosin phosphatase pathway, PKC and ROCK, are activated by pathways involved in immune responses and inflammation. Activation of PKC and extracellular signal-related kinases (ERKs) during enteropathogenic E. coli (EPEC) infection of intestinal epithelial cells leads to activation of NF-kB and the proinflammatory response (Savkovic et al., 2003). In addition, endothelial cell permeability is increased by lipopolysaccharide (LPS) and TNF-alpha which activate p38 and ERK1/2 MAP kinases, increase NF-kB signaling and ROCK mediated phosphorylation of MYPT1, and lead to the accumulation of phosphorylated MLC2 (Xing and Birukova, 2010). The upstream events leading to the phosphorylation of MYPT1 and MLC2 at the chlamydial inclusion microdomains remain to be verified but activation of upstream regulators of the myosin phosphatase pathway during an immune or inflammatory response to chlamydial infection would be expected to shift the mechanism of host-cell exit to favor extrusion. The dissemination of membrane-bound infectious EBs to distant sites to avoid exposure to the harsh environment of local inflammatory responses would thus be favored. Future characterization of the pathway and regulatory components should provide insights leading to a greater understanding of how C. trachomatis forms extrusions and its role in chlamydial pathogenesis.

Experimental Procedures

Strains and Cell Culture

Chlamydia trachomatis serovars L2 (LGV 434), D (UW3-Cx), B (Jali20/OT), and A (HAR-13), C. muridarum (MoPn), C. pneumoniae (AR-39), and C. caviae (GPIC) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., 1981). HeLa 229 cells were grown in RPMI 1640 medium with 10% fetal bovine serum (FBS). Yeast strain Y187 was purchased from Clontech and AH109 was provided by M.A. Scidmore.

Antibodies

Monoclonal antibody L2-I-45 against C. trachomatis L2 major outer membrane protein (MOMP) was kindly provided by H. D. Caldwell. GAL4-DNA BD fusions of CT228 were detected with anti-Myc antibody (Cell Signaling, Danvers, MA). Anti-MYPT1 (US Biologicals, Massachusetts, MA) and anti-T853 MYPT1 (Abnova, Walnut, CA) were used to detect pan-MYPT1 and Thr 853 phosphorylated MYPT1 by indirect immunofluorescence. Anti-MYPT1, anti-pT853 MYPT1, anti-pT696 MYPT1, anti-Myc, and anti-GAPDH (Cell signaling, Danvers, MA) were used for immunoblotting. Anti-phospho (pSer-19)-MLC2 was purchased from Abcam (Cambridge, MA). Anti- Src-pTyr419 (clone 9A6) (Millipore, Billerica, MA) was used to detect active Src-family kinases. Anti-14-3-3β (Clone H8), anti-MLCK (pTyr471) and anti-MLCK (pTyr464) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Chlamydia LPS (Thermo Scientific, Rockville, MD) was used to detect all species of chlamydiae and a rabbit polyclonal anti-EB serum was used to detect C. trachomatis. Anti-myosin IIA and anti-myosin IIB were purchased from Thermo Scientific (Thermo Scientific, Rockville, MD). Anti-mouse or anti-rabbit DyLight 594 and DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondaries for indirect immunofluorescence and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for immunoblotting and immunoelectron microscopy as described (Scidmore-Carlson et al., 1999).

Antibody production to CT228

Overnight cultures of BL21 (DE3) were sub-cultured 1:200 into 600 mL of Luria-Bertani broth and grown to mid exponential phase at 37°C. CT228-His expression was induced by addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a final concentration of 1mM with further 4 hrs growth at 37°C. The culture was harvested by centrifugation at 5000 rpm for 10 minutes and protein purification was carried out using the ProBond Purification System (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. Purified protein was quantitated using the Bradford protein assay (Bio-Rad, Hercules, CA) and dialyzed overnight against 100 mM NaCl. The purified protein was emulsified in Sigma Adjuvant System (Sigma) and rabbits immunized according to the manufacturer's instructions.

Yeast two-hybrid library screening

Yeast two-hybrid assays were performed according to the Matchmaker Gold yeast Two-Hybrid System (Clontech). The Y187 yeast strain pre-transformed with Normalized Mate and Plate HeLa S3 Library constructed in pGADT7-RecAB was purchased from Clontech. pGBKT7–CT228 was transformed into AH109 and mated overnight with Y187 containing Normalized Mate and Plate HeLa S3 Library. Matings were screened for interacting clones by selecting diploids on SD plates lacking leucine (Leu), tryptophan (Trp), and histidine (His) (low stringency). Positive diploids were re-screened for the ability to grow on SD plates lacking His, Leu, Trp, and adenine (Ade) + X-α Galactosidase (high stringency). Plasmid DNA was isolated from blue diploid colonies using PrepEase Yeast Plasmid Isolation Kit (Affymetrix, Santa Clara, CA), transformed into E. coli XL1-blue (Stratagene, Santa Clara, CA), re-extracted using a Qiagen plasmid extraction kit, and sequenced at the RML Genomics, Research Technologies Section, NIAID. Bait dependencies and targeted screens were performed by directed mating between AH109 carrying pGBKT7-CT228 and Y187 carrying prey plasmids from selected diploids. Matings were plated on high stringency plates and monitored for diploid growth. See Extended Experimental Procedures for a detailed description of plasmid constructions.

Protein preparation and Western blotting

Protein extracts from AH109 carrying pGBKT7-CT228 were prepared according to Urea/SDS protein extraction method provided in the Yeast Protocols Handbook (Clontech) prior to SDS-PAGE and electrophoretic transfer to nitrocellulose. HeLa cells were infected with C. trachomatis at a multiplicity of infection (MOI) of ~1. At appropriate times postinfection, the cells were rinsed in phosphate-buffered saline (PBS, pH 7.4) and lysed in Laemmli buffer (Laemmli, 1970) with 5% β-mercaptoethanol plus PhosSTOP inhibitor cocktail (Roche, Indianapolis, IN) and Complete Mini protease inhibitor cocktail (Roche, Indianapolis, IN).. Protein extracts and cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked in 5% nonfat dry milk/2%BSA in 50 mM Tris-HCl, pH 7.4- 150 mM NaCl, 0.1% Tween 20 (TBS-T) and incubated with protein specific primary antibody overnight in blocking buffer overnight. Unbound antibody was removed with by 3 rinses in TBS-T and bound antibody was detected with HRP-conjugated donkey anti-rabbit IgG secondary. Blots were then rinsed 3 times in TBS-T and developed with Amersham ECL selection western blotting detection reagent (GE Healthcare Life Sciences, Pittsburgh, PA) and exposed to CL-X posure film (Thermo Scientific, Rockville, MD).

siRNA knockdown

Monolayers of HeLa 229 cells (50% confluence) were plated on glass coverslips or Costar Cell bind (Corning, Lowell, MA) 24-well plates and incubated overnight with MYPT1, MLC2, myosin IIA, myosin IIB, MLCK or Scramble Targetplus Smartpool siRNA (Dharmacon) complexed in DharmaFECT1 in RPMI with 10% FBS. At 72 hrs post-transfection, cells were infected with C. trachomatis L2 at an MOI of 1 for 48 hrs. Knockdown efficiency based upon transcript level was determined by QuantiGene analysis per manufacturer's instructions (Panomics, Fremont, CA). Lysates were probed with MYPT1, MLC2, myosin IIA, myosin IIB, MLCK, or actin specific RNA probes and the luminescence for each sample and probe was measured using a Synergy4 plate reader (BioTek Instruments, Inc.). Each transcript was normalized to the actin signal as a loading control.

Coimmunoprecipitation

HeLa cells were either mock infected or infected with C. trachomatis L2. At 48 hrs post infection, cells were washed 3 times with PBS, scraped into 0.75 mls of RIPA buffer plus PhosSTOP inhibitor cocktail (Roche) and Complete Mini protease inhibitor cocktail (Roche) and incubated 20 minutes on ice. Cell debris was removed by centrifugation (13,000 rpm, 5 minutes, 4C). Cell-free lysates were incubated with anti-CT228 or anti-Rickettsia for one hour with rotation at 4C. Protein-A beads (Thermo Scientific, Rockville, MD) were added and rotation continued overnight at 4C. The beads were washed 3 times with PBS and bound proteins eluted into Laemmli buffer (Laemmli, 1970) with 5% β-mercaptoethanol, separated by SDS-PAGE and electrophoretically transferred to nitrocellulose for detection with MYPT1 antibody.

Infectious progeny and inclusion development assays

Following siRNA knockdown and 48 hrs of C. trachomatis L2 infection both supernatant and cell lysates were examined for inclusion forming units (IFUs). Supernatant and cells lysed in distilled water were serially diluted in Hank's balanced salt solution and plated onto HeLa cell monolayers. After 24 hrs infection, monolayers were fixed in methanol, stained with rabbit anti-EB sera followed by anti-rabbit DyLight 488. The number of inclusions per field of view were counted on 20 fields for each sample using a Nikon Eclipse 80i fluorescent microscope. Total IFUs/mL were calculated for each sample.

Extruded inclusion enumeration

Extruded inclusions were enumerated by a method similar to that of Chin et al (Chin et al., 2012). HeLa cells were infected with C. trachomatis L2 at an MOI ~1. At 24 hrs post-infection the medium was replaced with RPMI 1640 medium with 10% FBS supplemented with DMSO, blebbistatin (50 µM, EMD Millipore), or jasplakinolide (1 µM, Life Technologies). At the indicated times, the supernatants were removed and gently pelleted by centrifugation at 1000 rpm. The resulting pellet was resuspended in 100 µL of media, mixed with trypan blue (Life Technologies) and stained with NucBlue Hoechst live cell stain (Life Technologies). Extruded inclusions free of host cell nuclei were enumerated using a Hausser Bright-line Phase hemacytometer and a Zeiss LSM 510 Meta laser confocal scanning microscope.

Statistics

Statistical analysis was performed in Prism 5.0 using a one way analysis of variance and a Newman-Keuls posttest.

Supplementary Material

01

Chlamydia trachomatis CT228 recruits MYPT1 to the inclusion membrane

MYPT1 assumes an inactive phosphorylated form in microdomains late in infection

Active MLCK, MLC2, Myosin IIA and IIB are present in these microdomains

The phosphorylation state of myosin phosphatase controls chlamydial egress

Acknowledgements

This work was supported by the Intramural Research Program of the NIAID/NIH. We thank J. Sager and C. Dooley for excellent technical assistance, Drs. K. Hybiske, A. Omsland, L. Bauler, and D. Bublitz for critical review of the manuscript, and Heather Murphy and Anita Mora for graphic arts. We thank Dr. Ian Clarke for providing C. trachomatis B/Jali20.

Footnotes

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SUPPLEMENTAL INFORMATION

Supplemental information includes Extended Experimental Procedures and one figure and can be found online at

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