INTRODUCTION
Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections worldwide. From 2014 to 2018, the CDC reported a substantial 19.4% increase in chlamydial infections in the United States, with the number of these infections being disproportionately higher in women of reproductive age. Due to the asymptomatic nature of infections in the majority of cases, many are undiagnosed and, therefore, untreated, which can cause significant damage to female reproductive health, resulting in pelvic inflammatory disease, ectopic pregnancy, and infertility (
1).
As an obligate intracellular pathogen,
Chlamydia is highly evolved to survive within a eukaryotic host, where it primarily infects mucosal epithelial cells (
2). Within the host cell,
Chlamydia undergoes a unique developmental cycle, where organisms grow within a membrane-bound vacuole, called the chlamydial inclusion, and alternate between two morphological forms, the elementary body (EB) and the reticulate body (RB) (reviewed in reference
3). The chlamydial developmental cycle is characterized by three main temporal stages: early (∼2 to 10 h postinfection [hpi]), middle (∼12 to 28 hpi), and late (∼30 to 48 hpi), where the transcription of specific genes peaks within the time frames of each distinct developmental stage (
4–6).
Within the chlamydial inclusion,
Chlamydia is protected from host cell defenses. The inclusion is an ideal environment that allows efficient progression through the developmental cycle, arguably because the bacteria are never in direct contact with the host cytosol during infection. Rather, all host-pathogen interactions occur via the inclusion membrane (IM). Therefore, the IM is thought to serve at least two important functions for the survival of
Chlamydia: (i) to protect
Chlamydia from intracellular innate host defenses and (ii) to serve as a scaffold for host-pathogen interactions that allow it to scavenge nutrients from the host (
7). The latter function presumably allows the inclusion to interact with a variety of host cell compartments, such as the Golgi apparatus and endoplasmic reticulum (ER). The composition of the IM is derived from both the host and the pathogen, as it acquires host lipids like sphingomyelin and cholesterol for eventual incorporation into bacterial membranes (
8,
9) and is also studded with a class of chlamydial type III secreted effectors called inclusion membrane proteins, or Incs (
10–12).
C. trachomatis carries more than 50 genes for predicted Inc proteins, which account for ∼7% of
C. trachomatis’s highly reduced genome (
5,
10,
13–15); however, the specific functions for most of these Incs are unknown. The hallmark feature of an Inc protein is the presence of one or more bilobed transmembrane domains that anchor these proteins in the IM, with the N and C termini of these Incs facing the host cytosol (
16,
17). It is widely hypothesized and accepted in the field that one of the general functions of Incs is to mediate host-
Chlamydia interactions (
7).
As all chlamydial genes are temporally expressed,
inc genes are also transcribed in a developmentally regulated manner. For example,
inc gene transcription has been quantified during the immediate-early stages of the developmental cycle or during the mid-developmental cycle. The latter is a time when
Chlamydia is rapidly dividing, the inclusion is expanding, and large amounts of nutrients are being scavenged from the host cell (
4,
6,
11).
Interactions between certain Incs and eukaryotic binding partners have been identified. However, the experimental designs for these studies assumed that these interactions are very robust and stable throughout the developmental cycle; in other words, once an Inc is expressed it binds its eukaryotic partner for the remainder of the developmental cycle. To illustrate, some studies examined only one Inc protein to find interactions with host proteins (
18,
19), whereas others examined interactions of one Inc protein or one host protein at a single time point during the chlamydial developmental cycle (
20–30). This type of experimental design, while informative and helping to advance the field, likely misses the more dynamic and less robust Inc-host interactions that allow
Chlamydia to interact with multiple different host compartments via the chlamydial IM. A significant limitation to studying Incs is the lack of antibodies against endogenous forms, which is why many of these studies have relied on creating epitope-tagged Inc constructs for exogenous expression either from
Chlamydia or within uninfected eukaryotic host cells (
19,
25,
31). However, creating a transformed
Chlamydia strain that expresses a specific Inc with an epitope tag is time-consuming and requires extensive expertise. Thus, a list of candidate Incs winnowed by other methods assists studies that examine if host protein-Inc interactions occur at the chlamydial IM.
Overall, Inc proteins share little sequence or structural homology with annotated proteins, but bioinformatic predictions reveal potential domains similar to those found in specific eukaryotic proteins (
24,
25,
27,
32,
33). For example, three Inc proteins, IncA, CT223, and CT813, have been shown to contain domains that are similar to eukaryotic SNARE proteins (
32). SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are a conserved family of eukaryotic proteins that promote membrane fusion (
34,
35). SNARE proteins are classified by their central amino acid in the characteristic SNARE motif, arginine (R-SNARE) or glutamine (Q-SNARE). Together, 3 Q-SNARE motifs and 1 R-SNARE motif form a stable four-helical bundle called a SNARE complex to provide the necessary energy for membrane fusion (
36). Of the chlamydial Inc proteins containing a eukaryotic SNARE motif, IncA encodes two Q-SNARE domains that are required for the homotypic fusion of
C. trachomatis inclusions in cells infected with multiple organisms (
37,
38). This demonstrates a potential function for the chlamydial IncA SNARE motifs in effecting membrane fusion.
Relatedly, previous studies have demonstrated that multiple host SNARE proteins are recruited to chlamydial inclusions. Syntaxin 6, VAMP4, and syntaxin 10 are localized to inclusions during infection with
C. trachomatis serovar L2 (
39–41). The localization of syntaxin 6 is conserved in serovar D,
C. caviae,
C. pneumoniae, and
C. muridarum (
41). VAMP4 is also recruited to inclusions containing
C. muridarum but not to the inclusions of other serovars and chlamydial species tested (
39), suggesting different SNARE proteins perform different roles during infection with
Chlamydia. Further, VAMP4 is involved in sphingomyelin trafficking to the chlamydial inclusions to which it localizes, suggesting dedicated functions of SNAREs in host nutrient acquisition by
Chlamydia (
39). Other studies have demonstrated the recruitment of VAMP3, VAMP7, and VAMP8 to serovar D inclusions, and IncA was shown to interact with these SNAREs, including VAMP3, using an
in vitro liposome assay (
32). Lastly, dual ectopic expression in uninfected cells of the Inc, CT813, and VAMP7 and -8, followed by coimmunoprecipitation (co-IP), demonstrated that CT813 can interact with these SNARE proteins in this artificial system (
32). However, biochemical evidence for host-
Chlamydia interactions involving SNARE domains and whether these Incs function within SNARE complexes at the IM is lacking.
In this study, we examine the mechanism for the recruitment of VAMP3 and VAMP4 to the chlamydial inclusion by hypothesizing that Incs mediate this process. Short interfering RNA (siRNA) knockdown of VAMP3 and/or VAMP4 suggests that these VAMP proteins contribute to the expansion of the IM during chlamydial development (
39). To best understand time frames in which interactions were most likely to happen, we used confocal microscopy to characterize the localization of endogenous VAMP3 and VAMP4 over the course of the chlamydial developmental cycle and in the presence of chloramphenicol, a bacterial protein synthesis inhibitor. We found that the localization of VAMP4, but not VAMP3, is highly dependent on the Golgi structure and that localization of VAMP3 is dependent on
de novo chlamydial protein synthesis during the mid-developmental cycle. To identify candidate Inc proteins with which these VAMP proteins may interact, we used two
in vivo screening tools: the bacterial adenylate cyclase-based two-hybrid (BACTH) system and transient cotransfection of uninfected eukaryotic cells followed by coimmunoprecipitation. With a streamlined list of possible Inc-VAMP interactions, we then performed coimmunoprecipitation assays with chlamydial strains that we developed to inducibly express Inc-FLAG constructs in infected cells over several time points postinfection. These studies demonstrate that VAMP3 interacts with IncF, IncG, CT442, CT449, and CT813 in a temporal and transient manner. We were unable to validate any VAMP4-Inc interactions during infection with
C. trachomatis serovar L2, suggesting that VAMP4’s interactions at the IM are unique compared to VAMP3’s interactions. Further, VAMP3, but not VAMP4, localization to inclusions is altered in
incA or
ct813 mutant strains, where endogenous levels of VAMP3 at the IM are increased or decreased, respectively. By taking a systematic and temporal approach to identifying host-pathogen interactions at chlamydial inclusions, we provide novel insights into the dynamic nature of the interactions at the chlamydial inclusion that enables
Chlamydia to survive within its intracellular niche.
DISCUSSION
In this study, we examined the localization of endogenous eukaryotic SNARE proteins, VAMP3 and VAMP4, during infection with
C. trachomatis serovar L2. Both VAMP3 and VAMP4 are localized to
C. trachomatis serovar L2 inclusions at distinct time points within the developmental cycle. VAMP4, but not VAMP3, recruitment is heavily reliant on an intact Golgi structure, suggesting that there are different recruitment mechanisms of VAMP3 and VAMP4 to chlamydial inclusions (
Fig. 1; see also Fig. S1 in the supplemental material). Knockdown of either VAMP3 or VAMP4 with siRNA prior to infection with
C. trachomatis serovar L2 results in a decreased circumference of chlamydial inclusions (Fig. S2B), and our data revealed a statistically significant reduction in infectious progeny in single VAMP4 knockdown, but not VAMP3 knockdown or a double VAMP3/VAMP4 knockdown, compared to nontargeting control siRNA (Fig. S2C).
De novo chlamydial protein synthesis is required for VAMP3 and VAMP4 localization to the chlamydial inclusion (
Fig. 2) (
39), indicating the requirement of a chlamydial binding partner or a process requiring chlamydial protein synthesis.
To better understand the mechanism of chlamydial recruitment of these eukaryotic VAMPs to inclusions, we wanted to identify chlamydial binding partners, with Incs ostensibly being the most likely targets of VAMP3/4 recruitment to the inclusion. We initially used two screening methods, BACTH and coectopic expression in uninfected eukaryotic cells, to identify candidate Inc binding partners. By BACTH, we found that VAMP3 interacted with 9 different Incs, and VAMP4 interacted with 13 different Incs. Eight Incs interacted with both VAMP3 and VAMP4 (
Fig. 3). Coectopic expression of these VAMPs with candidate Incs in uninfected HeLa cells further narrowed the list of possible interactors to 6 Incs for VAMP3 and 7 Incs for VAMP4, with 5 of these Incs being common to both VAMP3 and VAMP4 (Fig. S6). Based on these results, we created inducible expression strains of
C. trachomatis serovar L2 encoding these candidate Incs and assessed interactions between these Incs and VAMP3 or VAMP4 by harvesting lysates for coimmunoprecipitation every ∼30 min during the mid-developmental cycle. For these studies, we transfected HeLa cells with 6×His-VAMP3 or 6×His-VAMP4, infected them with the indicated strains, induced the expression of Inc-FLAG constructs with aTc, and examined interactions between 15 and 24 hpi. With this approach, we were unable to confirm an Inc binding partner for VAMP4 (Fig. S10). However, we did observe interactions between VAMP3 and IncF, IncG, CT449, and CT813 at discrete times during the mid-developmental cycle of
C. trachomatis serovar L2, with most interactions occurring between 16 and 19 hpi (
Fig. 4). VAMP3 was also found to interact with CT442 between 28 and 30 hpi (
Fig. 4). Lastly, we observed that VAMP3, but not VAMP4, recruitment is altered to chlamydial inclusions deficient in certain Inc proteins, with an increase and decrease in VAMP3 recruitment to chlamydial inclusions lacking IncA or CT813, respectively (
Fig. 5). These data are consistent with VAMP3’s interaction with CT813 during infection with
C. trachomatis serovar L2 (
Fig. 4), indicating CT813 functions in VAMP3 recruitment by
Chlamydia. These data highlight the temporal and dynamic nature of certain Inc-host protein interactions at the chlamydial IM.
A previous study predicted that VAMP3 was part of the interactome surrounding the chlamydial inclusion (
58). Similarly, another study relying on the ectopic expression of Incs in uninfected HeLa cells to identify eukaryotic binding partners indicated that VAMP3 was a candidate binding partner for IncB and IncE (
19). In our current study, we confirmed that VAMP3 interacted with IncB, as assessed by BACTH or dual ectopic expression in uninfected cells followed by co-IP (
Fig. 3 and Fig. S6). However, we did not detect an interaction between VAMP3 and IncE via BACTH (Fig. S3); therefore, we did not study that potential interaction further. We were unsuccessful in creating a
C. trachomatis serovar L2 strain that could express IncB-FLAG, so we were unable to test this VAMP3-Inc interaction in the context of a chlamydial infection. However, a previous affinity purification mass spectrometry study, using an IncB-APEX2 construct, did not identify VAMP3 at 18 or 24 h postinfection (
59). The negative results in the previous study are not surprising given the dynamic nature of the observed VAMP3 interactions with specific Incs in this study. Regardless, we cannot exclude the possibility that IncB and VAMP3 interact in chlamydial infected cells.
Our data demonstrate a direct link between VAMP3 recruitment to chlamydial inclusions treated with chloramphenicol (CM) and the time points postinfection that VAMP3 interacts with Inc proteins in chlamydial infected cells. VAMP3 recruitment to chlamydial inclusions is abolished when organisms were treated with CM at 15.5 hpi. In contrast, CM treatment at 23.5 hpi resulted in polarized VAMP3 recruitment to the inclusion (
Fig. 2) in a manner similar to what is observed during later time points in the developmental cycle (
Fig. 1A). Our corresponding VAMP3-Inc interaction studies in chlamydial infected cells revealed that VAMP3 temporally interacts with 4 distinct Inc proteins between 15 and 22.5 hpi (
Fig. 4). Interestingly, these interactions are no longer detectable at 24 hpi, with the exception of CT442 at 28 to 30 hpi (
Fig. 4). Thus, we have established a window during the developmental cycle in which VAMP3 is recruited to the IM as well as to which Inc proteins it can bind during this time frame.
There are two distinct, but not mutually exclusive, possibilities for how VAMP3 is recruited by Incs to the IM. First, multiple Incs could be recruiting VAMP3 (and other host proteins) through redundant mechanisms or, second, some Incs could be acting as scaffolds for host protein recruitment to then allow for subsequent interactions with other Incs (summarized in
Fig. 6). When we induced expression of CT442-FLAG during the mid-developmental cycle, we also detected an interaction between VAMP3 and CT442-FLAG (Fig. S7), indicating that interactions between VAMP3 and target Incs are quite promiscuous during this time frame.
Of note, we observed VAMP3 at the IM throughout the developmental cycle, but it is unknown if the VAMP3 that is localized at the IM later in the developmental cycle is the same population of VAMP3 that is initially recruited or if there is continual recycling of VAMP3 to and from chlamydial inclusions. Live-cell imaging studies following VAMP3’s trafficking patterns in chlamydial infected cells would directly address these unknowns. We also do not understand how or if turnover of Incs on the IM contributes to the recruitment of eukaryotic proteins. This is a particularly relevant consideration when studying eukaryotic proteins where the localization pattern changes over the course of the developmental cycle.
Although we did not detect an Inc interaction with VAMP4 in the context of a chlamydial infection using the methods described in this study, we gained a better appreciation for the way VAMP4 may be interacting at the IM. We have previously demonstrated the importance of VAMP4 during infection with
C. trachomatis serovar L2 in that VAMP4 plays a role in inclusion expansion and chlamydial lipid acquisition (
39). Here, we have shown again that VAMP4 plays a role in inclusion expansion that is greater than that played by VAMP3 (Fig. S2B). We also show VAMP4’s localization at the inclusion is strongly dependent on Golgi structure, as BFA treatment to collapse the Golgi structure abolished most of the VAMP4 observed at the inclusion (
Fig. 1B and Fig. S1B). If very little VAMP4 is stably localized to the inclusion, then this likely contributes to our inability to confirm an Inc binding partner for VAMP4 (Fig. S10). This also suggests that VAMP4’s localization/role at the inclusion is linked with the Golgi structure instead of a direct interaction with an Inc protein. Another potential explanation of our inability to determine an Inc binding partner for VAMP4 is that we limited our focus to examining Inc proteins that were positive for interactions with VAMP4 via BACTH and coectopic expression pulldown experiments and, in doing so, may not have examined the “correct” Incs or used the optimal time frame in which to capture these interactions. Further, VAMP4’s recruitment to the IM could be more indirect. For example, VAMP4 recruitment could be mediated by another host protein that forms a bridge between it and an Inc, i.e., a cofactor, as described in our model for VAMP-Inc interactions at the IM (
Fig. 6). It is also feasible that VAMP4 is recruited by a lipid-driven mechanism that requires chlamydial protein synthesis.
Chlamydia intercepts certain host lipids, like sphingomyelin (
9) and cholesterol (
8), from the Golgi compartments, and these processes require chlamydial protein synthesis. Consistent with this hypothesis, siRNA knockdown of VAMP4 prevents sphingomyelin trafficking to chlamydial inclusions containing
C. trachomatis serovar L2 and
C. muridarum (
39). Thus, the recruitment mechanism of VAMP4 to
C. trachomatis serovar L2 inclusions remains elusive, and methods other than those described in this study will be needed to understand it. Specifically, studies that look more closely into the association of VAMP4, the Golgi structure, and lipid recruitment to the chlamydial inclusion are required (
Fig. 6).
Even though we established Inc binding partners for VAMP3, the function of VAMP3 at the chlamydial inclusion remains unknown. In uninfected cells, VAMP3 localizes to early and recycling endosomes in eukaryotic cells, where it functions in recycling and retrograde trafficking between the
trans-Golgi network and plasma membrane (
45). VAMP3 regulates the recycling of integrins and the transferrin receptor to the plasma membrane (
60,
61) while also participating in the retrograde transport of mannose-6 phosphate receptor to the Golgi membrane (
62). Additionally, VAMP3 has been heavily implicated in the formation and maintenance of vacuoles supporting survival of several intracellular pathogens. For example, during infection with
Yersinia pseudotuberculosis, VAMP3 is recruited early to the
Yersinia-containing vacuole (YCV), where it then acts as a checkpoint for the YCVs to preferentially become single-membrane, as opposed to LC3-positive double-membrane, to prevent autophagy (
63). Further, VAMP3 has also been found to be associated with
Mycobacterium tuberculosis-containing vacuoles, where the C terminus of VAMP3 is cleaved, which, in turn, alters traffic to and from the mycobacterial phagosome for the benefit of the bacteria (
64). Lastly, VAMP3 has been shown to aid in the clearance of group A
Streptococcus (GAS) by the fusion of GAS-containing autophagosome-like vacuoles with recycling endosomes to promote autophagy (
65). These studies demonstrate the role VAMP3 plays in regulating autophagic pathways to promote either clearance or maintenance of intracellular bacterium-containing vacuoles. Our studies have not yet identified a clear function for VAMP3 in chlamydial pathogenesis. However, by further exploring the implications of how VAMP3 engages with specific Inc proteins, and perhaps using less permissive cell lines than HeLa cells, we will gain a better understanding of why VAMP3 is recruited to the chlamydial inclusion.
In this study, we have successfully created, to our knowledge, the first
inc knockout strain of
C. trachomatis serovar L2 (Δ
incA) using allelic exchange mutagenesis (Fig. S11A). All other
inc-deficient strains of
C. trachomatis serovar L2 have been created using the TargeTron system to inactivate the
inc gene with the insertion of an intron (
66), which could lead to the production of a truncated protein depending on where the intron is inserted. Using the
C. trachomatis serovar L2 Δ
incA mutant, we report that the loss of IncA in the IM increased the amount of VAMP3 localized at chlamydial inclusions (
Fig. 5). A previous study indicated that IncA can act as an inhibitory SNARE protein (
67). However, as we did not detect a positive interaction between IncA and VAMP3 via BACTH, we did not explore this interaction further (Fig. S3). Another potential explanation for the increased VAMP3 recruitment to
C. trachomatis serovar L2 Δ
incA inclusions is that the total loss of an Inc protein in the IM can influence how other Inc proteins are organized within it (Fig. S13). Although the availability of antibodies against endogenous Inc proteins is limited, we examined three Inc proteins, IncE, CT223, and CT813, to observe their IM organization via confocal microscopy during infection with the
C. trachomatis serovar L2 Δ
incA mutant to compare it to WT
C. trachomatis serovar L2. The loss of IncA drastically impacts CT223 organization. In WT inclusions, CT223 is organized in microdomains (
33), whereas in IncA-deficient inclusions, CT223 is organized uniformly around the entire IM. There are no observable differences in the localization of IncE or CT813 when comparing WT and IncA-deficient inclusions, as these proteins are both uniformly localized throughout the IM of both strains (Fig. S13). These data raise the need to further understand how Inc proteins are organized in the IM and how interactions with other Incs may influence not only the composition of the IM but also the coordinated functions of Inc proteins to facilitate interactions with the host. These areas are poorly understood but likely play major roles in chlamydial IM pathogenesis.
There are conflicting reports on whether CT442 should be classified as an Inc protein (
13,
16,
17,
49,
68) or a chlamydial outer membrane protein, CrpA (
69–71). CT442 is expressed late in the developmental cycle, which would make it the only known late developmental cycle Inc protein (
4–6,
11). This potentially complicates the interpretation of our data demonstrating that VAMP3 interacts with CT442-FLAG. We examined the localization of CT442-FLAG within the IM and on fibers extending from the IM that colocalize with IncA-positive fibers (Fig. S8). To resolve these disparate classifications, future studies will need to examine if inhibiting chlamydial type III secretion also inhibits the localization of CT442-FLAG to the IM. Ultrastructural analysis using immunogold electron microscopy may also be useful in determining the localization of CT442, whether in the outer membrane of
Chlamydia or within the IM. An antibody against endogenous CT442 could resolve whether its localization on the IM is polarized in nature, similar to what is observed with VAMP3 localization to the IM at later time points postinfection.
In conclusion, our data highlight the dynamic nature of the interactions occurring at the chlamydial inclusion, demonstrating the sophisticated mechanisms employed by Chlamydia to maintain their intracellular niche. Deciphering the dedicated functions underlying these interactions will provide novel insights into how Chlamydia orchestrates their unique effectors in modulating host proteins for an intrinsic demand (e.g., nutrient acquisition) or the responses to certain intracellular environmental stimuli. Importantly, this study substantiates the necessity to investigate Chlamydia-host interactions in a temporal manner that combines multiple approaches to comprehensively dissect the unique pathogenesis of the chlamydial IM.
MATERIALS AND METHODS
Cell culture.
HeLa 229 cells (CCL-2.1; American Type Culture Collection [ATCC], Manassas, VA) and McCoy cells (ATCC CRL-1696) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) plus GlutaMAX supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone) at 37°C and 5% CO2.
Cultivation of Chlamydia.
All strains of
Chlamydia trachomatis were propagated and purified in HeLa cells using established protocols (
72,
73). Chlamydial titers were determined based on the number of inclusion-forming units (IFUs) in HeLa cells (
73,
74).
Chlamydial infection of eukaryotic cells.
HeLa cells were infected by centrifugation at 400 ×
g for 15 min at room temperature (or by rocking for 15 min at room temperature when using 10-cm dishes) at a multiplicity of infection (MOI) of 0.5 for siRNA knockdown and assessing endogenous VAMP3 localization using indirect immunofluorescence experiments or an MOI of 1 or 2 for affinity purification experiments. To collapse the Golgi structure,
Chlamydia-infected HeLa cells were treated 2 h prior to fixation with 1 μg/ml Brefeldin A (BFA). To halt chlamydial protein synthesis, organisms were treated with 200 μg/ml chloramphenicol, as previously described (
75), at either 15.5 or 23.5 hpi. When using transformed chlamydial strains expressing Inc-FLAG constructs, growth medium was supplemented with 1 U/ml penicillin for plasmid maintenance in
Chlamydia. To induce expression of Inc-FLAG constructs, organisms were treated the indicated amounts of aTc at the indicated times postinfection.
Transformation of Inc-FLAG-expressing strains of C. trachomatis serovar L2.
pBOMB4-tet_
inc-FLAG-transformed strains of
C. trachomatis serovar L2 were generated as previously described using WT
C. trachomatis serovar L2-pL2 (
22). Briefly, approximately 1 × 10
6 McCoy or HeLa cells were seeded in 6-well plates. The following day, purified WT
C. trachomatis serovar L2-pL2 EBs were mixed with Tris-CaCl
2 and 2 μg of various pBOMB4-tet_
inc-FLAG plasmids (see Table S1 in the supplemental material). The EB-CaCl
2-pDNA mixture was incubated for 30 min at room temperature (RT) and then added to the McCoy or HeLa cells containing 2 ml/well Hanks’ balanced salt solution (HBSS) supplemented with Ca
2+ and Mg
2+. The 6-well plates were centrifuged at 400 × g for 15 min at RT for infection and then were incubated at 37°C, 5% CO
2, for an additional 15 min. Medium then was aspirated and replaced with 2 ml/well DMEM plus 10% FBS and incubated at 37°C, 5% CO
2. At 7 hpi, tissue culture medium was removed and replaced with DMEM plus 10% FBS supplemented with 1 μg/ml cycloheximide and 1 U/ml penicillin as a selection agent. Every subsequent ∼48 hpi, infected monolayers were passaged 2 to 3 times onto fresh monolayers of McCoy or HeLa cells until a population of
C. trachomatis serovar L2 stably maintained the plasmid. The transformed strains were expanded and IFUs were enumerated in HeLa cells, and the optimal Inc-FLAG expression was titrated using various concentrations of the inducer anhydrotetracycline (aTc), 0 to 5 nM, to determine the concentration that allowed for
inc expression that did not disrupt inclusion size and Inc organization in the IM for each strain generated (
53).
Allelic exchange mutagenesis to delete incA in C. trachomatis serovar L2.
The original pSU vector for allelic exchange as described previously (
55) was modified to introduce KpnI and NcoI sites flanking the
bla cassette. Additionally, the
gfp cassette was removed. Approximately 1.1-kbp genomic fragments directly flanking the
incA gene were PCR amplified using the indicated primers (Table S1) and sequentially inserted into the KpnI (IncA 3′) and NcoI (IncA 5′) sites using the HiFi DNA assembly kit (NEB) by following the manufacturer’s instructions and transformed into chemically competent NEB-10beta
E. coli. A plasmid isolated from individual colonies was verified by restriction enzyme-mediated digestion, followed by sequencing into the flanking regions. The plasmid was then demethylated by transforming an
E. coli dam dcm mutant (NEB), and a plasmid midiprep was prepared and verified as described above. The resulting plasmid was used to transform
C. trachomatis serovar L2 lacking its endogenous plasmid (-pL2) as described previously (
55) until inclusions lacking mCherry fluorescence and resistant to penicillin were harvested. The phenotype of inclusions lacking IncA is multiple inclusions per cell when more than one EB infects a given cell (
76).
siRNA knockdown of VAMP proteins in HeLa cells.
Silencer select small interfering RNAs (Ambion) against VAMP3 (s17856), VAMP4 (s16525), and nontargeting controls (negative control 1 siRNA) were diluted to 10 nM in Opti-MEM (Gibco). Diluted siRNA was mixed with 1.5 μl Lipofectamine RNAiMax reagent (ThermoFisher Scientific) and incubated for 10 min at RT with rocking in a 24-well plate. A total of 4.8 × 104 HeLa cells were reverse transfected by seeding cells on top of the siRNA transfection mixture onto glass coverslips for indirect immunofluorescence to measure inclusion circumferences or enumeration of primary infections or directly into tissue culture wells for infectious progeny assays or Western blot analysis to confirm knockdown. Transfection medium was removed and replaced with fresh DMEM plus 10% FBS 18 h posttransfection. Knockdown cells were then infected with WT C. trachomatis serovar L2 (434/BU) by addition at an MOI of 0.5 for 30 h. At 30 hpi, either cells were fixed and processed for indirect immunofluorescence (see “Indirect immunofluorescence,” below) or proteins were collected for Western blot analysis.
To collect proteins to analyze knockdown efficiency via Western blotting, infected HeLa cells were trypsinized and pelleted. Cell pellets were lysed directly in 200 μl SDS sample buffer containing universal nuclease (Pierce) and 5% β-mercaptoethanol and then boiled at 95°C for 5 min. Samples were loaded on 12% SDS-polyacrylamide gels, electrophoresed 150 V for 55 min, and then transferred to PVDF. PVDF membranes were incubated overnight in appropriate primary antibodies (Table S2) in 5% carnation milk phosphate-buffered saline-Tween 20 (PBST) overnight at 4°C. Membranes were then incubated with NIR fluorescent secondary antibodies (LICOR) for 1 h at RT. Images were acquired using the NIR function on an Azure c600.
Infectious progeny determination.
HeLa cells were reverse transfected with siRNA to knock down either VAMP3, VAMP4, VAMP3, and VAMP4 together or nontargeting controls as described above. Knockdown cells then were infected with WT C. trachomatis serovar L2 at an MOI of 0.4 by a rocking infection. At 30 hpi, infected cells were fixed in methanol to enumerate the primary infection or lysed, titrated, and reinfected onto a fresh monolayer of HeLa cells. Secondary infections were fixed in methanol at 28 hpi. All fixed coverslips from the primary and secondary chlamydial infections were processed via indirect immunofluorescence to detect chlamydial organisms (guinea pig anti-L2 antibody). Coverslips were imaged on a Zeiss ApoTome.2 fluorescence microscope at ×40 magnification to enumerate both the primary infection and secondary infection, where a single inclusion represented a single EB. Inclusions were counted and the secondary infection/infectious progeny were normalized to the primary infection by dividing the IFU count of the secondary infection by the average IFU of the primary infection. Progeny counts were graphed and analyzed using GraphPad v.7.0. An ordinary one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons was performed to determine statistical significance, where two asterisks indicates a P value of <0.01 and four asterisks indicates a P value of <0.0001.
Indirect immunofluorescence.
HeLa cells were seeded onto glass coverslips in 24-well plates at a density of 1 × 105 cells/ml in 1 ml/well DMEM plus 10% FBS. Cells were fixed in 4% paraformaldehyde for 15 min at RT and then permeabilized with 0.5% Triton X-100 for 5 min at RT. Coverslips were then processed using indirect immunofluorescence (IF) using appropriate primary antibodies (Table S2), followed by secondary antibodies conjugated to specific fluorophores. All coverslips were mounted using Prolong Gold antifade mounting medium (Life Technologies). Images were acquired at ×63 magnification using a Zeiss LSM 800 or Nikon spinning disk confocal microscope at ×60 magnification and were processed using Adobe Photoshop version 21.1.
Inclusion circumference measurement.
Coverslips from knockdown experiments were processed for indirect immunofluorescence, as described above, and images were taken on a Zeiss ApoTome.2 fluorescence microscope at ×40 magnification. Inclusion circumferences were measured in Fiji/ImageJ with calibrated measurements, and resulting data were graphed using GraphPad Prism.
VAMP3 inclusion localization intensity measurement.
Coverslips from endogenous VAMP3 localization to inc mutant strains at 30 hpi were processed for indirect immunofluorescence, as described above, and imaged on a Zeiss LSM 800 at ×63. The intensity of VAMP3 localization to individual inclusions was measured in Fiji using the RawIntDen feature, which is a sum of the total pixel intensities. For each strain, we measured a minimum of 124 inclusions: WT C. trachomatis serovar L2, 175 inclusions; C. trachomatis serovar L2 ΔincA mutant, 125 inclusions; C. trachomatis serovar L2 ct813::bla mutant, 124 inclusions; and C. trachomatis serovar L2 ct005::bla mutant, 205 inclusions. The RawIntDen values then were divided by the area to account for heterogeneity in inclusion sizes. Images were taken using slightly different gain settings; thus, VAMP3 intensity was normalized to the gain setting used for each image acquired to most accurately measure VAMP3 intensity. The majority of images were taken using 732 V as the gain setting, so that was used as the normalization gain. The final equation used was (RawIntDen/area) × (gain used in image/normalization gain or 732 V). See Table S3 for raw data. The resulting numbers demonstrate VAMP3 inclusion intensity in arbitrary units and were plotted using GraphPad prism as the means with SEM. Statistical significance was determined using an ordinary one-way ANOVA with Dunnett’s post hoc test for multiple comparisons of each inc mutant strain to WT C. trachomatis serovar L2.
In vivo screening for protein-protein interactions of human VAMPs and chlamydial Incs by the BACTH system.
To create BACTH constructs, the human VAMP3 and VAMP4 genes were amplified from the pCMV7.1-3×FLAG-VAMP3 and pCMV7.1-3×FLAG-VAMP4 vectors (
39), and
inc genes were amplified from
C. trachomatis serovar L2 genomic DNA using designed primers that harbor overlapping sequences for each pST25 and pUT18C vector (Table S1). The resulting amplicons were subsequently cloned into either pST25 or pUT18C using the NEBuilder HiFi assembly cloning kit (NEB) and transformed into chemically competent NEB-10beta
E. coli. Isolated plasmid from individual colonies was verified by restriction enzyme-mediated digestion and then confirmed by DNA sequencing. pUT18C-CT288, -CT226, -CT223, -IncA, -IncF, and –IncE were described previously (
22). BACTH assays were performed as previously reported (
22,
47,
77). Briefly, plasmids were cotransformed into
E. coli DHT1(Δ
cyaA) CaCl
2 competent cells by heat shock at 42°C for 30 s. The transformed
E. coli cells were subsequently pelleted, washed, and resuspended in 1× M63 minimal medium. The resuspended
E. coli DHT1 cells were then plated on 1× M63 minimal medium plates containing 0.2% maltose, isopropyl β-
d-1-thiogalactopyranoside (IPTG; 0.5 mM), 5-bromo-4-chloro-3-indolyl-β-
d-galactopyranoside (X-Gal; 0.04 mg/ml), Casamino Acids (0.04%), spectinomycin (25 g/ml), and ampicillin (50 g/ml). The interaction results were observed 3 to 5 days after incubated at 30°C. The appearance of blue colonies indicates a positive interaction between proteins, since both the
lac and
mal operons require reconstituted cyclic AMP production from interacting T25 and T18 fragments to be expressed. The experiments were performed with three independent replicates. The positive control for VAMP3 and -4 was testing interactions with syntaxin 6, as these are known interactors (
78,
79).
To quantify interactions by a β-galactosidase assay, eight random colonies (or streaks from negative plates) were set up for an overnight culture in 1× M63 minimal medium containing 0.2% maltose, 0.5 mM IPTG, 0.04 mg/ml X-Gal, 0.01% Casamino Acids, spectinomycin (25 g/ml), and ampicillin (50 g/ml). The cultures were diluted after incubation for 20 to 24 h, and the optical density at 600 nm (OD
600) was measured. Simultaneously, a duplicate set of samples was permeabilized with SDS (0.05%) and chloroform prior to the addition of 0.4% o-nitrophenyl-β-
d-galactopyranoside in PM2 buffer (70 mM Na
2HPO
4·12H
2O, 30 mM NaH
2PO
4·H
2O, 1 mM MgSO
4, 0.2 mM MnSO
4; pH 7.0) with 100 mM 2-mercaptoethanol. The enzymatic reaction was terminated using 1 M Na
2CO
3 stop solution after precisely 20 min of incubation at RT. Absorbance at 405 nm was then recorded and normalized to bacterial growth (OD
600) and reported as relative units (RU). The results from three independent experiments were analyzed for each interaction, graphed by GraphPad Prism software, and consequently reported as the mean with the standard deviation. To identify common interactors, positive Incs from VAMP3 and VAMP4 BACTH assays were analyzed by Venny v2.1 (
80).
FLAG affinity purification.
For co-IP by cotransfection in uninfected cells, HeLa cells were seeded in a 6-well plate (5 × 10
5 cells/well) or a 100-mm dish (1 × 10
6 cells/dish) with coverslips in DMEM–10% FBS and allowed to grow overnight. The cells were cotransfected either with pCMV7.1-3×FLAG-VAMP3 or pCMV7.1-3×FLAG-VAMP4 and an individual pCMV7.1-Inc-6×His construct using jetPRIME (PolyPlus, New York, NY) by following the manufacturer’s instructions. The concentrations for pCMV7.1-3×FLAG-VAMP3 and 3×FLAG-VAMP4 were 1 μg/well (6-well plate) or 5 μg/100-mm dish, 2.5 μg (pCMV7.1-CT005-6×His), 2 μg (pCMV7.1-CT813-6×His), and 3 μg (pCMV7.1-IncB-6×His, pCMV7.1-IncF-6×His, pCMV7.1-IncG-6×His, pCMV7.1-CT006-6×His, pCMV7.1-CT179-6×His, pCMV7.1-CT442-6×His, and pCMV7.1-CT449-6×His) per well of a 6-well plate or 5 μg (pCMV7.1-IncA-6×His, pCMV7.1-CT222-6×His, pCMV7.1-CT223-6×His, and pCMV7.1-CT226-6×His) per 100-mm dish was used. At 4 h posttransfection, medium was aspirated and replaced with fresh DMEM–10% FBS medium and incubated overnight at 37°C, 5% CO
2. At 24 h, coverslips were removed and fixed (4% paraformaldehyde for 15 min at RT), and the remaining cells were collected and lysed for FLAG affinity purification as previously described (
22). Cells were harvested for lysis by scraping the transfected monolayers into Dulbecco’s PBS and pelleting the cells by centrifugation at 900 ×
g for 10 min at 4°C. Pellets were resuspended in 1 ml cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100 [Sigma, St. Louis, MO], 1 × Halt protease inhibitor cocktail [Thermo Scientific, Waltham, MA], universal nuclease [Pierce, Rockford, IL]) for a 6-well plate, while 1 ml lysis buffer was directly added into 100-mm dishes followed by scraping and collecting the entire contents in a 1.5-ml tube. The total lysates were incubated on ice for 30 min to 1 h with gentle vortexing every 10 min. Lysates were clarified by centrifugation at 17,000 ×
g for 10 min at 4°C. The clarified lysates were mixed with anti-FLAG magnetic beads (Sigma, St. Louis, MO), rotated overnight at 4°C. FLAG-tagged proteins and interacting partners then were eluted in 30 μl of lysis buffer containing FLAG peptide (200 μg/ml). The eluates from each sample were combined with 10 μl of 4× Laemmli sample buffer containing 5% β-mercaptoethanol, boiled at 95°C for 5 min, resolved by 12% SDS-polyacrylamide gels at 100 V for 1.5 h, and then transferred to a PVDF membrane (pore size, 0.45 μm; Thermo Scientific, Waltham, MA). The PVDF membranes were incubated overnight at 4°C in PSBT with 5% skim milk containing appropriate primary antibodies (Table S2). Membranes were then incubated with NIR fluorescent secondary antibodies (1:10,000; LICOR) in PBST with 5% skim milk for 1 additional hour at RT before imaging by an Azure c600 system (Azure Biosystems, Radnor, PA) and acquired by its NIR function. The data shown are representative from three biological replicates.
For co-IP in the context of chlamydial infected cells, HeLa cells were seeded for transfection in 6-well plates or 100-mm dishes as described above, using 2 μg or 5 μg, respectively, of either pCMV7.1-6×His-VAMP3 or pCMV7.1-6×His-VAMP4. At 4 h posttransfection, the transfected media were aspirated and then replaced with the DMEM–10% FBS medium containing 0.5 U/ml penicillin and 1 U/ml gentamicin, followed by an additional incubation for 2 h at 37°C, 5% CO2. The transfected HeLa cells were then infected with C. trachomatis serovar L2 transformed with pBOMB4 plasmids with incA-, incF-, incG-, ct005-, ct179-, ct222-, ct223-, ct226-, ct442-, ct449-, and ct813-flag at an MOI of 2 for 6-well plates or MOI of 1 for 100-mm dishes. At 7 h postinfection, the infected inc-flag expression was induced with 5 nM aTc (CT005-, CT179-, and CT442-FLAG) or 1 nM aTc for the remaining constructs. At the indicated time points postinfection, the coverslips were removed and processed for indirect immunofluorescence, while the remaining cells were collected and lysed. Affinity purification using anti-FLAG magnetic beads was performed as described above. The samples were then resolved by SDS-PAGE followed by Western blot analysis. Membranes were imaged using an Azure c600 system. All positive interactions identified from 6×His-VAMP3 and Inc-FLAG pulldowns were determined from three independent experiments.