Abstract
Studies of the chlamydial protease CPAF have been complicated by difficulties in distinguishing bona fide intracellular proteolysis from in vitro proteolysis. This confounding issue has been attributed to CPAF activity in lysates from Chlamydia-infected cells. We compared three methods that have been used to inhibit in vitro CPAF-mediated proteolysis: (1) pre-treatment of infected cells with the inhibitor clasto-lactacystin, (2) direct cell lysis in 8 M urea and (3) direct lysis in hot 1% SDS buffer. We identified a number of experimental conditions that reduce the effectiveness of each method in preventing CPAF activity during lysate preparation. The amount of in vitro proteolysis in a lysate was variable and depended on factors such as the specific substrate and the time in the intracellular infection. Additionally, we demonstrated for the first time that artifactual CPAF activity is induced before cell lysis by standard cell detachment methods, including trypsinization. Protein analysis of Chlamydia-infected cells therefore requires precautions to inhibit CPAF activity during both cell detachment and lysate preparation, followed by verification that the cell lysates do not contain residual CPAF activity. These concerns about artifactual proteolysis extend beyond studies of CPAF function because they have the potential to affect the analyses of host and chlamydial proteins from Chlamydia-infected cells.
Keywords: proteolysis, substrates, proteolytic activity
Novel factors contributing to artifactual CPAF proteolysis are identified and recommendations are made to prevent promiscuous CPAF activity when performing any protein analysis of Chlamydia-infected cells.
Novel factors contributing to artifactual CPAF proteolysis are identified and recommendations are made to prevent promiscuous CPAF activity when performing any protein analysis of Chlamydia-infected cells.
INTRODUCTION
CPAF is a conserved chlamydial protease that cleaves and degrades many host and chlamydial proteins (Paschen et al., 2008; Zhong 2009). However, the significance of this proteolysis to the bacterial infection has been called into question because of ongoing CPAF proteolytic activity during the analysis of Chlamydia-infected cells (Chen et al., 2012; Bavoil and Byrne 2014). Specifically, lysates of Chlamydia-infected cells, prepared under standard conditions, contain CPAF that is active at 4ºC and resistant to a standard protease inhibitor cocktail (Zhong et al., 2000; Chen et al., 2012). As a result, CPAF-dependent proteolysis of numerous host and chlamydial proteins appears to occur during lysate preparation, rather than in intact infected cells.
Importantly, the proteolysis of 12 published CPAF substrates was no longer detected by Western blot analysis when precautions were taken to inhibit CPAF activity during lysate preparation (Chen et al., 2012; Grieshaber and Grieshaber 2014). Methods to inhibit CPAF activity include pre-treatment of Chlamydia-infected cells with the CPAF inhibitor clasto-lactacystin prior to cell detachment and lysis, and direct lysis of cells in 8 M urea or hot 1% SDS buffer (Chen et al., 2012; Snavely et al., 2014). Using the hot 1% SDS method, Snavely et al. (2014) reported that vimentin is still cleaved and identified LAP1 as a new CPAF substrate. Vimentin cleavage was at greatly reduced levels and occurred at much later times during the intracellular infection than previously published (Kumar and Valdivia 2008). Thus, CPAF activity in infected cell lysates can misrepresent the extent and timing of proteolysis that may have occurred in an infected cell.
How then should one interpret protein cleavage or degradation that is detected in lysates when efforts are made to inhibit CPAF during lysate preparation? Depending on whether there is residual CPAF activity in a lysate, such proteolysis could represent bona fide effects on a substrate in the infected cell, or there could still be in vitro proteolysis during lysate preparation. To address this issue, we compared the effectiveness of three methods that can prevent CPAF-mediated proteolysis during lysate preparation. We also examined if experimental variables, such as the time in the infection, the cell collection procedure and the protein substrate being analyzed, can limit the effectiveness of these methods in inhibiting in vitro CPAF activity. Based on our findings, we outline an approach for preventing and checking for CPAF activity during protein analysis of Chlamydia-infected cells.
MATERIALS AND METHODS
Antibodies
The following antibodies were used in this study: mouse anti-vimentin (Sigma–Aldrich); mouse anti-Erk 1/2 (Cell Signaling Technology); mouse anti-p65/RelA (Santa Cruz Biotechnology); rabbit anti-RFX5 (Rockland Immunochemicals); rabbit anti-α-tubulin (Abcam); rabbit anti-HsSAS-6 (generous gift from Dr Pierre Gönczy, École Polytechnique Fédéral de Lausanne); goat anti-mouse 680LT and goat anti-rabbit 800CW (both from LI-COR); goat anti-mouse HRP and goat anti-rabbit HRP (both from Jackson ImmunoResearch Laboratories). For information about CPAF protein substrates used in this study, see Table 1.
Table 1.
Summary of CPAF substrates.
Cell culture
HeLa cells (ATCC) were grown in 6-well dishes in Advanced DMEM (4.5 g glucose L–1) (Invitrogen) supplemented with 2% fetal bovine serum (Hyclone/Thermo Fisher) and 2 mM GlutaMAX-I (Invitrogen). All cell lines were grown in 5% CO2 at 37°C and regularly screened for Mycoplasma contamination by PCR (Ossewaarde et al., 1996).
Chlamydia infections
Cell monolayers were infected with Chlamydia trachomatis serovar L2 (L2/434/Bu), LGV biovar, at a multiplicity of infection of 3 in sucrose-phosphate-glutamic acid (SPG). In parallel, uninfected control experiments were performed as mock infections in SPG alone. Infections were carried out by centrifugation at 700 × g in a Sorvall Legend Mach 1.6R centrifuge for 1 h at room temperature. After centrifugation, the inoculum was replaced by fresh cell culture medium without cycloheximide and monolayers were incubated at 37°C and 5% CO2. Chlamydial elementary bodies were verified to be free of Mycoplasma contamination by PCR (Ossewaarde et al., 1996).
Methods for cell collection and lysate preparation
Lysis in RIPA buffer: cells were harvested by trypsinization (TrypLE Express, Invitrogen) for 3–5 min at 37°C and transferred to a 15 mL conical tube on ice. The dish was washed twice with 1X PBS to recover remaining cells, and the washes were added to the 15 mL conical tube. The cells were pelleted by centrifugation at 1500 rpm for 3 min at 4°C and lysed on ice for 10 min in RIPA buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40] supplemented with protease inhibitors [2 mM pepstatin, 150 mM aprotinin (both from MP Biochemicals), 1 mM leupeptin (Calbiochem), 1 mM PMSF (Acros)]. The cells were resuspended by pipetting up and down in approximately 1 mL of ice-cold lysis buffer per 5 × 106 cells. Lysates were cleared by centrifugation at 13000 × g for 10 min at 4°C, and protein concentrations were determined by Bradford assay (Bio-Rad).
Direct lysis in urea: the monolayer of infected cells was washed with 1X PBS. A solution of 8 M urea (or 6–7 M urea where indicated) supplemented with 325 U mL–1 of Benzonase Nuclease (Sigma–Aldrich) was then directly added to the cell monolayer at a volume of 1 mL per 6 wells of a 6-well dish, and left for 10 min on ice. The resulting lysates were pooled and protein concentrations were determined by the DC protein assay (Bio-Rad).
Lysis in hot 1% SDS buffer: cells were washed with 1X PBS. 1% SDS buffer [50mM Tris (pH 7.5), 150mM NaCl, 1% SDS] was heated to 95°C and directly added to cell monolayers at a volume of 1 mL per 6 wells of a 6-well dish. Cells were scraped from the monolayers, and the resulting lysates from individual wells were pooled and supplemented with 325 U mL–1 of Benzonase Nuclease (Sigma–Aldrich). Protein concentrations were determined by the DC protein assay (Bio-Rad).
Cell collection methods (Fig. 4): Chlamydia-infected cells were collected by incubation in 500 μl trypsin (TrypLE Express, Invitrogen) or 500 μl accutase (Fisher Scientific) per well of a 6-well dish for 3–5 min at 37°C, or by scraping monolayers directly into 500 μl 1X PBS per well of a 6-well dish. Cells were then transferred to a 15 mL conical tube on ice. The dish was washed twice with 1X PBS, and the washes were added to the 15 mL conical tube. The cells were pelleted by centrifugation at 1500 rpm for 3 min at 4°C and lysed on ice for 10 min in 8 M urea supplemented with 325 U mL–1 of Benzonase Nuclease (Sigma–Aldrich).
Figure 4.
CPAF activity is induced by standard cell collection methods. (a) Uninfected and infected cells were detached at 48 hpi by treatment with trypsin or accutase, or by scraping, and then pelleted and lysed in 8 M urea. In parallel, cells were lysed directly in 8 M urea. Lysates were analyzed for vimentin proteolysis by Western blotting, with α-tubulin serving as a loading control. (b) Infected cell lysates from Fig. 4a were tested in the in vitro CPAF activity assay and reactions were examined by Western blot analysis with antibodies to vimentin. Expected cleavage products in the Western blots are indicated with arrows.
Clasto-lactacystin treatment
Clasto-lactacystin pre-treatment: clasto-lactacystin β-lactone (Cayman Chemical), dissolved in methyl acetate, was added to the cell culture medium at a final concentration of 150 μM for 60 min prior to cell processing. For example, samples of Chlamydia-infected cells at 48 hours post-infection (hpi) were treated with clasto-lactacystin at 47 hpi for 60 min and then processed. In parallel control experiments, methyl acetate as the solvent was added to the culture medium. Treated cells were collected by trypsinization followed by cell lysis in RIPA buffer (as described above).
Clasto-lactacystin in lysis buffer: Chlamydia-infected cells were harvested by trypsinization, and cell pellets were lysed on ice for 10 min in RIPA buffer containing protease inhibitors (as described above) and 150 μM of clasto-lactacystin β-lactone (Cayman Chemical). In parallel control experiments, methyl acetate was used instead of clasto-lactacystin in the RIPA buffer.
Western blot analysis
Cell lysates were diluted in Laemmli sample buffer [50 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.1% bromophenol blue] and incubated at 95°C for 5 min to denature proteins. Samples containing equal amounts of total protein were loaded and resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, blocked in 5% milk PBST (5% dry powdered milk, 0.1% Tween-20, 1X PBS) and incubated in primary antibodies followed by HRP-conjugated (Jackson ImmunoResearch) or IRDye-conjugated (LI-COR) secondary antibodies. Blots were imaged by enhanced chemiluminescence or the LI-COR Odyssey SA infrared imaging system.
In vitro CPAF activity assay
Chlamydia-infected HeLa cells at various times in the infection were processed by the three methods described above (trypsinization followed by lysis in RIPA buffer, direct lysis in 8 M urea, or direct lysis in hot 1% SDS). 4–8 μg of these infected cell lysates, as the source of CPAF, were incubated with 12.5 μg of uninfected HeLa cell lysate, as the source of host protein substrates, at 37°C for 30 min in CPAF reaction buffer [25 mM Tris (pH 8.0), 150 mM NaCl, 3 mM DTT]. Reactions were terminated by adding Laemmli sample buffer and boiling for 5 min. 16.5–20.5 μg of protein from these samples was analyzed by Western blotting with specific primary antibodies.
RESULTS AND DISCUSSION
We first examined the use of the CPAF inhibitor clasto-lactacystin to inhibit CPAF activity in cell lysates (Chen, et al. 2012). HeLa cells were infected with C. trachomatis L2 and collected at 36 hpi with a standard procedure involving trypsinization and lysis in RIPA buffer. We then tested the cell lysates for CPAF activity with an in vitro assay in which we incubated a small amount of each infected cell lysate, as a potential source of CPAF, with uninfected cell lysate as a source of host substrates. Without precautions, the infected cell lysate caused the complete cleavage of the host centrosomal protein HsSAS-6 in the in vitro activity assay, demonstrating that this lysate contained CPAF activity (Fig. 1a). However, pre-treatment of an infected cell monolayer with 150 μM clasto-lactacystin for 60 min before cell collection inhibited all CPAF activity in the lysate, as shown by the absence of a HsSAS-6 cleavage product in the in vitro activity assay (Fig. 1a). Shorter pre-treatment times, using the same concentration of clasto-lactacystin, did not eliminate CPAF activity in the lysate (Fig. 1a). Protease inhibitors do not typically require pre-treatment, and are usually added to the lysis buffer, but addition of 150 μM clasto-lactacystin to the RIPA lysis buffer, without pre-treating the cells, did not prevent artifactual proteolysis of the host protein p65 (Fig. 1b).
Figure 1.
Methods to inhibit CPAF activity during infected cell lysate preparation are not always effective. (a) Chlamydia-infected HeLa cells were pre-treated with 150 μM clasto-lactacystin for 30, 45 or 60 min prior to collection at 36 hpi by trypsinization and lysis in RIPA buffer. Infected cell lysates were tested for CPAF activity in an in vitro activity assay (outlined in Fig. 2a), which was analyzed by Western blotting with antibodies to the host protein HsSAS-6. The first lane with uninfected cell lysate alone shows uncleaved HsSAS-6. A cross-reacting band is marked with ‘*’. (b) Uninfected and infected cells were collected by trypsinization at 48 hpi, and lysed in RIPA buffer containing 150 μM clasto-lactacystin. Alternatively, infected cells were pre-treated with 150 μM clasto-lactacystin for 60 min prior to lysate preparation in RIPA buffer. Proteolysis of p65 as a substrate was monitored in the lysates by Western blot analysis with p65 antibodies, with α-tubulin serving as a loading control. (c) Uninfected and infected cells were pre-treated with two different batches of clasto-lactacystin at 150 μM for 60 min and then lysed in RIPA buffer. Lysates were assayed by Western blot for RFX5 degradation with antibodies to RFX5 or Erk 1/2 as a loading control. (d) Monolayers of uninfected and infected cells at 48 hpi were either collected by trypsinization and lysed in RIPA buffer or lysed directly in urea at the indicated concentrations. (e) Monolayers of uninfected and infected cells at 48 hpi were lysed directly in fresh or old solutions of 8 M urea. For (d) and (e), lysates were analyzed for vimentin proteolysis by Western blotting with vimentin antibodies. HsSAS-6, p65 and vimentin proteolysis products are indicated with arrows.
Our experiments also confirmed the reported batch-to-batch variability of clasto-lactacystin activity against CPAF (Snavely et al., 2014; Tan and Sütterlin 2014). Only some batches of clasto-lactacystin prevented degradation of the host protein RFX5 in infected cell lysates (Fig. 1c), even though we used the same concentration (150 μM) and pre-treatment time (60 min). Clasto-lactacystin batches that were ineffective against native CPAF did not inhibit recombinant CPAF either, although they retained their anti-proteasomal activity (data not shown). Thus, clasto-lactacystin can be an effective inhibitor of CPAF, but the specific batch, concentration and treatment time has to be carefully selected and optimized.
We also tested the denaturing agent urea, which has been used to non-specifically inhibit CPAF activity during lysate preparation (Chen, et al. 2012). Direct lysis of C. trachomatis-infected cells at 48 hpi in 8 M urea completely prevented vimentin proteolysis. In contrast, direct lysis in 6 or 7 M urea produced lysates containing vimentin cleavage products that were larger than the cleavage products seen in RIPA lysates. These partial cleavage products have been described before (Kumar and Valdivia 2008) and are consistent with proteolysis caused by residual CPAF activity in these lysates (Fig. 1d). Lysis in an old 8 M urea solution produced a cell lysate that also contained partially cleaved vimentin (Fig. 1e). Thus, fresh 8 M urea is required to effectively inhibit in vitro CPAF activity, and we recommend making the 8 M urea solution on the same day it is to be used.
These studies demonstrate the importance of confirming the effectiveness of the methods used to inhibit CPAF activity during lysate preparation. Lysates of Chlamydia-infected cells are typically examined for evidence of CPAF-mediated cleavage or degradation of specific proteins by Western blot analysis (‘Protein Analysis’, Fig. 2a). We propose that each infected cell lysate should also be tested for residual CPAF activity with an ‘In vitro CPAF Activity Assay’ (Fig. 2a). In this assay, we incubate infected cell lysate, as a potential source of CPAF, with uninfected HeLa cell lysate as a source of host proteins and analyze the reaction products by Western blot. Loss of the host protein being studied and/or appearance of cleavage products indicate that the infected cell lysate contains residual CPAF activity. We only use small amounts of this lysate to measure residual CPAF enzymatic activity, which makes it less likely that any detected cleavage products originate from the infected cell lysate before the in vitro assay, To confirm the absence of carry over, we routinely check if this amount of infected cell lysate has detectable cleavage products by Western blot analysis (Fig. 2). Residual CPAF activity can also be measured by performing the in vitro activity assay with a GFP-tagged substrate that is not present in the infected cell lysate (Fig. S1, Supporting Information). Ideally, this in vitro CPAF activity assay should be performed immediately after lysate preparation because freezing and thawing can decrease residual CPAF activity (data not shown).
Figure 2.
Analysis of infected cell lysates for substrate proteolysis and CPAF activity. (a) Procedure to examine proteolysis and CPAF activity in an infected cell lysate. ‘Protein Analysis’ refers to Western blot analysis of infected cell lysates for cleavage or degradation of a protein of interest. The ‘In vitro CPAF Activity Assay’ measures any residual CPAF activity present in the infected cell lysate. (b) At the indicated times in the Chlamydia infection, cells were lysed directly in 8 M urea, followed by protein analysis of the lysates by Western blotting with antibodies to p65. (c) Lysates from Fig. 2b were also assayed for CPAF activity against p65 using the in vitro assay. (d) Uninfected and infected cells were pre-treated with 150 μM clasto-lactacystin for 60 min prior to lysis in RIPA buffer at the indicated times in the Chlamydia infection. Lysates were examined for p65 proteolysis by Western blot analysis. (e) Lysates from the 54 hpi time point of Fig. 2d were tested in the in vitro CPAF activity assay for p65 cleavage. (f) Cells at the indicated times in the Chlamydia infection were lysed in hot 1% SDS buffer, followed by protein analysis of the lysates for p65 cleavage. (g) Lysates from Fig. 2f were tested for residual CPAF activity against p65 in the in vitro CPAF activity assay. Expected p65 cleavage products in the Western blots are indicated with arrows. For Fig. 2c, e and g, the same amounts of representative infected cell lysates that were used in the in vitro assay were included to demonstrate that it does not contain detectable amounts of the p65 cleavage product. Thus, the appearance of p65 cleavage products in the in vitro assays testing the clasto-lactacystin pre-treated (Fig. 2e) and hot 1% SDS (Fig. 2g) infected cell lysates indicate that these lysates contain residual CPAF activity.
Using this approach, we compared the effectiveness of the three methods reported to inhibit CPAF activity. Cell lysates generated by directly adding 8 M urea to a monolayer of Chlamydia-infected cells from 32 to 56 hpi did not contain any detectable p65 cleavage product (Fig. 2b). These cell lysates also lacked CPAF activity toward p65 in the parallel in vitro activity assay (Fig. 2c). Thus, urea was effective at inhibiting CPAF in the lysate, and p65 does not appear to be cleaved in intact infected cells up to 56 hpi. In contrast, pre-treatment of infected cells with clasto-lactacystin for 60 min was only effective in a chlamydial infection at time points up to 36 hpi because lysates prepared at 42 hpi and later showed progressive loss of full-length p65, and appearance of an ∼43 kDa cleavage product (Fig. 2d). Consistent with this observation, there was residual CPAF activity in these late lysates (shown for the 54 hpi lysate in Fig. 2e). Similarly, lysates prepared by direct lysis in hot 1% SDS buffer also showed evidence of p65 proteolysis between 32 and 56 hpi (Fig. 2f), and contained small amounts of residual CPAF activity toward p65 (Fig. 2g). We conclude that clasto-lactacystin pre-treatment and the hot SDS lysis method are less effective at inhibiting CPAF activity in lysates at late times, which may indicate that there is more CPAF in a late-stage Chlamydia-infected cell.
These results illustrate why the in vitro activity assay should be performed on each infected cell lysate. In this experiment, we had the benefit of knowing that the late proteolysis of p65 was an in vitro artifact because the 8 M urea lysates did not show any detectable p65 proteolysis as late as 56 hpi. Without this information, however, the protein analyses of lysates obtained with clasto-lactacystin pre-treatment or direct lysis in hot SDS would be interpreted as demonstrating CPAF-mediated proteolysis of p65 at late times in the infection. The in vitro activity assay, however, provided an alternative explanation for this proteolysis because it showed residual CPAF activity in these late lysates. Thus, the proteolysis detected in the lysate could have occurred in intact infected cells, during cell collection and lysate preparation, or both. These studies demonstrate that proteolysis of a host or chlamydial protein cannot be correctly interpreted unless the lysate is free of in vitro proteolytic activity against this protein.
Snavely et al. (2014) reported partial proteolysis of vimentin and LAP1 at late times in the intracellular Chlamydia infection. They took precautions to inhibit CPAF activity during lysate preparation by directly lysing Chlamydia-infected cells in hot 1% SDS, but the lysates were not verified to be free of CPAF activity. Interestingly, the temporal pattern of vimentin and LAP1 proteolysis resembles the p65 cleavage pattern that we observed in lysates prepared with the clasto-lactacystin pre-treatment and hot 1% SDS methods. It is therefore possible that the reported proteolysis of vimentin and LAP1 may have occurred during cell lysate preparation.
Over the course of our experiments, we noted that proteins differed in their susceptibility to CPAF-mediated proteolysis. The choice of substrates did not appear to make a difference when we examined methods to inhibit CPAF activity (data not shown), and thus we used a number of substrates in these studies. However, the specific substrate is relevant when the in vitro CPAF activity assay is used to help interpret the protein analysis of an infected cell lysate. To illustrate this point, we performed the in vitro CPAF activity assay on lysates prepared at 48 hpi with three different methods, and analyzed the reaction mixtures with antibodies to vimentin and p65. A lysate prepared with clasto-lactacystin pre-treatment caused partial cleavage of both vimentin and p65 (Fig. 3). However, lysates prepared with the 8 M urea and hot SDS methods caused some cleavage of p65, but not vimentin, indicating that the sensitivity of the in vitro CPAF assay depends on the substrate. We propose that the best approach is to evaluate the same substrate in both the protein analysis and the in vitro CPAF activity assay. Unlike in a previous experiment (Fig. 2b), 8 M urea did not fully prevent CPAF activity in this analysis (Fig. 3), showing that even this method is not always effective in inhibiting CPAF activity in a lysate.
Figure 3.
Protein substrates vary in their susceptibility to CPAF-mediated proteolysis. Lysates of infected cells at 48 hpi were prepared by three methods (pre-treatment with clasto-lactacystin followed by lysis in RIPA buffer, direct lysis in 8 M urea or direct lysis in hot 1% SDS) and were examined for residual CPAF activity in the in vitro activity assay. Reaction mixtures were examined for vimentin and p65 by Western blot analysis. Cleavage products in the Western blots are indicated with arrows.
We next tested if the method of cell detachment affects CPAF-mediated proteolysis. As an obligate intracellular bacterium, Chlamydia is commonly cultivated in a monolayer of host cells, which is removed from the plastic surface prior to analysis. At 48 hpi, we compared direct lysis in 8 M urea with three commonly used methods of cell collection, which are trypsin treatment, accutase treatment and mechanical scraping. The detached cells were then lysed in 8 M urea to inhibit CPAF activity in the lysate. We analyzed the cell lysates for the presence of vimentin cleavage products by Western blotting, and for CPAF activity against vimentin with the in vitro activity assay. Lysates from cells detached with trypsin, accutase or mechanical scraping each contained small amounts of cleaved vimentin (Fig. 4a). This vimentin proteolysis was unlikely to have occurred during lysate preparation, because the cells had been exposed to urea after cell detachment and all the lysates lacked CPAF activity in our in vitro assay (Fig. 4b). However, the proteolysis must have occurred at some point during cell collection because a lysate prepared by direct lysis in 8 M urea contained no detectable vimentin cleavage (Fig. 4a). Putting these unexpected findings together, it appears that artifactual CPAF-mediated vimentin cleavage in these samples occurred during cell collection but before cell lysis. These experiments provide worrisome evidence that CPAF activity can be induced by experimental manipulations and cause proteolysis within intact infected cells. We hypothesize that standard cell detachment methods, including trypsinization, induce some degree of CPAF-mediated proteolysis in unlysed cells. Subsequent cell lysis then exaggerates this artifact by allowing in vitro proteolysis that can be so extensive that it produces complete loss of specific host and chlamydial proteins in the lysate (Fig. 1D, RIPA samples).
This model of CPAF activation during cell collection may explain why some but not all methods are effective in preventing artifactual CPAF-dependent proteolysis. Direct lysis with the urea or hot SDS methods is likely to inhibit CPAF activity within seconds. However, clasto-lactacystin appears to require pre-treatment for 60 min in order to reach sufficient concentrations inside an infected cell to inhibit CPAF activity induced by cell collection. Although more convenient, addition of 8 M urea or a CPAF inhibitor to a trypsinized cell pellet is unlikely to be effective because it would be too late to prevent CPAF-mediated proteolysis that has already occurred during cell collection.
In summary, protein analysis of Chlamydia-infected cells requires precautionary methods to inhibit CPAF activity during cell collection, and an assay to reveal whether detected proteolysis could be due to residual CPAF activity in the cell lysate (Fig. 5). The recently described CPAF null mutant (Snavely et al., 2014) is an invaluable tool for determining if CPAF is necessary for the cleavage or degradation of a specific protein, but it does not address whether any CPAF-mediated proteolysis occurred in the course of the intracellular infection or during cell detachment and lysis. Of the methods that we examined, direct lysis of infected cells in 8 M urea appears to be more reliable than the other methods in its ability to inhibit CPAF activity during cell collection and lysate preparation. However, this method is not always 100% effective, and has the disadvantages that lysate samples have to be diluted for SDS-PAGE analysis and cannot be used for enzymatic assays. Clasto-lactacystin pre-treatment and direct lysis in hot 1% SDS can be effective, but not in all circumstances, especially late in the chlamydial infection. Ideally, infected cell lysates for protein analysis should be checked for residual CPAF activity toward the substrate being examined, because proteins differ in their susceptibility to CPAF.
Figure 5.
Recommended procedure for analysis of proteins in lysates from Chlamydia-infected cells. We propose that infected cell lysates, which are analyzed for cleavage or degradation of a protein of interest (‘Protein analysis’), should also be tested for the presence of residual CPAF activity (‘In vitro CPAF Activity Assay’). This parallel analysis should examine the same substrate as the protein analysis and be performed for each lysate. If the in vitro CPAF assay reveals residual CPAF activity in the infected lysate, any observed proteolysis in this lysate cannot be interpreted because it will be unclear whether the proteolysis occurred in the Chlamydia-infected cell, during lysate preparation, or both.
Artifactual CPAF-mediated proteolysis induced in an intact cell by standard cell detachment methods complicates protein and proteomic analyses of Chlamydia-infected cells. This unfortunate, and apparently unusual, property of Chlamydia-infected cells grown on a monolayer has broad implications for approaches such as flow cytometry, mass spectrometry, protein affinity chromatography and biochemical studies. Interpretation of past and future studies of host and chlamydial proteins will need to take into account whether the protein analysis truly reflects the situation in an infected cell prior to cell collection.
SUPPLEMENTARY DATA
Acknowledgments
We thank P. Gönczy for the HsSAS-6 antibodies and G. MacGregor for use of the LICOR OdysseySA.
Authors’ Contribution: K.A.J. and J.K.L. contributed equally to this work
Footnotes
equal contributions
Present address: Department of Microbiology Immunology and Molecular Genetics, UCLA, Los Angeles, CA 90095, USA.
FUNDING
This work was supported by a research scholar grant from the American Cancer Society (RSG-10–250–01-CCG) to CS. JKL was supported by a T32 training grant from NCI (T32CA009054).
Conflict of interest statement. None declared.
REFERENCES
- Bavoil PM, Byrne GI. Analysis of CPAF mutants: new functions, new questions (the ins and outs of a chlamydial protease) Pathog Dis. 2014;71:287–91. doi: 10.1111/2049-632X.12194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen AL, Johnson KA, Lee JK, et al. CPAF: a chlamydial protease in search of an authentic substrate. PLoS Pathog. 2012;8:e1002842. doi: 10.1371/journal.ppat.1002842. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christian J, Vier J, Paschen SA, et al. Cleavage of the NF-kappaB family protein p65/RelA by the chlamydial protease-like activity factor (CPAF) impairs proinflammatory signaling in cells infected with Chlamydiae. J Biol Chem. 2010;285:41320–7. doi: 10.1074/jbc.M110.152280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grieshaber SS, Grieshaber NA. The role of the chlamydial effector CPAF in the induction of genomic instability. Pathog Dis. 2014;72:5–6. doi: 10.1111/2049-632X.12207. [DOI] [PubMed] [Google Scholar]
- Kumar Y, Valdivia RH. Actin and intermediate filaments stabilize the Chlamydia trachomatis vacuole by forming dynamic structural scaffolds. Cell Host Microbe. 2008;4:159–69. doi: 10.1016/j.chom.2008.05.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lad SP, Li J, da Silva Correia J, et al. Cleavage of p65/RelA of the NF-kappaB pathway by Chlamydia. P Natl Acad Sci USA. 2007;104:2933–8. doi: 10.1073/pnas.0608393104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ossewaarde JM, de Vries A, Bestebroer T, et al. Application of a mycoplasma group-specific PCR for monitoring decontamination of mycoplasma-infected Chlamydia sp. strains. Appl Environ Microb. 1996;62:328–31. doi: 10.1128/aem.62.2.328-331.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paschen SA, Christian JG, Vier J, et al. Cytopathicity of Chlamydia is largely reproduced by expression of a single chlamydial protease. J Cell Biol. 2008;182:117–27. doi: 10.1083/jcb.200804023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Snavely EA, Kokes M, Dunn JD, et al. Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches. Pathog Dis. 2014;71:336–51. doi: 10.1111/2049-632X.12179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tan M, Sütterlin C. The Chlamydia protease CPAF: caution, precautions and function. Pathog Dis. 2014;72:7–9. doi: 10.1111/2049-632X.12213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhong G. Killing me softly: chlamydial use of proteolysis for evading host defenses. Trends Microbiol. 2009;17:467–74. doi: 10.1016/j.tim.2009.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhong G, Fan P, Ji H, et al. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med. 2001;193:935–42. doi: 10.1084/jem.193.8.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhong G, Liu L, Fan T, et al. Degradation of transcription factor RFX5 during the inhibition of both constitutive and interferon gamma-inducible major histocompatibility complex class I expression in Chlamydia-infected cells. J Exp Med. 2000;191:1525–34. doi: 10.1084/jem.191.9.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]