Complement deposition on Chlamydia trachomatis

Description Introduction Chlamydia trachomatis (C. trachomatis) is a Gram-negative bacterium and a common human pathogen. The World Health Organization (WHO) estimates that over 130 million people are infected with C. trachomatis globally each year and with increasing incidence. C. trachomatis causes long-lasting and recurrent infections that over time induce severe tissue damage in the female genital tract that can lead to ectopic pregnancy and infertility. Thus, the human immune system fails to control and eradicate C. trachomatis during primary infection and fails to develop protective immunity against secondary infections. In vivo infection models, using complement knock out mice, suggest that the complement system is critically involved in both anti-chlamydial immunity and infection-induced pathology. To increase our understanding of complement-mediated immunity against C. trachomatis we analyzed global complement deposition on serum-incubated C. trachomatis by mass spectrometry. Methods Purified C. trachomatis was incubated in seronegative normal human serum (NHS) or heat-inactivated normal human serum (HI-NHS) for 30 min, thoroughly washed, and processed for mass spectrometry. All samples were lysed, reduced and alkylated and digested with trypsin. Some samples were chemically modified to acetylate free amino groups (N-terminal and lysine amino groups) before trypsin digestion. Peptides were analyzed on a UltimateTM 3500 RSLCnano coupled to a Q Exactive HF-X mass spectrometer. Raw data files were searched against the Uniprot human reference proteome using MaxQuant. Results We demonstrate that C. trachomatis elicits potent complement activation demonstrated by deposition of both early and late complement factors together with several complement regulators. We further demonstrate proteolytically processing of complement C3b to “inactive” C3 cleavage fragments. Conclusion We demonstrate the deposition of several novel complement-associated proteins and -cleavage fragments on the surface of C. trachomatis. Sample Processing Protocol Bacterial organisms and propagation. C. trachomatis L2/434/Bu was obtained from the American Type Culture Collection (ATCC, VA USA) and propagated in McCoy cells (ATCC) according to Ripa and Mårdh [17]. C. trachomatis EB were purified by density gradient centrifugation essentially according to Caldwell. Serum isolation Serum was obtained from healthy volunteers at Aalborg University. Peripheral blood was collected by venipuncture in S-Monovette® serum tubes (Sarstedt, Nümbrecht, Germany) and serum was isolated according to manufacturer’s instructions. Aspirated serum was aliquoted and immediately stored on ice before freezing at -80°C. Serum heat-inactivation was carried out for 30 min at 56°C. The serum serostatus was tested by C. trachomatis-IgG-ELISA Plus (medac, Wedel, Germany) using a peptide from the variable domain IV of the major outer membrane protein (MOMP) as antigen. All protocols using human biological material was approved by the Ethics Committee of Region Nordjylland (case no. N-20150073) and all participants gave informed consent to participate in the study. The work was conducted in accordance with the 1964 Declaration of Helsinki. Sample preparation for mass spectrometry (MS) Purified C. trachomatis L2 EB were incubated in either 50% normal human serum (NHS) or 50% heat-inactivated human serum (HI-NHS) for 30 min at 37°C and thoroughly washed three times in PBS with centrifugation at 15,000 x g for 15 min at 4°C between each wash. Samples were processed for mass spectrometry using a filter-aided sample preparation protocol essentially according to Bennike et al.. Chlamydiae were lysed in 5% sodium deoxycholate (SDC) in 50 mM triethylammonium bicarbonate (TEAB) and vortexed thoroughly for 30 seconds and heated to 95°C for five min. The lysates were centrifuged for 10 min at 14,000 x g at the supernatant was transferred to Amicon® Ultra 30 kDa cut-off Centrifugal Filters (Merck Millipore, MA, USA). Lysates were washed twice in 0.5% SDC in 50 mM TEAB with centrifugation for 15 min at 14,000 x g between washes. Proteins were reduced and alkylated in 10 mM TCEP (Tris(2-carboxyethyl)phosphine) and 50 mM chloroacetamide in 50 mM TEAB for 30 min at 37°C and proteins were washed twice in 0.5% SDC in 50 mM TEAB. To differentiate between peptides generated by trypsin cleavage and peptides generated by complement cleavage, half of the samples were treated with 10 mM sulfo-N-hydroxysuccinimide (TermoFisher Scientific, MA, USA) for 90 min at 30°C to acetylate primary amines as described by Gevaert et al. [20]. Partial acetylation of serine and threonine residues was reversed using 2 µg/ml hydroxylamine (Sigma-Aldrich, MO, USA). Proteins were washed twice in 0.5% SDC in 50 mM TEAB washes and digested overnight at 37°C using PierceTM Trypsin Protease (ThermoFischer Scientific) 20 µg/ml in 0.5% SDC in 50 mM TEAB. Digested peptides were collected by centrifugation for 15 min at 14,000 x g and subjected to ethyl acetate extraction to remove remaining organic solvents. Ethyl acetate was added at a 3:1 ratio and the solution was acidified by adding 1% trifluoroacetic acid (TFA). Organic and aqueous phases were separated by centrifugation for one minute at 14,000 x g and the upper organic phase was removed. The extraction procedure was repeated three times in total and the peptides were dried down in a vacuum centrifuge. Finally, peptides were resuspended in 0.5% TFA and desalted using Pierce C18 Tips (ThermoFischer Scientific) according to manufacturer’s protocol. Eluted peptides were dried down in a vacuum centrifuge and kept at -80°C for further analysis. Ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis Peptides were reconstituted in 2% acetonitrile and 0.1% formic acid and separated by reverse phase liquid chromatography on a UltimateTM 3500 RSLCnano coupled to a QExactive HF-X mass spectrometer (ThermoFischer Scientific). Peptides were loaded onto a 2 cm AcclaimTM PepMapTM C18 trap column (ThermoFischer Scientific) and subsequently separated by reverse phase chromatography on a 75 cm AcclaimTM PepMapTM analytical C18 column (ThermoFischer Scientific): Samples were eluted with a gradient of 98% solvent A (0,1% formic acid) and 2% solvent B (0,1% formic acid in acetonitrile) from 0-10 min and solvent B was increased to 50% over 85 min on a ramp gradient. Solvent B was increased to 98% for 6 min and lowered to 2% on a 1 min ramp gradient and kept at 2% solvent B for 17 min. Data Processing Protocol Raw MS/MS data files were searched against the Uniprot human reference proteome (downloaded January 20, 2020, containing 20,362 entries) using MaxQuant (vers. 1.6.10.43). Standard search settings were used with minor modifications: three miss cleavages were allowed, carbamidomethyl (M) was set as fixed modification and acetylation (K), acetylation (N-term), oxidation (M) and deamidation (NQ) was set as variable modifications. Relative protein abundances were quantified using label-free quantification and all results were reported at a false discovery rate below 1%. Contact Tue Bjerg Bennike, Aalborg University Svend Birkelund, Head of Medical Microbiology and Immunology, Department of Health Science and Technology, Medical Faculty, Aalborg Universtity, Denmark (lab head) Submission Date 26/05/2020 Publication Date 23/11/2020 Project PXD01939