The role of an enzymatically inactive CPAF mutant vaccination in Chlamydia muridarum genital tract infection
Introduction
Chlamydia trachomatis is an obligate intracellular bacterium with a wide range of infection hosts and a complex pathogenesis [1]. In chlamydial biphasic cycle, the infectious elementary body (EB) invades host cells through adhesion and endocytosis, forming an inclusion body. In the inclusion body, EB differentiated into a reproductive reticulate body (RB), and then RB undergoes binary fission to produce more offspring EB. When the inclusion body matures and breaks, EB is released and then infects neighboring cells to form a new cycle [2]. In female, C. trachomatis asymptomatic lower genital tract infection often spread to the fallopian tubes, causing inflammatory pathologies, such as hydrosalpinx that result in infertility and increases the risk of co-infection with HPV and HIV [3,4]. Even though chlamydial infectious diseases can be treated with antibiotics such as tetracyclines, macrolides and quinolones, due to its hidden asymptomatic course in up to 80% of female patients and 50% of male patients, the health burden of chlamydial infections is high [5,6]. Therefore, it is urgent to develop a safe and effective preventive vaccine to reduce the spread of chlamydia disease.
To this end, chlamydial researchers have developed multiple vaccine strategies to prevent genital C. trachomatis infection. The failed clinical trials of human trachoma vaccine in 1950s [7,8], along with more and more new knowledge in immunology, suggested that subunit vaccines are likely to be developed as chlamydial clinical vaccines in the future [[9], [10], [11]]. Chlamydial protease-like activity factor (CPAF) is a serine protease that is secreted by chlamydia into the cytosol of infected cells [12]. A person who has a positive serological test for chlamydia, usually have higher antibody titers against CPAF than the exposed surface proteins (i.e., MOMP and Hsp 60), indicating the immunogenic dominance of this secreted protein [[13], [14], [15]]. Murthy et al. demonstrated that intranasal vaccination with CPAF enhances resolution of chlamydial genital infection and reduces oviduct pathology [16].
However, CPAF is an enzyme that is secreted by chlamydia to reach the cytosol for accumulation [12]. Ever since CPAF was discovered, several studies have revealed that lots of infected cell proteins can be targeted by CPAF for specific cleavage or degradation [17], including those involved in modulate apoptosis (BH3-only family [18]), DNA repair enzyme (PARP [19]), hypoxic inducible factor (HIF-1 [20]), MHC transcription factor (RFX5, USF-1, HMBG1 [[21], [22], [23]]), inflammation-related factor (NFκB p65 [24]), surface proteins (CD1D [25], nectin-1 [26] and cyclin B1 [19]), cytoskeleton remodeling proteins (keratin-8, keratin-18, vimentin [27,28]) and nutrient acquisition protein (golgin-84 [29]). In 2012, Chen et al. conducted a strict CPAF enzyme inactivation treatment on the lysates of chlamydia infected cells, they found that many host substrates confirmed by previous studies were false positive due to incomplete inactivation of CPAF enzyme during experimental operation [30]. More important, even the purified recombinant CPAF protein also exhibited broad and strong enzymatic activity to digest host proteins [31,32], these indicate that directly immunize with CPAF may cause tissue damage.
To promote clinical use of CPAF as vaccine, in this study, we tested the efficacy of vaccination with C. trachomatis CPAF mutant (Mut) in comparison to wild type (Wt) with a Th1 polarizing adjuvant CpG in a C. muridarum murine genital tract models. Both CPAF mutant and wild type immunization enhanced the chlamydia clearance in vaginal shedding and reduced the pathology in the oviduct of mice after the challenge. Immunization induces high CPAF specific antibody and IFN-γ production, it is CD4+ T lymphocyte but not neutralization antibody plays important role in this protection.
Section snippets
Bacteria
C. trachomatis serovar D and C. muridarum strains were grown on confluent HeLa 229 cells (ATCC, CCL-2.1). As described previouslythe infected HeLa cells were lysed by an ultrasonic machine, and chlamydial EBs were purified on Renografin gradients [33]. After suspension with sucrose-phospho-glutamine (SPG) buffer, EBs were aliquot and stored at 80°C.
CPAF (Wt) and CPAF (Mut)
The recombinant pET30a plasmids coding for C. trachomatis serovar D wild type CPAF enzyme and the mutant CPAF (E558A) were described elsewhere [31,32
Mutant CPAF loses its enzymatic activity
Recombinant CPAF wild type (Wt) and catalytic mutant E558A (Mut) were expressed as His-fusion protein and purified using Ni-resin and anion ion exchange as described previously (16). After SDS-PAGE electrophoresis, the CPAF (Wt) protein migrated into two main protein bands that corresponded to the molecular weights of two fragments, CPAFc-His of ~41 KDa and CPAFn of ~29 KDa. The CPAF (Mut) protein migrated into ~70 kDa band that corresponded to the molecular weights of full length CPAF-His (
Discussion
Since the discovery of chlamydial CPAF [12], the protein have aroused wide attention. With continuous in-depth study of CPAF, some of its biochemical and structural properties have been identified [31,35,36]. CPAF is a common and conserved protease in Chlamydiaceae and its enzyme activity can be detected in both chlamydia infected cells and purified recombinant CPAF protein [31,37]. After synthesized in the host cells or purified out from the prokaryotic expression system, CPAF zymogen (about
Declaration of competing interest
The authors declare that they have no known competing financial interests of personal relationships that could have appeared to influence the work reported in this paper.
Author contributions
Conceived and designed the experiments: GZ CW ZL ZS. Performed the experiments: HC BP XL XT LX SZ. Analyzed the data: HC CL ZL. Wrote the manuscript: HC CL BP.
Declaration of competing interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (no. 81471969 & 81202374); Innovation Platform Open Fund Project of Hunan Provincial Education Department (no. 20K108); the National Students' Innovation and Entrepreneurship Training Program (S202010555057) and Hunan Province Students' Innovation and Entrepreneurship Training Program (S202010555253).
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Hui Chen and Bo Peng contributed equally to this work.