Pathogenesis Study Of Virulence-related CT135 Paralog Of Chlamydia Psittaci

Chlamydia psittaci is an obligate intracellular zoonotic bacterium with a unique bi-phasic developmental cycle. According to the latest chlamydial taxonomy, C. psittaci belongs to Chlamydia genus, Chlamydiaceae family, Chlamydiales order. In 1930s, C. psittaci caused pandemic infections in at least 12 countries, and the 1930 pandemic in Maryland led to the foundation of the national institute of health of the United States. C. psittaci widely infects animals causing abortions and arthritis, et al. Human being is not a natural reservoir of C. psittaci, but human infections with C. psittaci often cause systemic symptoms and have a high mortality if left untreated. Due to its aerosol transmission and high morbidity, C. psittaci is widely accepted as a classic bioweapon and a bioterrorism agent.C. psittaci is an understudied pathogen mainly because of its difficulty to culture and its requirement of high-level containment. There is no reports on C. psittaci-specific virulence genes. Our understanding of C. psittaci pathogenesis mainly comes from studies of other chlamydial species, especially C. trachomatis. C. trachomatis is the pathogen of trachoma and most common bacterial sexually transmitted disease. The study focuses on CT135 paralog of C. psittaci, as CT135 is a newly discovered virulence gene of C. trachomatis. CT135 always remains intact in clinical samples, while it’s prone to be silenced in in vitro cultures. Intriguingly, two isogenic C. trachomatis strains, both having frameshift mutations in CT135, have distinct virulence in a female mouse genital tract infection model.Whether CT135 encodes a protein, whether CT135 paralogs of other chlamydial species have similar phenotypic instability, and the localization and function of CT135 are still unknown. The purpose of this study is to analyze the polymorphism of CT135 paralogs of C. psittaci epidemic strains in China, to analyze the gene stability, to determine CT135 protein expression and localization, and to construct CT135-paralog null mutants. In C. trachomatis, three hypothetical genes, CT134-136, likely function as an operon. They share 26%,21% and 65% amino acid identity with their C. psittaci paralogs B598₀589, B5980590 and B598₀557, respectively. Based on the possibility of functioning together, paralogs of CT134-136 are all included for analyses of protein expression and localization.This study firstly analyzed the polymorphism and in vitro stability of B5980590 of China C. psittaci isolates by using a Cel I based single-nucleotide-polymorphism detection technique. The Cel I enzyme was extracted from celery juices by ammonium sulfate precipitation, and was confirmed of having SNP detection activities. No polymorphisms were detected in nine China C. psittaci isolates by Cel â…  digestion, which was confirmed by sequencing. All nine isolates, having an intact B598＿0590, had been cultured in chicken embryonic eggs, not the routinely used cell cultures by foreign researchers. Therefore, we sought to speed the evolution of C. psittaci and then to detect gene polymorphisms. The C. psittaci strain CG1 was treated with ethyl methanesulfonate and was then passaged for continuous 20 times on Vero cells in 24-well plates. All cultures from 24 wells remain having an intact B5890590 albeit with their adaptation to Vero cells, which is clearly not consistent with the in vitro instability of CT135 in C. trachomatis.Chlamydia replicates in a specialized inclusion. Chlamydial proteins can locate inside the bacterium, on the bacterial surface, in the inclusion lumen, on the inclusion membrane or in the host cell cytosol. Proteins located in the cytosol and on the inclusion membrane have direct contacts with the host, thus are considered of regulating host cell signaling. This study expressed B598₀589, B5980590, B5980557, inclusion membrane protein IncA, major outer membrane protein MOMP and secreted protein CPAF of C. psittaci CG1 by using vector pGEX-4T1. Recombinant proteins were used to immunize mice for making anti-sera. under con-focal microscopy, B5980590 and IncA have similar immune-staining characteristic, suggesting B5980590 encodes an inclusion membrane protein. Different from IncA, anti-B598₀590 serum can stain the lumen in most inclusions, suggesting the possible bacterial or lumen localization of B5980590. The staining characteristics of B598₀589 and B598₀557 are similar to MOMP, suggesting they are outer membrane proteins. The specificities of all the anti-sera were confirmed by western blot and no post-translational modifications were detected in these assays.The discovery of B598₀590 encoding an inclusion membrane protein was further confirmed in assays of testing LpxC inhibitors and penicillin. In C. trachomatis, penicillin does not affect the inclusion growth, chromosomal replication, and does affect bacterial fission and the transformation of reticulate body to elementary body. LpxC inhibitors are novel antibiotics, can inhibit the synthesis of lipid A. In C. trachomatis, LpxC inhibitors do not affect the inclusion growth and fissions of reticulate bodies, but severely affect the maturation of elementary bodies. The effects of these two classes of drugs to C. psittaci are understudied. Possibly enlarged bacterial volume might help to analyze protein localization. We found both LpxC inhibitor and penicillin severely inhibit chlamydial fission and inclusion growth, and they have different effects to protein expression. Penicillin completely inhibits IncA secretion while LxpC inhibitor-treated CGI still secrets IncA into the inclusion membrane. Importantly, immunofluorescences of LpxC inhibitor-treated CGI with sera of anti-B598₀590 and anti-IncA show similar vacuole structures, which confirms that B5980590 encodes an inclusion membrane protein.The above discovery about B5980590 is consistent with bioinformatic analysis. According to literatures, most chlamydial inclusion membrane proteins have a typical bi-lobe of hydrophobic region of about 60 amino acids. All 12 species in Chlamydia genus have a CT135 paralog, and these paralogs have similar hydrophobic characteristics and same size of a hydrophobic bi-lobe. In C. trachomatis, CT134 and CT135 are predicted inclusion membrane proteins, but yet are to be identified. We found only the CT135 paralog-B5980590 encodes an inclusion membrane protein. To compare the homology of inclusion membrane proteins of C. psittaci and C. trachomatis, we did hydrophobic analyses to all hypothetical proteins and putative membrane proteins of C. psittaci GR9 and found 46 putative inclusion membrane proteins altogether. Among them, only IncA has a clear function of mediating inclusion fusion. Moreover, C. psittaci and C. trachomatis only share 21 homologous inclusion membrane proteins. These conserved homologous proteins likely have similar functions and relate to human pathogenesis, while other non-conserved proteins might relate to their host tropisms.This study also planned to knockout B5980590 by using the type â…¡ intron based TargeTron technique. We designed the type â…¡ intron insertion locations and primers for constructing pACD4K-C based knockout vectors, and tested their feasibilities in E. coli carrying the B598₀590 gene. Currently, P-lactamase is a widely used selection marker for C. trachomatis transformation. So the kanamycin resistance gene on pACD4K-C is removed by Mlu â…  digestion and β-lactamase is then inserted in this Mlu â…  site. Furthermore, promotors of B5980249 and ompA are added in the downstream of the original T7 phage promotor of the type â…¡ intron. Thus, the final two B5980590 knockout vectors have endogenous C. psittaci promotors for the intron expression, which is mandatorily required for future constructing C. psittaci mutants.For the purpose of preparing chlamydia genus specific lipopolysaccharide and antibody, we constructed an E. coli mutant expressing the chlamydia genus LPS epitopes by using the TargeTron technique. The chlamydia lipopolysaccharide is in essence a lipooligosaccharide which is very short and only consists of several Kdo molecules. Its a-Kdo-（2→8）-a-Kdo epitope is genus specific, and is synthesized by a multifunctional Kdo transferase KdtA. The C. trachomatis L2 kdtA was cloned into E. coli BL21（DE3）, then the type â…¡ intron was inserted inside the endogenous E. coli kdtA. The final E. coli kdtA::Gâ…¡ CtkdtA can express the chlamydia genus specific Kdo epitope. Formaldehyde inactivated E. coli can induce C. psittaci specific antibodies, suggesting recombinant Kdo epitopes have good immunogenicity and can be used for preparing chlamydia genus specific antibodies.In conclusion, a Cel I-based SNP detection technique was established and can be used in TILLING-based reverse genetics of C. psittaci; all China C. psittaci isolates have an intact CT135 paralog and this paralog may not relate to in vitro adaptive growth of C. psittaci strain CG1 on Vero cells; putative inclusion membrane proteins of C. psittaci strain GR9 were predicted by bioinformatic analyses; importantly, the CT135 paralog of C. psittaci-B598₀590 is identified as encoding an inclusion membrane protein by bioinformatic analysis, con-focal microscopy and LpxC inhibition assay, while paralogs of CT134 and CT136 likely encode membrane proteins; C. psittaci antigens, B5980589, B598₀590, B598₀557, IncA, MOMP, CPAF and Kdo epitopes, were expressed in E. coli; and two knockout vectors for B598₀590 were constructed; all the above will further our studying pathogenesis of CT135 paralogs.