Mechanism Of HrcA In Heat Shock Transcriptional Regulatory Network Of Chlamydia Trachomatis

Background:Heat shock response is an important protective mechanism,which is common in prokaryotes such as bacteria and chlamydia.Cells reprogram their transcriptomes in response to stress,such as heat shock.So heat shock response is essential for bacterial survival and adaptation to hostile environmental conditions.In bacteria,transcriptional regulation of heat-shock genes is mediated by specific alternative sigma factors of RNA polymerase enzyme and transcriptional repressors.By far,the roles and regulatory mechanisms of heat shock response in Chlamydia trachomatis（C.trachomatis）have not been elucidated.In addition,the transcriptome reprogramming and transcriptional regulatory network of Chlamydia trachomatis in response to heat shock have not yet been reported.Objective:This study aims to investigate the heat shock transcriptional regulatory network of Chlamydia trachomatis and the role of Hrc A in this process,which will provide theoretical basis for elucidating the pathogenic mechanism of C.trachomatis and finding new drug targets.Methods:1)Cultures of C.trachomatis Ct L2/RFP expressing a red fluorescent protein was stimulated by heat shock at 45°C for 2 hours and then returned to 37℃.Microscopic examination and detection of the relative genome copy number by q PCR were performed to determine the effects of temperature shifts on chlamydial growth.2)RNA-Seq analyses were performed to investigate heat shock transcriptomic response in C.trachomatis.RT-q PCR were carried out to validate the RNA-Seq results.COG functional classification was performed to determine the physiological significance of heat shock-induced transcriptomic changes.3)The FIMO（find individual motif occurrences）program was used to search for CIRCE（controlling inverted repeat of chaperone expression）elements andσ²⁸ promoters in C.trachomatis genome to predict differentially expressed genes in heat shock that may be regulated by Hrc A orσ²⁸.The STRING and Gephi programs were used to generate a heat shock transcriptional regulatory network of C.trachomatis.4)RNA-Seq and RT-q PCR analyses were performed to analyze the effect of Hrc A overexpression on the C.trachomatis transcriptome.Electrophoresis mobility shift assays（EMSA）were performed to determine the ability of Hrc A to bind the upstream regions of the new candidate target genes of Hrc A.5)Ch IP-Seq and Ch IP-q PCR were performed to systematically identify the binding sites of Hrc A in the C.trachomatis genome.Results:1)Red fluorescence protein-expressing C.trachomatis,which cultures incubated at 45°C for 2-hours following a 6-hours recovery at 37°C compared to cultures maintained at 45°C without a recovery,formed statistically significantly larger inclusions with more intense RFP signals.Likewise,quantitative PCR analysis detected a higher level of the bacterial genome in wild-type C.trachomatis cultures exposed to a 2-h incubation at 45°C followed by a 6?h recovery at 37°C relative to heat-shocked cultures without the recovery.C.trachomatis still partially replicate its genome after cultured at 45°C for 8 hours.These results indicated that C.trachomatis could recover from 45°C and replicate at a lower rate.2)Transcriptomic analyses showed that nearly one-third（303/907）of C.trachomatis genes underwent statistically significant（≥1.5-fold）expression changes30min after shifting from 37 to 45°C.Transcript copy numbers of 151 genesincreased while those of 152 genes decreased.3)Chlamydial chromosomal genes involved in energy metabolism enzymes,posttranslational modification,protein turnover,type III secretion proteins,as well as most plasmid-encoded genes,were disproportionately upregulated.In contrast,genes involved in amino acid and peptide transport and metabolism,and ribosomal structure and biogenesis wre disproportionately downregulated.4)Heat shock upregulated the expression of Hrc A,σ²⁸,σ⁶⁶ and Hrc A target genes grp E,dna K,gro ES and gro EL.Sequence analysis identified 13 and 33potential genes which response to heat shock were regulated by Hrc A orσ²⁸.5)Mostσ⁵⁴target genes are upregulated in the presence of downregulated rpo N and upregulated ato C and ato S following heat shock.6)Network analysis of the transcriptomic reprogramming in response to heat shock is accomplished through the functions of all three sigma factors,Hrc A,Pgp4,theσ⁵⁴ RNAP transcriptional activator Ato C,and additional transcription regulators.And constructed a heat shock transcriptional regulatory network of C.trachomatis.7)It was demonstrated that the promoter region of ctl0271,inc G and the ctl0505-ctl0506 operon contained potential CIRCE elements.These genes were upregulated in response to heat shock and might be regulated by Hrc A by binding to promoter region.8)Ch IP-Seq detected 94 potential Hrc A-binding sites in the C.trachomatis genome.Ch IP-q PCR and RT-q PCR preliminarily indicated that Hrc A might be bind the upstream regions of ftsk,mur F,opp A2,ctl0576,ctl0680 and ctl0819.These genes can be inhibited by Hrc A and induced by heat shock.Conclusions:1)C.trachomatis,which encodes only three sigma factors and a limited number of transcription factors,can mount a robust heat shock response that involves an extensive reprogramming of its transcriptome.And C.trachomatis can recover from 45°C and replicate at a lower rate.2)The reprogramming in heat shock promotes chlamydia increase energy production,increase communication with host cells,conservation of resources for protein synthesis,and retention of the fitness and virulence determinants of its plasmid.3)We show that the reprogramming is accomplished through the actions of all three sigma factors,Hrc A,Pgp4,theσ⁵⁴ RNAP transcriptional activator Ato C,and additional transcription regulators,and then a heat shock transcriptional regulatory network of C.trachomatis was constructed.4)It was found and confirmed that ctl0271,inc G and ctl0505-ctl0506 were new targets of transcriptional repressor Hrc A.Taken together,those findings reveal that C.trachomatis utilizes multiple novel survival strategies to cope with environmental stress.