Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia

Abstract

The developmental cycle of the obligate intracellular pathogen Chlamydia trachomatis serovar L2 is controlled in part by the small non-coding RNA (sRNA), IhtA. All Chlamydia alternate in a regulated fashion between the infectious elementary body (EB) and the replicative reticulate body (RB) which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of hctA until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens C. trachomatis serovars L2, D and L2b and C. pneumoniae. We show in this work that IhtA is also expressed by the animal pathogens C. caviae and C. muridarum. Expression of HctA in E. coli is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned hctA and ihtA from C. trachomatis serovar D, C. muridarum, C. caviae and C. pneumoniae and assayed for rescue of growth repression in E. coli co-expression studies. In each case, co-expression of ihtA with the cognate hctA resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of C. trachomatis serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with hctA message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.

Figure 1. IhtA is expressed in diverse chlamydial species.

(A) The IhtA containing genetic loci of C. trachomatis serovar L2, D and A and and the more disparate C. muridarum, C. caviae and C. pneumoniae were obtained and aligned by searching the complete genomes of the respective bacteria. (B) Northern analysis of IhtA expression during infection with C. trachomatis serovar D, C. caviae, C. trachomatis serovar L2 and C. muridarum. HeLa cell cultures were infected with C. trachomatis serovar L2, C. caviae, and C. muridarum for 24 h while C. trachomatis serovar D was grown for 48 h in 1 well of a 6 well plate prior to harvesting of sRNA. The entire sRNA sample was separated on a 10% TBE-Urea gel and probed with a species specific biotinylated PCR fragment.

Figure 2. IhtA sequence is conserved across species.

The sequence of ihtA of C. trachomatis serovar D, C. muridarum, C. caviae, and C. pneumoniae compared to C. trachomatis serovar L2. The TSS of serovars L2 and D and C. pneumoniae ihtA (indicated in bold) has been experimentally proven while the TSS of the other Chlamydia is predicted.

Figure 3. Structural prediction of species IhtA.

Structural predictions were made using the RNAfold server contained in the Vienna websuit (http://rna.tbi.univie.ac.at). The three stem:loops of L2 IhtA are indicted numerically. Only the minimal free energy (MFE) of C. trachomatis serovars L2 and D, C. muridarum and C. caviae are displayed as the centroid structures are identical to the MFE structures indicating high confidence in the predictions. Both the MFE and centroid structures of C. pneumoniae are shown as they differ significantly. The * indicates the region of IhtA complimentary to the first 6 nucleotides of hctA. Base pair probabilities calculated by the web server are color coded 0–1, with higher numbers corresponding to higher confidence.

Figure 4. IhtA of each species interacts with the cognate hctA target mRNA in vitro.

Run off hctA transcripts made from species specific PCR fragments were annealed to biotinylated oligo T and bound to BLI sensor tips. hctA bound tips were incubated with species specific native IhtA or antisense serovar L2 (scrambled control) IhtA and the change in reflected light indicating RNA:RNA binding was measured over time.

Figure 5. Repression of HctA translation by IhtA is a conserved mechanism.

(A) Northern analysis of expression of species IhtA in E. coli. The ihtA gene, including 5′UTR was PCR amplified from the genomes of the indicated chlamydial species, cloned into pLac and expressed in DH5alphaPRO E. coli. sRNA was isolated from overnight cultures using the mirVana miRNA Isolation kit (Ambion, Inc.). Northern analysis was performed on 2 µg of each sample separated on a 10% TBE-urea acrylamide gel which was transferred to BrightStar-Plus Nylon membrane and probed with a species specific biotinylated ihtA PCR fragment. (B) DRAQ5 DNA staining of E. coli induced to express the indicated species specific HctA and either empty vector (pLac) or the cognate IhtA. Condensed DNA appears as a central brightly fluorescent sphere (bar equals 2.5 um). C) Representative viability assay of E. coli expressing either species specific hctA alone (+pLac, light grey bars) or species specific hctA and IhtA (+cognate ihtA, dark grey bars). Each condition was performed in triplicate with a minimum of three repeats. The bars represent the S.E.M of each triplicate. The * indicates p value <0.001 and # indicates p value = 0.003.

Figure 6. Serovar L2 HctA expression is repressed by species IhtA.

A) Growth viability of E. coli co-expressing serovar L2 HctA and C. trachomatis serovar D, C. muridarum, C. caviae, or C. pneumoniae IhtA. E. coli co-expressing hctA and empty vector (pLac) or L2 IhtA served as negative and positive controls respectively. Each condition was performed in triplicate with a minimum of three repeats. The bars represent the S.E.M of each triplicate. B) Growth viability of E. coli co-expressing species hctA and C. trachomatis serovar L2 IhtA. Each condition was performed in triplicate with a minimum of three repeats. The bars represent the S.E.M of each triplicate. The * indicates a p value ≤0.001.