Identification of the Base-Pairing Requirements for Repression of <i>hctA</i> Translation by the Small RNA IhtA Leads to the Discovery of a New mRNA Target in <i>Chlamydia trachomatis</i>

<div><p>The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen <i>Chlamydia trachomatis</i> modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of <i>hctA</i>, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of <i>hctA</i>. Two additional genes in the chlamydial genome, <i>CTL0322</i> and <i>CTL0097</i>, contained some elements of the <i>hctA</i>:IhtA recognition sequence. The mRNA of both <i>CTL0322</i>and <i>CTL0097</i> interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of <i>CTL0322</i>. The proposed IhtA recognition site in the <i>CTL0322 </i>message contains significant G/C base pairing on either side of the initiation codon while <i>CTL0097</i> only contains G/C base pairing 3’ to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle.</p></div

iIhtA targets conserved sequences of <i>hctA</i>.

<p>(A) Mutations made across the conserved region of <i>hctA</i> resulted in the mutant <i>hctA</i>6–27. Each mutation was designed to maintain the amino acid structure whilst disrupting the linear RNA sequence of the potential target region for IhtA. (B) Mutant <i>hctA</i> constructs were co-transformed with empty pLac vector or wild type <i>ihtA</i> into <i>E</i>. <i>coli</i> and assayed for growth upon induction of HctA expression. Wild type <i>hctA</i> co-transformed with pLac or <i>ihtA</i> served as controls. Cell viability was expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. The bars represent the mean ± SEM of the triplicates in all experiments combined. * indicates P value < 0.01 using t-test statistical analysis.</p

iInteraction of IhtA with predicted mRNA targets <i>CTL0097</i> and <i>CTL0322</i>.

<p>(A) Predicted base-pairing by <i>TargetRNA</i> of additional IhtA target mRNAs which encode at least a partial G/C rich IhtA binding region (underlined). The location of loop 1 of IhtA is also indicated. (B) In vitro transcribed IhtA was incubated with <i>CTL0322</i> and <i>CTL0097</i> immobilized on a biosensor tip. IhtA incubated with in vitro transcribed <i>hctA</i> served as a positive control for binding and in vitro transcribed <i>hctB</i> served as a negative control. Binding was measured as a change in internally reflected light through the tip over time.</p

iLoop 1 of IhtA targets <i>hctA</i> mRNA.

<p>m(A) Schematic of the structure of IhtA and color coded base pair probability (color coded 1–0) as predicted by the <i>RNA</i>fold web server. The three stem:loops are indicated, stem:loop 3 is the rho-independent terminator. (B) <i>TargetRNA</i> prediction of interacting nucleotides between IhtA and <i>hctA</i>. Indicated are location of loop 1 and 2 of IhtA and the Shine-Dalgarno and start site of <i>hctA</i>. (C) Wild type <i>hctA</i> constructs were co-expressed with IhtA loop mutants <i>ihtA</i>L1, <i>ihtA</i>L2 and <i>ihtA</i>L3. Cell viability was expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. The bars represent mean ± SEM of the triplicates in all experiments combined. Statistical analysis performed using t-test, * indicates P value < 0.01 when compared to <i>ihtA</i>/<i>hctA</i>.</p

iIhtA represses the translation of <i>CTL0322</i> in vitro.

<p>(A) Schematic of <i>cheZ</i> constructs. Blue and red are chlamydial specific sequences, red indicates sequence that is translated. Green indicates <i>E</i>. <i>coli</i> cheZ. (B) <i>E</i>. <i>coli</i> lacking motility (MG1655 Δ<i>cheZ</i>) were co-transformed with either <i>ihtA</i>L1 (deficient in <i>hctA</i> translation repression) and <i>cheZ</i>, <i>hctBcheZ</i> or <i>hctAcheZ</i> (panels A,C and E) or <i>ihtA</i> and the aforementioned constructs (panels B, D and F). (C) <i>E</i>. <i>coli</i> lacking motility (MG1655 Δ<i>cheZ</i>) were co-transformed with <i>ihtA</i>L1 and <i>CTL0097cheZ</i> or <i>CTL0322cheZ</i> (panel A and C) and <i>ihtA</i> and <i>CTL0097cheZ</i> or <i>CTL0322cheZ</i> (panels B and D). All mobility assays were performed at least 3 times per strain.</p

iIhtA occludes the start site of <i>hctA</i>.

<p>(A) Schematic of the mutations made in IhtA and the corresponding compensatory mutations made in <i>hctA</i>. The start site of <i>hctA</i> is underlined. (B) <i>E</i>. <i>coli</i> were co-transformed with A/T rich mutant construct pairs <i>ihtA</i>#21 and <i>hctA</i>#22 and with G/C rich mutant pairs <i>ihtA</i>#9 and <i>hctA</i>#14 and cell viability was assayed. Wild type <i>hctA</i> co-transformed with pLac, <i>ihtA</i>, <i>ihtA</i>#21 or <i>ihtA</i>#9, and mutant <i>hctA</i>s co-transformed with pLac served as controls. (C) To determine importance of the G/C pairing 3’ of the <i>hctA</i> AUG, <i>E</i>. <i>coli</i> were co-transformed with A/T rich mutant construct <i>ihtA</i>#10 and its compensatory <i>hctA</i> partner <i>hctA</i>#16 or with G/C rich mutant <i>ihtA</i>#26 and the compensatory mutant <i>hctA</i>#25. Wild type <i>hctA</i> co-transformed with pLac, <i>ihtA</i>, <i>ihtA</i>#10 or <i>ihtA</i>#26, and mutant <i>hctA</i>s co-transformed with pLac served as controls. Cell viability in graphs B and C were expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. The bars represent the mean ± SEM of all samples. Statistical analysis using t-test, * indicates P value < 0.01 when compared to the relevant <i>hctA</i> control and ** indicates P value < 0.01 when compared to <i>ihtA/hctA</i> control.</p

iIhtA does not interact with the Shine-Dalgarno site.

<p>(A) Schematic of the UTR of pTet compared to the 5’ UTR, starting at the TSS of <i>C</i>. <i>trachomatis</i> serovar L2. The sequences in common are boxed and the predicted serovar L2 SD site is underlined. (B) Schematic of mutations made in the anti-SD of IhtA. (C) Wild type <i>hctA</i> was co-transformed with <i>ihtA</i> mutants #1–7 and assayed for growth upon induction of HctA expression. Co-transformation with pLac and wt <i>ihtA</i> served as controls. Statistical analysis was performed using t-test, * indicates P value < 0.01 when compared to the <i>ihtA</i>/<i>hctA</i> control (D) sRNA structure and base pair probabilities (color coded 0–1) predicted by the <i>RNA</i>fold web server of IhtA#3 and its intramolecular mutant IhtA#8. The * indicates mutated nucleotide/s. (E) Cell viability of strains co-transformed with intramolecular mutant <i>ihtA</i>#8 and <i>hctA</i>. Co-transformation of <i>hctA</i> with pLac, <i>ihtA</i> or <i>ihtA</i>#3 served as controls. * indicates P value < 0.01 when compared to <i>hctA</i> control and ** indicates P value < 0.01 when compared to <i>ihtA/hctA</i>. For graphs C and E, cell viability was expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. Bars represent the mean ± SEM of all experiments combined.</p