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

IhtA sequence is conserved across species.

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

IhtA sequence is conserved across species.

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

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

Translation Inhibition of the Developmental Cycle Protein HctA by the Small RNA IhtA Is Conserved across <em>Chlamydia</em>

<div><p>The developmental cycle of the obligate intracellular pathogen <em>Chlamydia trachomatis</em> serovar L2 is controlled in part by the small non-coding RNA (sRNA), IhtA. All <em>Chlamydia</em> 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 <em>hctA</em> 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 <em>C. trachomatis</em> serovars L2, D and L2b and <em>C. pneumoniae</em>. We show in this work that IhtA is also expressed by the animal pathogens <em>C. caviae</em> and <em>C. muridarum</em>. Expression of HctA in <em>E. coli</em> 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 <em>hctA</em> and <em>ihtA</em> from <em>C. trachomatis</em> serovar D, <em>C. muridarum, C. caviae</em> and <em>C. pneumoniae</em> and assayed for rescue of growth repression in <em>E. coli</em> co-expression studies. In each case, co-expression of <em>ihtA</em> with the cognate <em>hctA</em> resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of <em>C. trachomatis</em> serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with <em>hctA</em> message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.</p> </div

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

Repression of HctA translation by IhtA is a conserved mechanism.

<p>(A) Northern analysis of expression of species IhtA in <i>E. coli</i>. The <i>ihtA</i> gene, including 5′UTR was PCR amplified from the genomes of the indicated chlamydial species, cloned into pLac and expressed in DH5alphaPRO <i>E. coli.</i> sRNA was isolated from overnight cultures using the <i>mir</i>Vana 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 <i>ihtA</i> PCR fragment. (B) DRAQ5 DNA staining of <i>E. coli</i> 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 <i>E. coli</i> expressing either species specific <i>hctA</i> alone (+pLac, light grey bars) or species specific <i>hctA</i> and IhtA (+cognate <i>ihtA</i>, 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.</p

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

Serovar L2 HctA expression is repressed by species IhtA.

<p>A) Growth viability of <i>E. coli</i> co-expressing serovar L2 HctA and <i>C. trachomatis</i> serovar D, <i>C. muridarum, C. caviae</i>, or <i>C. pneumoniae</i> IhtA. <i>E. coli</i> co-expressing <i>hctA</i> 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 <i>E. coli</i> co-expressing species <i>hctA</i> and <i>C. trachomatis</i> 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.</p