Abstract
The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen Chlamydia trachomatis 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 hctA, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of hctA. Two additional genes in the chlamydial genome, CTL0322 and CTL0097, contained some elements of the hctA:IhtA recognition sequence. The mRNA of both CTL0322and CTL0097 interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of CTL0322. The proposed IhtA recognition site in the CTL0322 message contains significant G/C base pairing on either side of the initiation codon while CTL0097 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.
Highlights
Chlamydiaceae are obligate intracellular bacterial pathogens which, in a species dependent manner, infect epithelial cells of both human and animals resulting in a wide range of diseases
IhtA interacts with conserved sequences of hctA mRNA
As the small regulatory RNA (sRNA) IhtA is expressed and functional in the aforementioned species, and each species IhtA is capable of suppressing the translation of serovar L2 hctA [16], we hypothesized that IhtA interaction with hctA was limited to the conserved nucleotides of hctA
Summary
Chlamydiaceae are obligate intracellular bacterial pathogens which, in a species dependent manner, infect epithelial cells of both human and animals resulting in a wide range of diseases. Chlamydia are characterized by a tightly regulated developmental cycle which begins with the infection of the host cell by the spore-like elementary body (EB). HctA is expressed late in the developmental cycle, concurrent with RB to EB conversion and has been shown to shutdown transcription and translation by modulating genome topology and/or strongly binding to DNA or RNA [6,10,11,12,13,14]. IhtA interacts directly with the hctA mRNA and represses translation of the protein until the RB to EB transition point, at which time IhtA is down-regulated and HctA is expressed [15,16]. Repression of hctA translation by IhtA, which is itself regulated by an unknown mechanism, represents a critical "control point" in RB to EB differentiation
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