The tryptophan salvage pathway is dynamically regulated by the iron-dependent repressor YtgR in Chlamydia trachomatis

24 Mammalian hosts restrict cellular nutrient availability to starve invading pathogens and 25 successfully clear an infection by a process termed “nutritional immunity”. For the 26 obligate intracellular pathogen Chlamydia trachomatis, nutritional immunity likely 27 encompasses the simultaneous limitation of the amino acid tryptophan and the essential 28 biometal iron. Unlike other model bacteria, C. trachomatis lacks many global stress 29 response systems – such as “stringent response” homologs – adapted to these host 30 insults. However, a physiological response by Chlamydia that is common to both 31 stresses is the development of an aberrant, “persistent” state, suggesting that 32 tryptophan and iron starvation trigger a coordinated developmental program. Here, we 33 report that the trpRBA operon for tryptophan salvage in C. trachomatis serovar L2 is 34 regulated at the transcriptional level by iron. The expression of the tryptophan synthase 35 encoding genes, trpBA, is induced following iron limitation while that of the repressor 36 trpR is not. We show that this specific induction of trpBA expression initiates from a 37 novel promoter element within an intergenic region flanked by trpR and trpB. YtgR, a 38 DtxR-homolog and the only known iron-dependent transcriptional regulator in 39 Chlamydia, can bind to the trpRBA intergenic region upstream of the alternative trpBA 40 promoter to repress transcription. This binding also likely attenuates transcription from 41 the primary promoter upstream of trpR by blocking RNA polymerase read-through. 42 These data illustrate a dynamic and integrated method of regulating tryptophan 43 biosynthesis in an iron-dependent manner, which has not been described in any other 44 prokaryote, underscoring the uniqueness of Chlamydia. 45

historically implicated Trp starvation as the primary mechanism by which persistence 2 7 6 develops in C. trachomatis following exposure to IFN-γ. However, these studies have 2 7 7 routinely depended on prolonged treatment conditions that monitor the terminal effect of 2 7 8 persistent development, as opposed to the immediate molecular events which may 2 7 9 have important roles in the developmental fate of Chlamydia. As such, these studies 2 8 0 may have missed the contribution of other IFN-γ-stimulated insults such as iron 2 8 1 limitation.

8 2
To decouple Trp limitation from iron limitation and assess their relative 2 8 3 contribution to regulating a critical pathway for responding to IFN-γ-mediated stress, we 2 8 4 monitored the transcript expression of the trpRBA operon under brief Trp or iron 2 8 5 starvation by RT-qPCR. When starved for Trp for 6 hrs, we observed that the 2 8 6 expression of trpR, trpB and trpA were all significantly induced greater than 10.5-fold 2 8 7 relative to 12 hpi (p = 0.00077, 0.025 and 9.7e-5, respectively; Fig. 3B). All three ORFs 2 8 8 were also significantly elevated relative to the equivalent mock-treated time-point (p = 2 8 9 0.00076, 0.025 and 9.7e-5, respectively). This result was surprising with respect to the 2 9 0 relative immediacy of operon induction in response to our Trp starvation protocol, 2 9 1 confirming the relevant Trp-starved transcriptional phenotype. To induce Trp-deprived 2 9 2 persistence in C. trachomatis, many laboratories rely on compounded techniques of 2 9 3 IFN-γ pre-treatment to deplete host Trp pools in conjunction with culturing in Trp-2 9 4 depleted media, among other strategies. While the phenotypic end-point differs here, it 2 9 5 is nonetheless interesting to note that only 6 hrs of media replacement is sufficient to 2 9 6 markedly up-regulate trpRBA expression. This suggests that C. trachomatis has a 2 9 7 highly attuned sensitivity to even moderate changes in Trp levels.

9 8
We next performed the same RT-qPCR analysis on the expression of the trpRBA 2 9 9 operon in response to 6 hrs of iron limitation via Bpdl treatment (Fig. 3C). While we 3 0 0 observed that the transcript expression of all three ORFs was significantly elevated at 3 0 1 least 2.1-fold relative to the equivalent mock-treated time-point (p = 0.015, 0.00098 and 3 0 2 0.0062, respectively), we made the intriguing observation that only the expression trpB 3 0 3 and trpA was significantly induced relative to 12 hpi (p = 0.00383 and 0.0195, 3 0 4 respectively). The significant induction of trpBA expression, but not trpR expression, 3 0 5 suggested that trpBA are specifically regulated by iron availability. This result is 3 0 6 consistent with a recent survey of the iron-regulated transcriptome in C. trachomatis by 3 0 7 RNA sequencing, which also reported that iron-starved Chlamydia specifically up-3 0 8 regulate trpBA expression in the absence of altered trpR expression (37). Our results 3 0 9 expand on this finding by providing a more detailed investigation into the specific profile 3 1 0 of this differential regulation of trpRBA in response to iron deprivation. Taken together, 3 1 1 these findings demonstrated that an important stress response pathway, the trpRBA 3 1 2 operon, is regulated by the availability of both Trp and iron, consistent with the notion 3 1 3 that the pathway may be cooperatively regulated to respond to various stress 3 1 4 conditions. Notably, iron-dependent regulation of Trp biosynthesis has not been 3 1 5 previously documented in other prokaryotes.

1 6
Specific iron-regulated expression of trpBA originates from a novel alternative 3 1 7 transcriptional start site within the trpRBA intergenic region. We hypothesized that 3 1 8 the specific iron-related induction of trpBA expression relative to trpR expression may 3 1 9 be attributable to an iron-regulated alternative transcriptional start site (alt. TSS) 3 2 0 downstream of the trpR ORF. Indeed, a previous study reported the presence of an alt. 3 2 1 TSS in the trpRBA IGR, located 214 nucleotides upstream of the trpB translation start 3 2 2 position (20). However, a parallel study could not identify a TrpR binding site in the 3 2 3 trpRBA IGR (21). We reasoned that a similar alt. TSS may exist in the IGR that 3 2 4 controlled the iron-dependent expression of trpBA. We therefore performed Rapid 3 2 5 Amplification of 5'-cDNA Ends (5'-RACE) on RNA isolated from C. trachomatis L2-3 2 6 infected HeLa cells using the SMARTer 5'/3' RACE Kit workflow (Takara Bio). Given the 3 2 7 low expression of the trpRBA operon during normal development, we utilized two 3 2 8 sequential gene-specific amplification steps (nested 5'-RACE) to identify 5' cDNA ends 3 2 9 in the trpRBA operon. These nested RACE conditions resulted in amplification that was 3 3 0 specific to infected-cells (Fig. S3A). Using this approach, we analyzed four conditions: 3 3 1 12 hpi, 18 hpi, 12 hpi + 6 hrs of Bpdl treatment, and 12 hpi + 6 hrs of Trp-depletion ( Fig.  3 3 2 4A). We observed three RACE products that migrated with an apparent size of 1.5, 1.1 3 3 3 and 1.0 kilobases (kb). At 12 and 18 hpi, all three RACE products exhibited low 3 3 4 abundance, even following the nested PCR amplification. This observation was 3 3 5 consistent with the expectation that the expression of the trpRBA operon is very low 3 3 6 under normal, iron and Trp-replete conditions. However, we note that the 6-hr difference 3 3 7 in development did appear to alter the representation of the 5' cDNA ends, which may 3 3 8 suggest a stage-specific promoter utilization within the trpRBA operon. In our Trp 3 3 9 starvation condition, we observed an apparent increase in the abundance of the 1.5 kb 3 4 0 RACE product, which was therefore presumed to represent the primary TSS upstream 3 4 1 of trpR, at nucleotide position 511,389 (C. trachomatis L2 434/Bu). Interestingly, the 1.0 3 4 2 kb product displayed a very similar apparent enrichment following Bpdl treatment, 3 4 3 suggesting that this RACE product represented a specifically iron-regulated TSS. Both 3 4 4 the 1.5 and 1.0 kb RACE products were detectable in the Trp-depleted and iron-3 4 5 depleted conditions, respectively, during the primary RACE amplification, consistent 3 4 6 with their induction under these conditions (Fig. S3B).

4 7
If iron depletion was inducing trpBA expression independent of trpR, we 3 4 8 reasoned that we would observe specific enrichment of trpB sequences in our 5'-RACE 3 4 9 0 reports on YtgR repressor activity as a function of β-galactosidase expression (14). In 4 0 1 brief, a candidate DNA promoter element was cloned into the pCCT expression vector 4 0 2 between an arabinose-inducible pBAD promoter and the reporter gene lacZ. This  Fig. 5B). As expected, from the pCCT-EV reporter construct, ectopic YtgR 4 1 2 expression did not significantly reduce the activity of β-galactosidase. Additionally, 4 1 3 reporter gene expression from pCCT-lpdA, containing the promoter of iron-regulated 4 1 4 lpdA (37), which is not known to be YtgR-regulated, was not affected by ectopic 4 1 5 expression of YtgR. This demonstrated that the assay can discriminate between the 4 1 6 promoter elements of iron-regulated genes and bona fide YtgR targeted promoters. 4 1 7 Indeed, in the presence of pCCT-ytgABCD, induction of YtgR expression produced a 4 1 8 significant decrease in β-galactosidase activity (p = 0.03868) consistent with its 4 1 9 previously reported auto-regulation of this promoter (34). 4 2 0 Using this same assay, we then inserted into the pCCT reporter plasmid 1) the 4 2 1 trpR promoter element (pCCT-trpR), 2) the putative trpBA promoter element pCCT-trpR background, we observed no statistically distinguishable change in β-4 2 7 galactosidase activity. However, in the pCCT-trpBA background, ectopic YtgR 4 2 8 expression significantly reduced β-galactosidase activity at levels similar to those 4 2 9 observed in the pCCT-ytgABCD background (p = 0.01219). This suggested that YtgR 4 3 0 was capable of binding to the trpBA promoter element specifically. Interestingly, this 4 3 1 repression phenotype was abrogated in the pCCT-trpBAΔOperator background, where 4 3 2 we observed no statistically meaningful difference in β-galactosidase activity. We 4 3 3 subsequently addressed whether the region of the trpRBA IGR containing the YtgR 4 3 4 operator site was sufficient to confer YtgR repression in this assay (Fig. S5). Therefore, 4 3 5 we cloned three fragments of the trpRBA IGR into the pCCT reporter plasmid: the first 4 3 6 fragment represented the 5'-end of the IGR containing the operator site at the 3'-end 4 3 7 (pCCT-IGR1), the second fragment represented a central region of the IGR containing 4 3 8 the operator site at the 5'-end (pCCT-IGR2), and the third fragment represented the 3'-4 3 9 end of the IGR and did not contain the operator site (pCCT-IGR3). Surprisingly, we 4 4 0 observed that none of these fragments alone were capable of producing a significant 4 4 1 repression phenotype in our reporter system. This finding indicated that while the 4 4 2 operator site was necessary for YtgR repression, it alone was not sufficient. Together, 4 4 3 these data indicated that YtgR could bind to the trpBA promoter element and that this 4 4 4 binding was dependent upon an intact AT-rich palindromic sequence, likely representing 4 4 5 an YtgR operator, but that further structural elements in the trpRBA IGR may be 4 4 6 necessary for repression. Nonetheless, we demonstrated the existence of a functional 4 4 7 YtgR binding site that conferred iron-dependent transcriptional regulation to trpBA, 4 4 8 independent of the major trpR promoter. RNAP initiating transcription at the upstream trpR promoter. Similar systems of RNAP 4 5 5 read-through blockage have been reported; the transcription factor Reb1p "roadblocks" 4 5 6 RNAPII transcription read-through in yeast by a mechanism of RNAP pausing and 4 5 7 subsequent labelling for degradation (44). To investigate this question, we first identified 4 5 8 transcription termination sites (TTSs) in the trpRBA operon in C. trachomatis. We 4 5 9 utilized 3'-RACE to map the 3'-ends of transcripts using gene-specific primers within the 4 6 0 trpR CDS (Fig. 6A; bottom). We again utilized two RACE amplification cycles to 4 6 1 generate distinct, specific bands suitable for isolation and sequencing ( Fig. S6B-C). By 4 6 2 gel electrophoresis of the 3'-RACE products, we observed the appearance of four 4 6 3 distinct bands that migrated with an apparent size of 0.55, 0.45, 0.40 and 0.20 kb. In our 4 6 4 Trp-depleted condition, we observed only a very weak amplification of the 2.5 -3 kb 4 6 5 full-length trpRBA message by 3'-RACE (Fig. S6A). However, we did observe it across 4 6 6 all replicates. To confirm that the full-length product was relatively specific to the Trp-4 6 7 deplete treatment, we amplified the trpRBA operon by RT-PCR from the 3'-RACE cDNA 4 6 8 ( Fig. 6A; top). As expected, only in the Trp-deplete sample did we observe robust 4 6 9 amplification of the full-length trpRBA message. We note however that image contrast 4 7 0 adjustment reveals a very weak band present in all experimental samples.

7 1
To identify the specific TTS locations, we gel extracted the four distinct 3'-RACE 4 7 2 bands across all conditions and cloned them into the pRACE sequencing vector as was 4 7 3 done for the 5'-RACE experiments. We then sequenced the inserted RACE products 4 7 4 and mapped them to the C. trachomatis L2 434/Bu genome (Fig. 6B). This revealed a 4 7 5 highly dynamic TTS landscape contained almost exclusively within the trpRBA IGR, 4 7 6 which has not previously been investigated (For a full description of mapped 3'-RACE 4 7 7 products, see Dataset S2). The 0.20 kb RACE product mapped tightly to the 3'-end of 4 7 8 the trpR CDS, with a mean nucleotide position of 511,665 and a modal nucleotide 4 7 9 position of 511,667 (Fig. S7A). Contrastingly, the other three RACE products did not 4 8 0 map in such a way so as to produce specific, unambiguous modal peaks. Instead, their 4 8 1 distribution was broader and more even, with only a few nucleotide positions mapping 4 8 2 more than once. Accordingly, the 0.45 kb product mapped with an average nucleotide 4 8 3 position of 511,889, just downstream of the 1.1 kb 5'-RACE product (Fig. S7C), while 4 8 4 the 0.55 kb product mapped with an average nucleotide position of 511,986, upstream 4 8 5 of the 1.0 kb 5'-RACE product (Fig. S7D). Interestingly, the 0.40 kb product mapped to 4 8 6 a region directly overlapping the putative YtgR operator site, with a mean nucleotide 4 8 7 position of 511,811 (Fig. S7B). We therefore reasoned that this putative TTS may have 4 8 8 an iron-dependent function. 4 8 9 We next aimed to quantitatively analyze the possibility that iron-depletion, and 4 9 0 thus dissociation of YtgR from this region, may facilitate transcription read-through at 4 9 1 the operator site. Working from the 3'-RACE generated cDNAs, we utilized RT-qPCR to 4 9 2 monitor the abundance of various amplicons across the trpRBA operon in relation to a 4 9 3 "read-through" normalization amplicon that should only be represented when the full-4 9 4 length trpRBA message is transcribed (Fig. 6C). Therefore, as each amplicon is 4 9 5 increasingly represented as a portion of the full-length, read-through transcript, the 4 9 6 representation ratio of the specific amplicon to the normalization amplicon should 4 9 7 approach 1.0. We first analyzed an amplicon from nucleotide 511,416 -531 to monitor 4 9 8 transcript species associated with transcription initiating at the trpR promoter. We 4 9 9 observed that the representation of this amplicon was not significantly altered following 5 0 0 iron limitation relative to 12 hpi, suggesting that the depletion of iron was not affecting 5 0 1 initiation of transcription at the trpR promoter. Interestingly, at 18 hpi, the representation 5 0 2 ratio of this amplicon significantly shifted further away from 1.0 (p = 0.00358), indicating 5 0 3 that at 18 hpi this amplicon is represented less as a component of read-through 5 0 4