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Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in underdeveloped areas as well as some developed countries. Chlamydia carries genes that encode a limited number of known transcription factors. While Euo is thought to be critical for early chlamydial development, the functions of GrgA and HrcA in the developmental cycle are unclear.

Chlamydiae are common, important pathogens for humans and animals alike. Despite recent advancement in genetics, scientists are still searching for efficient tools to knock out or knock down the expression of chromosomal genes. We attempted to adopt a dCas9-based CRISPR interference (CRISPRi) technology to conditionally knock down gene expression in Chlamydia trachomatis using an anhydrotetracycline (ATC)-inducible expression system. Surprisingly, expression of the commonly used Streptococcus pyogenes dCas9 in C. trachomatis causes strong inhibition in the absence of any guide RNA (gRNA). Staphylococcus aureus dCas9 also shows strong toxicity in the presence of only an empty gRNA scaffold. Toxicity of the S. pyogenes dCas9 is readily observed with as little as 0.2 nM ATC. Growth inhibition by S. aureus dCas9 is evident starting at 1.0 nM ATC. In contrast, C. trachomatis growth was not affected by methionine-tRNA ligase overexpression induced with 10 nM ATC. We conclude that S. pyogenes and S. aureus dCas9 proteins in their current forms have limited utility for chlamydial research and suggest strategies to overcome this problem.

Cells reprogram their transcriptome in response to stress, such as heat shock. In free-living bacteria, the transcriptomic reprogramming is mediated by increased DNA-binding activity of heat shock sigma factors and activation of genes normally repressed by heat-induced transcription factors. In this study, we performed transcriptomic analyses to investigate heat shock response in the obligate intracellular bacterium Chlamydia trachomatis, whose genome encodes only three sigma factors and a single heat-induced transcription factor. Nearly one-third of C. trachomatis genes showed statistically significant (≥1.5-fold) expression changes 30 min after shifting from 37 to 45°C. Notably, chromosomal genes encoding chaperones, energy metabolism enzymes, type III secretion proteins, as well as most plasmid-encoded genes, were differentially upregulated. In contrast, genes with functions in protein synthesis were disproportionately downregulated. These findings suggest that facilitating protein folding, increasing energy production, manipulating host activities, upregulating plasmid-encoded gene expression, and decreasing general protein synthesis helps facilitate C. trachomatis survival under stress. In addition to relieving negative regulation by the heat-inducible transcriptional repressor HrcA, heat shock upregulated the chlamydial primary sigma factor σ66 and an alternative sigma factor σ28. Interestingly, we show for the first time that heat shock downregulates the other alternative sigma factor σ54 in a bacterium. Downregulation of σ54 was accompanied by increased expression of the σ54 RNA polymerase activator AtoC, thus suggesting a unique regulatory mechanism for reestablishing normal expression of select σ54 target genes. Taken together, our findings reveal that C. trachomatis utilizes multiple novel survival strategies to cope with environmental stress and even to replicate. Future strategies that can specifically target and disrupt Chlamydia’s heat shock response will likely be of therapeutic value.

The obligate intracellular bacterial pathogen has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary sigma factor, σ, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor σ In prior work, we identified GrgA as a specific transcription factor that activates σ-dependent transcription by binding DNA and interacting with a non-conserved region (NCR) of σ Here, we extend these findings by showing GrgA can also activate σ-dependent transcription through direct interaction with σ We measure the binding affinity of GrgA for both σ and σ, and we identify regions of GrgA important for σ-dependent transcription. Similar to results obtained with σ, we find that GrgA's interaction with σ involves a NCR located upstream of conserved region 2 of σ Our findings suggest GrgA is an important regulator of both σ- and σ-dependent transcription in and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms. is the number one sexually transmitted bacterial pathogen worldwide. A substantial proportion of -infected women develop infertility, pelvic inflammatory syndrome and other serious complications. is also a leading infectious cause of blindness in under-developed countries. The pathogen has a unique developmental cycle, which is transcriptionally regulated. The discovery of an expanded role for the specific transcription factor GrgA helps understand progression of the chlamydial developmental cycle.

The sexually transmitted obligate intracellular bacterial pathogen Chlamydia trachomatis has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary Chlamydia sigma factor, σ66, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor σ28. In prior work we identified GrgA as a Chlamydia-specific transcription factor that activates σ66-dependent transcription by binding DNA and interacting with a non-conserved region (NCR) of σ66. Here, we extend these findings by showing GrgA can also activate σ28-dependent transcription through direct interaction with σ28. We measure the binding affinity of GrgA for both σ66and σ28, and we identify regions of GrgA important for σ28-dependent transcription. Similar to results obtained with σ66, we find that GrgA’s interaction with σ28 involves a NCR located upstream of conserved region 2 of σ28. Our findings suggest GrgA is an important regulator of both σ66- and σ28-dependent transcription in C. trachomatis and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.

ABSTRACTThe obligate intracellular bacterium Chlamydia trachomatis is an important human pathogen whose biphasic developmental cycle consists of an infectious elementary body and a replicative reticulate body. Whereas σ66, the primary sigma factor, is necessary for transcription of most chlamydial genes throughout the developmental cycle, σ28 is required for expression of some late genes. We previously showed that the Chlamydia-specific transcription factor GrgA physically interacts with both of these sigma factors and activates transcription from σ66- and σ28-dependent promoters in vitro. Here, we investigate the organismal functions of GrgA. We show that GrgA overexpression decreased RB proliferation via time-dependent transcriptomic changes. Significantly, σ66-dependent genes that code for two important transcription repressors are among the direct targets of GrgA. One of these repressors is Euo, which prevents the expression of late genes during early phases. The other is HrcA, which regulates gene expression in response to heat shock. The direct regulon of GrgA also includes a σ28-dependent gene that codes for the putative virulence factor PmpI. Conditional overexpression of Euo and HrcA also inhibited chlamydial growth and affected GrgA expression. Transcriptomic studies suggest that GrgA, Euo, and HrcA have distinct but overlapping indirect regulons. Furthermore, overexpression of either GrgA leads to decreased expression of numerous tRNAs. These findings indicate that a GrgA-mediated transcriptional regulatory network controls C. trachomatis growth and development.IMPORTANCEChlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in under-developed areas as well as developed countries. Previous studies showed that the novel transcription factor GrgA activated chlamydial gene transcription in vitro, but did not addressed the organismal function of GrgA. Here, we demonstrate growth inhibition in C. trachomatis engineered to conditionally overexpress GrgA. GrgA overexpression immediately increases the expression of two other critical transcription factors (Euo and HrcA) and a candidate virulence factor (PmpI), among several other genes. We also reveal chlamydial growth reduction and transcriptomic changes including decreased GrgA mRNA levels in response to either Euo or HrcA overexpression. Thus, the transcription network controlled by GrgA likely plays a crucial role in chlamydial growth and pathogenesis.

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