Expression activation of over 70% of Chlamydia trachomatis genes during the first hour of infection.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance.

IMPORTANCE: Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance.

Chlamydia, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia-specific transcriptional regulator GrgA, previously shown to activate σ66 and σ28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression (DOPE) to create Chlamydia trachomatis with conditional GrgA-deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fail to maintain its virulence plasmid. Results of RNA-seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of σ54, the third and last sigma factor, and its activator AtoC, and thereby indirectly upregulating the expression of σ54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity.