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Euo is a developmental regulator that represses late genes and activates midcycle genes in C. trachomatis

ABSTRACT: The pathogenic bacterium Chlamydia reproduces via an unusual intracellular developmental cycle in which it converts from a dividing form (reticulate body or RB) to an infectious form (elementary body or EB). The transcription factor Euo has been proposed as a developmental regulator in C. trachomatis because it repressed a number of late chlamydial promoters, which are transcribed during RB-to-EB conversion. To define the Euo regulon, we performed a genome-wide study that combined Euo DNA Immunoprecipitation-seq (DIP-seq) studies with RNA-seq analysis of HeLa cells infected with an Euo-overexpressing C. trachomatis strain. We demonstrate that Euo directly regulates ~7% of C. trachomatis genes. However, only about half were downregulated (28/61; 45.9%) by Euo overexpression while paradoxically the other half were upregulated (33/61; 54.1%). Intriguingly, all downregulated genes were late genes, while the majority of upregulated genes were midcycle genes, which are transcribed during RB replication. DIP analysis showed that Euo occupancy sites were restricted to the core promoter region for downregulated genes, but were located over a wider region immediately upstream of the promoter for upregulated genes. We also found that Euo controls its own expression through a negative feedback mechanism. Electron microscopy analysis of cells infected with the Euo-overexpressing strain showed fewer EBs, consistent with a block in RB-to-EB conversion, as well as fewer and larger RBs. Together, these findings broaden the role of Euo as a developmental regulator that functions as both a transcriptional repressor of late genes and a transcriptional activator of midcycle genes in C. trachomatis.

ORGANISM(S): Chlamydia trachomatis

PROVIDER: GSE202416 | GEO | 2023/06/21

REPOSITORIES: GEO

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Project description:The bacteria in the chlamydiales order are obligate intracellular parasites of eukaryotic cells. They are reliant on an infectious cycle consisting of at least three phenotypically distinct cell forms termed the reticulate body (RB), the intermediate body (IB) and the elementary body (EB). The EB is infectious but does not replicate. The RB replicates in the host cell but is non-infectious, while the IB is an intermediate form that transitions to the EB form. Within this order, the genus Chlamydia contains the causative agents of a number of important pathogens of humans. C. psittaci causes zoonotic infections resulting in pneumonia, while C. pneumoniae is a human pathogen that causes respiratory disease and is linked to atherosclerosis. Biovars of C. trachomatis are the causative agents of trachoma, the leading cause of preventable blindness worldwide, as well as sexually transmitted infections with the potential to cause pelvic inflammatory disease and infertility. Irrespective of the resulting disease, all chlamydial species share the same obligate intracellular life cycle and developmental cell forms. In this study we investigated the role of four transcriptional regulators on chlamydial gene expression and completion of the developmental cycle. We ectopically expressed the transcriptional repressor Euo, each of the two nucleoid associated proteins HctA and HctB and the two component sensor kinase CtcB in the RB. All four blocked development of infectious EBs. Transcriptional analysis using RNA-seq and differential expression clustering revealed three classes of gene expression. FISH analysis demonstrated that these classes corresponded to distinct cell forms revealing the gene expression profile of the RB, IB and EB developmental forms. Additionally, these data revealed that the genes for the T3SS were cell type restricted. The structural genes for the T3SS were expressed primarily in the IB while the two translocons were expressed in distinct cell types one in the RB and the other in the EB. Overall these data show that the chlamydial developmental cycle produces three distinct regulons corresponding to the RB, IB and EB cell forms and that the T3SS is expressed in a cell form specific manner suggesting defined functional roles for the T3SS in specific cell forms.

Project description:The obligate intracellular, gram-negative bacterium, Chlamydophila pneumoniae (Cpn), has impact as an acute and chronic disease-causing pathogen. Little is known about changes in the Cpn transcriptome during its biphasic developmental cycle (the acute infection) and persistence stages that are linked to chronic diseases. To analyze Cpn CWL029 gene expression, we designed a pathogen-specific oligo microarray and optimized the extraction method for pathogen RNA. Throughout the acute infection, ratio expression profiles for each gene were generated using 48 hours post infection as a reference. Based on these profiles, significantly expressed genes were separated into 12 expression clusters using SOM clustering and manually into the ‘early’, ‘mid’, ‘late’ and ‘tardy’ cluster classes. The latter two were differentiated because the ‘tardy’ class showed steadily increasing expression at the end of the cycle. The transcriptome of the Cpn elementary body (EB) and published EB proteomics data were compared to the cluster profile of the acute infection. We found an intriguing association between ‘late’ genes and genes coding for EB proteins, whereas ‘tardy’ genes were mainly associated with genes coding for EB mRNA. It has been published that iron depletion leads to Cpn persistence. We compared the gene expression profiles during iron depletion-mediated persistence with the expression clusters of the acute infection. This led to the finding that establishment of iron depletion-mediated persistence is more likely a mid-cycle arrest in development rather than a completely distinct gene expression pattern. Taken together, here we describe the Cpn transcriptome during the acute infection with ‘late’ genes correlating to EB proteins and ‘tardy’ genes to EB mRNA. Expression profiles during iron mediated-persistence led us to propose the hypothesis that the transcriptomic ‘clock’ is arrested during acute mid-cycle. Keywords: time course, iron, persistence, Chlamydia, Chlamydophila, pneumoniae, recovery, pathogen, life cycle, developmental cycle

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Euo is a developmental regulator that represses late genes and activates midcycle genes in C. trachomatis

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