Identification of the alternative sigma factor regulons of Chlamydia trachomatis using multiplexed CRISPR interference

Abstract

Chlamydia trachomatis is a developmentally regulated, obligate intracellular bacterium that encodes three sigma factors: σ66, σ54, and σ28. σ66 is the major sigma factor controlling most transcription initiation during early- and mid-cycle development as the infectious elementary body (EB) transitions to the non-infectious reticulate body (RB) that replicates within an inclusion inside the cell. The roles of the minor sigma factors, σ54 and σ28, have not been well characterized to date; however, there are data to suggest each functions in late-stage development and secondary differentiation as RBs transition to EBs. As the process of secondary differentiation itself is poorly characterized, clarifying the function of these alternative sigma factors by identifying the genes regulated by them will further our understanding of chlamydial differentiation. We hypothesize that σ54 and σ28 have non-redundant and essential functions for initiating late gene transcription thus mediating secondary differentiation in Chlamydia. Here, we demonstrate the necessity of each minor sigma factor in successfully completing the developmental cycle. We have implemented and validated multiplexed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference techniques, novel to the chlamydial field to examine the effects of knocking down each alternative sigma factor individually and simultaneously. In parallel, we also overexpressed each sigma factor. Altering transcript levels for either or both alternative sigma factors resulted in a severe defect in EB production as compared to controls. Furthermore, RNA sequencing identified differentially expressed genes during alternative sigma factor dysregulation, indicating the putative regulons of each. These data demonstrate that the levels of alternative sigma factors must be carefully regulated to facilitate chlamydial growth and differentiation. IMPORTANCE Chlamydia trachomatis is a significant human pathogen in both developed and developing nations. Due to the organism's unique developmental cycle and intracellular niche, basic research has been slow and arduous. However, recent advances in chlamydial genetics have allowed the field to make significant progress in experimentally interrogating the basic physiology of Chlamydia. Broadly speaking, the driving factors of chlamydial development are poorly understood, particularly regarding how the later stages of development are regulated. Here, we employ a novel genetic tool for use in Chlamydia while investigating the effects of dysregulating the two alternative sigma factors in the organism that help control transcription initiation. We provide further evidence for both sigma factors' essential roles in late-stage development and their potential regulons, laying the foundation for deeper experimentation to uncover the molecular pathways involved in chlamydial differentiation.

Keywords: CRISPRi; Chlamydia; development; differentiation; gene regulation; sigma factor.

Fig 1

Validation of chlamydial sigma factor knockdown and overexpression strains via RT-qPCR. (A) Knockdown strains (x-axis) were induced or not with 10 nM aTc at 4 hpi. Samples were collected at 24 hpi and processed as described in the Materials and Methods. Uninduced ratios of cDNA:16S are set to 1, and induced samples’ cDNA:16S ratios were normalized as a relative proportion of the uninduced value. NT: non-targeting. (B) Overexpression strains (x-axis) were induced or not with 10 nM aTc at 14 hpi and collected at 18 hpi. All samples include three biological replicates. *P < 0.05 via ratio paired t-test.

Fig 2

Sigma factor dysregulation is detrimental to chlamydial developmental cycle completion. IFU assays were performed to assess the impact of sigma factor knockdown (A) or overexpression (B) on the ability to produce infectious progeny. Strains were induced or not with 10 nM aTc at 4 hpi (A) or 14 hpi (B) and collected at 24 hpi for reinfection and enumeration. Values are expressed as a ratio of EBs extracted from induced samples and their respective uninduced counterparts. NT: non-targeting and LD: below limit of detection. All samples include three biological replicates. *P < 0.05 via ratio paired t-test. (C and D) IFA was performed to assess inclusion size and organism morphology using the same induction conditions described in (A) and (B). At 24 hpi, cells were fixed with methanol and stained with a primary antibody against the C. trachomatis major outer membrane protein. Representative images of three biological replicates are shown. All images were acquired on an Axio Imager.Z2 with ApoTome.2 at 100× magnification. Scale bars represent 2 µm.

Fig 3

(Top) All significant hits observed during σ28 KD are listed alongside the fold changes observed during OE. “Name” includes the annotated name under the accession file used for annotation. Other common names may be given in parentheses. Y: Yu et al. found these to be regulated by σ28 in vitro. (Bottom) Promoter regions of tsp and hctB are shown as described by Yu et al. Identical nucleotides in the proposed −35 and −10 boxes are bolded. A ct(c)cct motif of unknown significance is underlined.

Fig 4

Model of a hypothetical σ28 regulation cascade. (Left) σ28 activity is inhibited during mid-cycle development by an unidentified anti-sigma factor. During the transition to late cycle, stimulation of the AtoS/C signaling cascade allows σ54 to begin transcribing genes within its regulon. (Right) Direct and indirect products of σ54 culminate in the modulation of existing T3SS apparatus and translation of an unidentified chaperone acting as an anti-anti-sigma factor, thereby releasing σ28 and guiding the anti-sigma factor to be secreted. The liberated σ28 is now free to associate with RNAP and begin transcribing the genes within its regulon.