Journal: bioRxiv
Article Title: FLI1 localization to the chlamydial inclusion involves multiple mechanisms
doi: 10.1101/2023.10.17.562819
Figure Lengend Snippet: (A) Diagram of the ct227 operon. Triangles indicate the binding site of the indicated CRISPR RNA (crRNA) in the intergenic region upstream of the targeted gene. (B) RT-qPCR analysis of knockdown strains. HEp2 cells were seeded, infected with the indicated strains, and induced as described in Materials and Methods. RNA was collected and processed for RT-qPCR. cDNA transcript levels of genes were normalized to 16s transcripts and reported as the ratio of uninduced to induced at 12hpi. A paired student’s t-test was used to determine statistically significant differences in transcript levels between uninduced and induced conditions for each strain. (C) Diagram of vector design for complementation downstream of dCas12 in pBOMBL12CRia vector. (D) RT-qPCR analysis of complement strains. Samples were collected and analyzed as described above. (E) Infectious progeny production in knockdown strains. Infectious progeny were measured in both uninduced and induced inclusions and reported as inclusion forming units (IFU) per mL and a paired student’s t-test was used to determine statistical significance between uninduced and induced conditions for each strain. (F) Infectious progeny production in complementation strains. (G-I) Indirect immunofluorescence confirming expression and localization of the complemented 3XFLAG-tagged Inc for all three complement strains. HEp2 cells were seeded on glass coverslips, infected with appropriate complement strain and induced as described in the Materials and Methods. At 24hpi, the coverslips were processed for indirect immunofluorescence to visualize the 3XFLAG-tagged Inc (anti-FLAG; red) and host and chlamydial DNA (DAPI; blue). Constitutively expressed GFP was used to visualize the chlamydial organisms (GFP; green). (G) Ct226-3XFLAG is dimly visible at the inclusion under un-inducing conditions, indicating some leaky expression of the complement. Under induced conditions, Ct226-3XFLAG was secreted and localized as expected to the chlamydial inclusion membrane. (H) Complemented Ct224-3XFLAG is secreted and localizes to the inclusion membrane, as anticipated. (I) Ct225-3XFLAG notably localizes to the chlamydial organisms, as opposed to the inclusion membrane. (J) Quantification of inclusion area in complementation strains by ImageJ. An ordinary two-way ANOVA test was performed to identify statistically significant differences in inclusion area between the complement strains compared to the negative control strain and the L2/ ct226 KD strain. Neither E.V. strain nor L2/ ct226 KD strain demonstrated a difference in inclusion size between un-inducing and inducing conditions. Complementation of either Ct226 or Ct225 led to a 77% or 37% decrease in inclusion area, respectively, while complementation of Ct224 did not impact inclusion area. Scale bar= 2μm. Statistical significance was reported as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns= not significant.
Article Snippet: Primary antibodies used in these studies were polyclonal rabbit anti-LRRF1 (Bethyl Laboratories), sheep anti-IncA, rabbit anti-FLI1 (Thermo Fisher), goat anti-MOMP (Meridian), mouse anti-FLAG (Sigma-Aldrich), rabbit anti-FLAG (Sigma-Aldrich), mouse anti-Ct225-GST (gift from Guangming Zhong, University of Texas Health Sciences Center-San Antonio), rabbit anti-Ct226 (gift from Erika Lutter, Oklahoma State University), mouse anti-AsCpf1 (dCas12; Sigma-Aldrich).
Techniques: Binding Assay, CRISPR, Quantitative RT-PCR, Infection, Plasmid Preparation, Immunofluorescence, Expressing, Membrane, Negative Control
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