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Dual RNA-seq analysis of in vitro infection multiplicity and RNA depletion methods in Chlamydia-infected epithelial cells.

ABSTRACT: Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host-pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

SUBMITTER: Hayward RJ

PROVIDER: S-EPMC8128910 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Comparative analysis using different multiplicity of infection ratios from Chlamydia-infected epithelial cells

Project description:Here we examined gene expression from an in-vitro dual-RNA-seq experiment using Chlamydia trachomatis-infected HEp-2 epithelial cells. The experimental design consisted of three different multiplicity of infection ratio's (0.1, 1 and 10) across two different timeframes (1 and 24hrs), in addition to applying two different depletion methods (rRNA and rRNA + PolyA depletion). The aim was to examine and outline which MOI and depletion methods are most suited for bacterial-based infections.

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Comparison of Poly(A) capture versus Ribosomal RNA depletion methods for RNA-seq

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Chromatin accessibility dynamics of Chlamydia-infected epithelial cells.

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Chlamydial Hsp60-2 is iron responsive in Chlamydia trachomatis serovar E-infected human endometrial epithelial cells in vitro.

Project description:Chlamydial 60-kDa heat shock proteins (cHsp60s) are known to play a prominent role in the immunopathogenesis of disease. It is also known that several stress-inducing growth conditions, such as heat, iron deprivation, or exposure to gamma interferon, result in the development of persistent chlamydial forms that often exhibit enhanced expression of cHsp60. We have shown previously that the expression of cHsp60 is greatly enhanced in Chlamydia trachomatis serovar E propagated in an iron-deficient medium. The objective of this work was to determine which single cHsp60 or combination of the three cHsp60 homologs encoded by this organism responds to iron limitation. Using monospecific polyclonal peptide antisera that recognize only cHsp60-1, cHsp60-2, or cHsp60-3, we found that expression of cHsp60-2 is responsive to iron deprivation. Overall, our studies suggest that the expression of cHsp60 homologs differs among the mechanisms currently known to induce persistence.

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Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads.

Project description:A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.

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