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.

Host Cell Amplification of Nutritional Stress Contributes To Persistence in Chlamydia trachomatis

“…Our experiment was designed to capture the presumably small fraction of chlamydial transcripts produced in persistently infected HeLa cells, and we accordingly used a multiplicity of infection (MOI) of 5 for each condition. In agreement with recent reports, we recovered high levels of chlamydial transcripts in each library ( 25 ), with no fewer than 7.5 × 10 6 mapped reads under any condition. We also note that across replicates, no more than 0.01% of the total library mapped to the chlamydial reference genome in any mock-infected sample, indicating a negligible influence of cross-aligned reads in our samples.…”

Host cell amplification of nutritional stress contributes to persistence in Chlamydia trachomatis

“…Our experiment was designed to capture the presumably small fraction of chlamydial transcripts produced in persistently infected HeLa cells, and we accordingly used a multiplicity of infection (MOI) of five for each condition. In agreement with recent reports, we recovered high levels of chlamydial transcripts in each library (Hayward et al, 2021), with no fewer than 7.5 x 10 6 mapped reads under any condition. We also note that across replicates, no more than 0.01% of the total library mapped to the chlamydial reference genome in any mock- Analysis of our RNA-seq datasets revealed that the transcriptional response of Ctr is largely conserved across conditions.…”

“…each condition. In agreement with recent reports, we recovered high levels of chlamydial transcripts in each library (25), with no fewer than 7.5 x 10 6 mapped reads under any condition. We also note that across replicates, no more than 0.01% of the total library mapped to the chlamydial reference genome in any mock-infected sample, indicating a negligible influence of cross-aligned reads in our samples.…”

A novel intergenic dSH3-irr insertion mutation in Bradyrhizobium diazoefficiens causes its symbiotic incompatibility with soybean

“…Dual RNA-seq determination captures the RNA transcripts from the intracellular pathogens or symbiotic bacteria and host cells simultaneously 15 , and it can provide insights into metabolic alternations and responses from both the included parties 16,17 , and was ever applied to establish bacterial infection model 15,18 . However, dual RNAseq has not been used to explore the transcriptional variation of symbiotic nitrogen fixation between legumes and rhizobia.…”