Developing probe-based transcriptional profiling of mucosal infections in humans

About This Grant

Rapid, cost-effective, and noninvasive tools to monitor host-pathogen-microbiome interactions during Chlamydia trachomatis (CT) infection are urgently needed to identify correlates of protection and predict risk for reproductive tract damage—critical for advancing vaccine development and clinical care. Chlamydia trachomatis is a globally impactful sexually transmitted bacterial pathogen for which no vaccine is available, and predictive biomarkers for adverse outcomes are lacking. Prior research and our recent study indicate that chlamydia-microbiome and microbiome-host interactions can influence establishment of infection and spread to the upper genital tract. We previously demonstrated that total RNAseq can transcriptionally profile host, pathogen and cervicovaginal microbiome activity during human genital tract infection. However, this method requires high-abundance, only moderately degraded RNA and is cost-prohibitive for large-scale use. To overcome these limitations, we developed a simple and cost-efficient, probe-based transcriptional profiling method, inspired by advancements in single-cell RNA sequencing technologies, and demonstrated its feasibility using cervical specimens from experimentally infected mice. The goals of this proposal are to: i) evaluate probe-based transcriptional profiling as an effective and cost-effective alternative to total RNAseq for characterizing human clinical cervical specimens; ii) determine if this technique can simultaneously and sensitively profile chlamydial gene expression during natural infection while iii) monitoring cervicovaginal microbiome composition. We will complete the following specific aims: (1) Compare performance of probe-based RNA sequencing with total RNAseq for cervical brush specimens collected from women at high risk for C. trachomatis infection. Using previously collected, cervical brush specimens currently undergoing total RNAseq in an R01 funded study, we will perform parallel, probe-based profiling and compare outcomes for sensitivity and robustness, particularly in the face of low RNA quality and/or abundance. (2) Profile pathogen gene expression during natural cervical infection using probes targeting the global chlamydial transcriptome. We will use probes targeting the global chlamydial transcriptome to profile gene expression during natural cervical infection, generating RNA-seq libraries from cervical brush samples with varying chlamydial loads. (3) Determine if incorporation of 16S-targeting probes will support coincident CVM characterization during chlamydial infection. Chlamydial 16S and 23S rRNAs were sensitively and robustly detected in murine genital infection samples so we will extend this approach to determine feasibility for coincidental monitoring of co-pathogen abundance and to profile resident mucosal microbiota without need for separate 16S rRNA gene amplicon library pipelines. This approach integrates emerging RNA profiling technologies into a customizable, scalable pipeline that supports unbiased studies of mucosal immunity, pathogen burden, and microbiome composition, even in low-yield or degraded clinical samples. The successful development of this platform would accelerate determination of molecular correlates of protection or immunopathogenesis in humans after infection and has significant promise for vaccine evaluation not only for chlamydia, but also for other sexually transmitted mucosal pathogens.