Novel high-resolution targeted sequencing of the cervicovaginal microbiome

BACKGROUND: The cervicovaginal microbiome (CVM) plays a significant role in women's cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Current sequencing methods, especially most commonly used multiplex 16S rRNA gene sequencing, struggle to fully clarify these changes because they generally fail to provide sufficient taxonomic resolution to adequately perform species-level associative studies. To improve CVM species designation, we designed a novel sequencing tool targeting microbes at the species taxonomic rank and examined its potential for profiling the CVM. RESULTS: We introduce an accessible and practical circular probe-based RNA sequencing (CiRNAseq) technology with the potential to profile and quantify the CVM. In vitro and in silico validations demonstrate that CiRNAseq can distinctively detect species in a mock mixed microbial environment, with the output data reflecting its ability to estimate microbes' abundance. Moreover, compared to 16S rRNA gene sequencing, CiRNAseq provides equivalent results but with improved sequencing sensitivity. Analyses of a cohort of cervical smears from hrHPV-negative women versus hrHPV-positive women with high-grade cervical intraepithelial neoplasia confirmed known differences in CST occurring in the CVM of women with hrHPV-induced lesions. The technique also revealed variations in microbial diversity and abundance in the CVM of hrHPV-positive women when compared to hrHPV-negative women. CONCLUSIONS: CiRNAseq is a promising tool for studying the interplay between the CVM and hrHPV in cervical carcinogenesis. This technology could provide a better understanding of cervicovaginal CST and microbial species during health and disease, prompting the discovery of biomarkers, additional to hrHPV, that can help detect high-grade cervical lesions

PQN-75 is expressed in the pharyngeal gland cells of Caenorhabditis elegans and is dispensable for germline development

In Caenorhabditis elegans, five pharyngeal gland cells reside in the terminal bulb of the pharynx and extend anterior processes to five contact points in the pharyngeal lumen. Pharyngeal gland cells secrete mucin-like proteins thought to facilitate digestion, hatching, molting and assembly of the surface coat of the cuticle, but supporting evidence has been sparse. Here we show pharyngeal gland cell expression of PQN-75, a unique protein containing an N-terminal signal peptide, nucleoporin (Nup)-like phenylalanine/glycine (FG) repeats, and an extensive polyproline repeat domain with similarities to human basic salivary proline-rich pre-protein PRB2. Imaging of C-terminal tagged PQN-75 shows localization throughout pharyngeal gland cell processes but not the pharyngeal lumen; instead, aggregates of PQN-75 are occasionally found throughout the pharynx, suggesting secretion from pharyngeal gland cells into the surrounding pharyngeal muscle. PQN-75 does not affect fertility and brood size in C. elegans but confers some degree of stress resistance and thermotolerance through unknown mechanisms

Longitudinal analysis on the ecological dynamics of the cervicovaginal microbiome in hrHPV infection

The cervicovaginal microbiome (CVM) is a dynamic continuous microenvironment that can be clustered in microbial community state types (CSTs) and is associated with women’s cervical health. Lactobacillus-depleted communities particularly associate with an increased susceptibility for persistence of high-risk human papillomavirus (hrHPV) infections and progression of disease, but the long-term ecological dynamics of CSTs after hrHPV infection diagnosis remain poorly understood. To determine such dynamics, we examined the CVM of our longitudinal cohort of 141 women diagnosed with hrHPV infection at baseline with collected cervical smears at two timepoints six-months apart. Here we describe that the long-term microbiome dissimilarity has a positive correlation with microbial diversity at both visits and that women with high abundance and dominance for Lactobacillus iners at baseline exhibit more similar microbiome composition at second visit than women with Lactobacillus-depleted communities at baseline. We further show that the species Lactobacillus acidophilus and Megasphaera genomosp type 1 associate with CST changes between both visits. Lastly, we also observe that Gardnerella vaginalis is associated with the stability of Lactobacillus-depleted communities while L. iners is associated with the instability of Megasphaera genomosp type 1-dominated communities. Our data suggest dynamic patterns of cervicovaginal CSTs during hrHPV infection, which could be potentially used to develop microbiome-based therapies against infection progression towards disease

Expression of PRC2 members at 1 dpf.

<p>Lateral view of the spatio-temporal expression assessed by whole mount <i>in situ</i> hybridization of the PRC2 members: <i>ezh2</i>, <i>ezh1</i>, <i>eed</i>, <i>suz12a</i>, <i>phc2a</i>, and <i>rbbp4</i>, at 1 dpf zebrafish embryos.</p

Expression of PcG genes at 2 and 3 dpf.

<p>(A) Spatio-temporal expression assessed by whole mount <i>in situ</i> hybridization of <i>ezh2</i>, <i>ezh1</i>, <i>eed</i>, <i>suz12a</i>, <i>phc2a</i>, <i>rbbp4</i>, and <i>pcgf6</i> at 2 dpf. Lateral views are shown for all genes. Dorsal views of <i>ezh2</i>, <i>ehz1</i>, <i>eed</i>, <i>phc2a</i>, and <i>rbbp4</i>. Ventral view of <i>pcgf6</i>. (B) Spatio-temporal expression assessed by whole mount <i>in situ</i> hybridization of <i>ezh2</i>, <i>ezh1</i>, <i>eed</i>, <i>phc2a</i>, <i>rbbp4</i>, <i>bmi1a</i>, and <i>pcgf6</i> at 3 dpf. Lateral views are shown for all genes. Dorsal views of <i>ezh2</i>, <i>ezh1</i>, <i>eed</i>, <i>phc2a</i>, and <i>rbbp4</i>. in: intestine, pf: pectoral fin (buds), HB: hindbrain, MHB: mid-hind brain boundary, pep: presumptive epiphysis, pa: pharyngeal arches 3–7, ot: optic tectum, re: retina, ob: olfactory bulb, n.d. = no data.</p

Whole mount <i>in situ</i> hybridization probes from plasmids.

<p>Whole mount <i>in situ</i> hybridization probes from plasmids.</p

Expression of PRC2 members at early stages of embryonic development.

<p>Spatio-temporal expression assessed by whole mount <i>in situ</i> hybridization of <i>ezh2</i>, <i>ezh1</i>, <i>eed</i>, <i>suz12a</i>, <i>phc2a</i> and <i>rbbp4</i>, at the 2-cell stage (0.75 hpf) and 50% epiboly (5.3 hpf). Scale bar: 200 μm.</p

Expression of the majority of the PcG genes that were tested is enriched in the adult germ line.

<p>(A) Relative expression assessed by RT-qPCR for <i>ezh2</i>, <i>eed</i>, <i>suz12a</i>, <i>rnf2</i>, <i>bmi1a</i>, <i>pcgf6</i>, and <i>rbbp4</i> and for the lower expressed genes <i>ezh1</i>, <i>suz12b</i>, <i>pcgf5a</i>, and <i>pcgf5b</i> in the adult ovary and testis and two somatic tissues: muscle and eye. Relative expression to the reference gene <i>rsp18</i> is shown. Data based on three biological replicates and three technical replicates. Error bars indicate standard deviation. (B) Spatio-temporal expression of <i>ezh2</i>, <i>ehz1</i>, <i>eed</i>, <i>suz12a</i>, <i>phc2a</i>, <i>rbbp4</i>, <i>rnf2</i>, <i>pcgf1</i>, <i>bmi1a</i>, <i>pcgf5a</i>, <i>pcgf5b</i>, and <i>pcgf6</i> in adult ovaries. Lower right panel shows a schematic representation of stage IB, II, and III of oogenesis. Stages of oogenesis are assessed according to Selman <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200316#pone.0200316.ref032" target="_blank">32</a>]. Scale bar: 100 μm. (C) Expression of the PcG genes that were tested in different stages of oogenesis. Purple boxes indicate expression of the corresponding gene at that stage of oogenesis. Stages of oogenesis are assessed according to Selman <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200316#pone.0200316.ref032" target="_blank">32</a>]. (D) Spatio-temporal expression of <i>ezh2</i>, <i>eed</i>, <i>suz12a</i>, <i>phc2a</i>, and <i>bmi1a</i> in adult testes. Lower right panel shows examples of the four stages of spermatogenesis. Scale bar: 100 μm. (E) Expression of the PcG genes that were tested in different stages of spermatogenesis. Purple boxes indicate positive expression of the corresponding gene at that stage of spermatogenesis. Stages of spermatogenesis are determined according to Leal <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200316#pone.0200316.ref033" target="_blank">33</a>].</p