The Alterations of Vaginal Microbiome in HPV16 Infection as Identified by Shotgun Metagenomic Sequencing

Qian Yang, Yaping Wang, Xinyi Wei, Jiawei Zhu, Xinyu Wang, Xing Xie, Weiguo Lu, Qian Yang, Yaping Wang, Xinyi Wei, Jiawei Zhu, Xinyu Wang, Xing Xie, Weiguo Lu

Abstract

The association of microbiome imbalance with cancer development is being one of the research hotspots. Persistent HPV infection is a causal event in cervical cancer initiation, but, little is known about the microbiome composition and function in HPV infection. Here we identified the compositional and functional alterations on vaginal samples from 27 HPV16 positive women and 25 age-matched HPV negative controls using shotgun metagenomic sequencing, to provide a comprehensive investigation describing the microbial abundances and enriched metabolic functions in cervicovaginal metagenomes. We further employed qPCR assays to evaluate two selected gene markers of HPV16 infection in an independent validation cohort consisting of 88 HPV16 positive women and 81 controls, and six selected species markers in a subset of validation cohort of 45 HPV16 positive women and 53 controls. We found that the relative abundance of dominant Firmicutes was lower, Actinobacteria, Fusobacteria and viruses phyla were significantly higher in the HPV16-positive group; 77 genera including Gardnerella, Peptostreptococcus, and Prevotella were higher, and 20 genera including Lactobacillus and Aerococcus were lower in the HPV16-positive women. Abundance of 12 genes, 17 genera, and 7 species biomarkers showed an excellent predictive power for the HPV16-positive individuals, with 0.861, 0.819, and 0.918, respectively, of the area under the receiver-operating characteristic curve (AUC). We further characterized the microbial function, and revealed that HPV16-positive women were enriched in metabolism and membrane transport, and depleted by glycan biosynthesis and metabolism, and replication and repair. Quantitative PCR measurements validated that one gene marker and three species were significantly enriched in HPV16-positive women. These results highlight a fundamental fact that there are altered composition and function of the vaginal microbiome in HPV16-positive women, suggesting that vaginal dysbiosis may be associated with HPV infection in the female genital tract.

Keywords: HPV infection; cervical cancer; metabolism; shotgun metagenomic sequencing; vaginal microbiome.

Copyright © 2020 Yang, Wang, Wei, Zhu, Wang, Xie and Lu.

Figures

Figure 1
Figure 1
Flowchart of the study.
Figure 2
Figure 2
Phylogenetic profiles in vaginal microbes between HPV16-positive women and controls. Composition of vaginal microbiota in two groups at the genus level (A) and species level (B). Comparison of differentially abundant phylotypes identified by the Wilcoxon rank-sum test, at phyla (C), genera (D), and species (E) level, respectively. Only the top 2 phyla, top 10 genera and top 20 species are shown. The phylotypes enriched in the control group are colored with red. The relative abundances are shown by boxplot. Boxes represent the interquartile ranges, lines inside the boxes denote medians, and “+” denotes means.
Figure 3
Figure 3
Vaginal microbiome as HPV16-infection markers. (A) Heatmap showing the relative fold change of bacterial species in HPV16 infection. The species enriched in controls are colored with red. (B) Histogram of the LDA scores computed for species differentially abundant between HPV16-positive women and controls. The LDA scores (log10) > 2 are listed.
Figure 4
Figure 4
Presence of non-bacterial microbial taxa, archaea (A, left), eukaryote (B, left), and viruses (C, left) within the vaginal microbiome. Histogram of the LDA scores computed for species differentially abundant between HPV16-positive women and healthy controls, archaea (A, right), eukaryote (B, right), and viruses (C, right) biomarkers are shown. The LDA scores (log10) > 2 are listed.
Figure 5
Figure 5
A predictive model of importance based on the gene/genus/species-level abundance profile using random forests (RF). The relative importance of each gene (A)/genus (B)/species (C) in the predictive random forest model using the mean decreasing accuracy. ROC curve generated by the RF using 12 genes (A)/17 genus (B)/7 species (C) in the vaginal microbiota. The plots shown in the ROC represent the corresponding optimal threshold.
Figure 6
Figure 6
Functional predictions for the vaginal microbiome of the HPV16-positive and control groups. (A) The abundance of each sample at level 2 metabolic pathway. (B) The KOs with significantly different abundances in the vaginal microbiome identified by Metastats analysis (P, FDR < 0.05). (C) Comparison between the HPV16-positive group-enriched and the control-enriched KO markers on level 2 of the KEGG functional category.
Figure 7
Figure 7
The abundance of gene and species markers in validation cohort by qPCR. Abundance of two gene markers (GI_0004362, C69 family dipeptidase from Gardnerella vaginalis; GI_0014455, GBSi1, group II intron, maturase from multispecies) were measured in validation cohort of 88 cases and 81 controls (A,B). Abundance of six species, Atopobium vaginae (C), Peptostreptococcus anaerobius (D), Candida albicans (E), Gardnerella vaginalis (F), Lactobacillus iners (G) and Chlamydia trachomatis (H) were measured in a subset of validation cohort (53 cases and 45 controls). The y-axis represents the relative abundance of the corresponding genes and species in all samples. Statistical comparison by Wilcoxon rank-sum test.

References

    1. Anahtar M. N., Gootenberg D. B., Mitchell C. M., Kwon D. S. (2018). Cervicovaginal microbiota and reproductive health: the virtue of simplicity. Cell Host Microbe 23, 159–168. 10.1016/j.chom.2018.01.013
    1. Boris S., Barbés C. (2000). Role played by lactobacilli in controlling the population of vaginal pathogens. Microbes Infect. 2, 543–546. 10.1016/s1286-4579(00)00313-0
    1. Briselden A. M., Moncla B. J., Stevens C. E., Hillier S. L. (1992). Sialidases (neuraminidases) in bacterial vaginosis and bacterial vaginosis-associated microflora. J. Clin. Microbiol. 30:663.
    1. Brotman R. M., Shardell M. D., Gajer P., Tracy J. K., Zenilman J. M., Ravel J., et al. . (2014). Interplay between the temporal dynamics of the vaginal microbiota and human papillomavirus detection. J. Infect. Dis. 210, 1723–1733. 10.1093/infdis/jiu330
    1. Brown R. G., Marchesi J. R., Lee Y. S., Smith A., Lehne B., Kindinger L. M., et al. . (2018). Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin. BMC Med. 16:9. 10.1186/s12916-017-0999-x
    1. Bruni L., Diaz M., Castellsagué X., Ferrer E., Bosch F. X., de Sanjosé S. (2010). Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J. Infect. Dis. 202, 1789–1799. 10.1086/657321
    1. Chen C., Song X., Wei W., Zhong H., Dai J., Lan Z., et al. . (2017). The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases. Nat. Commun. 8:875. 10.1038/s41467-017-00901-0
    1. Chen Y., Hong Z., Wang W., Gu L., Gao H., Qiu L., et al. . (2019). Association between the vaginal microbiome and high-risk human papillomavirus infection in pregnant Chinese women. BMC Infect. Dis. 19:677. 10.1186/s12879-019-4279-6
    1. de Sanjose S., Quint W. G. V., Alemany L., Geraets D. T., Klaustermeier J. E., Lloveras B., et al. . (2010). Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol. 11, 1048–1056. 10.1016/S1470-2045(10)70230-8
    1. Di Paola M., Sani C., Clemente A. M., Iossa A., Perissi E., Castronovo G., et al. . (2017). Characterization of cervico-vaginal microbiota in women developing persistent high-risk Human Papillomavirus infection. Sci. Rep. 7:10200. 10.1038/s41598-017-09842-6
    1. Elovitz M. A., Gajer P., Riis V., Brown A. G., Humphrys M. S., Holm J. B., et al. . (2019). Cervicovaginal microbiota and local immune response modulate the risk of spontaneous preterm delivery. Nat. Commun. 10:1305. 10.1038/s41467-019-09285-9
    1. Freitas A. C., Bocking A., Hill J. E., Money D. M. (2018). Increased richness and diversity of the vaginal microbiota and spontaneous preterm birth. Microbiome 6:117. 10.1186/s40168-018-0502-8
    1. Gajer P., Brotman R. M., Bai G., Sakamoto J., Schütte U. M. E., Zhong X., et al. . (2012). Temporal dynamics of the human vaginal microbiota. Sci. Transl. Med. 4:132ra52. 10.1126/scitranslmed.3003605
    1. Gopinath S., Iwasaki A. (2015). Cervicovaginal microbiota: simple is better. Immunity 42, 790–791. 10.1016/j.immuni.2015.05.006
    1. Kanehisa M., Goto S., Kawashima S., Okuno Y., Hattori M. (2004). The KEGG resource for deciphering the genome. Nucleic Acids Res. 32, D277–D280. 10.1093/nar/gkh063
    1. Kostic A. D., Chun E., Robertson L., Glickman J. N., Gallini C. A., Michaud M., et al. . (2013). Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 14, 207–215. 10.1016/j.chom.2013.07.007
    1. Kroon S. J., Ravel J., Huston W. M. (2018). Cervicovaginal microbiota, women's health, and reproductive outcomes. Fertil. Steril. 110, 327–336. 10.1016/j.fertnstert.2018.06.036
    1. Lee J. E., Lee S., Lee H., Song Y.-M., Lee K., Han M. J., et al. . (2013). Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort. PLoS ONE 8:e63514. 10.1371/journal.pone.0063514
    1. Lewis W. G., Robinson L. S., Gilbert N. M., Perry J. C., Lewis A. L. (2013). Degradation, foraging, and depletion of mucus sialoglycans by the vagina-adapted Actinobacterium Gardnerella vaginalis. J. Biol. Chem. 288, 12067–12079. 10.1074/jbc.M113.453654
    1. Li R., Li Y., Kristiansen K., Wang J. (2008). SOAP: short oligonucleotide alignment program. Bioinformatics 24, 713–714. 10.1093/bioinformatics/btn025
    1. Li R. E., van Vliet S. J., van Kooyk Y. (2018). Using the glycan toolbox for pathogenic interventions and glycan immunotherapy. Curr. Opin. Biotechnol. 51, 24–31. 10.1016/j.copbio.2017.11.003
    1. Long X., Wong C. C., Tong L., Chu E. S. H., Ho Szeto C., Go M. Y. Y., et al. . (2019). Peptostreptococcus anaerobius promotes colorectal carcinogenesis and modulates tumour immunity. Nat. Microbiol. 136:E359. 10.1038/s41564-019-0541-3
    1. Luo R., Liu B., Xie Y., Li Z., Huang W., Yuan J., et al. . (2012). SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1:18. 10.1186/2047-217X-1-18
    1. Nené N. R., Reisel D., Leimbach A., Franchi D., Jones A., Evans I., et al. . (2019). Association between the cervicovaginal microbiome, BRCA1 mutation status, and risk of ovarian cancer: a case-control study. Lancet Oncol. 20, P1171–P1182. 10.1016/S1470-2045(19)30340-7
    1. Noguchi H., Park J., Takagi T. (2006). MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic Acids Res. 34, 5623–5630. 10.1093/nar/gkl723
    1. Nowak R. G., Randis T. M., Desai P., He X., Robinson C. K., Rath J. M., et al. . (2018). Higher levels of a cytotoxic protein, vaginolysin, in lactobacillus-deficient community state types at the vaginal mucosa. Sex. Transm. Dis. 45, e14–e17. 10.1097/OLQ.0000000000000774
    1. Qin N., Yang F., Li A., Prifti E., Chen Y., Shao L., et al. . (2014). Alterations of the human gut microbiome in liver cirrhosis. Nature 513, 59–64. 10.1038/nature13568
    1. Quince C., Walker A. W., Simpson J. T., Loman N. J., Segata N. (2017). Shotgun metagenomics, from sampling to analysis. Nat. Biotechnol. 35, 833–844. 10.1038/nbt.3935
    1. Raman R., Tharakaraman K., Sasisekharan V., Sasisekharan R. (2016). Glycan-protein interactions in viral pathogenesis. Curr. Opin. Struct. Biol. 40, 153–162. 10.1016/j.sbi.2016.10.003
    1. Randis T. M., Zaklama J., LaRocca T. J., Los F. C. O., Lewis E. L., Desai P., et al. . (2013). Vaginolysin drives epithelial ultrastructural responses to Gardnerella vaginalis. Infect. Immun. 81, 4544–4550. 10.1128/IAI.00627-13
    1. Ranjan R., Rani A., Metwally A., McGee H. S., Perkins D. L. (2016). Analysis of the microbiome: advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem. Biophys. Res. Commun. 469, 967–977. 10.1016/j.bbrc.2015.12.083
    1. Schnaar R. L. (2016). Glycobiology simplified: diverse roles of glycan recognition in inflammation. J. Leukoc. Biol. 99, 825–838. 10.1189/jlb.3RI0116-021R
    1. Segata N., Izard J., Waldron L., Gevers D., Miropolsky L., Garrett W. S., et al. . (2011). Metagenomic biomarker discovery and explanation. Genome Biol. 12:R60. 10.1186/gb-2011-12-6-r60
    1. Shah M. S., DeSantis T. Z., Weinmaier T., McMurdie P. J., Cope J. L., Altrichter A., et al. . (2018). Leveraging sequence-based faecal microbial community survey data to identify a composite biomarker for colorectal cancer. Gut 67, 882–891. 10.1136/gutjnl-2016-313189
    1. Shannon B., Yi T. J., Perusini S., Gajer P., Ma B., Humphrys M. S., et al. . (2017). Association of HPV infection and clearance with cervicovaginal immunology and the vaginal microbiota. Mucosal. Immunol. 10, 1310–1319. 10.1038/mi.2016.129
    1. Smith S. B., Ravel J. (2017). The vaginal microbiota, host defence and reproductive physiology. J. Physiol. 595, 451–463. 10.1113/JP271694
    1. Stanley M. (2010). Pathology and epidemiology of HPV infection in females. Gynecol. Oncol. 117, S5–10. 10.1016/j.ygyno.2010.01.024
    1. Tachedjian G., Aldunate M., Bradshaw C. S., Cone R. A. (2017). The role of lactic acid production by probiotic Lactobacillus species in vaginal health. Res. Microbiol. 168, 782–792. 10.1016/j.resmic.2017.04.001
    1. Tsoi H., Chu E. S. H., Zhang X., Sheng J., Nakatsu G., Ng S. C., et al. . (2017). Peptostreptococcus anaerobius induces intracellular cholesterol biosynthesis in colon cells to induce proliferation and causes dysplasia in Mice. Gastroenterology 152, 1419–1433.e5. 10.1053/j.gastro.2017.01.009
    1. van de Wijgert J. H. H. M. (2017). The vaginal microbiome and sexually transmitted infections are interlinked: consequences for treatment and prevention. PLoS Med. 14:e1002478. 10.1371/journal.pmed.1002478
    1. Walboomers J. M. M., Jacobs M. V., Manos M. M., Bosch F. X., Kummer J. A., Shah K. V., et al. . (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189, 12–19.
    1. White J. R., Nagarajan N., Pop M. (2009). Statistical methods for detecting differentially abundant features in clinical metagenomic samples. PLoS Comput. Biol. 5:e1000352. 10.1371/journal.pcbi.1000352
    1. Zou J., Cao Z., Zhang J., Chen T., Yang S., Huang Y., et al. . (2016). Variants in human papillomavirus receptor and associated genes are associated with type-specific HPV infection and lesion progression of the cervix. Oncotarget 7, 40135–40147. 10.18632/oncotarget.9510

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