Bacteriophage-host dynamics as a factor in the etiology of bacterial vaginosis              

This project is a collaboration with Dr. Eric Wommack, at the University of Delaware

Bacterial vaginosis (BV) is typified by vaginal discharge, discomfort, and malodor, and constitutes the most common vaginal complaint of reproductive-age women, resulting in millions of health care visits annually in the United States alone. The prevalence of BV ranges from 29% in the US to over 50% of women in rural Ugandan villages. The disease is characterized by an elevated pH and vaginal communities with reduced proportions of Lactobacillus sp. and increased proportions of strict anaerobes including species of Atopobium, Gardnerella, Prevotella, and other taxa of the order Clostridiales (8). A wealth of evidence indicates that BV and high Nugent scores (used in the diagnosis of BV) are independent risk factors for severe reproductive tract and obstetric sequelae, including preterm delivery and low birth weight (12-14, 23, 24) as well as the acquisition of sexually transmitted infections, including HIV (4, 21, 30, 41). Despite the high prevalence of this condition and the associated risk for severe adverse outcomes, the etiology of BV remains unknown. Interventions to treat BV focus on the use of antibiotics, but the effectiveness of such treatments is disappointingly low, and relapse is common. Our recent work characterizing the temporal dynamics of the vaginal microbiota demonstrated that vaginal bacterial communities are highly dynamic and undergo rapid fluctuations in and out of BV-like states (9, 31). The frequencies and duration of these fluctuations do not appear to correlate with specific members of the community or behaviors, and the underlying biological causes of these fluctuations remain unknown.

Extensive work by the PI and others has demonstrated the omnipresence and high abundance of viruses within microbial communities. Recent work has demonstrated that the human lung (43), gut (32) and mouth (42) contain diverse communities of viruses many of which are bacteriophages . Frequently, the viral communities within human body sites appear to be more genetically distinguishable between individuals and more sensitive to perturbations such as changes in diet (25). What we have learned from environmental and laboratory studies is that bacteriophages are efficient and highly specific predators which have the potential to quickly decimate a population of sensitive bacterial hosts and consequently alter the composition of co-existing microbial communities. One hallmark of BV is rapid changes in the abundance of bacterial populations leading to a vaginal bacterial community responsible for disease outcomes. Thus, the proposed research will leverage a unique combination of scientific expertise to address the hypothesis that bacteriophage infection is a contributing underlying biological causative factor capable of rapidly altering the composition of vaginal bacterial communities, changes that ultimately lead to BV. To test this hypotheses, we will capitalize on a prospective longitudinal clinical study of 135 women who self-collected daily vaginal swabs and associated metadata for 10 weeks. In this study we will assess the dynamics of phage and bacterial communities within 10 women from the larger study who experienced clinically-diagnosed symptomatic BV. Using samples from these women, obtained prior to, the day of disease onset, and during BV events, we propose to test our hypothesis by achieving the following specific aims:

Aim I) Characterize fluctuations in the activity of CRISPR/Cas systems and the diversity of CRISPR spacer elements prior to, the day of disease onset, and during BV events.

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated (Cas) genes, i.e., the CRISPR/Cas system is an acquired immunity system in Archaea and Bacteria that defends the host cell against invading foreign DNA — phages and plasmids (15). The CRISPR/Cas defense mechanism is somewhat analogous to RNAi in that short DNA spacer elements within CRISPR arrays are processed into small RNAs (CRISPR RNAs, crRNAs) that ultimately interfere with the expression of their target gene. An important characteristic of CRISPR spacers is that they effectively form a genetic record of phage and host interactions. With regards to the vaginal microbiome, we hypothesize that heightened occurrence and expression of CRISPR/Cas systems occurs in response to phage infection pressure within the vaginal microbiome.

Aim II) Determine whether changes in the abundance and composition of free vaginal bacteriophage communities are linked to the onset of BV.

To date, studies of bacteriophage communities within the vagina have exclusively used cultivation- dependent approaches to isolate temperate or virulent phages infecting Lactobacillus spp. (39, 40). Microbial ecological investigations using cultivation-independent approaches, such as those conducted by co-PI Ravel and others are investigating connections between vaginal bacterial communities and BV. No parallel studies have been conducted for high-resolution examination of the dynamics of vaginal bacteriophage communities during BV events. Recognizing the real possibility that phage infection plays an important role in the ecology of vaginal bacterial populations, we hypothesize that shifts in the compositional features of vaginal bacteriophage communities are a bellwether to changes in the bacterial communities occurring with BV.

This research reported is supported by National Institute of Allergy and Infectious Diseases, of the National Institutes of Health under award number R21AI109555.