The vaginal microbiome is believed to protect women against Chlamydia trachomatis, the etiological agent of the most prevalent sexually transmitted infections (STIs) in developed countries.
New research by the University of Maryland School of Medicine (UMSOM) shows how the microbiome can either protect or make a woman more susceptible to these serious infections.
The research is important amid a rising number of cases of chlamydia worldwide.
In the U.S. alone, 1.7 million cases of chlamydia were reported in 2017, a 22% increase since 2013, according to data from the Centers for Diseases Control and Prevention (CDC).
“Chlamydia is a major growing health issue in the U.S., and more work is needed to understand why some women are apparently naturally protected while other are not,” commented Jacques Ravel, Ph.D., Professor of Microbiology and Immunology, Associate Director and Senior Scientist at the Institute for Genome Sciences (IGS) at UMSOM. Dr. Ravel is also a Principal Investigator for this research.
“Our novel research aims to decipher the mechanistic and functional underpinnings of communication between the host and the cervicovaginal microbiome to better understand resistance and susceptibility to this infection.”
An Important Mechanism in Vaginal Microbiome
While Lactobacillus-dominated microbiota in a woman’s vagina has long been suspected to provide a protective barrier against STIs like chlamydia, investigators at IGS and the University of Maryland School of Dentistry (UMSOD) are reporting for the first time a mechanism enabling specific types of cervicovaginal microbiome to predispose cells in the vagina and cervix to resist chlamydial infection.
“We will now be able to leverage these microbiomes to identify women at risk of infections, but more importantly to develop improved strategies to restore an optimal protection when it is lacking.
Unlike our genes, the vaginal microbiome can be modulated to increase protection against chlamydia, but also against other sexually transmitted infections, including HIV,” said Dr. Ravel of the research, which was published today in mBio, “Cervicovaginal Microbiota-Host Interaction Modulates Chlamydia trachomatis Infection.”
The investigators have shown previously that five major types of vaginal microbiome exist, four of which are dominated by a different species of Lactobacillus, while the fifth has very low numbers of Lactobacillus bacteria and is associated with an increased risk of adverse outcomes including STIs, such as HIV, and even premature births.
The current research showed that Lactobacillus iners, a bacterium actually commonly found in the vagina did not optimally protect human cells against chlamydial infection, while products of Lactobacillus crispatus, another Lactobacillus species frequently found in the vagina, did.
Previously published research has hinted at L. iners being a risk factor for STI; however, the mechanism by which these bacteria were specifically suboptimal at protecting women against STI has remained elusive.
Like other Lactobacillus, L. iners produces lactic acid, but only the L isoform.
The researchers found that D-lactic acid, not L-lactic acid, down-regulates cell cycling through epigenetic modifications thus blocking C. trachomatis entry into the cell, one of the pathogen key infectious process, among other processes.
Thus, a rather unexpected result of this study is that the vaginal microbiome does not affect the pathogen per se, but drives susceptibility or resistance to infection, by modifying the cells that line up the cervicovaginal epithelium.
The researchers further demonstrated that exposure to optimal vaginal microbiota provided long term protection, which has major implication on how a woman is protected.
These mechanisms are now being exploited to develop strategies to optimize protection against C. trachomatis infections but also other STIs.
Patrik Bavoil, Ph.D., Professor & Chair, Department of Microbial Pathogenesis, University of Maryland School of Dentistry, a well-known expert in C. trachomatis biology and pathogenesis, is a Co-Principal Investigator with Dr. Ravel on the NIH funding that supported this study. The investigators also collaborated with Larry Forney, Ph.D. at the University of Idaho.
“Chlamydia is reputed to be a most difficult microorganism to study.
By hiding inside cells, the pathogen routinely avoids antimicrobial host defenses.
By causing mostly asymptomatic infection, it often escapes detection by both the infected host and the physician alike,” said Dr. Bavoil.
“What we have done in this study through several years of hard work by dedicated researchers is to provide, for the first time, a huge, new stepping stone on which future translational research to exploit the microbiome in the fight against chlamydial infection and disease, can be based.”
Chlamydia trachomatis (CT) represents the agent of the most common bacterial sexually transmitted infection (STI) worldwide (ECDC, 2015). In women, urogenital CT infections are often asymptomatic, thus remaining unnoticed and untreated. This can lead to complications and sequelae including pelvic inflammatory disease, tubal infertility, and ectopic pregnancy (Price et al., 2013; Menon et al., 2015).
A normal vaginal microbiota, dominated by lactobacilli, is crucial for the prevention of several urogenital and sexually transmitted infections, including Chlamydia (Gupta et al., 1998; Spurbeck and Arvidson, 2008; Parolin et al., 2015; Nardini et al., 2016; Foschi et al., 2017; Ñahui Palomino et al., 2017).
This aspect is strengthened by the demonstration that in case of bacterial vaginosis, a clinical condition characterized by the depletion of lactobacilli, a higher risk of STI transmission and acquisition is reported (Taha et al., 1998; Martin et al., 1999; Wiesenfeld et al., 2003; Abbai et al., 2015).
The protective role of lactobacilli against urogenital pathogens is exerted through different mechanisms including the production of various antibacterial compounds (lactic acid, hydrogen peroxide, bacteriocins, and biosurfactants), the competitive exclusion for epithelial adhesion and the immunomodulation (Kaewsrichan et al., 2006; Borges et al., 2014; Parolin et al., 2015; Younes et al., 2018).
In this context, the use of probiotic lactobacilli for the prevention and treatment of several urinary and vaginal tract infections has been extensively evaluated, with different results depending on the Lactobacillus species, the strain origin, the concentrations used and the outcome considered (Barrons and Tassone, 2008; Bolton et al., 2008; Spurbeck and Arvidson, 2011; Vitali et al., 2016).
Until now, only a few studies have focused on the in vitro interaction between lactobacilli and CT and many aspects remain to be elucidated (Gong et al., 2014; Mastromarino et al., 2014; Nardini et al., 2016).
Considering that CT is an obligate intracellular bacterium, characterized by a unique biphasic developmental cycle alternating between the extracellular infectious ‘elementary body’ (EB) and the intracellular ‘reticulate body’ (RB) (Moulder, 1991), lactobacilli can interfere with CT infectivity acting on the different steps of its cycle.
Previous studies shed light on the metabolic interaction between CT and lactobacilli, mimicking what happens in the acid environment of the vaginal niche (Gong et al., 2014; Nardini et al., 2016), but they did not evaluate the ability of lactobacilli cells to compete and interfere with CT EBs infectivity in epithelial cells.
It has also been reported that the interaction of lactobacilli with cervical cells results in changes in the structure/functions of the plasma membrane of epithelial cells, especially at the level of α5β1 integrin exposure (Calonghi et al., 2017).
The integrin family of receptors is a major target for bacterial pathogens that colonize human tissues or invade specific cell types (Hoffmann et al., 2011; Hauck et al., 2012). Integrins are heterodimeric transmembrane receptors that mediate cell–cell and cell–extracellular matrix adhesion and, as a result, regulate many aspects of cell behavior.
In addition to providing a physical transmembrane link between the extracellular environment and the cytoskeleton, they are capable of transducing bi-directional signals across the cell membrane (Hynes, 2002).
In this context, the interaction of chlamydial Ctad1 adhesin with β1 integrin subunit has been proposed as one mechanism for EBs binding, invasion, and signaling during entry into host epithelial cells (Elwell et al., 2016; Stallmann and Hegemann, 2016).
The aim of this study was to identify vaginal Lactobacillus strains capable of interfering with the infectious process of CT in cervical cells (HeLa cell line) and to understand the rationale of this interaction. A L. crispatus strain was chosen as a model to study the molecular mechanisms underlying the anti-Chlamydia activity, with particular reference to the modulation of plasma membrane properties and integrin role in HeLa cell line.
Results
Interference of Lactobacilli With CT Infection
In the present work we investigated the capability of vaginal lactobacilli to counteract the infection process of C. trachomatis in HeLa cells, chosen as an in vitro epithelial model of cervical infection. Specifically, we tested the inhibitory activity of 15 Lactobacillus strains, previously isolated from vaginal swabs of healthy premenopausal women (Parolin et al., 2015) through an exclusion assay.
The effects of lactobacilli cells against C. trachomatis infection are reported in Figure Figure11 and the raw data are listed in Supplementary Table S1. All the Lactobacillusstrains significantly reduced the chlamydial infection. The most active strains were L. crispatusBC1, BC3, BC4, BC5, BC6, L. gasseri BC9, BC11, BC14, and L. vaginalis BC16.
We excluded that the observed anti-CT activity was related to an acidic environment, since the pH values of the culture media were not modified by the addition and incubation with lactobacilli cells: in particular, pH of DMEM without lactobacilli ranged between 8.0 and 8.1, whereas the pH values at 1 and 2 h post-incubation with lactobacilli ranged between 8.0 and 8.3.
In order to verify if the observed anti-Chlamydia activity was restricted to Lactobacillus strains, exclusion experiments were also performed with other Gram-positive microorganisms, namely S. agalactiae, E. faecalis, and B. subtilis. No reduction of Chlamydia infectivity was noticed for these control strains (Figure (Figure1).1). In particular, as B. subtilis has similar shape and size of lactobacilli, we can suppose that the inhibition exerted by lactobacilli is not due to a physical barrier created by the large rod-shaped bacteria, but to a specific interaction with HeLa cells membrane.
Dose–Response Effect of Lactobacilli on CT Infectivity
We sought to investigate the effect of different doses of lactobacilli cells on the level of inhibition of CT infection. For this purpose, we selected three strains among the most active ones (L. crispatus BC4, L. crispatus BC5, and L. gasseri BC14) and we evaluated the inhibitory effects of two dilutions, corresponding to the doses of 5 × 106 and 5 × 105 cells (Figure (Figure2).2). All Lactobacillus strains significantly reduced Chlamydia infectivity, confirming the strong antagonistic effect of Lactobacillus cells toward the infectious process of Chlamydia. Despite the antagonist activity was maintained even at lower doses, the level of inhibition was clearly dose dependent for all the lactobacilli.
Dose–response effect of Lactobacillus cells on C. trachomatis infectivity in HeLa cells. Exclusion experiments were performed at different doses (5 × 107, 5 × 106, and 5 × 105 cells) for L. crispatus BC4, L. crispatus BC5, and L. gasseri BC14 strains. C. trachomatis infectivity was evaluated as number of IFUs/microscopic field. Results were expressed in percentage compared with control, taken as 100% (black bars). Bars represent median values, error bars represent median absolute deviations. Statistical significance was calculated vs. control. ∗P ≤ 0.01.
More information: Vonetta L. Edwards et al. The Cervicovaginal Microbiota-Host Interaction Modulates Chlamydia trachomatis Infection, mBio (2019). DOI: 10.1128/mBio.01548-19
Journal information: mBio
Provided by University of Maryland School of Medicine
