When a foreign object (such as a microbe) first invades our bodies, there are two broad niches it may land on: wet or dry. The dry option, our skin, allows direct microbial interaction with our cells, though the outermost layer is dead or dying cells that will be lost by desquamation. The wet option, our mucous membranes, is covered by a layer of viscous mucins, glycoproteins, and water (namely, mucus), which acts as a barrier between invading microbes and the underlying cells. Almost anything that isn’t skin – eyes, oral cavity, GI tract, genital tract, lungs – is part of this more hydrated environment. And like skin, it is sloughed off and constantly replenished: the human body makes 1 liter of mucus each day.
That substance, mucus, has long been considered a first line of defense against microbial invasion, based on its ability to trap and remove pathogens. But how many people think of the changing properties of mucus? Dr. Samuel Lai of University of North Carolina says that despite being thought of as a static substance, “there are a lot of emerging insights that have really shown that not only is mucus the first line of defense, but also that it’s dynamic.” This dynamism is addressed in the recent findings from Lai’s lab published this week in mBio.
Lai’s group focuses on the properties of cervicovaginal mucus. “We don’t understand the vaginal mucus barrier well,” explains Lai. “We know it has the capability of possibly blocking infectious disease by trapping pathogens in mucus. But one of the puzzling findings is that the ability to trap seems to vary quite a lot between different women, and sometimes between different mucus obtained from the same woman at different times.” By characterizing the barrier properties of vaginal mucus, Lai hopes to discover ways to better reinforce this barrier to improve women’s health.
To test this barrier property, lead author Kenetta Nunn used a variation on a previously-established fertility assay. One of the tests administered to couples with difficulty conceiving is to test whether the man’s sperm can swim across the woman’s mucus. Rather than investigating sperm, Nunn looked at HIV viruses. These engineered viruses “contained a fluorescent protein in the capsid of the virus,” says Lai. “The fluorescent proteins are therefore not exposed to the surface, which ensures we maintain virus integrity and yet can see them. Using very high-resolution microscope that allows us to follow individual viruses quickly – 15 frames/sec frame-rate – we can accurately follow the movement of the viruses.” The resulting data produces traces, as seen in the image at right (taken from the paper).
Right away, the researchers observed that vaginal mucus with high levels of D-lactic acid trapped HIV. This was expected, Lai says, because gynecologists have known for a long time that lactic acid presence indicates a protective environment. Microbes living in vaginal mucus can influence a woman’s susceptibility to infections. For example, bacterial vaginosis is a condition resulting from an overgrowth of non-lactate producing bacteria that makes women markedly more susceptible to infections such as HIV, herpes, gonorrhea, chlamydia, and even preterm birth. The presence of Lactobacilli, which produces D-lactic acid, has been the primary condition for assessing whether a woman has a healthy microflora that would confer lowered infection risk. Lactic acid is the primary means of acidifying the vagina and is protective (in part) because few bacteria can survive at this lowered pH.
Not all Lactobacilli are created equal
There are two conventional methods for testing the abundance of lactobacilli. One, a method called Nugent Scoring, involves counting the number of Gram-positive, rod-shaped bacteria observed in a given sample (like the sample on the far left). However, this method doesn’t distinguish between different Lactobacilli species; it simply enumerates bacteria with the lactobacilli morphology. The other method involves measuring the bacterially produced D-lactic acid, which indirectly suggests the presence of lactobacilli. Not until the ability of ribosomal DNA sequencing could investigators detail the specific species of the lactobacilli present.
The Lai lab collaborated with Jacques Ravel at the University of Maryland to use 16S ribosomal DNA sequencing to identify the prominent species within different patient samples of cervicovaginal mucus. This allowed them to differentiate three groups: women with either L. crispatus-dominated, L. iners-dominated, or Gardnerella vaginalis-dominated (indicating bacterial vaginosis) vaginal microflora. When the mucus from these groups was tested for virus trapping, the L. crispatus-dominated mucus had many fewer mobile HIV virions, and those virions traveled more tightly than virions in mucus from the other two groups. “We don't know exactly what the different Lactobacilli are doing; all we can tell for now is that L. crispatus seems to confer a mucoprotective barrier property whereas L. iners seems to be correlated to a compromised mucus that cannot trap HIV or other enveloped viruses,” explains Lai.
Could the difference be due to lactic acid secretion? Lai doesn’t think so. “There is very little correlation with pH. Both [species] are capable of secreting lactic acid, the primary driver of the pH in the vaginal mucus.” While the mechanism behind the difference remains unknown, Lai stressed an important take-home point: “We could not have inferred this either by pH, by Nugent Scoring, or by quantifying the total amount of lactic acid present. By all the conventional means of characterizing the vaginal mucus, we could not have detected this difference.”
The findings could have clinical ramifications in terms of diagnostics. The data show that standard Nugent Scoring techniques misclassify many women with L. iners-dominated vaginal microbiota into a protected group, when they may not receive protection in terms of mucus barrier properties. And protection of women with L. crispatus-dominated microbiota may actually increase if examined in relation to all others, including those with L. iners.
Furthermore, these findings may help address major global public health concerns. Most women in Africa do not have L. crispatus. There are far greater populations with either BV or intermediate microflora – often times intermediate microflora has an abundance of L. iners. If the populations of these women can be shifted to L. crispatus, it may confer greater protection and act synergistically with other methods being pioneered to control STIs, speculates Lai. “The interest in altering vaginal microbiota certainly hasn't caught up to the interest of people in altering the gut microbiota,” he says. “This is very much still an emerging field.”
-- Julie Wolf
Photo credits: Vaginal Gram stain,