Research Project 2
PI: Kimberly Jefferson, Ph.D.
The risk for very preterm birth (<32 weeks gestation) in African American women is three times as high as the risk in Caucasian women. Sub-clinical intrauterine infections correlate strongly with very preterm birth, and chorioamniotic tissues from the majority of women presenting in spontaneous labor at <32 weeks are infected with bacteria. Vaginal bacteria are a source for intrauterine infections especially in women with bacterial vaginosis (BV). BV is the most common vaginal disorder worldwide, affecting ~9% of Caucasian and ~23% of African American pregnant women and it more than triples the risk for preterm delivery in African American women. Many bacterial species can be isolated from the vagina during BV, but a small subset of these species is actually detected in fetal membranes and/or amniotic fluid in cases of preterm birth. We have evidence that this subset of bacteria is equipped with virulence factors including mucinases (e.g. sialidase), and collagenases that contribute to uterine invasion and preterm birth. We hypothesize that mucinases enable traversal of the cervical mucus plug and ascension into the uterus and that collagenases degrade chorionic and amnionic collagen, contributing to invasion and preterm premature rupture of membranes (PPROM). We have evidence that African American race significantly increases the relative risk for vaginal colonization by certain “utero-invasive” bacteria and we hypothesize that this contributes significantly to the racial disparity in preterm birth and preterm premature rupture of membranes. The goals of this project are to identify bacterial virulence determinants that play a role in intrauterine invasion, or the “virulome of infectious preterm birth”, and to determine whether colonization by bacteria equipped with this virulome is associated with the elevated risk for preterm birth in African American women.
Specific Aim 1. Characterize mucinase and collagenase activities of utero-invasive bacterial isolates.
- Perform whole genome sequencing on amniotic fluid isolates from preterm deliveries. Because bacteria vary tremendously in their virulence potential, even within a given species, we will sequence genomic DNA from invasive isolates. Sequencing will facilitate prediction and identification of virulence factor genes.
- Analyze mucinase, and collagenase activities. The goal is to test whole bacteria and spent medium as a preliminary screen for mucin- and collagen-degrading activity.
Specific Aim 2. Identify genes encoding mucinase and collagenase activities.
- Clone genes for predicted enzymes and express and purify recombinant proteins.
- Analyze the enzymatic activity in vitro. The goal is to confirm the identity of virulence factors that play a role in intra-uterine invasion.
- Confirm the role of key genes in mucus degradation and invasion/weakening of fetal membranes. We will use targeted deletion mutagenesis to confirm the roles of key genes.
Specific Aim 3. Determine the prevalence of utero-invasive strains in African American vs. Caucasian women.
- Compare the vaginal virulomes of African American versus Caucasian women. The goal is to determine which virulence factors are associated with preterm birth and which are associated with African American race.
- Analyze the microbiomes and virulomes of the amniotic sac from African American versus Caucasian women. We will analyze amniotic membranes from full term and preterm births using culture-based and culture-independent (PCR) techniques to identify invasive species and virulence genes.
Machado A, Jefferson KK, Cerca N. (2013) Interactions between Lactobacillus crispatus and Bacterial Vaginosis (BV)-Associated Bacterial Species in Initial Attachment and Biofilm Formation. International Journal of Molecular Sciences. Jun 5;14(6):12004-12. PMCID:PMC3709769