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Research
Community Behavior of the Microbiome
Much focus has been put into research of the human microbiomes with investigators working to determine specific bacterial species that are present and correlate them with disease state. However, one must always take into account that bacteria do not function independently and often coordinate their behavior across their community via quorum sensing (QS), a complex system of gene regulation dependent upon small chemical molecules known as quorum sensing molecules (QSMs). QS controls a plethora of bacterial processes across differing species, most notably biofilm formation and production of virulence factors. QS has been shown to be a form of interkingdom signaling, with bacterial QSMs being able to directly interact with receptors on cells in its human host, potentially even initiating oncogenic pathways in mammalian cells.
Thus, my research group has shifted its focus on the microbiome beyond the metagenomics approach to also include investigations into bacterial QSMs as a way to better understand the community behavior.
Biosensors for Quorum Sensing Molecules
Bacterial quorum sensing (QS), a cell-cell communication system based on small molecules known as quorum sensing molecules (QSMs), controls processes reliant on the synchronized behavior of large communities of bacteria. QS hinges on the timely production, release, and detection of QSMs. As bacterial density increases, so does the concentrations of QSMs; once a threshold of QSMs is surpassed molecular cascades are triggered, resulting in population-wide gene regulation. A recent study identified a novel QSM, 3,5-dimethylpyrazin-2-ol (DPO) employed by pathogenic Vibrio cholerae to inhibit biofilm formation. DPO is synthesized by threonine dehydrogenase (Tdh). Tdh is highly conserved across bacteria, archaea, and eukarya, suggesting a possible role for DPO in other unidentified QS circuits. Studies that have evaluated microbiome composition and function in the context of health and disease have thus far relied on the use of costly metagenomic sequencing, metabolomic profiling, and bioinformatic analysis. These investigations have resulted in our understanding that the healthy microbiome is highly diverse and that this community of microbes is in an altered state in a variety of disorders. The transition into an altered state with profound effects on the host physiology is not fully understood, however it is known that these massive shifts in bacterial populations involve a complex interplay between distinct members of the community and their environment, implicating QS in population-wide gene regulation. Our group is interested in expanding our understanding of the microbiome beyond metagenomic and metabolomic sequencing by enabling the study of bacterial communication systems as it relates to host-microbiome interactions.
There is currently an unmet need for tools enabling quantification of QSM’s, and whole-cell biosensors (WCB’s) can help to fill that void. WCBs integrate molecular recognition with signal generating proteins to create a sensing cell that can be integrated in a device to detect a target molecule(s). The goal of this proposal is to investigate DPO’s currently unexplored relevance to host health and disease by developing a new biosensing tool capable of the specific and selective quantitative detection of DPO in biological samples. The central hypothesis of this project is that the development of a WCB for detection of the novel AI, DPO, will provide insight into and highlight the relevance of bacterial communication systems within the microbiome in the context of health and disease states.
Quorum Sensing as a Biomarker in Inflammatory Bowel Disease
Crohn’s Disease (CD), a type of Inflammatory Bowel Disease (IBD), is an immune-mediated, chronic inflammatory disease affects approximately 565,000 individuals in the United States. Although considered a disease of the Western world, CD incidence is on the rise in the developing world as well as in younger populations; thus, there is increasing need for better diagnosis and management of CD. The field continues to rely on colonoscopy (see image below of a healthy and inflamed colon) for a diagnosis of CD and to assess mucosal healing in response to therapy. Unfortunately, this is an invasive procedure carrying risk and is unpleasant for patients to have performed frequently.
The microbiome plays a critical role in the initiation and perpetuation of intestinal inflammation in Crohn's disease patients with studies of the microbiota in CD patients revealing a decrease in overall diversity. With such changes in the composition of the bacterial community, in collaboration with Dr. Maria T. Abreu, Director of the Crohn’s and Colitis Center at the University of Miami Miller School of Medicine, we wished to determine how this impacts bacterial quorum sensing (QS), the community communication system employed by bacteria. As such, we determined the levels of specific classes of QSMs in the blood serum of individuals with CD and active intestinal inflammation. Our results, shown below, indicate that bacterial QSMs act as a serum marker for intestinal inflammation in individuals with CD and may prove a biomarker of disease state.
The Role of Quorum Sensing in Colon Cancer
Dysbiosis is a consistent finding in IBD and colitis associated cancer (CAC), however, actionable strategies to reduce inflammation and/or CAC risk through microbiome manipulation are not yet available. Quorum sensing molecules (QSMs) are secreted by bacteria and are responsible for bacterial communication, biofilm formation, and activation of virulence systems. Recently, specific QSMs have been shown to stimulate oncogenic pathways in mammalian cells including pancreatic cancer and colonic mucosa.
To determine how bacterial QSM levels in serum correlate with CAC development, in collaboration with Dr. Maria T. Abreu, we assessed a sub-population of individuals at high risk for development of CAC. Individuals selected had been diagnosed with Ulcerative Colitis (UC) >10 years at the point of evaluation and either had active colonic inflammation or quiescent disease. As a comparison, we selected individuals who had been diagnosed with UC for <5 years and either had active or quiescent disease. As shown below, we found that patients with UC >10 years and active disease had elevated levels of certain classes of QSMs compared to the other groups, indicating their potential role as a biomarker or underlying initiator of CAC.
Host-Microbiome Interactions in Spinal Cord Injury
In the United States each year approximately 11-12,000 new spinal cord injuries (SCI) occur resulting in a spectrum of neurological impairments and disabilities. Currently, there are approximately 250,000 people living in the US with the devastating neurological deficits and somatic and autonomic reflexes that develop with chronic SCI. Chronic SCI causes interrelated secondary complications that significantly compromise health and quality of life after injury, including pathological dysfunction in the autonomic nervous system, immune system, and gastrointestinal tract.
Work by our group, in collaboration with Dr. Dalton Dietrich, Scientific Director of the Miami Project to Cure Paralysis and the University of Miami Miller School of Medicine, has determined that SCI causes short- and long-term alterations to intestinal inflammation, the gut microbiome, and bacterial QSM levels in a rat model of SCI. Ongoing studies in our laboratory are working to translate these findings into individuals living with SCI, with the intent to determine the molecular mechanisms underlying the GI dysfunction within these individuals and thus potentially develop therapeutics to restore function. As shown in the figure below, alterations to the gut microbiome (beta diversity) have been determined in individuals living with SCI as compared to control.
Effect of the Microbiome in the Gut-Brain Axis
Key to unlocking the role of the gut microbiome is to understand the interactions with its environment. A potential link between the gut and the brain is the mammalian neurotransmitter serotonin. More than 90% of the body’s monoamine serotonin is synthesized by gut enterochromaffin cells. However, the molecular mechanism that dictates the levels of serotonin produced and its metabolism is not fully elucidated. This is of utmost importance given that gut-derived serotonin is responsible for regulation of functions such as bone development, immune responses, gut motility, and platelet aggregation. Perhaps more interesting is the role that dysregulation of serotonin plays in the pathogenesis of certain intestinal diseases such as irritable bowel syndrome (IBS).
To this end, our research has demonstrated that serotonin can act as a bacterial communication molecule for pathogenic bacteria, specifically Pseudomonas aeruginosa. Serotonin increases Pseudomonas virulence factor and biofilm production in vitro and enhances virulence in infected mice. These findings are beneficial in understanding the impact of the microbiome on human health and may open the door for novel treatments for human disease.
The Role of Quorum Sensing in Depression
Major depressive disorder is a debilitating disease with a lifetime incidence of ~20% with current treatments often lacking efficacy and taking several weeks to be effective. Recent evidence has pointed toward a role of the gut microbiome composition in exacerbating psychiatric disorders, including major depression. The bidirectional communication between the gut and the brain is thought to involve neural, hormonal, and immunological routes, including the sympathetic and parasympathetic arms of the autonomic nervous system and the enteric nervous system. Dysregulation of this communication often leads to pathophysiological effects.
As part of ongoing studies in our group, in collaboration with Dr. Eleonore Beurel, we have investigated alterations in the microbiome and quorum sensing molecules in a murine model of depressive-like behaviors (learned helplessness) and found that the specific quorum sensing molecule, autoinducer-2 (AI-2), can induce depressive-like behaviors. Further, these effects can be alleviated through inhibition of the AI-2 pathway. Continued investigations will allow us to better understand the role bacterial quorum sensing plays in depression and potentially lead to development of therapeutic interventions.
Examining the Association between the reproductive tract Microbiome and successful implantation of embryos
In collaboration with Dr. Jonah Bardos, we are researching whether the microbiome inside a woman’s reproductive tract (vaginal, cervical, and uterine) have an effect on the success of an in vitro fertilization (IVF) cycle. In addition, we are evaluating a new method to see if we can predict the success or failure of a cycle before we even transfer the embryo. We are sampling discarded instruments after they are used as well as obtaining samples from the lower reproductive tract in order to determine the presence and levels of bacterial quorum sensing molecules (QSMs). This is very exciting and cutting-edge research that has the potential to change the way IVF is practiced. Currently, we have confirmed the presence of a category of QSMs within cervical swabs from healthy individuals as shown below.
Understanding the Role of the Microbiome in Endometrial Cancer
Endometrial cancer (EC) is the most common gynecologic malignancy in the United States, yet there is no screening test for it. The incidence of this disease has increased, such that it is now one of the leading causes of cancer death in women. Metagenomics, or the comprehensive evaluation of the microbial genome, is a novel way to understand the vaginal microbial environment and has revealed differences in diversity and phyla abundance between benign and tumor conditions, and across tumor grades, which also correlated with tumor genomics. As such, in collaboration with Drs. Matthew Schlumbrecht, Vice Chair of Global and Community Health at the University of Miami Miller School of Medicine, and Sophia George, we have begun investigations into bacterial quorum sensing (QS) in EC. As shown below, classes of quorum sensing molecules from endometrial swabs were found to have different levels depending upon low-grade (LG) EC, high-grade (HG) EC, or from healthy controls.
Effect of Environmental and Occupational Exposure to PAHs on the Microbiome
The routine exposure of firefighters to numerous toxic agents may affect their reproductive health, which can lead to miscarriages and infertility. An overall challenged reproductive potential has been associated with altered vaginal microbiome composition and dysbiosis. In this project we evaluate the vaginal microbiota of active firefighters and controls via Shotgun Metagenomic sequencing, while also assessing the levels of polycyclic aromatic hydrocarbons in vaginal swabs, with the aim of investigating a potential association between environmental and occupational exposures, and alterations of the microbial profile.