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Modulating the Action of Cylindrical Proteases to Eliminate Neisseria Gonorrhea and Chlamydia Trachomatis Infections
Project Summary/Abstract Sexually transmitted bacteria diseases caused by Chlamydia trachomatis (Ctr) and Neisseria gonorrhoeae (NG) are the two most common sexually transmitted bacterial diseases. The infections caused by these pathogens may result in infertility, ectopic pregnancy, blindness, and perinatal mortality. Over 1.70 M cases of chlamydia and 0.65 M cases of drug-resistant gonorrhea are reported yearly in the US. Women with gonorrhea are co- infected with chlamydia in 17.6%–57.9% of cases, while women with chlamydia are co-infected with gonorrhea in 2.1%–17.2% of cases. These infections are treated with broad spectrum antibiotics, which can favor the development of resistance on NG/CTr but also in other bacteria, or damage the microbiota, diminishing its protective function and allowing bacteria and viruses to infect the patient. The Caseinolytic protease (ClpP) proteolytic machinery regulates protein turnover and homeostasis and is key in bacterial growth and development The machinery consists of the proteolytic unit (the ClpP) and its chaperone (ClpX), which transports proteins to be degraded, and it is termed the ClpXP. Our theory is that molecules that inhibit the action of the ClpX chaperone can become efficient antibacterial agents against both pathogens. We have found that the dihydrothiazepines can erradicate both pathogens and prevent the action of the ClpXP complex. Our goal is to advance the dihydrothiazepines as selective agents against Ctr and NG infections. To develop these therapeutic agents, we have envisioned four specific aims. Specific Aim 1. Synthesis and Optimization of the Pharmacophore. Our goal is to use computational models to design dihydrothiazepines molecule that will be synthesized, purified, and characterized using chemical techniques. The molecules will be tested against Ctr and NG and their toxicity against human cells evaluated. Also, we will determine their effect in other bacterial, including those from the microbiota. Specific Aim 2. Assessment of Stability and In Vivo Activity. We will study the stability of the most active molecules under various conditions. Then, we will study the pharmacokinetics, biodistribution , and antibacterial activity against Ctr and NG in mice. Specific Aim 3. Target Validation and Effect. We will study the ability of the compounds to inhibit the activity of ClpX using a luciferase assay and to block protein degradation. We will try grow crystal of the protein and the molecule and will study if the molecules prevent the assembly of the ClpXP system. Finally, we will assess the ability of the bacteria to develop resistance to the molecules.
Host-pathogen-microbiome interactions in Mycoplasma genitalium pathology and treatment: experiments in a 3D organotypic cervical epithelium model to strengthen clinical guidelines
ABSTRACT Mycoplasma genitalium (MG) is an emerging sexually transmitted pathogen whose clinical outcomes in women are poorly understood. Unlike other bacterial sexually transmitted infections (STI), the CDC does not recommend MG screening for asymptomatic women because it is unclear how often asymptomatic MG leads to adverse reproductive outcomes like cervicitis, which can lead to further adverse outcomes, including pelvic inflammatory disease, infertility, and ectopic pregnancy. Epidemiologic data on MG and cervicitis are mixed, and mechanistic data primarily come from models that did not faithfully recapitulate in vivo cervical microphysiological conditions. Key elements they lacked are cervical mucus, which mediates host-pathogen interactions, and the cervicovaginal microbiota. The microbiota appears to contribute to MG outcomes, and our preliminary epidemiologic data indicate that MG and bacterial vaginosis (BV) may synergize to promote cervicitis. MG care is further complicated by its ongoing rise in antibiotic resistance. Resistance-guided therapy and novel antibiotics improve treatment outcomes, but these are not available in the US. Recent clinical and in vitro data indicate that metronidazole and tinidazole, two antibiotics that are available in the US and used to treat BV, may hold promise for improving MG treatment outcomes. The overall objective of this R21 is to generate robust experimental data to clarify MG pathology, evaluate potential therapies, and inform more thorough and actionable clinical recommendations. We developed an innovative in vitro 3D organotypic model of the cervical epithelium that is ideally suited for investigating MG pathology, host-MG-microbiota interactions, and potential therapies. The model uses primary human cervical cells and better recapitulates cervical epithelial structure and physiology (including cervical mucus production) than prior 2D models. It also allows for simultaneous STI infection and co- culture of live cervicovaginal microbiota. Using the 3D organotypic cervical epithelium model, we will determine if MG causes microbiota-dependent cervical epithelial damage, a hallmark of cervicitis (Aim 1), and we will test if metronidazole and tinidazole arrest MG infection (Aim 2). In both Aims, we will interrogate the potential mediating role of the microbiota by inoculating models with live representative cervicovaginal microbiota, and we will assess host-MG-microbiota interactions via transcriptomics. We hypothesize that a polymicrobial BV-like microbiota will exacerbate MG-induced cervical epithelial damage, and removal of a polymicrobial BV microbiota will partially mediate metronidazole’s and tinidazole’s anti-MG activity. The proposed Aims have high translational potential and will provide crucial pre-clinical evidence to inform more thorough and actionable MG testing and treatment guidelines and improve reproductive health outcomes. This R21 will generate some of the first experimental data on MG-host and MG-microbiota interactions, which we will use to support an R01 to validate these interactions during in vivo MG infection and identify novel therapeutic targets for MG.
Personalized medicine and predictive health and wellness: Adding the chemical component
Wearable sensors that detect and quantify biomarkers in retrievable biofluids (e.g., interstitial fluid, sweat, tears) provide information on human dynamic physiological and psychological states. This information can transform health and wellness by providing actionable feedback. Due to outdated and insufficiently sensitive technologies, current on-body sensing systems have capabilities limited to pH, and a few high-concentration electrolytes, metabolites, and nutrients. As such, wearable sensing systems cannot detect key low-concentration biomarkers indicative of stress, inflammation, metabolic, and reproductive status. We are revolutionizing sensing. Our electronic biosensors detect virtually any signaling molecule or metabolite at ultra-low levels. We have monitored serotonin, dopamine, cortisol, phenylalanine, estradiol, progesterone, and glucose in blood, sweat, interstitial fluid, and tears. The sensors are based on modern nanoscale semiconductor transistors that are straightforwardly scalable for manufacturing. We are developing sensors for >40 biomarkers for personalized continuous monitoring (e.g., smartwatch, wearable patch) that will provide feedback for treating chronic health conditions (e.g., perimenopause, stress disorders, phenylketonuria). Moreover, our sensors will enable female fertility monitoring and the adoption of more healthy lifestyles to prevent disease and improve physical and cognitive performance.
Hypothalamic episode generators underlying the neural control of fertility
The hypothalamus controls diverse homeostatic functions including fertility. Neural episode generators are required to drive the intermittent pulsatile and surge profiles of reproductive hormone secretion that control gonadal function. Studies in genetic mouse models have been fundamental in defining the neural circuits forming these central pattern generators and the full range of in vitro and in vivo optogenetic and chemogenetic methodologies have enabled investigation into their mechanism of action. The seminar will outline studies defining the hypothalamic “GnRH pulse generator network” and current understanding of its operation to drive pulsatile hormone secretion.
Brain-body interactions in the metabolic/nutritional control of puberty: Neuropeptide pathways and central energy sensors
Puberty is a brain-driven phenomenon, which is under the control of sophisticated regulatory networks that integrate a large number of endogenous and environmental signals, including metabolic and nutritional cues. Puberty onset is tightly bound to the state of body energy reserves, and deregulation of energy/metabolic homeostasis is often associated with alterations in the timing of puberty. However, despite recent progress in the field, our knowledge of the specific molecular mechanisms and pathways whereby our brain decode metabolic information to modulate puberty onset remains fragmentary and incomplete. Compelling evidence, gathered over the last fifteen years, supports an essential role of hypothalamic neurons producing kisspeptins, encoded by Kiss1, in the neuroendocrine control of puberty. Kiss1 neurons are major components of the hypothalamic GnRH pulse generator, whose full activation is mandatory pubertal onset. Kiss1 neurons seemingly participate in transmitting the regulatory actions of metabolic cues on pubertal maturation. However, the modulatory influence of metabolic signals (e.g., leptin) on Kiss1 neurons might be predominantly indirect and likely involves also the interaction with other transmitters and neuronal populations. In my presentation, I will review herein recent work of our group, using preclinical models, addressing the molecular mechanisms whereby Kiss1 neurons are modulated by metabolic signals, and thereby contribute to the nutritional control of puberty. In this context, the putative roles of the energy/metabolic sensors, AMP-activated protein kinase (AMPK) and SIRT1, in the metabolic control of Kiss1 neurons and puberty will be discussed. In addition, I will summarize recent findings from our team pointing out a role of central de novo ceramide signaling in mediating the impact of obesity of (earlier) puberty onset, via non-canonical, kisspeptin-related pathways. These findings are posed of translational interest, as perturbations of these molecular pathways could contribute to the alterations of pubertal timing linked to conditions of metabolic stress in humans, ranging from malnutrition to obesity, and might become druggable targets for better management of pubertal disorders.
Central SELENOT expression regulates gonadotrope axis function, sexual behavior, and fertility in male and female mice
FENS Forum 2024
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