Research

Drug use and substance use disorders (SUDs) affect more than 27 million people in the United States, leading to an enormous burden to society; and yet, no successful evidence-based treatments have been developed. In the past years, the gut-to-brain axis has proven to be a crucial regulator of brain maturation, plasticity and function. Our research seeks to identify clear and mechanistically defined relationships between drug exposure, gut microbiota composition and vulnerability to develop SUDs. It is our goal to generate highly valuable data to develop systemic evidence-based interventions and effective therapeutic strategies for better manage drug abuse and SUDs. The uniqueness of our approach stems from combining strong microbiology and drug abuse research. Our main findings have shown that that cocaine exposure increases intestinal levels of norepinephrine, which is sensed through the bacterial-adrenergic receptor QseC to promote intestinal colonization of Proteobacteria (E. coli). This shift in microbiota composition leads to depletion of glycine in the gut and cerebrospinal fluid and enhance host cocaine-induced behaviors. Importantly, repletion of glycine reversed this exacerbated response. Moreover, intestinal colonization by Proteobacteria (E. coli) unable to uptake glycine did not alter the host response to cocaine. Microbiota-modulated glycine levels changed cocaine-induced pathways in the brain leading to the enhanced addiction behaviors observed. Together, these results introduce a mechanism by which intestinal bacteria alter the host’s metabolic landscape and brain responses and drug-associated behaviors.

We described an inter-kingdom chemical signaling system between bacteria and host that is exploited by deadly pathogens such as enterohemorrhagic E. coli (EHEC) to activate its virulence genes. This work described a novel chemical signaling molecule produced by the human gastrointestinal (GI) microbiota named AI-3; it described the connection between bacterial intercellular signaling and host neurotransmitter signaling (specifically the host stress hormones epinephrine and norepinephrine). We found that an a-adrenergic antagonist (phentolamine; PE) can specifically block the QseC response to these signals. We also showed that QseC directly binds to norepinephrine, and that this binding is inhibited by PE. Furthermore, we demonstrated that a qseC mutant is attenuated for virulence in animals, underscoring the importance of this signaling system to bacterial virulence “in vivo”. Finally, an in silicosearch found that QseC is conserved among several bacterial species. These “in vitro” and “in vivo” data suggest a pivotal role for QseC in bacterial pathogenesis and inter-kingdom signaling. The QseC sensor constitutes an example of a receptor for both a bacterial and a host signals, thereby integrating bacterial-host signaling at the biochemical level.

There are further links of QseC signaling with the gut-brain-axis that include the host endocannabinoid system (in the gut) and the intestinal microbiota. Compositional manipulations of the gut microbiota through antibiotic treatment or through dietary interventions have been shown to alter endocannabinoid tone in intestinal tissues. Similarly, abrogation of host endocannabinoid signaling has also been associated with changes in the GI microbiota. We have been building on this body of work to define a molecular mechanism by which a host-derived endocannabinoid can be sensed by gut bacteria and potentially modulate bacterial behavior. We have shown that 2-Arachidonoylglycerol (2-AG), an endocannabinoid and endogenous agonist of the CB₁ receptor and primary ligand for the CB2 ligand, inhibits virulence-associated type III secretion activity in gut pathogens, resulting in reduced pathogen burdens and attenuated colitis. This occurs through 2-AG antagonism of the pro-virulence bacterial receptor QseC, which is required for full activation of essential type III secretion-dependent virulence programs in enteric pathogens. Our findings are consistent with the anti-colitic effects that have associated with augmented host endocannabinoid signaling. More broadly, because QseC is encoded within the core genomes of Enterobacteriaceae species, it is tempting to speculate that 2-AG may also modulate additional aspects of bacterial function in both pathogenic and commensal bacteria. Thus, our findings introduce the possibility that intestinal bacteria may serve as an additional and potentially “druggable” signaling node within the enteric endocannabinoid system that impacts GI physiology, immunity, and susceptibility to infection.

Further studies in the gut-brain-axis concern the integration of sensing of bacteria and host-derived Tryptophan derivatives. The enteric nervous system (ENS) highly innervates the GI tract, ensuring prominence of neurotransmitters. The serotonin neurotransmitter is primarily synthesized in the GI tract, where it is secreted into the lumen and subsequently removed. We showed that serotonin decreases virulence gene expression from EHEC and Citrobacter rodentium (extensively used a surrogate murine infection model for EHEC). We also identified the membrane bound histidine sensor kinase CpxA as a bacterial serotonin receptor. Using knockout animals and pharmacological inhibitors, we showed that the presence of higher levels of serotonin in the intestine of mice decreased virulence in C. rodentium, whereas decreased levels are conducive to pathogenesis. Importantly, the fact that bacteria have receptors to neurotransmitters opens a broad array of possibilities as to how the gut-brain axis functions as a two-way street between microbes and their mammalian hosts. This knowledge is especially compelling given that various serotonin agonists and antagonists have been developed to treat diarrhea-predominant inflammatory bowel disease (IBS) and/or constipation. The fact that one can conceptually co-opt these drugs already used clinically (e.g. Prozac) to treat infectious diseases is potentially exciting. CpxA also recognizes the microbiota-derived signal indole. It is also noteworthy that indole produced by the gut microbiota can accumulate in the brain and thus impact host behavior. This integration of inter-kingdom signaling by one bacterial histidine kinase (HK) sensing a host neurotransmitter and a bacterial signal seems to be a recurring theme in biology. Importantly, these inter-kingdom signaling systems that engage neurotransmitters may not be restricted to impacting the pathogenesis of EHEC and C. rodentium. Many GI pathogens, such as Salmonella, Yersinia enterocolytica, Shigella disenteria, and others, encode the CpxA serotonin/indole receptor.

Our lab has also shown that enteric pathogens exploit nutritional cues (carbon and/or nitrogen sources) made available by the gut microbiota as signals to coordinate virulence regulation and modify the pathogen’s metabolism (allowing for efficient host colonization). We described a novel two-component system that we named FusKR. The sensor kinase FusK, an eight transmembrane protein, senses fucose released from the mucus by the microbial flora (Bacteroidetes). Our findings suggest that EHEC uses fucose from the microbiota to modulate EHEC pathogenicity and reveals an added layer of complexity in the inter-kingdom signaling that underlies EHEC pathogenicity.

We have also been performing many high throughput screens to catalogue regulatory pathways that influence expression of the type III secretion system encoded within EHEC’s locus of enterocyte effacement (LEE) pathogenicity island. This has established a dataset of regulatory pathways that control EHEC virulence expression under anaerobic conditions. Major findings have included that amino-acid and fatty acid metabolism converge with redox sensing, linking two transcription factors, CutR and FadR, in modulating virulence expression. This high throughput approach proved to be a powerful tool to map the web of cellular circuits that allows an enteric pathogen to monitor the gut environment and adjust the levels of expression of its virulence repertoire.

In the footsteps of screening efforts, we also described yet another example of microbiota-pathogen “collaboration”. Galacturonic-acid is a carbon source made available in the gut by saccharolytic members of the microbiota, such as Bacteroides thetaiotamicron, through the digestion of dietary pectin. Galacturonic-acid is used by EHEC and C. rodentium in the gut as a carbon source, aiding these pathogens’ initial expansion. Galacturonic-acid is sensed through ExuR (identified in screens) which, in the absence of this sugar acid, acts as a transcriptional activator of the LEE as the infection progresses. Importantly this ExuR-galacturonic-acid regulation plays a key role in the establishment and progression of C. rodentium murine infection. In this scenario, the pathogen’s ability to gage the concentration of galacturonic-acid, and its ability to moonlight it as a nutrient and a signal, plays a key role in its adaptation to the GI environment.

Also derived from HTS screens came our discovery that exogenous arginine is sensed by enteric pathogens in the colon to enhance virulence gene expression. identified the arginine sensor ArgR as a regulator of virulence genes in EHEC and C. rodentium (extensively used as a surrogate murine infection model for EHEC)) and showed that ArgR senses arginine fluctuations and regulates virulence both in vitro and during murine infections.  At peak C. rodentiuminfection, increased arginine concentrations in the colon correlated with downregulation of the host SLC7A2 arginine transporter. These findings suggest that a delicate balance exists between host and pathogen responses to arginine during disease progression.