Circadian rhythms regulate our physiological, metabolic, and behavioral activity in a 24-hour cycle to maintain organismal homeostasis. In virtually all tissues, the intrinsic circadian clock is governed by a canonical molecular transcriptional-translational feedback mechanism that is self-sustained. Whether additional functions for the molecular circadian clock exist remains largely unexplored. Here we investigate an unprecedented role for the circadian clock protein, BMAL1, in the nucleolus. We identified novel BMAL1 interactions with resident nucleolar proteins using biochemical fractionation and pull-down assays. Moreover, through use of molecular assays we reveal a critical role in pre-ribosomal RNA processing and maturation. Instead, environmental-mediated synchronization of peripheral tissues is regulated in part by external cues including light-dark, sleep-wake, and feed-fasting cycles. The degree to which non-photic environmental factors perturb the circadian clock in the brain continues to be a major point of interest. We studied an in vivo model to elucidate the effects of drugs of abuse as stressors on the striatal circadian clock. Mice treated with an acute dose of cocaine were sampled throughout a full circadian cycle. Using high-throughput RNA sequencing, our findings show cocaine drastically reprograms the circadian transcriptional clock in the nucleus accumbens. Furthermore, we reveal dopamine signaling is required for the cocaine-mediated PPARgamma activation that leads to de novo circadian transcriptional rhythmicity. Taken together, in the studies presented, we implement two discrete approaches to further our knowledge on the versatility and reprogrammability of the molecular clock. Our novel findings provide previously unexplored connections between RNA regulation or drugs of abuse and the circadian clock, and uncover new possible therapeutic targets for treatment development for disorders such as addiction.

Enterobacteriaceae are a family of Gram-negative bacteria that cause morbidity and mortality worldwide. Infections with these organisms may cause foodborne diarrheal illnesses, urinary tract infections, and sepsis. These pathogens pose a serious global public health threat that is exacerbated by the increase of antibiotic resistance, the dearth of new antibiotics in the drug pipeline, and the limited availability of vaccines. To slow the emergence of antibiotic resistance and reduce the incidence of secondary infections, narrow-spectrum therapeutic strategies that limit the disruption of the host microbiota, which comprises beneficial microbes that provide colonization resistance to pathogens, are needed.

Iron is an essential nutrient for almost all organisms, and bioavailable iron is limiting in the vertebrate host. Thus, scavenging iron from the host environment using small molecule iron chelators called siderophores is an essential process for the pathogenesis of infection. Therefore, bacterial iron acquisition represents an appealing target for the development of new therapeutic approaches that are needed in the wake of antibiotic resistance. In this work, we have developed two new possible strategies to target pathogens belonging to the Enterobacteriaceae family that are siderophore-producers.

In particular, we provide the first evidence that antimicrobial proteins termed microcins are important for inter- and intraspecies competition among enterobacteria during intestinal inflammation. Indeed, we demonstrate that E. coli Nissle 1917, a prominent probiotic organism originally isolated from the human gut, utilizes microcins to inhibit a variety of competitors: commensal E. coli, the pathobiont (a commensal strain that can become pathogenic in certain conditions) adherent invasive E. coli, and the pathogen Salmonella. Microcins produced by E. coli Nissle 1917 are post-translationally modified at the C-terminus with a siderophore moiety. Therefore, these microcins can target competing bacteria through siderophore receptors, a mechanism described as a “Trojan horse” mode of action. In this work I show for the first time that this mechanism plays a role in vivo, as these microcins coupled to siderophores promote competition among enterobacteria in the inflamed gut, where iron is limited.

As a second way to target pathogens that utilize siderophore to acquire iron, we employed a novel immunization strategy to target siderophores with antibodies. Specifically, in collaboration with Dr. Elisabeth Nolan and Dr. Phoom Chariatana at MIT, we synthesized the enterobactin siderophore to be then linked to an immunogenic carrier protein. We found that our immunization strategy induced the development of specific anti-siderophore antibodies and successfully reduced Salmoenlla gut colonization. Moreover, we found that this siderophore-capture mechanism mediated by the adaptive immune system provided narrow-spectrum growth inhibitory activity, as other member of the microbiota were unaffected by our immunization.

Altogether, we demonstrated that microcins are a source of narrow-spectrum antimicrobials that target siderophore-producing Enterobacteriaceae in the inflamed gut. In addition, we provide first evidence of a successful immunization against bacterial siderophores in order to reduce pathogens’ growth.