Novel strategies to inhibit the growth of enteropathogens and pathobionts

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