Lac operon-dependent metabolism in Enterobacteriaceae and its role in liver disease-associated infections

PROJECT SUMMARY Dysbiosis, an alteration of the gut microbiome associated with chronic diseases, has been previously described in patients with cirrhosis. Common features of dysbiosis include reduced bacterial diversity and the outgrowth of the family of human pathogenic bacteria Enterobacteriaceae, including Escherichia coli. Infectious complications, namely spontaneous bacterial peritonitis (SBP) and bacteremia, are deadly for patients with cirrhosis with up to 50% mortality. E. coli is one of the most common causes of these infections, thought to derive from bacterial translocation across the intestinal epithelium from reduced barrier function. While this suggests a connection between dysbiosis and disseminated infection, the underlying risk factors of disseminated infection are not well understood in this population. Lactulose, a simple carbohydrate which is the first line treatment for the cirrhosis complication hepatic encephalopathy, has been associated with higher abundance of E. coli in the gut microbiota. Our work demonstrates that lactulose also increases colonization of Enterobacteriaceae, by overcoming carbon limitation in the colon for these pathogens. My preliminary data show that E. coli acquire mutations allowing it to utilize lactulose as a carbohydrate source both in culture and in the mouse gut, thus increasing its fitness. This phenotype is dependent upon constitutive expression of the lactose (lac) operon via deactivation of the transcriptional regulatory protein LacI, a repressor of the lac operon, which encodes the lactulose metabolizing enzyme β-galactosidase. I therefore hypothesize that lactulose treatment selects for mutant Enterobacteriaceae capable of metabolizing lactulose, thereby increasing colonization and the risk of disseminated infection in patients with liver disease. My objectives in this proposal are to characterize the gain of function mutations that enable lactulose metabolism by E. coli, determine the competitive advantage imparted by these mutations, and assess the impact of lactulose on disseminated infection in mice and humans with liver disease. My hypothesis will be tested through two inter-related Specific Aims that will evaluate the adaptive mutations and competitive fitness imparted by lactulose treatment to E. coli (Aim 1) and test the impact of lactulose on disseminated infection (Aim 2). This proposal takes advantage of several innovative techniques and unique resources including a novel mouse model of disseminated infection in liver disease and creation of a human disseminated infection strain library. The University of Pennsylvania is an ideal research environment for these studies given the local expertise in gut microbiome, pathogen biology, and comparative bacterial genomics. The candidate will acquire critical skills for his career development as an independent investigator, including in bioinformatics, bacterial genomics, and mouse disease modeling. Successful completion of this proposal will enable the investigator to reach their career goal to become an independently funded tenure-track faculty member, identifying the dietary and bacterial factors involved in gastrointestinal diseases.