Mechanisms of hypervirulent Klebsiella pneumoniae gastrointestinal colonization and dissemination

Parent Project Abstract. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae (Kpn), a gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat as Kpn has become multi-drug resistant. Epidemiological studies suggest that gastrointestinal (GI) colonization of Kpn is a major reservoir through which Kpn can cause disease manifestations either in the colonized host or transmit from host to host. This site of Kpn colonization has not been the focus of previous studies as a tractable model of Kpn GI colonization, and host-to-host transmission did not exist. We have recently developed a murine model that allows for the study of Kpn mucosal (oropharynx and GI) colonization, shedding within feces, and transmission through the fecal-oral route. Using an oral route of inoculation and fecal shedding as a marker for GI colonization, we show that Kpn can asymptomatically colonize the GI tract of immunocompetent mice and modifies the host GI microbiota. We premise that specific Kpn genes contribute to its GI colonization, and the products of these genes could serve as novel targets for the prevention of the establishment of GI colonization. More recently, we used our murine model to screen a library of Kpn random transposon mutants (In-seq) to identify the complete set of "GI colonization" genes from a single isolate. A metagenomics sequencing analysis further identified bacterial species and the metabolic pathways affected by Kpn in the GI tract. Herein, we will focus on two sets of pathways identified through In-seq whose products allow Kpn to overcome colonization resistance provided by the resident gut microbiota. Thus, in Aim#1, we will focus on the ethanolamine utilization pathway genes (eut) of Kpn that allow it to utilize ethanolamine (EA), a byproduct of cellular membranes and diet in the gut that can serve as an alternative nutrient source. Unlike many other enteric pathogens that contain a single eut operon, Kpn has two genetically distinct eut operons. We will identify the role of each eut locus in EA metabolism and determine the underlying molecular mechanism through which EA metabolism provides Kpn with a fitness advantage against members of the microbiome. Aim#2 will take a different approach by focusing on the contact-dependent killing machinery of the Kpn (Type 6 secretion system [T6SS]) in overcoming colonization resistance provided by the resident microbiota. We will focus on the unique regulatory mechanism that modulates the expression of Kpn T6SS in the GI tract and provide it with a selective and competitive advantage against the resident gut microbiota. Results from these studies would provide us with a fundamental understanding of the molecular mechanisms involved in the establishment of GI colonization by an incoming pathogen. These studies will also lay the groundwork for developing potential strategies to reduce the Kpn disease burden.