A major gap in our understanding of infectious diseases is the lack of information about molecular interaction networks between an infecting pathogen and the human host, which has yet to be accomplished for a bacterial infection in humans. The purpose of this application is to define an interactome on a transcript level for the primary skin pathogen, Haemophilus ducreyi (HD), in experimentally infected human volunteers using RNA sequencing (RNA-seq) and metabolomics. HD causes chancroid, a genital ulcer (GU) disease that facilitates HIV transmission and is as a major cause of cutaneous ulcers (CU) in children in yaws-endemic countries. Efforts to eradicate HD-associated CU with antibiotics failed due to environmental reservoirs. To study the biology of HD, we developed a model in which the HD GU strain 35000HP and its derivatives are inoculated into the skin of the upper arm of adult volunteers. Whole genome sequencing shows that most CU strains are nearly identical to 35000HP, indicating that our model is highly relevant to GU and CU. In the model and in natural chancroid, HD resides in an abscess where it is surrounded by polymorphonuclear leukocytes and macrophages and remains extracellular by evading phagocytosis. In our new preliminary data, we show that an interaction network exists between HD and the host and that differential host and bacterial transcript expression correlates with metabolomic changes at infected vs. wounded sites. HD primarily upregulates the expression of genes involved in adapting to anaerobiosis and uptake and utilization of metal ions and alternative carbon sources, such as ascorbic acid, consistent with the idea of ?nutritional virulence.? Our new metabolomics data show that ascorbic acid pathways are upregulated in lesions. Thus, we hypothesize that the host regulates its gene transcription to phagocytize and limit nutrients to HD, that HD regulates its gene transcription to counteract these host defenses, that host gene expression correlates with metabolomic profiles at infected sites, and that bacterial genes that are involved in adaptation to the metabolic niche created by the host response will be required for virulence.
Our specific aims are 1) to define the metabolome and the interactome between HD and the human host in infected tissue and to correlate the host transcriptional response to metabolic changes in lesions; 2) to identify the cells responsible for the host transcriptional response using single cell RNA-seq; 3) to determine whether the HD genes involved in exploitation of the host niche are required for virulence in humans and the mechanism(s) by which these genes contribute to virulence. The importance of this study is that we will be the first to determine an interactome at a site of a bacterial human infection, define the host cells responsible for the transcriptional response, correlate the host response to metabolomic changes in lesions, and study how HD exploits these metabolites. We will provide new insights into the interaction between a model extracellular bacterium and the human host and unmask novel strategies to control HD-associated CU.
Definition of the how the host and an infecting bacterium interact with each other on a molecular level offers a new strategy to broaden our understanding of infectious diseases but has yet to be done in humans. The purpose of this application is to determine such a network for the skin pathogen Haemophilus ducreyi in experimentally infected human volunteers.