Role of TraJ in Neonatal E. coli Sepsis and Meningitis

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DESCRIPTION (provided by applicant): Despite the use of modern therapeutics and diagnostic measures, neonatal bacterial sepsis and meningitis continues to be a disease with unacceptable rates of morbidity and mortality. The fatality rate of this disease is 15-75 percent; furthermore, approximately 50 percent of meningitis survivors have significant neurological and developmental abnormalities. The primary, route of infection for E. coli K1 (the most common causative agent of gram-negative meningitis) is oral. Following intestinal colonization, bacteria translocate through the GI tract to extra-intestinal sites of mesenteric lymph nodes (MLN), liver, spleen, and the blood. E. coli K1 then multiply systemically within the bloodstream reaching a necessary threshold of bacteremia to gain access to the central nervous system (CNS). Recently we identified an E. coli K1 plasmid-encoded blood-brain barrier (BBB) invasion gene, traJ, with homology to traJ of various F-like plasmid tra operons. Our preliminary data indicate that the traJ mutation specifically attenuates dissemination from the MLN to the liver, spleen, blood, and the CNS in the neonatal rat. In addition, although animals orally infected with the traJ mutant demonstrated a decrease or no recoverable bacteria in the liver or spleen, these tissues showed a significant inflammatory response. In vitro studies determined that the traJ mutant is taken-up less by macrophages and shows a loss of a 55 kDa-secreted protein. The central hypothesis of this application is that TraJ controls the expression of a set of genes whose products (i.e., 55 kDa secreted protein) are involved in E. coli K1 dissemination, systemic infection and crossing the BBB, and these events within the disease process occur via TraJ-dependent interactions with professional phagocytes. The following proposed experiments are designed to test and substantiate our hypotheses. We aim to 1) Elucidate the genetic and molecular characteristics of the traJ region and plasmid, evaluate the ability of the endogenous plasmid to self-transfer, and determine the potential role of the traJ-containing plasmid in E. coli K1 neonatal rat virulence, 2) Evaluate the function of the TraJ-regulated proteins (i.e., 55 kDa-secreted protein) in E. coli K1 systemic dissemination and meningitis, and 3) Determine the mechanism of TraJ-dependent host inflammatory response in the neonatal rat. Improved knowledge of molecular mechanisms for early systemic dissemination and the exact interplay of the host inflammatory response during these events will assist in achieving our long-term goal of identifying novel rational approaches to development of new treatments and preventive strategies for E. coli K1 sepsis and meningitis.