Genetic Manipulation of Unreaplasma spp. Infecting Humans using Synthetic Genomic

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DESCRIPTION (provided by applicant): Ureaplasma urealyticum and Ureaplasma parvum are sexually transmitted human pathogens. Infection is widespread. As many as 40-80% of women are carriers. While most infections are asymptomatic, they cause invasive diseases in some patients. Although they have been associated with a wide range of diseases, their largest impact on society is as a cause of adverse pregnancy outcomes and infection of newborns, leading to higher risk of newborn mortality. Virtually all very-low-birth-weight newborns (less than 2.5 kg or 5.5 lbs) have respiratory ureaplasma infections. Ureaplasma infections resistant to antibiotics are increasingly common and the best drugs for ureaplasma treatment, fluoroquinolones and tetracyclines, are not recommended for pregnant women or children. Efforts to understand ureaplasma biology and pathogenesis have been inhibited by the lack of means to modify the DNA of these bacteria. The capacity to modify organisms' genomes has been a prerequisite in understanding host-pathogen interaction and factors involved in pathogenicity in many well studied pathogens. Development of genetic tools for the creation of designed mutant ureaplasmas will give scientists the power to identify factors making ureaplasmas pathogenic and how to better prevent disease due to ureaplasma infection. Recently our J. Craig Venter Institute research team created a bacterial cell with a chemically synthesized genome. To reach this synthetic cell milestone we developed foundation tools for the new science of Synthetic Genomics. One of those tools called "genome transplantation" enabled us to take a genome out of one species of bacteria called Mycoplasma mycoides, and install it into another species of bacteria. With another tool, we cloned the same M. mycoides genome inside a yeast cell, where it sits quietly parked. Once inside that yeast cell we can use well developed genetic tools used by yeast biologists to modify that genome by deleting or adding genes. These two methods in concert allowed us to make an altered M. mycoides strain. This new M. mycoides could not have been made using any existing technology. The goal of this project is to apply this Synthetic Genomics technology to develop genetic tools for creation of modified ureaplasma strains that will aid in the understanding of ureaplasma pathogenicity. Our specific aims are first to adapt our genome transplantation method for installation of a U. urealyticum genome into a U. parvum or U. diversum recipient cell. The second aim, which is of little use until aim one is achieved, is to clone that same ureaplasma genome in a yeast cell. The result of the two aims is the capacity to modify ureaplasma gene content and expression so that we can test hypotheses about ureaplasma pathogenesis and develop new therapies. Beyond the immediate advances for these poorly understood pathogens, there could be other even more important consequences. Currently we only know how to transplant the genome of one bacterial species. Learning how to transplant ureaplasma genomes could lead to our being able to do this for many other species including bacteria with unrealized pharmaceutical or industrial potential, for which we have no genetic tools. PUBLIC HEALTH RELEVANCE: Ureaplasmas are sexually transmitted pathogens that are important causes of adverse pregnancy outcomes and increased risk of low birthweight infant morbidity. Research into causes of ureaplasma pathogenesis and possible therapies has been hindered by the absence of genetic tools that would enable hypothesis driven studies. Methods invented for the new field of Synthetic Genomics to create a "synthetic bacterial cell" will be adapted for modification of ureaplasma genomes.