A Mycobacterial Systems Resource for the Research Community.

Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulceransIMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.

Fetal hematopoietic stem cell transplantation into beta-thalassemic mice.

The transplantation of fetal-derived hematopoietic stem cells (HSCs) may potentially be used to treat hemoglobinopathies, immunodeficiencies, and storage diseases. The levels of donor cell engraftment needed to reconstitute the recipient's hematopoietic system are disease-dependent and remain unknown for most deficiencies. We have explored the application of fetal hematopoietic stem cell transplants for the amelioration of hemolytic disease in a murine model of beta-thalassemia. Nonirradiated neonatal homozygous beta-thalassemic mice were transplanted intraperitoneally (IP) with 10(6) fetal liver cells from syngeneic nonthalassemic murine fetal donors (14 to 16 days gestation). Donor hemoglobin was demonstrated in the peripheral blood of 9 of 14 transplant recipients at levels ranging from 8.8% to 27.1% at 30 days. The levels of engraftment in 6 of these 9 transplant chimeras remained stable or increased up to 150 days after transplantation, with levels ranging from 13.6% to 54.6% at 280 days. Three chimeras have demonstrated gradually decreasing engraftment after 200 days. The degree of engraftment correlated with clinically relevant improvement: decreased reticulocyte counts (8.4% to 15.7% in chimeras [n = 9] v 17.1% to 19.1% in controls [n = 8], P = .01), increased mean RBC deformability, and the significant reduction in extramedullary hematopoiesis and iron deposits seen on histological examination of chimeric liver and spleen. These data demonstrate that fetal HSC transplants results in significant long-term chimerism with favorable alterations in red cell characteristics, and decreased hemolytic anemia in beta-thalassemia.