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.

Characterization of the effect of AG337, a novel lipophilic thymidylate synthase inhibitor, on human head and neck and human leukemia cell lines.

Effects of lipophilic thymidylate synthase (TS) inhibitor AG337 on human head and neck squamous cell carcinoma (HNSCC) cell lines and CCRF-CEM human leukemia cells and sublines with acquired methotrexate (MTX) resistance were assayed using continuous or intermittent drug exposure. During 120-h continuous exposure, HNSCC cell lines A253 and FaDu are equally MTX sensitive (EC50 equals approximately 15nM); AG337 is less potent (EC50 approximately equals 1 microM). A253 is intrinsically resistant to 24-h intermittent MTX exposure (EC50 equals approximately 17 microM; FaDu, EC50 equals approximately 0.3 microM); both HNSCC cell lines are resistant to 24-h AG337 exposure (EC50 >100 microM). CCRF-CEM shows MTX (EC50 =14 nM) and AG337 (EC50 equals approximately 0.6 microM) sensitivity in continuous exposure similar to HNSCC; however, AG337 retains potency against CCRF-CEM cells in intermittent exposure (24-h, EC50 equals approximately 2 microM; 6-h, EC50 equals approximately 48 microM). The reduced folate leucovorin (LV) at > or = 0.1 microM fully protects from growth inhibition by continuous MTX exposure, but growth inhibition by AG337 is reversed only slightly by < or = 100 microM LV. Thymidine fully protects A253 and FaDu against growth inhibition by AG337, while hypoxanthine alone is without effect, suggesting inhibition is TS-specific. CCRF-CEM sublines with acquired MTX-resistance resulting from DHFR overexpression, defective MTX transport, or defective MTX polyglutamylation retain full sensitivity to AG337 in continuous exposure (all EC50 =0.4 microM). These data indicate that AG337 may be useful in therapy of tumors that have acquired resistance to MTX by most common mechanisms.

Thymidylate synthase as a target for growth inhibition in methotrexate-sensitive and -resistant human head and neck cancer and leukemia cell lines.

Thymidylate synthase (TS) inhibitor effects on growth of human head and neck squamous cell carcinoma (HNSCC) cell lines and CCRF-CEM human leukemia cells and sublines with acquired methotrexate (2,4-diamino-10-methylpteroylglutamic acid) (MTX) resistance were studied. During 120-h treatment, HNSCC cell lines A253 and FaDu are equally sensitive to MTX, whereas the polyglutamylatable TS inhibitors ZD1694 and BW1843U89 are 5- to 35-fold more potent than MTX and the lipophilic AG331 is approximately 10(2)-fold less potent than MTX. A253 is intrinsically resistant to intermittent (24 h) MTX and BW1843U89 exposure (higher EC50 values and shallower slopes of concentration-response curves relative to FaDu); AG331 and ZD1694 largely overcome this intrinsic resistance to intermittent exposure. Thymidine (TdR) protects against growth inhibition by these inhibitors, confirming that TS is their target in HNSCC; at high AG331 levels, TdR only partially protects, implying that a second site of action exists. Growth inhibition of HNSCC by ZD1694 and BW1843U89 is protected by leucovorin (LV) at > or = 10(-7) and > 10(-3) M, respectively; 10(-4) M LV cannot protect HNSCC cells against AG331. Results similar to protection studies are obtained if LV addition is delayed < or = 24 h after ZD1694 or BW1843U89 exposure. CCRF-CEM sublines with acquired MTX resistance resulting from dihydrofolate reductase (DHFR) overexpression, defective MTX transport, or defective MTX polyglutamylation retain full sensitivity to AG331. Cells with defective MTX transport are highly cross-resistant to ZD1694 and BW1843U89, implicating the reduced folate/MTX carrier in their transport. Minor cross-resistance of the DHFR overexpressing line to ZD1694 and BW1843U89 is observed. A subline with highly defective MTX polyglutamylation is cross-resistant to 120-h exposure to ZD1694, but not to BW1843U89, suggesting a profound contribution of polyglutamylation to the mechanism of action of ZD1694.