Fig 1.
Chemo-genomic characterization of antifolate resistance determinants in M. smegmatis.
(A) Simplified enzymatic conversions of folate derivatives in de novo biosynthesis and the one-carbon metabolic network in bacteria. Abbreviations: H4PteGlun, tetrahydrofolate (green) serves as carrier for one-carbon groups. AICART, aminoimidazolecarboxamide ribonucleotide transferase; DHFS, dihydrofolate synthase; DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthase; FTD, 10-formyltetrahydrofolate dehydrogenase; FTS, 10-formyltetrahydrofolate synthetase; Gly, glycine; GTP, guanosine triphosphate; H2PteGlun, dihydrofolate; Hcy, homocysteine; Met, methionine; MS, methionine synthase; MTCH, methylenetetrahydrofolate cyclohydrolase; MTD, methylenetetrahydrofolate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase; MTHFS, 5,10-methenyltetrahydrofolate synthetase; pABA, para-aminobenzoic acid; PGT, phosphoribosyl glycinamide transferase; Pte, pteroate; PteGlu1, folic acid; Ser, serine; SHMT, serine hydroxymethyltransferase; TS, thymidylate synthase. Two different types of TS have been described: ThyA and ThyX. While most organisms contain either ThyA or ThyX, some organisms including M. tuberculosis have both. Reactions directly involved in the methylfolate trap (MS) and thymineless death (TS) are highlighted in yellow and red, respectively. (B) Genome distributions of antifolate resistance determinants in M. smegmatis. Laboratory assigned catalog numbers (n = 1–50, S1 Table) were plotted against their corresponding locus tags (msmeg_No.). (C) A typical SULFA susceptibility and chemical complementation assay of M. smegmatis strains. A pool of antifolate sensitive mutants was replicated onto NE plates, in top-down order: (i) control, (ii) SCP, (iii) SCP plus PteGlu1, (iv) SCP plus 5-CHO-H4PteGlu1, (v) SCP plus 5-CH3-H4PteGlu1, and (vi) SCP plus pABA. SCP was used at 10.5 μg/ml while supplements were used at 0.3 mM final concentration. Colonies marked with “C” were from the parental strain mc2155, which was used as a control. Colonies marked with asterisks were from the “white” mutants.
Fig 2.
Methylfolate trap in Mycobacterium smegmatis.
(A) A model depicting the chemical conversions and factors involved in the methylfolate trap-mediated SULFA sensitivity. The CH3- group in 5-CH3-H4PteGlun is first transferred to the B12 cofactor, which further transfers it to homocysteine (Hcy) to make methionine (Met). The MetH reaction thereby recycles 5-CH3-H4PteGlun back to free H4PteGlun which continues the flow of the one-carbon network. (B) Chemical complementation of M. smegmatis “white” mutants mapped to metH or cobIJ. The strains exhibited increased SULFA susceptibility and impaired 5-CH3-H4PteGlu1 utilization. Approximately 5x103 cells were spotted onto NE medium added with 10.5 μg/ml SCP with or without exogenous supplements. Unlike wild type and other mutants, these mutants were unable to use 5-CH3-H4PteGlu1 to antagonize SCP. Exogenous B12 restored 5-CH3-H4PteGlu1 utilization and SCP resistance to cobIJ but not metH mutants. (C) Effect of metH and cobIJ on the folate pool in M. smegmatis. Growing cultures of M. smegmatis strains were treated with 285 μg/ml SCP for 30 min followed by folate extraction and LC-MS/MS analysis. Data shows the combined levels of all 5-CH3-H4PteGlun species (top), all non-methyl folate species (middle), and the total folate (bottom). Bars represent means of biological triplicates with standard deviations. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference between the indicated strains. (D) Targeted mutagenesis confirms the roles of metH and cobIJ in methylfolate trap-induced SULFA sensitivity and 5-CH3-H4PteGlu1 utilization in M. smegmatis. Paper discs were embedded with 0.5 mg SCP and placed at the center of the medium surface, seeded with bacterial strains. Exogenous B12 and 5-CH3-H4PteGlun were used at 0.3 and 1 mM, respectively. Genetic complementation was achieved by in trans expression of metH or cobIJ. B12 alone restored wild type SULFA resistance level to MsΔcobIJ, whereas the combination of 5-CH3-H4PteGlu1 and B12 completely abolished SULFA resistance to all strains but MsΔmetH.
Table 1.
Susceptibility of bacterial strains to antifolates.
Fig 3.
Methylfolate trap in Mycobacterium tuberculosis.
(A) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 104 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. (B) H37Rv-derived strains were grown to OD600 of 1 and 5 μl cultures were spotted onto 7H10-OADC or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B12, and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10−6 dilution plates provide a visual comparison between strains in viability (top). (C) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE (http://prosite.expasy.org). Domains are labeled as the cofactors to which they bind. (D) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B12 and methionine was analyzed using the MTT method. Cultures growing at an OD600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 104 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. (E) Survival of H37Rv (Red), its derived metH mutant (RvΔmetH, Blue) and the complemented strain (RvΔmetH/metH, Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p<0.01; ns, no significant difference compared to corresponding H37Rv sample. The data presented is the representative of four independent experiments. (F) Survival of H37Rv (Red), CDC1551 (Blue), and the CDC1551 strain in trans expressing the intact metH gene from H37Rv (CDC1551/metH, Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p<0.01; ns, no significant difference compared to H37Rv.
Fig 4.
Methylfolate trap and its role in Gram-negative bacteria.
(A) SULFA susceptibility in Escherichia coli strains was analyzed by 10-fold serial dilutions. 5 μl of 10X diluted cell suspensions starting from OD1 were spotted on LB agar in the absence or presence of 125 μg/ml SMZ. Exogenous B12 was added at 2 nM final concentration. Growth was recorded after 48 h of incubation at 37°C. (B) Effect of metH and btuB on the folate pool of E. coli. Growing cultures (OD1) of E. coli strains were treated with 2.5 mg/ml SMZ for 15 min followed by folate extraction and LC-MS/MS analysis. Data shown, from top to bottom, are the combined levels of all 5-CH3-H4PteGlun species, all non-methylated folate species, and the total folate, respectively. Bars represent means of biological triplicates with standard deviations. ns, no significant difference between the indicated mutant and wild type E. coli. (C) Role of the methylfolate trap in SULFA sensitivity of Pseudomonas aeruginosa strains. Cultures underwent 10-fold serial dilutions, and 5 μl of diluted cultures were spotted onto solid media in the absence or presence of 150 μg/ml SMZ. Exogenous B12 was added at 2 nM final concentration. Growth was recorded after 48 h of incubation at 37°C. (D) Effect of metH, btuB and cobI on the folate pool of P. aeruginosa. Growing cultures (OD1) of P. aeruginosa strains were treated with 2.5 mg/ml SMZ for 15 min followed by folate extraction and LC-MS/MS analysis. Data shown, from top to bottom, are the combined levels of mono- and di-glutamylated methyl folate species (5-CH3-H4PteGlu1-2), tri- and tetra-glutamylated methyl folate species (5-CH3-H4PteGlu3-4), all non-methylated folate species, and the total folate. Bars represent means of biological triplicates with standard deviations. ns, no significant difference between the indicated mutant and wild type P. aeruginosa.
Fig 5.
Metabolic dynamics of the methylfolate trap in Salmonella typhimurium SULFA resistance.
(A) Wide-spectrum SULFA susceptibility of S. typhimurium metH(+) and metH(-) analyzed by 10X serial dilution. Cultures were diluted starting with OD1 and 5 μl cell suspensions were spotted onto LB agar in the absence (control) or presence of different SULFAs, used at the indicated concentrations. These SULFA drugs are classified into all four subgroups, in left-right order: short-acting (blue), intermediate-acting (yellow), long-acting (green), and ultra-long-acting (pink), respectively. Growth was recorded after 48 h at 37°C. (B) Viability of S. typhimurium metH(+) (red) and metH(-) (blue) on LB agar 24 h post-SMZ addition (125 μg/ml). Colony forming units (c.f.u.) were determined and normalized to c.f.u. values of the inoculation input (0 h). The y-axis represents c.f.u. fold-change on a log10 scale of SMZ-treated (+SMZ, hatched bars) and control non-treated samples (-SMZ, empty bars). Error bars represent standard deviations from biological triplicates. **** p<0.0001; ns, no significant difference. (C) SULFA susceptibility of S. typhimurium strains in liquid LB medium. Cultures of metH(+) (red) and metH(-) (blue) growing at OD1 was added with 2.5 mg/ml SMZ (arrow). Growth was monitored by measuring OD600. (D) Dynamics of the folate pool in S. typhimurium metH(+) (red) and metH(-) (blue) strains. At selected time points following SULFA treatment, cells were collected and folate extracted and analyzed by LC-MS/MS. Bars represent the combined levels of all 5-CH3-H4PteGlun species (top), all non-methylated folate species (middle), and total folate (bottom) following SMZ addition. s, significant difference between metH(-) and corresponding metH(+) samples; ns, no significant difference. (E) Dynamics of 41 metabolites in metH(+) (upper) and metH(-) (lower) strains. Metabolites are shown with their fold change over time (0–8 hours post SMZ addition). At selected time points following SMZ treatment, cells were collected and metabolites extracted and analyzed by LC-MS/MS. Signal intensity was normalized to OD600nm at each time point. Relative levels are expressed as the log ratio of the normalized signal intensity of SMZ-treated cells at each time point to the normalized signal intensity of the no drug control sample at t = 0 (n = 3).
The data shown in all figures represents the mean of biological repeats (n ≥ 3) with standard deviations. In the experiments demonstrated in Fig 5C–5E, SMZ was added at 2.5 mg/ml when cultures reached OD1.
Fig 6.
Methylfolate trap-mediated thymineless death.
(A) Cellular levels of methionine, SAM, and SAH in S. typhimurium cells following SMZ treatment. metH(-) (blue triangle) displayed a lower level of methionine but higher levels of SAM and SAH than its parent metH(+) (red circle). (B) Higher level of glycine in metH(-) (blue triangle) compared to metH(+) (red circle). (C) Dynamics of nucleotide pool in S. typhimurium cells during SMZ treatment. While the levels of nucleotides and intermediates were sharply reduced in response to SMZ in metH(+) (red circle), cells of metH(-) (blue triangle) failed to deplete these metabolites. (D) Thymine abolishes SULFA-induced cell death and restores growth in metH(-). Salmonella cultures were 10X serially diluted and 5 μl of diluted cultures were spotted on LB agar in the absence or presence of 125 μg/ml SMZ and 2 mM thymine. Growth on test plates (top panel) was recorded after 24 h of incubation at 37°C. Corresponding 24-hour viability of colonies grown from spotted OD0.001 cell suspensions (arrow) was determined by measuring c.f.u. and normalized to c.f.u. values of the input inoculum (lower panel). The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from biological triplicates. *** p<0.001; **** p<0.0001.
Fig 7.
Genetic and chemical induction of the methylfolate trap during Salmonella infection of macrophages.
(A) Survival of Salmonella strains in macrophages treated with SULFAs. Macrophages J774A.1 were infected for 1 h followed by 18 h chase, during which cells were untreated or treated with 1 mg/ml SMZ. Colony forming units (c.f.u.) were determined by serial dilution and plating method. (B) Cellular uptake and conversion of exogenous B12 in mammalian cells requires transcobalamin (TC) and CblC proteins, respectively. Antivitamin B12 molecules such as EtPhCbl inhibit transcobalamin and CblC, thereby restricting B12 bioavailability to intracellular bacteria. (C) Depletion of CblC expression, detected by Western Blot using a specific antibody (top), caused B12 starvation (middle) and increased SULFA sensitivity (bottom) of intracellular Salmonella. siRNA transfected THP-1 macrophages were infected with S. typhimurium cells expressing β-galactosidase from a B12 starvation-responsive promoter for 1 h, followed by 18 h chase, during which the infected macrophages were treated without or with 1 mg/ml SMZ. B12 starvation was estimated by determining β-galactosidase activity while Salmonella survival measured by c.f.u. counting. (D) Chemical restriction of B12 sensitizes intracellular S. typhimurium to SULFA treatment. Macrophages J774A.1 were infected with S. typhimurium cells harboring a B12 molecular probe for 1 h followed by 18 h chase, during which cells were untreated or treated with 1 mg/ml SMZ or/and 50 nM EtPhCbl. B12 starvation was estimated through measuring enzymatic activity (top) and expression of β-galactosidase by Western Blot (middle). Salmonella survival from the corresponding macrophages was measured through c.f.u. counting (bottom). Error bars represent standard deviations from biological triplicates. ns, no significant difference compared to control groups.