Tuberculosis
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Sulfatase-activated fluorophores for rapid discrimination of mycobacterial species and strains Kimberly E. Beattya,1, Monique Williamsb,c, Brian L. Carlsona, Benjamin M. Swartsa, Robin M. Warrenb, Paul D. van Heldenb, and Carolyn R. Bertozzia,d,2 aDepartments of Chemistry and Molecular and Cell Biology, and dHoward Hughes Medical Institute, University of California, Berkeley, CA 94720-1460; bDivision of Molecular Biology and Human Genetics, National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research/Medical Research Council Centre for Molecular and Cellular Biology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town 7505, South Africa; and cMedical Research Council/National Health Laboratory Service/University of Cape Town Molecular Mycobacteriology Research Unit, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town 7925, South Africa Edited by Laura L. Kiessling, University of Wisconsin-Madison, Madison, WI, and approved June 7, 2013 (received for review January 4, 2013) Most current diagnostic tests for tuberculosis do not reveal the colorimetric sulfatase substrate phenolphthalein disulfate (17). species or strain of pathogen causing pulmonary infection, which Species and strain identification using this and other sulfatase can lead to inappropriate treatment regimens and the spread of probes [e.g., p-nitrophenyl sulfate (p-NPS) (18) and 4-methyl- disease. Here, we report an assay for mycobacterial strain assign- umbelliferyl sulfate (4-MUS) (19); structures are shown in SI ment based on genetically conserved mycobacterial sulfatases. We Appendix, Fig. S1] have been largely ineffective due to slow re- developed a sulfatase-activated probe, 7-hydroxy-9H-(1,3-dichloro- action kinetics, a high detection limit with these substrates, and 9,9-dimethylacridin-2-one)–sulfate, that detects enzyme activity in the lack of a simple platform to resolve strain-specific enzymes. native protein gels, allowing the rapid detection of sulfatases in In this paper, we describe a method for discriminating myco- fl mycobacterial lysates. This assay revealed that mycobacterial strains bacterial species and strains using the sulfatase-activated uo- fi rophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) have distinct sulfatase ngerprints that can be used to judge both – the species and lineage. Our results demonstrate the potential (DDAO) sulfate as an enzyme probe (Fig. 2). We separated mycobacterial lysates on native polyacrylamide gels and found, of enzyme-activated probes for rapid pathogen discrimination for fl fi infectious diseases. using DDAO uorescence, unique sulfatase ngerprints that were dependent on the mycobacterial species and lineage. This in-gel assay was used to distinguish among different pathogenic Mycobacterium tuberculosis | chemical biology | fluorescence | species and M. tuberculosis strains rapidly, including a selection enzyme assay | hydrolase of M. tuberculosis strains from different geographical lineages. uberculosis (TB) is caused by members of the Mycobacterium Results and Discussion Ttuberculosis complex (MTBC), which include Mycobacterium Genetic Conservation of Mycobacterial Sulfatases. Bioinformatics bovis, Mycobacterium africanum, Mycobacterium microti, and the analysis of mycobacterial genomes suggests the presence of sulfa- hundreds of related strains of Mycobacterium tuberculosis. Pul- tases from all three known classes. Types I and III were the most monary disease can also be caused by other mycobacterial spe- attractive enzyme targets to us because they catalyze the hydrolysis cies (e.g., Mycobacterium avium, Mycobacterium intracellulare, of sulfate esters (20–22) (Table 1) rather than the iron-dependent, Mycobacterium septicum, Mycobacterium kansasii) (1, 2). There is oxidative process characteristic of the type II sulfatases (23, 24). an emerging appreciation of the phenotypic variation across Type I sulfatases have an unusual amino acid modification in mycobacterial species and strains (3, 4), including the many fi their active site that confers catalytic activity. The formylglycine- signi cant differences in virulence and immune responses to generating enzyme (25–28) converts a cysteine residue within the different members of the MTBC (4, 5). Identification of the consensus sequence -C-X-P-X-R- to a formylglycine residue. In infecting species is critical because therapeutics have variable water, the formylglycine aldehyde hydrates to form a geminal efficacy against the various pathogenic mycobacteria. For ex- diol, which serves as a nucleophile during hydrolysis of the sul- ample, most strains of M. bovis are resistant to pyrazinamide (6), fi and pulmonary disease caused by the M. avium complex requires fate ester (20) (SI Appendix, Fig. S2). There are ve putative type different treatment than TB caused by members of the MTBC I sulfatases encoded in the M. tuberculosis H37Rv genome: (2). New assays for TB should be sought that enable various Arylsulfatase (Ats)A (Rv0711), AtsB (Rv3299), AtsD (Rv0663), AtsF (Rv3077), and AtsG (Rv0296c). species and strains to be distinguished unambiguously based on 2+ β easily detected variations in phenotype. Type III sulfatases are Zn -dependent metallo- -lactamases. Enzyme probes have been developed to study human patho- Enzymes of this type encode a conserved metal binding motif CHEMISTRY gens (7, 8); however, to date, they have not been explored as (21, 22), and there are three predicted in the genome of M. tuberculosis (Rv2407, Rv3796, and Rv3762c) (26). Rv3796 a means for mycobacterial strain assignment. Of particular use 2+ are probes that are enzyme-activatable and fluorogenic, going [zinc-dependent sulfatase-A (ZnSulf-A)] has a Zn -binding from a dim to bright fluorescent state on substrate turnover. motif, but it was annotated previously as a type I sulfatase Sensitivity is augmented because one enzyme can modify many (13). Computational prediction of the protein structure using probes, dramatically increasing the fluorescence signal that can be measured in complex samples. Furthermore, enzyme-acti- vated probes can be used in a variety of assay platforms, in- Author contributions: K.E.B. and C.R.B. designed research; K.E.B., M.W., B.L.C., and B.M.S. cluding molecular imaging and high-throughput screens (9–11). performed research; K.E.B., M.W., B.L.C., B.M.S., R.M.W., P.D.v.H., and C.R.B. contributed MICROBIOLOGY In our research, we have targeted the mycobacterial sulfo- new reagents/analytic tools; K.E.B., M.W., B.L.C., B.M.S., R.M.W., P.D.v.H., and C.R.B. an- transferases and sulfatases, which control the sulfation states of alyzed data; and K.E.B. and C.R.B. wrote the paper. biomolecules (12–14) (Fig. 1). Sulfated compounds have estab- The authors declare no conflict of interest. lished roles in metabolism and virulence in mycobacteria (13– This article is a PNAS Direct Submission. 15). Sulfatases are conserved across mycobacterial genomes (13), 1Present address: Department of Physiology and Pharmacology, Oregon Health and Sci- although their substrates, structures, and functions are largely ence University, Portland, OR 97239. unknown. Since the 1950s, there have been efforts to use sulfa- 2To whom correspondence should be addressed. E-mail: [email protected]. tase activity to detect mycobacteria (16). One assay uses a 2-wk This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. incubation with live mycobacteria in media containing the 1073/pnas.1222041110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1222041110 PNAS | August 6, 2013 | vol. 110 | no. 32 | 12911–12916 Downloaded by guest on September 23, 2021 We validated DDAO-sulfate as an enzyme-activated probe by determining the kinetic parameters for this compound with commercially available sulfatases (SI Appendix, Table S1). Sul- fatase activity from Aerobacter aerogenes was evaluated in buffer [50 mM Tris, 100 mM NaCl, 2.5 mM MgCl2, 250 μM CaCl2 (pH = 7.5)] at 37 °C, and the formation of DDAO was detected in real time. Hydrolysis of DDAO-sulfate in the absence of en- zyme was negligible over the course of the experiment. The Km value for DDAO-sulfate was determined to be 164 ± 14 μM. For Fig. 1. Sulfation is controlled by two classes of enzymes, sulfotransferases comparison, A. aerogenes sulfatase was evaluated using p-NPS and sulfatases. Sulfotransferases catalyze sulfuryl group transfer to bio- ± μ ′ ′ and 4-MUS. The Km value for p-NPS was 1,800 120 M, and molecules using an activated donor called 3 -phosphoadenosine-5 -phos- that for 4-MUS was 700 ± 160 μM (32). phosulfate (PAPS). Sulfate ester, or N-sulfate, hydrolysis is catalyzed by sulfatases. Sulfated biomolecules include steroids, lipids, carbohydrates, and The pKa of DDAO is 5.0 (41), and the formation of DDAO proteins. PAP, 3′-phosphoadenosine-5′-phosphate. can be monitored continuously at any pH above this value. This is an important feature for a sulfatase probe because many sul- fatases have an acidic pH optimum (12). In contrast, the pKa of Phyre (protein homology/analogy recognition engine) (29) 4-MU is 7.8 (42), and acidic enzyme reactions must be quenched fi fl analysis supports the reclassification of ZnSulf-A as a member by basi cation for detection of uorescence. The high pKa of of the metallohydrolase superfamily. 4-MU prevents its use in continuous assays of enzymes at acidic pH. Sulfatases from three other sources were examined at their Development of a Sulfatase-Activated Fluorophore. The majority of