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Distribution of DHPS Mutations Among ITS Subtypes of P. carinii
STEVEN R. MESHNICK,a PAUL A. HOSSLER, a KYLE S. ENGER, a POWEL
KAZANJIAN, a JOSHUA REST, a DAVID MINDELL, a B.Z. LI, b CHAO H. LEE, b
L. F. NIMRI, JANE L. CARTER, c C. BENJAMIN BEARD, c CARLOS DEL RIO, e
aUniversity of Michigan, bIndiana University, cCDC, dSan Francisco General Hospital,
Sulfa drugs are widely used in the prophylaxis and treatment of Pneumocystis carinii
pneumonia. Co-trimoxasole, the first-line agent, is a combination of sulfamethoxazole plus trimethoprim but functions as sulfa monotherapy since trimethoprim appears to be inactive. Dapsone, a sulfone, is a commonly used second line agent. Both sulfamethoxazole and dapsone act by binding to the dihydropteroate synthase (DHPS) (reviewed in ref [2, 16] (Fig. 1). Sulfa-resistant Pneumocystis carinii
may be an emerging public health problem. However, since in vitro cultivation of human-derived organisms is not possible, molecular tools for determining resistance are needed. Genetic polymorphisms in the P. carinii
DHPS were first reported by us in 1997 . Two of the mutations, at positions 55 and 57, appear to be in the enzyme active site (Fig. 1, based on the E. coli structure ). These mutations have been associated with prophylaxis break-through in a number of studies [4, 6, 8, 10, 12-15]. These mutations appear to have occurred recently, and then to have spread over time. In several studies, the mutations were infrequent in patient samples obtained before 1992-4, and then became more frequent as the sulfa drugs became used more widely in prophylaxis [4, 8, 9]. Also, the prevalence of these mutations varies geographically, with higher prevalences at sites with older epidemics [6, 9]. How did the mutations evolve? There are two possibilities. The mutations might have arisen only once and then spread as a result of selection on a population level. Alternatively, Mutations could arise independently in each treated patient and increase as a result of selection within host. Obviously, intermediate scenarios (where the mutations arose independently but infrequently) are also possible. In order to understand how these mutations arose, we asked the question of whether DHPS mutations were associated with individual strains of human-derived P. carinii
. Strains were defined by sequencing the Internally Transcribed Spacer (ITS) regions. Human-derived P. carinii
contain >15 ITS1 types (A-O) and >14 ITS2 types (a-n) More than 50 different combinations have been identified [5, 7, 11]. We postulated that, if mutations have arisen once or only a few times, then DHPS mutations should be associated with specific ITS types. However, we did not anticipate that the associations would be perfect, since ITS and DHPS genes are probably unlinked And there may be horizontal genetic transfer (sexual, parasexual, etc.) in P. carinii.
We obtained ITS and DHPS genotyping data from 57 patient isolates retrospectively and prospectively. These patients were from Indiana, Atlanta, San Francisco, Los Angeles, Seattle and were a subset of patients used in previous studies [3, 6, 8-10]. We then selected all 37 isolates with monoclonal infections (only single ITS type and DHPS type) Characteristics of 37 monoclonal patient isolates are shown in Table 2. There were 22 WT, 14 double mutants and 1 single mutant (position 55). Mutations were more common in Atlanta and the West Coast than Indiana, and more common in samples obtained more recently, but these differences were not statistically significant. One ITS type, Eg, was common in all 3 sites (Table 3). Three other types, Ea, Eb, and Ne, were found in two of the 3 sites. The other 6 ITS types were only found in single sites. The distribution of DHPS mutations among ITS types is shown in Figure 2. Mutations are very common in some ITS types (such as Ne), but not found in others. We next asked the question of whether the ITS types that contained mutant DHPS genes were related evolutionarily. A cladogram (Fig. 3) were constructed using the consensus sequences for ITS 1 and ITS2, combined in the same manner as the isolates used in our study. Monkey-derived P. carinii
ITS types were used as outgroups. The cladogram was bootstrapped, with values < 50 not well supported. No evolutionary relatedness among ITS types containing mutations was observed. We then asked post-hoc, whether certain groups of ITS types contained statistically higher numbers of DHPS mutants than other ITS types using the Fishers’ exact test. Among isolates from Indiana, Ee isolates were significantly more likely to be DHPS mutants than all others. Among all isolates, Ne, Eg and Ee isolates were more likely to contain DHPS mutants than all others. This is not an artefact associated with date of acquisition, since Ee and Eg are among the oldest of the isolates collected. These data are consistent with the possibility that DHPS mutations arose infrequently. Acknowledgements We would like to thank Marilyn Bartlett, Jim Smith, Wendy Armstrong, Jeffrey Duchin, James Richardson, David Rimland, Jeff Duchin, Paul Simon for access to patient samples. This work was supported by NIH grant RO1 AI 46966. 1.
Achari, A., D.O. Somers, J.N. Champness, P.K. Bryant, J. Rosemond, and D.K.
Stammers. 1997. Crystal structure of the anti-bacterial sulfonamide drug target
dihydropteroate synthase. Nat Struct Biol 4
Armstrong, W., S. Meshnick, and P. Kazanjian. 2000. Pneumocystis carinii
mutations associated with sulfa and sulfone prophylaxis failures in
immunocompromised patients. Microbes Infect 2
Beard, C.B., J.L. Carter, S.P. Keely, L. Huang, N.J. Pieniazek, I.N. Moura, J.M. Roberts, A.W. Hightower, M.S. Bens, A.R. Freeman, S. Lee, J.R. Stringer, J.S.
Duchin, C. del Rio, D. Rimland, R.P. Baughman, D.A. Levy, V.J. Dietz, P.
Simon, and T.R. Navin. 2000. Genetic variation in Pneumocystis carinii isolates
from different geographic regions: implications for transmission. Emerg Infect
Helweg-Larsen, J., T.L. Benfield, J. Eugen-Olsen, J.D. Lundgren, and B.
Lundgren. 1999. Effects of mutations in Pneumocystis carinii dihydropteroate
synthase gene on outcome of AIDS-associated P. carinii pneumonia. Lancet 354
Helweg-Larsen, J., C.H. Lee, S. Jin, J.Y. Hsueh, T.L. Benfield, J. Hansen, J.D.
Lundgren, and B. Lundgren. 2001. Clinical correlation of variations in the internal
transcribed spacer regions of rRNA genes in Pneumocystis carinii f.sp. hominis.
Huang, L., C.B. Beard, J. Creasman, D. Levy, J.S. Duchin, S. Lee, N. Pieniazek,
J.L. Carter, C. del Rio, D. Rimland, and T.R. Navin. 2000. Sulfa or sulfone
prophylaxis and geographic region predict mutations in the Pneumocystis carinii
dihydropteroate synthase gene. J Infect Dis 182
Jiang, B., J.J. Lu, B. Li, X. Tang, M.S. Bartlett, J.W. Smith, and C.H. Lee. 1996.
Development of type-specific PCR for typing Pneumocystis carinii f. sp. hominis
based on nucleotide sequence variations of internal transcribed spacer region of
rRNA genes. J Clin Microbiol 34
Kazanjian, P., A.B. Locke, P.A. Hossler, B.R. Lane, M.S. Bartlett, J.W. Smith, M.
Cannon, and S.R. Meshnick. 1998. Pneumocystis carinii mutations associated
with sulfa and sulfone prophylaxis failures in AIDS patients. Aids 12
Kazanjian, P., W. Armstrong, P.A. Hossler, W. Burman, J. Richardson, C.H. Lee,
L. Crane, J. Katz, and S.R. Meshnick. 2000. Pneumocystis carinii mutations are
associated with duration of sulfa or sulfone prophylaxis exposure in AIDS
patients. J Infect Dis 182
Lane, B.R., J.C. Ast, P.A. Hossler, D.P. Mindell, M.S. Bartlett, J.W. Smith, and
S.R. Meshnick. 1997. Dihydropteroate synthase polymorphisms in Pneumocystis
carinii. J Infect Dis 175
Lee, C.H., J. Helweg-Larsen, X. Tang, S. Jin, B. Li, M.S. Bartlett, J.J. Lu, B.
Lundgren, J.D. Lundgren, M. Olsson, S.B. Lucas, P. Roux, A. Cargnel, C. Atzori,
O. Matos, and J.W. Smith. 1998. Update on Pneumocystis carinii f. sp. hominis
typing based on nucleotide sequence variations in internal transcribed spacer
regions of rRNA genes. J Clin Microbiol 36
Ma, L., L. Borio, H. Masur, and J.A. Kovacs. 1999. Pneumocystis carinii
dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate
with prior trimethoprim- sulfamethoxazole or dapsone use. J Infect Dis 180
Mei, Q., S. Gurunathan, H. Masur, and J.A. Kovacs. 1998. Failure of co-
trimoxazole in Pneumocystis carinii infection and mutations in dihydropteroate
synthase gene. Lancet 351
Santos, L.D., P. Lacube, S. Latouche, G. Kac, C. Mayaud, M. Marteau, J.L.
Poirot, E. Maury, J. Guillot, and P. Roux. 1999. Contribution of dihydropteroate
synthase gene typing for Pneumocystis carinii f.sp. hominis epidemiology. J
Eukaryot Microbiol 46
Visconti, E., E. Ortona, P. Margutti, S. Marinaci, M. Zolfo, P. Mencarini, L.P.
Celentano, A. Siracusano, and E. Tamburrini. 1999. Identification of
dihydropteroate (DHPS) gene mutant in Pneumocystis carinii in respiratory
samples of HIV+ patients from 1992 to 1997. J Eukaryot Microbiol 46
Walker, D.J., A.E. Wakefield, M.N. Dohn, R.F. Miller, R.P. Baughman, P.A.
Hossler, M.S. Bartlett, J.W. Smith, P. Kazanjian, and S.R. Meshnick. 1998.
Sequence polymorphisms in the Pneumocystis carinii cytochrome b gene and
their association with atovaquone prophylaxis failure. J Infect Dis 178
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