Genetic Divergence at the Mitochondrial Small Subunit Ribosomal RNA Gene among Isolates of Pneumocystis carinii from Five Mammalian Host Species

5 Biodiversity has been demonstrated among isolates of Pneurnocystis carinii from the infected lungs of different host species, by comparison of antigenic proteins, isoenzymes, chromosomal karyotypes and DNA sequence. These cumulative data, including the results of crossinfectivity experiments demonstrating the lack of transmission of the parasite between different mammalian species, strongly suggest that P. carinii infection is host-species specific. Analysis of DNA sequence divergence among different isolates of P. carinii has been reported at a number of genetic loci, including the genes encoding mitochondrial large subunit rRNA, the nuclear rRNA operon, the AROM protein, thymidylate synthase, the major surface glycoprotein, and aand ptubulin [for review, see 41. The mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of P. carinii sp. f. carinii has been cloned and sequenced 131. In this paper we report the analysis of genetic divergence at a portion of this gene among isolates of P. carinii from five different mammalian host species. MATERIALS AND METHODS. P. carinii infection was induced in Sprague-Dawley rats and Balb/c mice by steroid immunosuppression. Ferret-derived P. carinii (P. carinii sp.f. mustelae) was kindly provided by Dr F Gigliotti and Dr W T Hughes, rabbit-derived P. carinii (P. cariniisp.f. oryctolagi) by Dr E Dei-Cas, and human-derived P. carinii (P . carinii sp.f. horninis) by Dr R F Miller. Oligonucleotide primers, pAZl12-10F (5’-TAGACGGTCACAGAGATCAG-3’) and pAZl1210R (5’-GAACGATTACTAGCAATTCC-3’), designed to the sequence of the mt SSU rRNA gene from P. carinii sp.f. carinii (prototype ratderived P. carinii) were used. Amplification was performed for 40 cycles with denaturation at 94°C for 1.5 min, annealing at 55°C for 1.5 min and extension at 72°C for 2.0 min, in a reaction mixture with a final concentration of 50mM KCI, lOmM Tris (pH8.0), 0.1 % Triton X-1O0,3mMMgCI2, 400pM (each) deoxynucleoside triphosphate, lpM oligonucleotide primer and 0.025 U/pl Taq polymerase (Promega). The amplification products were cloned into the plasmid TA vector pCRTMII (Invitrogen), and the DNA sequence of the recombinants determined [l]. The primers pAZl12-10F and pAZl12-10R efficiently amplified P. carinii-specific DNA from organisms isolated from the infected lungs of rat, human, mouse, ferret and rabbit. A striking variation in the length of the amplification products was observed, ranging from 388 bp in mouse-derived P. carinii (P. carinii sp.f. muris) and P. carinii sp.f. horninis, 423 bp in P. carinii sp.f. oryctolagi, 445 bp in P. carinii sp.f. mustelae, 605 bp in P. carinii sp.f. rartus (variant rat-derived P. carinii) and 706 bp in P. curinii sp.f. carinii (prototype rat-derived P. carinio. The amplification products were cloned and sequenced, and the sequences were aligned (Figure I ) . Some regions of the sequence displayed high levels of homology whereas other regions were highly divergent. The P. carinii sp.f. carinii sequence contained an intervening sequence (approximately 250 bp) which was not present in any of the other sequences. Another intervening sequence was found at a position approximately 90 bp downstream of the first. This intervening RESULTS AND DISCUSSION. 12 8 18 17 16