RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro.

The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

Inhibitors of delta24(25) sterol methyltransferase block sterol synthesis and cell proliferation in Pneumocystis carinii.

Detailed analysis of the endogenous sterol content of purified Pneumocystis carinii preparations by gas-liquid chromatography coupled to mass spectrometry suggested that this parasite can both synthesize de novo steroid skeletons (to produce delta7 sterols) and take them from the infected host (leading to delta5 sterols). In both cases the final products are 24-alkyl sterols, resulting from the action of delta24(25) and delta24(24') sterol methyltransferases, enzymes not present in vertebrates. To investigate the physiological significance of these sterols, cultures of P. carinii in embryonic lung cells were exposed to 22,26-azasterol (20-piperidin-2-yl-5alpha-pregnan-3beta-20(R)-diol), a compound previously shown to inhibit both enzymes and to halt cell proliferation in fungi and protozoa. This compound produced a dose-dependent reduction in the parasite proliferation, with a 50% inhibitory concentration of 0.3 microM and 80% reduction of growth after 96 h at 10 microM. Correspondingly, parasites treated with the azasterol at 10 microM for 48 h accumulated 24-desalkyl sterols such as zymosterol (cholesta-8,24-dien-3beta-ol) and cholesta-8,14,24-trien-3beta-ol to ca. 40% of the total mass of endogenous sterols. This is the first report on the antiproliferative effects of a sterol biosynthesis inhibitor on P. carinii and indicate that sterol methyltransferase inhibitors could be the basis of a novel and specific chemotherapeutic approach to the treatment of P. carinii infections.