AUTHOR=Valero Clara , Buitrago María José , Gits-Muselli Maud , Benazra Marion , Sturny-Leclère Aude , Hamane Samia , Guigue Nicolas , Bretagne Stéphane , Alanio Alexandre 

TITLE=Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.01413

DOI=10.3389/fmicb.2016.01413

ISSN=1664-302X

ABSTRACT=<p><italic>Pneumocystis jirovecii</italic> is an unculturable fungus and the causative agent of <italic>Pneumocystis</italic> pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of <italic>mtLSU rRNA</italic> mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (<italic>mtSSU rRNA</italic>, <italic>mtLSU rRNA</italic>, <italic>NAD1</italic>, and <italic>CYTB</italic>) in comparison to nuclear genome (<italic>DHPS</italic> and <italic>HSP70</italic>) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with <italic>mtSSU rRNA</italic> gene being more represented (37 copies) than <italic>NAD1</italic> (23 copies), <italic>mtLSU rRNA</italic> (15 copies) and <italic>CYTB</italic> (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (<italic>p</italic> = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of <italic>mtLSU rRNA</italic>, <italic>NAD1</italic>, and <italic>CYTB</italic> but not <italic>mtSSU rRNA</italic> differed according to <italic>P. jirovecii</italic> physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of <italic>P. jirovecii</italic> mtDNA copy number that fluctuates according to <italic>P. jirovecii</italic>’s physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene.</p>