Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens.
Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA 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 (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) 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 mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii 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 P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii's physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene.
Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III Madrid, Spain.
Clara
C
Valero
Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III Madrid, Spain.
María José
MJ
Buitrago
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique - Hôpitaux de ParisParis, France; Sorbonne Paris Cité, Université Paris DiderotParis, France.
Maud
M
Gits-Muselli
Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses Invasives et Antifongiques, Institut Pasteur Paris, France.
Marion
M
Benazra
Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses Invasives et Antifongiques, Institut Pasteur Paris, France.
Aude
A
Sturny-Leclère
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique - Hôpitaux de Paris Paris, France.
Samia
S
Hamane
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique - Hôpitaux de Paris Paris, France.
Nicolas
N
Guigue
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique - Hôpitaux de ParisParis, France; Sorbonne Paris Cité, Université Paris DiderotParis, France; Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses Invasives et Antifongiques, Institut PasteurParis, France; CNRS URA3012Paris, France.
Stéphane
S
Bretagne
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique - Hôpitaux de ParisParis, France; Sorbonne Paris Cité, Université Paris DiderotParis, France; Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses Invasives et Antifongiques, Institut PasteurParis, France; CNRS URA3012Paris, France.
Alexandre
A
Alanio
DNA quantification; PcP; Pneumocystis jirovecii; carriage; copy number variation; mitochondria; real-time PCR
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