Dual localization of Mdj1 in pathogenic fungi varies with growth temperature

Medical Mycology, 2014, 52, 187–195 doi: 10.1093/mmy/myt002 Advance Access Publication Date: 19 February 2014 Original Article Original Article Dual localization of Mdj1 in pathogenic fungi varies with growth temperature Ítala Bruna Z. Dourado1 , Wagner L. Batista2 , Larissa V. G. Longo1 , Renato A. Mortara1 and Rosana Puccia1,∗ 1 Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, EPM-UNIFESP and 2 Departamento de Ciências Biológicas, Universidade Federal de São Paulo, UNIFESP/Campus Diadema, São Paulo, Brazil *To whom correspondence should be addressed. Rosana Puccia, Department of Microbiology, Immunology and Parasitology, EPM-UNIFESP, Rua Botucatu, 862, oitavo andar, 04023-062, São Paulo, SP, Brazil. Tel: + 55 11 5576 4551, ext 1508; E-mail: Received 20 May 2013; Revised 25 July 2013; Accepted 25 October 2013 Abstract Paracoccidioides brasiliensis and P. lutzii are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis (PCM). Previously, we characterized the PbMDJ1 gene. This gene encodes P. brasiliensis chaperone Mdj1, which in yeast is a mitochondrial member of the J-domain family, whose main function is to regulate cognate Hsp70 activities. We produced rabbit polyclonal antibody antirecombinant PbMdj1 (rPbMdj1), which labeled the protein not only in mitochondria but also at the cell wall of P. brasiliensis yeasts of isolate Pb18. Here we used anti-rPbMdj1 in confocal microscopy to localize Mdj1 in Pb18 and other fungal isolates grown at different temperatures. Dual intracellular and cell surface pattern were initially seen in yeast-phase P. brasiliensis Pb3, Pb18 (control), P. lutzii Pb01, and Histoplasma capsulatum. Pb18 and Aspergillus fumigatus hyphae as well as Pb3 pseudo hyphae formed at 36◦ C were labeled predominantly along the cell surface. Preferential surface localization was observed by 72 h of yeast-mycelium thermotransition. It was interesting to observe that anti-rPbMdj1 concentrated at the surface tip and branching points of A. fumigatus hyphae grown at 36◦ C, suggesting a role in growth, whereas at 23◦ C, anti-rPbMdj1 was distributed along the hyphal surface. In Pb3, Pb18, and Pb01 mitochondrial extracts, the antibodies revealed a specific 55-kDa band, which corresponds to the processed Mdj1 size. The presence of Mdj1 on the fungal cell wall suggests that this protein could also play a role in the interaction with the host. Key words: Mdj1, cell wall, mitochondria, pathogenic fungi, temperature. Introduction Paracoccidioides species are temperature-dependent dimorphic human pathogens that cause human paracoccidioidomycosis (PCM), an endemic fungal disease that is common in Latin America [1]. Paracoccidioides species grow as multibudding yeasts in host tissues and when cultivated at 36◦ C, and as mycelia when incubated in vitro below 28◦ C. Environmental fungal conidia reach the pulmonary alveoli upon inhalation and transform to the yeast pathogenic phase to initiate the most common form of PCM – a  C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: 187 188 ates cleavage of misfolded or unassembled proteins [9], while Mdj1 is essential for substrate degradation [12,13]. PbMDJ1 and PbLON are adjacent, inversely oriented, and separated by a common 5 intergenic region that ranges between 400 and 485 nucleotides, which contain heat shock and AP-1 functional motifs [6]. PbMdj1 identity with its orthologs varies from 85% in H. capsulatum and B. dermatitidis to 61% in A. nidulans. In our previous work, we were the first to describe a J-domain protein on the cell surface, specifically on fungal cell walls [7], where its function is presently unknown. We observed that the sera from patients with PCM sera reacted with rPbMdj1; however, anti-rPbMdj1 antibodies were unable to interfere with P. brasiliensis yeasts growth in vitro. The aim of study reported here was to use anti-rPbMdj1 in confocal microscopy analysis to localize Mdj1 in several pathogenic fungal isolates/species grown at different temperatures. We analyzed P. brasiliensis Pb18, Pb3; P. lutzii Pb01; H. capsulatum; and also A. fumigatus, which is a filamentous species that causes an opportunistic mycosis [14]. We observed that dual intracellular and cell surface labeling occurred in both yeasts and mycelia, but the labeling patterns unexpectedly varied with growth temperatures. Material and methods Fungal isolates and growth condition We analyzed clinical isolates of P. brasiliensis (Pb18 and Pb3) and P. lutzii (Pb01), H. capsulatum (bat strain M-240/06 isolated in the São Paulo Zoonosis Center, Brazil), and A. fumigatus AF293. Paracoccidioides strains were isolated from mice lungs and maintained in the yeast phase in slants of modified yeast peptone dextrose (YPD) (modYPD: 0.5% yeast extract, 0.5% casein peptone, 1.5% glucose; pH 6.3) for about two months at 4◦ C before use. For yeast-phase confocal studies, one loop full of stored cultures was transferred to liquid modYPD (30 ml) and incubated for 5 days at 36◦ C under shaking (120 rpm). To obtain hyphae, 1 ml was transferred from this culture to fresh medium (50 ml) and cultivated at 23◦ C for 10 to 15 days under shaking until complete transition to mycelium. For experiments in which phase transition forms were used, 3 to 5 × 103 Paracoccidioides yeasts were transferred to modYPD (50 ml) and incubated at 23◦ C under shaking. For mycelium-to-yeast transition, 1 ml of mycelium culture was transferred to 50 ml of modYPD and incubated at 36◦ C under shaking. Cells were collected for analysis at different transition times for 12 days. Contamination and cell viability were checked microscopically using trypan blue. H. capsulatum yeasts were cultivated in brain heart infusion (BHI)/cys (42 µg cysteine/ml) and maintained at −70◦ C in the same medium containing 25% glycerol. For pulmonary infection. Genetic variation among clinical, animal, and environmental Paracoccidioides isolates allowed recognition of three P. brasiliensis phylogenetic lineages (S1, PS2, and PS3) [2] and the proposal of a new species, P. lutzii (said to be “Pb01-like”) [3]. PS2 corresponds to a cryptic small group of P. brasiliensis isolates represented by Pb3 that cause regressive infections in mice, in contrast with S1 isolates (e.g., Pb18) [4]. Other thermodimorphic pathogenic fungi that are agents of human infections with general characteristics similar to those of PCM [5] include Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis/posadasii. Previously we characterized P. brasiliensis PbMdj1 as a heat shock protein [6] that is sorted to both yeast mitochondria and cell wall, notably to budding regions [7]. Mdj1 is a type I J-domain/Hsp40 chaperone that is localized in the mitochondria of Saccharomyces cerevisia (...truncated)