The iron-responsive microsomal proteome of Aspergillus fumigatus

Abstract

The opportunistic fungal pathogen Aspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus siderophore as a 'Trojan horse' to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome.

Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria

“…Samples were acidified by the addition of 1 µ L trifluoroacetic acid (TFA). Peptide solutions were then dried in a centrifugal evaporator, resuspended in 0.5% (w/v) TFA and desalted using C 18 Ziptips (Millipore Ziptips C18) [91][92][93][94] .…”

Functional Investigation of Iron-Responsive Microsomal Proteins, including MirC, in Aspergillus fumigatus

“…RP-HPLC purified siderophores were applied to a Zorbax SB-C18 HPLC-Chip with a 40 nl enrichment column and a 75 μm × 43 mm (5 μm particle and 300 Å pore size) analytical column (Collins et al, 2013; Owens et al, 2014; Moloney et al, 2016). For label-free quantitative (LFQ) comparative proteomics, A. fumigatus WT, Δ mirC, or Δ mirC 2 were cultured for 72 h in either iron-deplete or iron-replete conditions ( n = 4 for iron-deplete samples, n = 3 for iron-replete samples).…”

“…Using this approach, MirA was found to be specific for enterobactin and MirB for TAFC +Fe (Haas et al, 2003). The A. fumigatus genome encodes seven putative SITs (Haas et al, 2008), which include five transporters induced under iron limitation (Moloney et al, 2016); Sit1, AFUA_3G03670, MirA/D, MirB, and MirC. Though MirA and MirB have been implicated in siderophore transport in A. nidulans , a role for MirC has yet to be demonstrated (Haas et al, 2003).…”

Proteomic analysis of Aspergillus fumigatus – clinical implications

“…Targeting siderophore uptake via SITs represents a promising anti-fungal strategy, but requires further dissection [104,105]. Recently, the iron-starved microsomal proteome of A. fumigatus has been characterized [16]. Mycelial lysates were successfully enriched for membrane proteins via cellular fractionation with ultracentrifugation [15].…”

“…Fractionation according to specific properties can provide further insight into a proteome. Shotgun proteomics has facilitated analysis of proteins previously intractable to 2-DE, such as membrane proteins through gel-free approaches [15,16]. However, 2-DE still plays an important role in analysing post-translational modifications and the immunoproteome [17][18][19][20][21][22][23][24].…”