The high affinity iron permease is a key virulence factor required forRhizopus oryzaepathogenesis

Abstract

SummaryRhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first-line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron-depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidosis (DKA) mice. In addition, we disrupted FTR1 by double cross-over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti-Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus, FTR1 is a virulence factor for R. oryzae, and anti-Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis.

Iron starvation induces apoptosis in rhizopus oryzae in vitro

“…oryzae transformed with RNAi plasmid targeting FTR1 expression (FTR1::RNAi, iron permease mutant with >90% inhibited FTR1 expression) or empty plasmid (control) were described before. 3 Spores of R. oryzae strains RNAi empty or FTR1::RNAi were collected from 4-5 d old plates that have been incubated at 37 o C on yeast nitrogen base (YNB) agar plates supplemented with amino acids without uracil and containing either 1 mM ascorbic acid and ferrozine (iron-starved conditions) or 0.125% FeCl-3 (iron-rich conditions), as previously described. 1,19 Spores were washed in phosphate buffer saline (PBS) and enumerated in a hemocytometer.…”

“…1,2 Unfortunately, despite surgical debridement and systemic antifungal therapy, the overall mortality rate for mucormycosis remains high and may approach 100% in patients with disseminated disease, or persistent neutropenia. 2,3,4 Thus, new strategies to prevent and treat mucormycosis are urgently needed.…”

“…Iron starvation causes the rapid expression of FTR1 in R. oryzae, while excess iron in the form of ferric chloride reduces this expression. 1,3 Apoptosis has been studied in many higher eukaryotes but also have been observed in lower eukaryotes, including yeasts and filamentous fungi. [8][9][10][11] Rhizopus exhibit apoptotic markers that are similar to those of mammalian cells, including phosphatidylserine externalization, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential dissipation, and DNA condensation and fragmentation.…”

Human Mucormycosis

“…In last decades, a link has been reported between infection with the Rhizopus species and resistant gram-negative bacteria which can produce endosymbiotic toxin [4,5]. Hemodialysis patients under treatment with deferoxamine, due to high concentration of free iron in plasma and tissues are at risk for mucormycosis [6]. The common presentations are rhino-orbitalcerebral involvement, pulmonary and gastrointestinal infection, and invasive mucormycosis (central nervous system infection).…”

“…Therefore, diabetic patients with high serum iron level and glucose concentrations are more susceptible to mucormycosis than other high risk patients. Mucorales could invade the tissues by proteolytic, lipolytic, and glycosidic enzymes and also with metabolite products like mycotoxins such as agroclavine [6,8].…”