An Antimicrobial Peptidomimetic Induces Mucorales Cell Death through Mitochondria-Mediated Apoptosis

The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections

Mitochondrial Respiratory Pathways Inhibition in <i>Rhizopus oryzae</i> Potentiates Activity of Posaconazole and Itraconazole via Apoptosis

<div><p>The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against <i>Rhizopus oryzae</i>. We observed cellular changes characteristic of apoptosis in <i>R. oryzae</i> cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against <i>R. oryzae</i> was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of <i>R. oryzae</i> cells. These apoptotic features were prevented by the addition of the ROS scavenger <i>N</i>-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes <i>R. oryzae</i> exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents.</p></div

Iron starvation induces apoptosis in rhizopus oryzae in vitro

Mortality associated with mucormycosis remains high despite current antifungals. Iron-starvation strategies have been shown to have promising activity against Mucorales. We hypothesized that iron starvation enhances apoptosis in Rhizopus oryzae. Apoptosis was characterized in R. oryzae transformed with RNAi plasmid targeting FTR1 expression (iron permease mutant) or empty plasmid grown in iron rich (0.125% FeCl3) and iron depleted media (YNB+1mM ferrozine and 1 mM ascorbic acid). Increased apoptosis was observed with dihydrorhodamine-123 and rhodamine-123 staining in the iron starved mutant FTR1 when compared to empty plasmid, followed by increased extracellular ATP levels. In addition, DNA fragmentation and metacaspase activity were prominent in FTR1. In contrast, Rhizopus strains grown in iron-rich medium displayed minimal apoptosis. Our results demonstrate a metacaspase dependent apoptotic process in iron deprived condition and further support the role of iron starvation strategies as an adjunct treatment for mucormycosis, a mechanism by which iron starvation affects R. oryzae

Iron starvation induces apoptosis in rhizopus oryzae in vitro

Mortality associated with mucormycosis remains high despite current antifungals. Iron-starvation strategies have been shown to have promising activity against Mucorales. We hypothesized that iron starvation enhances apoptosis in Rhizopus oryzae. Apoptosis was characterized in R. oryzae transformed with RNAi plasmid targeting FTR1 expression (iron permease mutant) or empty plasmid grown in iron rich (0.125% FeCl(3)) and iron depleted media (YNB+1mM ferrozine and 1 mM ascorbic acid). Increased apoptosis was observed with dihydrorhodamine-123 and rhodamine-123 staining in the iron starved mutant FTR1 when compared to empty plasmid, followed by increased extracellular ATP levels. In addition, DNA fragmentation and metacaspase activity were prominent in FTR1. In contrast, Rhizopus strains grown in iron-rich medium displayed minimal apoptosis. Our results demonstrate a metacaspase dependent apoptotic process in iron deprived condition and further support the role of iron starvation strategies as an adjunct treatment for mucormycosis, a mechanism by which iron starvation affects R. oryzae

Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation

Diet Modification and Metformin Have a Beneficial Effect in a Fly Model of Obesity and Mucormycosis

<div><p>In an experimental model of obesity and hyperglycemia in <i>Drosophila melanogaster</i> we studied the effect of diet modification and administration of metformin on systemic infection with <i>Rhizopus</i>, a common cause of mucormycosis in diabetic patients. Female <i>Wt</i>-type <i>Drosophila</i> flies were fed regular (RF) or high-fat diet (HFD; 30% coconut oil) food with or without metformin for 48 h and then injected with <i>R. oryzae</i>. Survival rates, glucose and triglyceride levels were compared between 1) normal-weight flies (RF), 2) obese flies (HFD), 3) obese flies fed with RF, 4) flies continuously on HFD + metformin, 5) flies fed on HFD + metformin, then transferred to RF, and 6) obese flies administered metformin after infection. Glucose levels were compared across groups of non-infected flies and across groups of infected flies. Survival was significantly decreased (<i>P</i> = 0.003) in obese flies, while post-infection glucose levels were significantly increased (<i>P</i> = 0.0001), compared to normal-weight flies. Diet and administration of metformin led to weight loss, normalized glucose levels during infection, and were associated with decreased mortality and tissue fungal burden. In conclusion, diet and metformin help control infection-associated hyperglycemia and improve survival in <i>Drosophila</i> flies with mucormycosis. Fly models of obesity bear intriguing similarities to the pathophysiology of insulin resistance and diabetes in humans, and can provide new insights into the pathogenesis and treatment of infections in obese and diabetic patients.</p></div

Sub-minimum inhibitory concentrations of statins attenuate the virulence of Rhizopus oryzae.

International audience Mucormycosis is a destructive invasive mold infection afflicting patients with diabetes and hematologic malignancies. Patients with diabetes are often treated with statins, which have been shown to have antifungal properties. We sought to examine the effects of statins on Rhizopus oryzae, a common cause of mucormycosis.  Clinical strains of R. oryzae were exposed to lovastatin, atorvastatin, and simvastatin and the minimum inhibitory concentrations (MICs) were determined. R. oryzae germination, DNA fragmentation, susceptibility to oxidative stress, and ability to damage endothelial cells were assessed. We further investigated the impact of exposure to lovastatin on the virulence of R. oryzae.  All statins had MICs above 64 µg/mL against R. oryzae. Exposure of R. oryzae to statins decreased germling formation, induced DNA fragmentation, attenuated damage to endothelial cells independently of the expression of GRP78 and CotH. Additionally, R. oryzae exposed to lovastatin showed macroscopic loss of melanin, increased susceptibility to the oxidative agent peroxide and had attenuated virulence in both fly and mouse models of mucormycosis.  Exposure of R. oryzae to statins at sub-MIC concentrations decreased virulence both in vitro and in vivo. Further investigation is warranted into the use of statins as adjunctive therapy in mucormycosis

Statin Concentrations below the Minimum Inhibitory Concentration Attenuate the Virulence of Rhizopus oryzae

Background. Mucormycosis is a destructive invasive mold infection afflicting patients with diabetes and hematologic malignancies. Patients with diabetes are often treated with statins, which have been shown to have antifungal properties. We sought to examine the effects of statins on Rhizopus oryzae, a common cause of mucormycosis. Methods. Clinical strains of R. oryzae were exposed to lovastatin, atorvastatin, and simvastatin and the minimum inhibitory concentrations (MICs) were determined. R. oryzae germination, DNA fragmentation, susceptibility to oxidative stress, and ability to damage endothelial cells were assessed. We further investigated the impact of exposure to lovastatin on the virulence of R. oryzae. Results. All statins had MICs of >64 μg/mL against R. oryzae. Exposure of R. oryzae to statins decreased germling formation, induced DNA fragmentation, and attenuated damage to endothelial cells independently of the expression of GRP78 and CotH. Additionally, R. oryzae exposed to lovastatin showed macroscopic loss of melanin, yielded increased susceptibility to the oxidative agent peroxide, and had attenuated virulence in both fly and mouse models of mucormycosis. Conclusions. Exposure of R. oryzae to statins at concentrations below their MICs decreased virulence both in vitro and in vivo. Further investigation is warranted into the use of statins as adjunctive therapy in mucormycosis

Membrane damaging action of <i>P</i>. <i>aeruginosa</i> biofilm culture filtrates (non-CF, Muc-CF and NMuc-CF) on preformed <i>A</i>. <i>fumigatus</i> biofilms, as shown by the morbidity stain DiBAC.

<p>(A) Fluorescence images of <i>A</i>. <i>fumigatus</i> preformed biofilms stained with DiBAC. (B) Relative fluorescence of preformed <i>A</i>. <i>fumigatus</i> biofilms stained with DiBAC. Bars represent fungicidal action as measured by DiBAC staining using spectrophotometry at 490 nm. Assays were performed in triplicate and results are presented as the mean of three replicates. Error bars represent the standard deviation of the mean. DIC: differential interference contrast. *<i>p</i> <0.05, ***<i>p</i> <0.0001 (compared with untreated controls).</p