Stress response in medically important Mucorales - Wiley Online Library

mycoses

Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Original article

Stress response in medically important Mucorales Pankaj Singh,a Saikat Paul,a M. Rudramurthy Shivaprakash, Arunaloke Chakrabarti and Anup K. Ghosh Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India

Summary

Mucorales are saprobes, ubiquitously distributed and able to infect a heterogeneous population of human hosts. The fungi require robust stress responses to survive in human host. We tested the growth of Mucorales in the presence of different abiotic stress. Eight pathogenic species of Mucorales, including Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, Apophysomyces elegans, Licthemia corymbifera, Cunninghamella bertholletiae, Syncephalastrum racemosum and Mucor racemosus, were exposed to different stress inducers: osmotic (sodium chloride and D-sorbitol), oxidative (hydrogen peroxide and menadione), pH, cell wall and metal ions (Cu, Zn, Fe and Mg). Wide variation in stress responses was noted: R. arrhizus showed maximum resistance to both osmotic and oxidative stresses, whereas R. pusillus and M. indicus were relatively sensitive. Rhizopus arrhizus and R. microsporus showed maximum resistance to alkaline pH, whereas C. bertholletiae, L. corymbifera, M. racemosus and A. elegans were resistant to acidic pH. Maximum tolerance was noted in R. microsporus to Cu, R. microsporus and R. arrhizus to Fe and C. bertholletiae to Zn. In contrast, L. corymbifera, A. elegans and M. indicus were sensitive to Cu, Zn and Fe respectively. In conclusion, R. arrhizus showed high stress tolerance in comparison to other species of Mucorales, and this could be the possible reason for high pathogenic potential of this fungi.

Key words: Mucorales, stress response, pH.

Introduction During stress condition, organism maintains its internal homeostasis to survive.1 However, the external environment is very dynamic in nature and result in a variety of perturbations that disturb the internal homeostasis.2 The evolution of fungi in heterologous environmental host like amoeba, plant, insects, etc., may help in its adaptation.3 Studies have correlated the evolution of stress adaptation in environment with Correspondence: Assoc. Prof. Dr. Anup K Ghosh, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh 160012, India. Tel.: +91 172 275 5156. Fax: +91 172 274 4401. E-mail: [email protected] a

Co-first authors.

Submitted for publication 19 January 2016 Revised 8 April 2016 Accepted for publication 13 April 2016

© 2016 Blackwell Verlag GmbH

the evolution of pathogenic fungi.3 In addition, certain reports have also suggested the potential association between stress response and expression of virulence gene in pathogen.3 The evaluation of the stress response therefore may be helpful to elucidate virulence potential of pathogenic fungi. Fungi have different lifestyle ranging from saprobe in the environment to commensals or pathogenic existence in human being.2 For pathogenic fungi, the capability to respond to stress is crucial to adapt in host environment.4 In nature, fungi survive in diverse ecological niches and are exposed to quite variable abiotic stresses (extreme temperature, pH, etc.).3 However, while causing infection in human host, the fungi have to adapt the high temperature of the human body, low oxygen concentration in tissue and lack of essential nutrients.2 The mechanism of stress faced by fungi under Mucorales is intriguing, as the hyphae are fragile and aseptate. However, a notable increase in the incidence

doi:10.1111/myc.12512

P. Singh et al.

of mucormycosis has been observed.5 Rhizopus arrhizus is the predominant isolate (70% cases) from patients, and other species, including Rhizopus microsporus, Rhizomucor pusillus, Apophysomyces elegans, Licthemia corymbifera, Cunninghamella bertholletiae, Syncephalastrum racemosum and Mucor indicus, were isolated at variable proportion from the patients.5 Possibly, high growth rate and thermo tolerance help them to adapt to stress.5,6 In this study, we evaluated the effect of abiotic stresses (osmotic, oxidative, pH, cell wall and metal ions) on the growth of Mucorales.

50 mmol l 1) and menadione sodium bisulphate (0.05–5 mmol l 1) for oxidative stress; media with range of pH from 1.5 to 12 for pH stress; Calcofluor white and Congo red (200–800 lg ml 1) for cell wall stress; CuSO4 (0.1–5 mmol l 1), ZnSO4 (0.5– 6 mmol l 1), FeSO4 (0.5–11 mmol l 1) and MgSO4 (50–500 mmol l 1) for metal stress were used. About 5 ll of each dilutions of fungal spores (10 1 to 10 6 spores ml 1) were spotted on plates containing stress inducing agent. Plates were incubated at 37 °C and growth was examined after 48 h incubation.9–11 Semi-quantitative analysis of stress resistance

Material and methods Fungal strains (Mucorales)

Eight different species of Mucorales used in this study were obtained from National Culture Collection of Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. These isolates included R. arrhizus (NCCPF no: 710222), R. microsporus (NCCPF 710189), R. pusillus (NCCPF 720004), A. elegans (NCCPF 102063, originally CBS 477.78), C. bertholletiae (NCCPF 890001), L. corymbifera (NCCPF 700002), S. racemosum (NCCPF 610007), M. indicus (NCCPF 690005). The isolates were revived and sub-cultured fresh on sabouraud dextrose agar (SDA) at 37 °C for 3–5 days. Preparation of inocula

Mucorales other than A. elegans were grown on SDA at 37 °C for sporulation. Water agar was used for sporulation in A. elegans.7,8 To harvest the spores, mycelial mats were removed and suspended in normal saline containing 0.03% Triton X-100. After a brief vortex, the mycelial mats were allowed to settle by keeping the tubes at room temperature for 10 min. The supernatant was carefully transferred to another tube and spores were collected by centrifugation at 3074 g for 5 min. Spores (106 spores ml 1) were counted by haemocytometer and serial dilutions (10 1 to 10 6) were prepared in normal saline except A. elegans, 105 spores ml 1 were used.9 Stress sensitivity assay

SDA with range of concentration of stress inducers was used to evaluate stress sensitivity of Mucorales. Sodium chloride (NaCl) (1–3 mol l 1) and D-sorbitol (1–5 mol l 1) for inducing osmotic stress; H2O2 (3–

2

To evaluate the stress sensitivity of Mucorales, we visually compared the growth of each species relative to their non-stress control for each dilution (10 1 to 10 6). The growth was observed in all dilutions for each stress condition tested and expressed as percentages of those on the corresponding control plates.9

Results To determine the stress sensitivity of Mucorales, eight clinical isolates were used. Stress sensitivity was examined by evaluating the effect on spore germination and growth of fungi. Fungal spores (1 9 106 conidia ml 1) were counted by haemocytometer except for A. elegans (105 spores ml 1 were used) and serial dilutions were (10 1 to 10 6) prepared in normal saline. No growth was observed at higher dilutions (10 4, 10 5 and 10 6). Osmotic stress sensitivity

Our result revealed that R. microsporus, R pusillus, A. elegans and M. indicus were sensitive to NaCl and failed to grow above 1 mol l 1, whereas, R. arrhizus, L. corymbifera and C. bertholletiae exhibited high resistance and were able to grow up to 1.5 mol l 1 NaCl, but no growth was observed in 2 mol l 1 NaCl (Table 1 and Fig. 1). The isolates showed varied sensitivity to sorbitol-induced osmotic stress. Rhizomucor pusillus and M. indicus were more sensitive to sorbitol and no growth was observed above 2 mol l 1. Rhizopus arrhizus was the most resistant species and grew even at 4 mol l 1 sorbitol. Other species including A. elegans, L. corymbifera, C. bertholletiae and S. racemosum showed intermediate sensitivity and tolerated up to 3 mol l 1 sorbitol stress (Table S1 and Fig. S1).


Stress response in pathogenic Mucorales

Table 1 Relative sensitivity of Mucorales to sodium chloride

(NaCl). NaCl (mol l 1)

Relative growth %1 Species

Control

1

1.5

2

R. arrhizus R. microsporus R. pusillus A. elegans L. corymbifera C. bertholletiae S. racemosum M. indicus

100 100 100 100 100 100 100 100

92 48 40 56 92 40 56 32

32 8 0 0 16 8 16 0

0 0 0 0 0 0 0 0

1

The fungal growth was observed after 48 h. NaCl stress sensitivities were quantified by calculating the percentage growth under each condition relative to the corresponding non-stress control for that species.

Oxidative stress sensitivity

Rhizomucor pusillus, A. elegans and S. racemosum were relatively more sensitive to hydrogen peroxide and no growth was observed above 5 mmol l 1. Rhizopus microsporus, L. corymbifera, C. bertholletiae and M. indicus showed intermediate resistance and grew up to 10 mmol l 1 (Table 2 and Fig. 2). Rhizomucor pusillus, L. corymbifera and S. racemosum were relatively more sensitive to menadione and no growth was observed above 1 mmol l 1; R. microsporus and C. bertholletiae withstand to 2 mmol l 1 menadione (Table S2). Rhizopus arrhizus was resistant to both oxidative stress and grew even at 20 mmol l 1 H2O2 and 3 mmol l 1 menadione.

Table 2 Relative sensitivity of Mucorales to hydrogen peroxide

(H2O2). Hydrogen peroxide (H2O2, mmol l 1)


Control

3

5

10

20

30

R. arrhizus R. microspores R. pusillus A. elegans L. corymbifera C. bertholletiae S. racemosum M. indicus

100 100 100 100 100 100 100 100

100 100 100 100 100 100 100 100

100 100 64 48 92 100 92 100

100 64 0 0 8 92 0 100

24 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

pH stress sensitivity

Rhizopus arrhizus and R. microsporus were most resistant to alkaline pH and could grow up to pH 12. While A. elegans, L. corymbifera, C. bertholletiae and M. indicus showed higher resistance to acidic pH and grew up to pH 2 (Table 3). Cell wall stress sensitivity

Mucorales tested in this study were resistant to both Calcofluor white and Congo red (grew up to 800 lg ml 1) and no difference was noted in their resistance (data not shown). Metal stress sensitivity

Iron (Fe) Apophysomyces elegans was sensitive to Fe and growth was observed till 7 mmol l 1. Rhizomucor pusillus, L. corymbifera, S. racemosum and M. indicus showed intermediate resistance and growth (50%) was observed at 11 mmol l 1 Fe. In contrast, R. arrhizus and R. microsporus were resistant and showed confluent growth even at 11 mmol l 1 Fe (Table 4 and Fig. S2). Copper (Cu) Licthemia corymbifera was sensitive to Cu and could grow only up to 1.5 mmol l 1. Rhizomucor pusillus, A. elegans, C. bertholletiae and M. indicus showed intermediate resistance and grew up to 2 mmol l 1. Rhizopus arrhizus, S. racemosum and R. microsporus were resistant and grew till 3 mmol l 1 Cu (Table S3). Zinc (Zn) Apophysomyces elegans was sensitive to Zn and tolerated only up to 3 mmol l 1. Rhizopus arrhizus, R. pusillus, L. corymbifera, S. racemosum and M. indicus showed intermediate resistance and grew at 4 mmol l 1 Zn. While R. microsporus and C. bertholletiae were relatively more resistant and grew at 5 mmol l 1 Zn (Table S4 and Fig. S3). Magnesium (Mg) All Mucorlaes were resistant to Mg stress and showed growth at all concentration of Mg (data not shown).

Discussion

1

The fungal growth was observed after 48 h. H2O2 stress sensitivities were quantified by calculating the percentage growth under each condition relative to the corresponding non-stress control for that species.


Fungi are ubiquitous and exposed to different abiotic stresses including oxidative stress, osmotic stress, pH, cell wall and metal ions in nature. The adaptation of

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P. Singh et al.

Figure 1 Comparison of Mucorales NaCl sensitivities. Growth of fungi on media containing various NaCl concentrations; the control

plates lack NaCl.

Figure 2 Comparison of Mucorales H2O2 sensitivities. Growth of fungi on media containing various H2O2 concentrations; the control plates lack H2O2.

fungi to these stresses in dynamic ecological niche determines their evolution in an environment. In this study, the sensitivities of Mucorales to abiotic stresses

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were evaluated by observing the effect on growth. Different species showed wide variation in stress sensitivity.



Table 3 Relative sensitivity of Mucorales

to pH.

pH


Control

1.5

2

3

4

8

9

10

11

12

R. arrhizus R. microspores R. pusillus A. elegans L. corymbifera C. bertholletiae S. racemosum M. indicus

100 100 100 100 100 100 100 100

0 0 0 0 0 0 0 0

48 48 48 92 92 90 48 90

90 90 92 92 92 90 90 90

10 100 100 84 56 92 16 100

100 100 100 84 100 100 92 100

100 100 100 100 100 100 100 100

100 100 90 92 100 100 100 100

100 100 81 64 100 100 100 100

92 88 44 24 24 40 42 16

1

The fungal growth was observed after 48 h. pH stress sensitivities were quantified by calculating the percentage growth under each condition relative to the corresponding non-stress control for that species.

Table 4 Relative sensitivity of Mucorales to iron (Fe).

FeSO4 (mmol l 1)


Control

0.5

1

3

5

7

9

11

R. arrhizus R. microspores R. pusillus A. elegans L. corymbifera C. bertholletiae S. racemosum M. indicus A. elegans

100 100 100 100 100 100 100 100 100

100 100 100 84 100 100 100 92 84

100 100 95 72 100 100 100 72 72

100 100 91 64 100 92 100 64 64

100 100 82 48 100 92 100 48 48

100 100 55 24 92 64 84 32 24

100 100 36 0 64 48 50 24 24

84 100 18 0 40 48 40 0 24

1 The fungal growth was observed after 48 h. Iron sulphate (FeSO4) stress sensitivities were quantified by calculating the percentage growth under each condition relative to the corresponding non-stress control for that species.

Osmotic stress induces the loss of water, causing shrinkage of cells leading to cell death.12 During such stress, fungi activate the high osmolarity glycerol (HOG) 1 response pathway, which in turn regulate the osmotic pressure by accumulation of glycerol.12 The majority of microorganism grows well at 0.85% (0.15 mol l 1) NaCl. Mucorales could withstand 0.5–1 mol l 1 NaCl.12 Optimal growth was observed at 0.5 mol l 1 NaCl, though isolates could tolerate up to 1.5 mol l 1 NaCl. The isolates were relatively more resistant to sorbitol (3 mol l 1) than NaCl (1.5 mol l 1). The reason for the difference in tolerability may be linked to non-ionic stress by sorbitol, while both ionic and osmotic stress by NaCl.9 A parasitic adaptation of fungi may also help in increased resistance to sorbitol. Mucorales cause diseases in both plants and animals.2 In human host, Mucorales are exposed to osmotic stress in phagocytic cells (k+ flux), kidney (NaCl, 600 mmol l 1) and skin (NaCl, 10 mol l 1).13 The inactivation of HOG 1 also


attenuates the virulence in fungi.12 Hence, during fungal–host interaction, the osmotic adaptation of fungi play major role in infection. Yeast and filamentous fungi withstand oxidative damage by production of catalases, peroxidases, glutathione peroxidase, melanin, etc.2,14,15 The expression profile of these enzymes and pigments in fungi increased during oxidative stress.16 Mutation and deletion of these genes and increased susceptibility to oxidative stress were demonstrated in fungi.2,14,17 Mucorales could grow under oxidative stress; R. arrhizus was relatively more resistant to menadione (3 mmol l 1) and H2O2 (20 mmol l 1) than other Mucorales, indicating the presence of well-developed oxidative stress-sensing signalling pathway. During infection, fungi exposed to oxidative stress, the phagocytic cells use reactive oxygen species (ROS) to kill the pathogens.14 The production of antioxidants and ROS neutralising enzymes by fungi may help to evade

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P. Singh et al.

oxidative damage by phagocytic cells.14–16 These enzymes convert the superoxide to peroxide and the peroxide is neutralised by catalases. Transition metals may convert H2O2 into hydroxyl radical and cause inhibition of fungal growth.2,9,14 The higher resistance to oxidative stress by R. arrhizus may be the reason of its association with human infection. Mucorales were completely resistant to both cell wall stress inducers (Congo red and Calcoflour white). Calcofluor white and Congo red induce cell wall stress by interacting with different cell wall components of fungi. Calcofluor white inhibits the formation of chitin and Congo red inhibits b-1, 3-glucan synthesis.18,19 Hence, the sensitivity of fungi to Calcoflour white and Congo red may vary with the difference in chitin and b-glucan content of the cell wall. The cell wall of Mucorales is composed of chitin–chitosan and protein.20 They showed similar response like other filamentous fungi. Confluent growth was observed at all concentration of Calcoflour white and Congo red. The absence of b-glucan in Mucorales, probably account for Congo red resistance. However, Chitin ranges from 22% to 44% in Mucorales cell wall, suggesting some other mechanisms may be responsible for the Calcofluor white resistance in fungi.9,20 In addition, the absence of b-glucan in Mucorales also make them intrinsically resistant to antifungal drugs like echinocandin in human host during infection.2,14 Fungi can sense and adapt to a wide range of pH in environment. pH serves as an external stimulus and provides information about the local environment to fungi. The pH signalling pathways play an important role in adaptation of fungi.2,13,14 Fungi sense and respond to ambient pH level via cell surface sensor, PalH and activation of a transcription factors, PacC in filamentous fungi and Rim 101 in yeast.2,13,14 Mucorales showed wide variation in sensitivity to the pH stress and were relatively resistant to both alkaline and acidic pH. During infection, Mucorales come across a variety of sites from skin to oral cavity to respiratory tract to urogenital tract with varied ranges in pH.21 Environmental pH also serves as a potent inducer of fungal development.22 pH is also supposed to have profound effect on drug therapy as well.23 The fluctuations in pH also modulate the activity of immunological agents.24 Thus, the adaptation of Mucorales to pH stress is essential for fungal pathogenicity.2 Metal ions at low concentration are necessary for the growth of fungi, but high concentrations of ions adversely affect the spore germination and

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growth.13,25,26 Fungi respond to metal stress by different ways like pump out across cell membrane, biosorption, entrapment in extracellular capsules, precipitation and transformation of metals.27 The chitin, chitosan and glucan present in the cell wall of Mucorales are known to be efficient metal ion biosorbents,20,28 which possibly leads to the metal resistance in this group of fungi. Iron (Fe) is required by most living organisms.26 Presence of free Fe is very low (10 14 mol l 1) in aerobic environment and cannot meet the optimum requirement of microbes (10 8 to 10 6 mol l 1).26,29,30 In addition, Fe can also induce free radical formation by Haber-Weiss-Fenton reaction.31 The main problem with Fe metabolism is acquisition and storage. In order to overcome this problem, fungi develop Fe uptake and storage system.26,27,29–31 Mucorales produce a-hydroxycarboxylate, siderophores, rhizoferrin to absorb Fe and zygoferritin for Fe storage.2,14,32 These Fe binding proteins make the fungi relatively resistant to Fe toxicity and may be responsible for increased Fe resistance in R. arrhizus and R. microsporus. During infection, pathogens have to compete with host for Fe, and the production of siderophores and Fe-binding protein increase the virulence potential of fungi during infection.2,13,14,29,30,33 Rhizopus arrhizus, R. microsporus and S. racemosum were relatively more resistant to Cu. This suggests that these fungi possibly detoxify free radicals by enzymes (catalase, peroxidase, superoxide dismutase, etc.) or accumulate excess Cu in cell wall and polyphosphate granules.27 Toxic concentration of Cu induces ROS generation in cells, causing lipids, DNA and proteins damage, leading to cellular injury.26 This may be responsible for increased sensitivity of some fungi to Cu. During infection, immune cells prevent the fungal growth by reducing the metal (Cu) availability.2,14 The higher Cu resistance in R. arrhizus, R. microsporus and S. racemosum indicates the presence of well-developed Cu-sensing signalling pathway and higher pathogenic potential of these fungi. Zinc acts as a cofactor for many enzymatic processes in eukaryotic cells. During infection, host cells increased production of Zn binding proteins (calprotectin) in fungal-infected cells to reduce Zn levels, causing inhibition of fungal growth.34 Zn is an essential element for cellular activity, but toxic at higher concentrations.34 Host phagocytic cells also use Zn overloading mechanism to produce free radicals for killing of pathogens in phagosome.34 The higher resistance in R. microsporus and C. bertholletiae to Zn



indicate the abilities of these fungi to detoxify the Zn.27 The adaptation of Mucorales to Zn toxicity suggests the presence of well-developed signalling pathway for Zn metabolism in fungi. Magnesium (Mg) is a macronutrient, required by microbes for the maintenance of integrity of cell membrane, nucleic acids, chromosomes, etc.35 This suggest Mg homeostasis must be tightly regulated in all cells.35 In fungi, the Mg regulation involves the localisation, compartmentalisation and sequestration.35 During infection, macrophage transport metals out of phagosome and mutational disruption of the gene also results in increased susceptibility to infection by intracellular pathogens.36 This suggests the important role of nutritional immunity in human infection. In this study, Mucorales were relatively resistant to Mg, suggesting the presence of well-developed signalling pathway for Mg hemostasis. Although the present study has yielded some preliminary findings, the major limitation of this study was that all the experiments were performed only with a single strain per species.

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12 13 14

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Conclusion Mucorales exist in nature as saprobes. The disease in human depends on host, pathogens and environmental interaction. The ability of stress response in fungi determines its virulence. The higher stress resistance by R. arrhizus compared to other Mucorales is the possible reason of its comparatively higher association with human infection.

Acknowledgments We duly acknowledge Prasanna Honnavar for scientific input and Indian Council of Medical Research (ICMR), New Delhi, India for the financial support for this research.

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Conflicts of interest

24

There are no conflicts of interest to declare for any of the authors in the study.

25 26 27

References 1 2 3

Woods HA, Wilson JK. An information hypothesis for the evolution of homeostasis. Trends Ecol Evol 2013; 28: 283–9. Cooney NM, Klein BS. Fungal adaptation to the mammalian host: it is a new world, after all. Curr Opin Microbiol 2008; 11: 511–6. Casadevall A. Amoeba provide insight into the origin of virulence in pathogenic fungi. Adv Exp Med Biol 2012; 710: 1–10.


28

29

Bahn YS, Xue C, Idnurm A, Rutherford JC, Heitman J, Cardenas ME. Sensing the environment: lessons from fungi. Nat Rev Microbiol 2007; 5: 57–69. Chakrabarti A, Singh R. Mucormycosis in India: unique features. Mycoses 2014; 57(Suppl. 3): 85–90. Binder U, Maurer E, Lass-Florl C. Mucormycosis–from the pathogens to the disease. Clin Microbiol Infect 2014; 20(Suppl. 6): 60–6. King AD Jr, Hocking AD, Pitt JI. Dichloran-rose bengal medium for enumeration and isolation of molds from foods. Appl Environ Microbiol 1979; 37: 959–64. Padhye AA, Ajello L. Simple method of inducing sporulation by Apophysomyces elegans and Saksenaea vasiformis. J Clin Microbiol 1988; 26: 1861–3. Nikolaou E, Agrafioti I, Stumpf M, Quinn J, Stansfield I, Brown AJ. Phylogenetic diversity of stress signalling pathways in fungi. BMC Evol Biol 2009; 9: 44. Reetha S, Bhuvaneswari G, Selvakumar G, Thamizhiniyan P, Pathmavathi M. Effect of temperature and pH on growth of fungi Trichoderma harzianum. J Chem Biol Phys Sci 2014; 4: 3287– 92. Baidya S, Duran RM, Lohmar JM et al. VeA is associated with the response to oxidative stress in the aflatoxin producer Aspergillus flavus. Eukaryot Cell 2014; 13: 1095–103. Hohmann S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 2002; 66: 300–72. Brown AJ, Budge S, Kaloriti D et al. Stress adaptation in a pathogenic fungus. J Exp Biol 2014; 217: 144–55. Hartmann T, Sasse C, Schedler A, Hasenberg M, Gunzer M, Krappmann S. Shaping the fungal adaptome–stress responses of Aspergillus fumigatus. Int J Med Microbiol 2011; 301: 408–16. Jamieson DJ. Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 1998; 14: 1511–27. Angelova MB, Pashova SB, Spasova BK, Vassilev SV, Slokoska LS. Oxidative stress response of filamentous fungi induced by hydrogen peroxide and paraquat. Mycol Res 2005; 109: 150–8. Kim JH, Chan KL, Faria NC, Martins Mde L, Campbell BC. Targeting the oxidative stress response system of fungi with redox-potent chemosensitizing agents. Front Microbiol 2012; 3: 88. Herth W. Calcofluor white and Congo red inhibit chitin microfibril assembly of Poterioochromonas: evidence for a gap between polymerization and microfibril formation. J Cell Biol 1980; 87: 442–50. Roncero C, Duran A. Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization. J Bacteriol 1985; 163: 1180–5. Zamani A, Edebo L, Sjostrom B, Taherzadeh MJ. Extraction and precipitation of chitosan from cell wall of zygomycetes fungi by dilute sulfuric acid. Biomacromolecules 2007; 8: 3786–90. Morace G, Borghi E. Invasive mold infections: virulence and pathogenesis of mucorales. Int J Microbiol 2012; 2012: 349278. Ernst JF. Transcription factors in Candida albicans – environmental control of morphogenesis. Microbiology 2000; 146(Pt 8): 1763–74. Pd R. pH and multidrug resistance. Novartis Found Symp 2001; 240: 232–47. Lardner A. The effects of extracellular pH on immune function. J Leukoc Biol 2001; 69: 522–30. Prentice AM, Ghattas H, Cox SE. Host-pathogen interactions: can micronutrients tip the balance? J Nutr 2007; 137: 1334–7. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem 2005; 12: 1161–208. de Lima MA, Franco Lde O, de Souza PM et al. Cadmium tolerance and removal from Cunninghamella elegans related to the polyphosphate metabolism. Int J Mol Sci 2013; 14: 7180–92. Wang J, Chen C. Chitosan-based biosorbents: modification and application for biosorption of heavy metals and radionuclides. Bioresour Technol 2014; 160: 129–41. Hissen AH, Wan AN, Warwas ML, Pinto LJ, Moore MM. The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding

7

P. Singh et al.

30

31 32

33

34

35 36

8

L-ornithine N5-oxygenase, is required for virulence. Infect Immun 2005; 73: 5493–503. Schrettl M, Bignell E, Kragl C et al. Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence. J Exp Med 2004; 200: 1213–9. Kehrer JP. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 2000; 149: 43–50. de Locht M, Boelaert JR, Schneider YJ. Iron uptake from ferrioxamine and from ferrirhizoferrin by germinating spores of Rhizopus microsporus. Biochem Pharmacol 1994; 47: 1843–50. Schrettl M, Bignell E, Kragl C et al. Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection. PLoS Pathog 2007; 3: 1195–207. Winters MS, Chan Q, Caruso JA, Deepe GS Jr. Metallomic analysis of macrophages infected with Histoplasma capsulatum reveals a fundamental role for zinc in host defenses. J Infect Dis 2010; 202: 1136–45. Hartwig A. Role of magnesium in genomic stability. Mutat Res 2001; 475: 113–21. Lang T, Prina E, Sibthorpe D, Blackwell JM. Nramp1 transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on antigen processing and presentation. Infect Immun 1997; 65: 380–6.

Supporting Information Additional Supporting Information may be found online in the supporting information tab for this article: Figure S1. Comparison of Mucorales sorbitol sensitivities. Figure S2. Comparison of Mucorales iron (Fe) sensitivities. Figure S3. Comparison of Mucorales zinc (Zn) sensitivities. Table S1. Relative sensitivity of Mucorales to sorbitol. Table S2. Relative sensitivity of Mucorales to menadione. Table S3. Relative sensitivity of Mucorales to copper (Cu). Table S4. Relative sensitivity of Mucorales to zinc (Zn).
