Pathogenesis of Zygomycosis Is There Anything New?

- is there anything new?

Innsbruck Medical University ESCMID OnlineDivision of Hygiene Lecture and Medical Microbiology Library

2nd ESCMID Conference on Invasive Fungal Infections, Rome, 2013 Pathogenesis of Zygomycosis

Overview Topics…

…is there anything new?

Kontoyannis et al:“Explosion in the number of published studies“ © by author ESCMID Online Lecture Library

Kontoyiannis DP et.al. Future directions in mucormycosis research. Clin Infect Dis. 2012 Feb;54 Suppl 1:S79-85. Pathogenesis of Zygomycosis

Overview Topics

The pathogens – organisms involved in zygomycosis/new classification The infection clinical manifestation of mucormycosis risk factors

Virulence traits & host defense role of iron in pathogenesis angioinvasion and© cell by damage author – endothelial receptor host defense – differences to other fungal pathogens ESCMIDSummary / Conclusion Online Lecture Library

Pathogenesis of Zygomycosis – organisms involved very diverse group of fungi classification in change => based on morphologic similarities of sexual reproductive organs, life cycle, ultrastructure, nutritional modes etc.

mucorales, entomophthorales and 8 other orders now => taxonomy based on evolutionary relationships reflected in genomes of species sequence analysis of more than 9 regions (e.g.: rRNA subunits, tubulins, …)

Zygomycota = polyphyletic group© by author ESCMID Online Lecture Library

Kwon-Chung K J Clin Infect Dis. 2012;54:S8-S15 Pathogenesis of Zygomycosis – organisms involved

⇒ Zygomycota = polyphyletic group ⇒ phylum Glomeromycota  subphyla; Mucoromycotina & Entomophthoromycotina 2 different clades; still further revision

Rhizopus Lichtheimia

A proposed new classification scheme of the kingdom Fungi.

⇒ clinically relevant: Mucoromycotina & Entomophthoromycotina ⇒ differences between infections caused© by by Mucoromycotina author & Rhizomucor Cunighamella Entomophthoromycotina ⇒ name of the disease under discussion : zygomycosis = mucormycosis ESCMID Online Lecture Libraryzygospore of ⇒ most commonly found in human infections: Rhizopus Mucor Rhizopus spp., Lichtheimia (Absidia) spp., Mucor spp., Rhizomucor spp., Cunninghamella spp.

Kwon-Chung K J Clin Infect Dis. 2012;54:S8-S15 Pathogenesis of Zygomycosis

Characteristics of clinically most important orders of (formerly) „Zygomycota“

Mucorales – Entomophthorales – Mucormycosis Entomophthoromycosis

− common saprobes on decaying material; − „naturally“ insect pathogens worldwide distribution − tropical and subtropical − rapid growth − restricted growth − rapidly progressive and invasive − chronic and subcutanous − tissue necrosis − tissue infiltration − invasion of blood vessels © by author− infectious granuloma − primarily in immunosuppressed − also in immunocompetent − often rhinocerebral − sinusitis, hardly ever cerebral ESCMID Online Lecture Library

Chokka et al. Ind J Pathology & Mikrobiol,2010 Kwon-Chung K J Clin Infect Dis. 2012;54:S8-S15 Perusquia-Ortiz et al. JDDG, 2012 Pathogenesis of Mucormycosis – clinical manifestations

rare, but acute and highly destructive

hallmarks : angioinvasion => thrombosis => tissue necrosis affinity to CNS high mortality

Classification based on anatomic site

classification occurance mortality (%) (%) rhinocerebral 39 (sinuses) - pulmonary © by24 author 76 cutaneous 19 - gastrointestinal - 85 disseminated 23 96 uncommonESCMID (e.g. renal Online Lecture- - Library infection)

Lidor et al. New Engl J Med 1997; Petrikkos et al. CID 2012, 54(S1); Morace et al.Int J Micorbiol 2012 Pathogenesis of Mucormycosis – risk factors

in general: rare => in healthy individuals: intact immune system is effective almost always: underlying compromising condition  opportunistic disease

Conditions predisposing to mucormycosis:

- malignant hematological disease – highest in AML (acute myelogenous leukemia) - solid organ malignancies & solid organ transplantation - prolonged leukopenia (neutropenia and lymphopenia) - poorly controlled diabetes mellitus => hypergycemia, complicated by ketoacidosis - prolonged corticosteroid treatment - immunosuppression (therapy) - burns, major trauma - iron overload – chelation© therapy by with author deferoxamine (DFO) - voriconazole/caspofungin treatment => breakthrough zygomycosis - neonatal prematurity ESCMID- malnutrition Online Lecture Library - hospital stay/nosocomial

Petrikkos et al. CID 2012, 54(S1) Sun et al. Lancet Infect Dis 2011, 11 (4) Pathogenesis of Mucormycosis – virulence traits and host defense Potential virulence factors of opportunistic fungal pathogens: for Aspergillus fumigatus many known/discussed – what about Mucorales?

conidial size species dependent – 3-11µm – bigger than Aspergillus conidia temperature max. growth temperature for clin. relevant > 37°C growth speed of different Lichtheimia spp. (Schwartze et al.) => no obvious influence nutrient aquisition and utilization/metabolic flexibility Lichtheimia spp.- differences in nutrient/carbon utilization but no correspondance with virulence potential stress tolerance Lichtheimia spp.- differences in stress resistance (osmotic/oxidative and CW stress) but no correspondance with virulence potential secretion of proteases © by author larger numbers of genes for lytic enzymes than other fungi: SAPs (28), subtilases (23) iron aquisition/iron metabolism interactionESCMID with endothelial cellsOnline Lecture Library evading host defense downregulation of host genes (Drosophila model) involved in pathogen recognition, innate immune defense mechanisms and tissure repair mechanisms Alastruey-Izquierdo et al. J Clin Microbiol. 2010; Morace et al. Int J Micorbiol 2012; Schwartze et al. Plos ONE 2012; Ibrahim Curr. Opin.Microbiol 2011 Ma et al. Plos GENET: 2009; Chamilos et al. Proc Natl Acad Sci USA 2008 Pathogenesis of Mucormycosis – virulence traits and host defense

Potential virulence factor : iron uptake host defense strategy: low iron levels in serum /bound iron ⇒fungal pathogens possess special systems for iron uptake

⇒A. fumigatus: 2 strategies when iron depleted conditions reductive iron assimilation (reductase and permease (FTR1)) siderophore-mediated

Schrettl & Haas. Curr Opin Microbiol. 2011

⇒R.oryzae : no genes for nonribosomal peptides – no hydroxamate siderophores – depends on rhizoferrin (not efficient) reductive assimilation (FTR1)© and hemeoxygenasesby author (homologues found to C. albicans) => FTR1 is expressed during infection in DKA mice – also required for full virulence => can use xenosiderophores – iron chelators deferoxamine (DFO) => other chelators (DFX deferasirox, DFP deferiprone) ESCMID higher affinity Online to Fe-ions, fungicidal Lecture Library

Lewis et al. CID 2012, 54(S1); Roilides et al. CID 2012:54 Ibrahim et al. Mol Microbiol 2010; Ibrahim et al. CID 2012; 54 (S1) Symeonidis Clin Microbiol Infect 2009

Pathogenesis of Mucormycosis – virulence traits and host defense

Combination therapy of mucormycosis with chelators – in vitro and in vivo studies deferoxamine (DFO) increases susceptibility to mucormycosis – xenosiderophore deferiprone (DFP) as effective as liposomal amB in a murine model, synergistic effect if combined deferasirox (DFX) fungicidal effect species dependent, lowest MICs Rhizopus synergistic effect in combi with liposomal amB in murine studies, enhanced in triple therapy with micafungin  first clinical trial: DEFEAT Mucor study: randomized, double blinded, placebo-controlled trial 20 patients with probable or ©proven by mucormycosis author randomly treated with LAmB plus deferasirox, or LAmB plus placebo  death more frequent in deferasirox than in placebo arm at 30 days, also higher mortality rate ESCMIDat 90 days Online Lecture Library  reasons: small population, imbalanced

Ibrahim et al. AAC 2011; Lewis et al. AAC 2011 Ibrahim et al. J Antimicrob Chemother 2006 Spellberg et al. J Antimicrob Chemother 2012 Spellberg et al. Med Mycol 2012 Pathogenesis of Mucormycosis – virulence traits and host defense Angioinvasion as a pathogenic strategy of Mucorales

⇒ during angioinvasion pathogen invades (by inducing endocytosis) and damages vascular endothelial cells ⇒ identification of specific receptor responsible for invasion: GRP78 (glucose-regulated protein 78) ⇒ binds to germlings but not spores of Rhizopus and other pathogenic Mucorales, not A.fumigatus or C.albicans ⇒ invasion is two step approach: adhesion (receptor still unknown) and GRP78 triggered invasion ⇒ iron is involved in regulating cell damage (chelation with phenanthroline – prevents invasion less cell damage) © by author ⇒ iron and glucose (as in DKA patients) are inducers of GPR 78 (both on transcriptional and protein level)  possibly explains high susceptibility to mucormycosis in DKA patients ESCMID Online Lecture Library in vivo: diabetic ketoacidose (DKA) model: elevated iron and glucose – increased expression of GPR 78 in mice with DKA protection by blocking of GPR 78 by antibodies when given 2 h before infection Liu et al. J Clin Invest. 2010 Pathogenesis of Mucormycosis – virulence traits and host defense

Host defense – similarities and differences to other fungal pathogens natural immunity against zygomycosis is high in healthy humans usually opportunistic infections in patients with underlying disease

1st barrier – depending on entry site : skin bronchial cells/mucus/ciliary movement maintaining „anti-micorbial environment“ – e.g. low iron levels – bound to proteins

2nd : innate immunity: macrophages and neutrophils  phagocytosis  DKA & corticosteroid treatment PAMPs (pathogen associated molecular patterns)/soluble PRRs (pattern recognition receptors) release of cytokines, chemokines and growth factors Aspergillus: IL-18,© IL -12,by TNF αauthor => immunregulatory pathways and effector cells not much known for Mucorales cells ESCMIDthat also play a role: Onlineplatelets decrease Lecture germination and fungal Library growth in vitro NK cells - only damage Rhizopus hyphae (perforin), not conidia possible immunsuppressive effect

Roilides et al. CID 2012; Ibrahim et al. CID 2012 Perkhofer et al. J Infect Dis. 2009; Schmidt et al. Immunobiology 2012 Pathogenesis of Mucormycosis – virulence traits and host defense

Host defense – similarities and differences to other fungal pathogens differences* to Aspergillus – infections: phagoytosis of conidia: less efficient than for A. fumigatus also damage by phagocytes less, but species-dependent cytokine release varies: more pronounced proinflammatory response to Rhizopus than Aspergillus species (IL-6, TNFα)  differences in CW composition recognition: TLR2 only differentially expressed genes in monocytes of healthy donors

Roilides et al. CID 2012; Warris et al. Med Mycol 2005 *most studies carried out with R.oryzae Chamilos et al. Proc Natl Acad Sci USA 2008 Pathogenesis of Mucormycosis – conclusion

The key points of mucormycosis… …and future aims to increase our understanding of the disease

 rapidly growing pathogens with high affinity for blood vessels  unique susceptibility to mucormycosis of DKA patients  identification of one rezeptor involved (GRP78) and its increased expression in high iron and high glucose levels sheds light on the unique susceptibility of DKA patients to mucormycosis – also offers therapeutic option (anti-GRP78 Ab)  iron plays a central role in mucormycosis – iron chelation as a therapeutic strategy?  virulence properties need to be investigated in more detail  deciphering interaction between fungus and lung or sinus epithelial cells is necessary to understand how infection occurs – identification of new therapeutic targets  better understanding of host response© - especiallyby author with regard to different underlying conditions  genome sequencing of other species might help to decipher these virulence properties ESCMID Online Lecture Library

ESCMID Online Lecture LibraryUlrike Binder

Innsbruck Medical University Division of Hygiene and Medical Microbiology

2nd ESCMID Conference on Invasive Fungal Infections, Rome, 2013