Correlation of phospholipase and proteinase production of Candida with in vivo pathogenicity in Galleria mellonella

Rossoni, Rodnei Dennis; Barbosa, Júnia Oliveira; Vilela, Simone Furgeri Godinho; Santos, Jéssica Diane dos; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

Abstract

An essential factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. AIM:This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experimental model. METHODS: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. RESULTS: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. CONCLUSIONS: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain.

Candida; virulence factors; invertebrates

References

¹ Pathak AK, Sharma S, Shrivastva P. Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate. J Appl Oral Sci. 2012; 20: 70-5.
² Machado AG, Komiyama EY, Santos SS, Jorge AO, Brighenti FL, Koga-Ito CY. In vitro adherence of Candida albicans isolated from patients with chronic periodontitis. J Appl Oral Sci. 2011; 19: 384-7.
³ Noumi E, Snoussi M, Hentati H, Mahdouani K, del Castillo L, Valentin E, et al. Adhesive properties and hydrolytic enzymes of oral Candida albicans strains. Mycopathologia. 2010; 169: 269-78.
⁴ Hube B, Naglik J. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology. 2001; 147: 1997-2005.
⁵ Seneviratne CJ, Wong SS, Yuen KY, Meurman JH, Pärnänen P, Vaara M, et al. Antifungal susceptibility and virulence attributes of bloodstream isolates of Candida from Hong Kong and Finland. Mycopathologia. 2011; 172: 389-95.
⁶ Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Adherence and biofilm formation of non-Candida albicans Candida species. Trends. Microbiol. 2011; 19: 241-7.
⁷ Allen CM. Animal models of oral candidiasis: a review. Oral Surg Oral Med Oral Pathol. 1994; 78, 216:21.
⁸ Junqueira JC, Martins JS, Faria RL, Colombo CED, Jorge AOC. Photodynamic therapy for the treatment of buccal candidiasis in rats. Lasers Med Sci. 2009; 24: 877-84.
⁹ Samaranayake YU, Samaranayake LP. Experimental oral candidiasis in animal models. Clin Microbiol. Rev. 2001; 14: 398-429.
¹⁰ Takakura N, Sato Y, Ishibashi H, Oshima H, Uchida K, Yamaguchi H, et al. A novel murine model of oral candidiasis with local symptoms characteristic of oral thrush. Microbiol Immunol. 2003; 47: 321-6.
¹¹ Teichert MC, Jones J.W, Usacheva MN, Biel MA. Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002; 93: 155-60.
¹² Junqueira JC. Galleria mellonella as a model host for human pathogens: recent studies and new perspectives. Virulence. 2012; 3: 474-6.
¹³ Mylonakis E. Galleria mellonella and the study of fungal pathogenesis: making the case for another genetically tractable model host. Mycopathologia. 2008; 165: 1-3.
¹⁴ Cotter G, Doyle S, Kavanagh K. Development of an insect model for the in vivo pathogenity testing of yeasts. FEMS Immunol Med Microbiol. 2000; 27: 163-9.
¹⁵ Fuchs BB, Mylonakis E. Using non-mammalian hosts to study fungal virulence and host defense. Curr Opin Microbiol. 2006; 9: 346-51.
¹⁶ Naglik JR, Fidel PL Jr, Odds FC. Animal models of mucosal Candida infection. FEMS Microbiol Lett. 2008; 283: 129-39.
¹⁷ Brennan M, Thomas DY, Whiteway M, Kavanagh K. Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae. FEMS Immunol Med Microbiol. 2002; 34: 153-7.
¹⁸ Ghannoum MA. Potential role of phospholipases in virulence and fungal pathogenesis. Clin Microbial Rev. 2000; 13: 122-43.
¹⁹ Junqueira JC, Vilela SF, Rossoni RD, Barbosa JO, Costa AC, Rasteiro VM, et al. Oral colonization by yeasts in HIV-positive patients in Brazil. Rev Inst Med Trop São Paulo. 2012; 54: 17-24.
²⁰ Price M.F, Wilkinson ID, Gentry LO. Plate method for detection of phospholipase activity in Candida albicans Sabouraudia. 1982; 20: 7-14.
²¹ Pereira CA, Costa AC, Machado AK, Beltrame Júnior M, Zöllner MS, Junqueira JC, et al. Enzymatic activity, sensitivity to antifungal drugs and Baccharis dracunculifolia essential oil by Candida strains isolated from the oral cavities of breastfeeding infants and in their mothers' mouths and nipples. Mycopathologia. 2011; 171: 103-9.
²² Ruchell R, Tegeller R, Trost MA. A comparison of secretory proteinases from different strains of Candida albicans Sabouraudia. 1982; 20: 233-44.
²³ Fuchs BB, Eby J, Nobile CJ, El Khoury JB, Mitchell AP, Mylonakis E. Role of filamentation in Galleria mellonella killing by Candida albicans Microbes Infect. 2010; 12: 488-96.
²⁴ Junqueira JC, Fuchs BB, Muhammed M, Coleman JJ, Suleiman JM, Vilela SF, et al. Oral Candida albicans isolates from HIV-positive individuals have similar in vitro biofilm-forming ability and pathogenicity as invasive Candida isolates. BMC Microbiol. 2011; 11: 247.
²⁵ Abaci O. Investigation of extracellular phospholipase and proteinase activities of Candida species isolated from individual dentures wearers and genotypic distribution of Candida albicans strains. Curr Microbiol. 2011; 62: 1308-14.
²⁶ Martins JS, Junqueira, JC, Faria RL, Santiago NF, Rossoni RD, Colombo CE, et al. Antimicrobial photodynamic therapy in rat experimental candidiasis: evaluation of pathogenicity factors of Candida albicans Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011; 111: 71-7.
²⁷ Mane A, Pawale C, Gaikwad S, Bembalkar S, Risbud A. Adherence to buccal epithelial cells, enzymatic and hemolytic activities of Candida isolates from HIV-infected individuals. Med Mycol. 2011; 49: 548-51.
²⁸ Mesa-Arango AC, Forastiero A, Bernal-Martínez L, Cuenca-Estrella M, Mellado E, Zaragoza O. The non-mammalian host Galleria mellonella can be used to study the virulence of the fungal pathogen Candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast. Med Mycol. 2013; 51: 461-72.
²⁹ Naglik JR, Moyes DL, Wachtler B, Hube B. Candida albicans interactions with epithelial cells and mucosal immunity. Microbes Infect. 2011; 13: 963-76.
³⁰ Bulushova NV, Elpidina EN, Zhuzhikov DP, Lyutikova LI, Ortego F, Kirillova NE, et al. Complex of digestive proteinases of Galleria mellonella caterpillars: composition, properties, and limited proteolysis of Bacillus thuringiensis endotoxins. Biochemistry (Mosc). 2011; 76: 581-9.

Publication Dates

Publication in this collection
28 Oct 2013
Date of issue
Sept 2013

History

Received
12 June 2013
Accepted
13 Sept 2013

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] ¹ Pathak AK, Sharma S, Shrivastva P. Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate. J Appl Oral Sci. 2012; 20: 70-5.

[2] ² Machado AG, Komiyama EY, Santos SS, Jorge AO, Brighenti FL, Koga-Ito CY. In vitro adherence of Candida albicans isolated from patients with chronic periodontitis. J Appl Oral Sci. 2011; 19: 384-7.

[3] ³ Noumi E, Snoussi M, Hentati H, Mahdouani K, del Castillo L, Valentin E, et al. Adhesive properties and hydrolytic enzymes of oral Candida albicans strains. Mycopathologia. 2010; 169: 269-78.

[4] ⁴ Hube B, Naglik J. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology. 2001; 147: 1997-2005.

[5] ⁵ Seneviratne CJ, Wong SS, Yuen KY, Meurman JH, Pärnänen P, Vaara M, et al. Antifungal susceptibility and virulence attributes of bloodstream isolates of Candida from Hong Kong and Finland. Mycopathologia. 2011; 172: 389-95.

[6] ⁶ Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Adherence and biofilm formation of non-Candida albicans Candida species. Trends. Microbiol. 2011; 19: 241-7.

[7] ⁷ Allen CM. Animal models of oral candidiasis: a review. Oral Surg Oral Med Oral Pathol. 1994; 78, 216:21.

[8] ⁸ Junqueira JC, Martins JS, Faria RL, Colombo CED, Jorge AOC. Photodynamic therapy for the treatment of buccal candidiasis in rats. Lasers Med Sci. 2009; 24: 877-84.

[9] ⁹ Samaranayake YU, Samaranayake LP. Experimental oral candidiasis in animal models. Clin Microbiol. Rev. 2001; 14: 398-429.

[10] ¹⁰ Takakura N, Sato Y, Ishibashi H, Oshima H, Uchida K, Yamaguchi H, et al. A novel murine model of oral candidiasis with local symptoms characteristic of oral thrush. Microbiol Immunol. 2003; 47: 321-6.

[11] ¹¹ Teichert MC, Jones J.W, Usacheva MN, Biel MA. Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002; 93: 155-60.

[12] ¹² Junqueira JC. Galleria mellonella as a model host for human pathogens: recent studies and new perspectives. Virulence. 2012; 3: 474-6.

[13] ¹³ Mylonakis E. Galleria mellonella and the study of fungal pathogenesis: making the case for another genetically tractable model host. Mycopathologia. 2008; 165: 1-3.

[14] ¹⁴ Cotter G, Doyle S, Kavanagh K. Development of an insect model for the in vivo pathogenity testing of yeasts. FEMS Immunol Med Microbiol. 2000; 27: 163-9.

[15] ¹⁵ Fuchs BB, Mylonakis E. Using non-mammalian hosts to study fungal virulence and host defense. Curr Opin Microbiol. 2006; 9: 346-51.

[16] ¹⁶ Naglik JR, Fidel PL Jr, Odds FC. Animal models of mucosal Candida infection. FEMS Microbiol Lett. 2008; 283: 129-39.

[17] ¹⁷ Brennan M, Thomas DY, Whiteway M, Kavanagh K. Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae. FEMS Immunol Med Microbiol. 2002; 34: 153-7.

[18] ¹⁸ Ghannoum MA. Potential role of phospholipases in virulence and fungal pathogenesis. Clin Microbial Rev. 2000; 13: 122-43.

[19] ¹⁹ Junqueira JC, Vilela SF, Rossoni RD, Barbosa JO, Costa AC, Rasteiro VM, et al. Oral colonization by yeasts in HIV-positive patients in Brazil. Rev Inst Med Trop São Paulo. 2012; 54: 17-24.

[20] ²⁰ Price M.F, Wilkinson ID, Gentry LO. Plate method for detection of phospholipase activity in Candida albicans Sabouraudia. 1982; 20: 7-14.

[21] ²¹ Pereira CA, Costa AC, Machado AK, Beltrame Júnior M, Zöllner MS, Junqueira JC, et al. Enzymatic activity, sensitivity to antifungal drugs and Baccharis dracunculifolia essential oil by Candida strains isolated from the oral cavities of breastfeeding infants and in their mothers' mouths and nipples. Mycopathologia. 2011; 171: 103-9.

[22] ²² Ruchell R, Tegeller R, Trost MA. A comparison of secretory proteinases from different strains of Candida albicans Sabouraudia. 1982; 20: 233-44.

[23] ²³ Fuchs BB, Eby J, Nobile CJ, El Khoury JB, Mitchell AP, Mylonakis E. Role of filamentation in Galleria mellonella killing by Candida albicans Microbes Infect. 2010; 12: 488-96.

[24] ²⁴ Junqueira JC, Fuchs BB, Muhammed M, Coleman JJ, Suleiman JM, Vilela SF, et al. Oral Candida albicans isolates from HIV-positive individuals have similar in vitro biofilm-forming ability and pathogenicity as invasive Candida isolates. BMC Microbiol. 2011; 11: 247.

[25] ²⁵ Abaci O. Investigation of extracellular phospholipase and proteinase activities of Candida species isolated from individual dentures wearers and genotypic distribution of Candida albicans strains. Curr Microbiol. 2011; 62: 1308-14.

[26] ²⁶ Martins JS, Junqueira, JC, Faria RL, Santiago NF, Rossoni RD, Colombo CE, et al. Antimicrobial photodynamic therapy in rat experimental candidiasis: evaluation of pathogenicity factors of Candida albicans Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011; 111: 71-7.

[27] ²⁷ Mane A, Pawale C, Gaikwad S, Bembalkar S, Risbud A. Adherence to buccal epithelial cells, enzymatic and hemolytic activities of Candida isolates from HIV-infected individuals. Med Mycol. 2011; 49: 548-51.

[28] ²⁸ Mesa-Arango AC, Forastiero A, Bernal-Martínez L, Cuenca-Estrella M, Mellado E, Zaragoza O. The non-mammalian host Galleria mellonella can be used to study the virulence of the fungal pathogen Candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast. Med Mycol. 2013; 51: 461-72.

[29] ²⁹ Naglik JR, Moyes DL, Wachtler B, Hube B. Candida albicans interactions with epithelial cells and mucosal immunity. Microbes Infect. 2011; 13: 963-76.

[30] ³⁰ Bulushova NV, Elpidina EN, Zhuzhikov DP, Lyutikova LI, Ortego F, Kirillova NE, et al. Complex of digestive proteinases of Galleria mellonella caterpillars: composition, properties, and limited proteolysis of Bacillus thuringiensis endotoxins. Biochemistry (Mosc). 2011; 76: 581-9.