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

ntial 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

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

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

Competitive interactions between C. albicans, C. glabrata and C. krusei during biofilm formation and development of experimental Candidiasis

In this study, we evaluated the interactions between Candida albicans, Candida krusei and Candida glabrata in mixed infections. Initially, these interactions were studied in biofilms formed in vitro. CFU/mL values of C. albicans were lower in mixed biofilms when compared to the single biofilms, verifying 77% and 89% of C. albicans reduction when this species was associated with C. glabrata and C. krusei, respectively. After that, we expanded this study for in vivo host models of experimental candidiasis. G. mellonella larvae were inoculated with monotypic and heterotypic Candida suspensions for analysis of survival rate and quantification of fungal cells in the haemolymph. In the groups with single infections, 100% of the larvae died within 18 h after infection with C. albicans. However, interaction groups achieved 100% mortality after 72 h of infection by C. albicans-C. glabrata and 96 h of infection by C. albicans-C. krusei. C. albicans CFU/mL values from larvae hemolymph were lower in the interacting groups compared with the monoespecies group after 12 h of infection. In addition, immunosuppressed mice were also inoculated with monotypic and heterotypic microbial suspensions to induce oral candidiasis. C. albicans CFU/mL values recovered from oral cavity of mice were higher in the group with single infection by C. albicans than the groups with mixed infections by C. albicans-C. glabrata and C. albicans-C. krusei. Moreover, the group with single infection by C. albicans had a higher degree of hyphae and epithelial changes in the tongue dorsum than the groups with mixed infections. We concluded that single infections by C. albicans were more harmful for animal models than mixed infections with non-albicans species, suggesting that C. albicans establish competitive interactions with C. krusei and C. glabrata during biofilm formation and development of experimental candidiasis

Quantification of fungal cells recovered from the buccal cavity of mice.

<p>(A) Mean and standard deviation of the CFU/mL (Log) of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>krusei</i> recovered from the buccal cavity of immunosuppressed mice with single and mixed infections. Student <i>t</i> test. *CFU/mL of <i>C</i>. <i>albicans</i>: comparison between single infection by <i>C</i>. <i>albicans</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>krusei</i> (<i>p</i> = 0.012); **CFU/mL of <i>C</i>. <i>krusei</i>: comparison between single infection by <i>C</i>. <i>krusei</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>krusei</i> (<i>p</i> = 0.004). (B) Mean and standard deviation of the CFU/mL (Log) of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>glabrata</i> recovered from the buccal cavity of immunosuppressed mice with monotypic and heterotypic infections. Student <i>t</i> test. *CFU/mL of <i>C</i>. <i>albicans</i>: comparison between single infection by <i>C</i>. <i>albicans</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>glabrata</i> (<i>p</i> = 0.079); **CFU/mL of <i>C</i>. <i>glabrata</i>: comparison between single infection by <i>C</i>. <i>glabrata</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>glabrata</i> (<i>p</i> = 0.001).</p

Macroscopic analysis of candidiasis lesions.

<p>Scores and medians obtained from the macroscopic examination of the tongue dorsum of groups infected with <i>C</i>. <i>albicans</i> monospecies, <i>C</i>. <i>krusei</i> monospecies, <i>C</i>. <i>glabrata</i> monospecies, <i>C</i>. <i>albicans</i>-<i>C</i>. <i>krusei</i> multi-species and <i>C</i>. <i>albicans</i>-<i>C</i>. <i>glabrata</i> multi-species. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (<i>p</i> = 0.0001), with similarities between <i>C</i>. <i>albicans</i> monospecies and multi-species groups, and variations when compared with <i>C</i>. <i>krusei</i> and <i>C</i>. <i>glabrata</i> monospecies groups.</p

Quantification of hyphae, yeast and epithelial changes.

<p>(A) Scores and medians of the number of hyphae and yeast derived from single and mixed infection. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (<i>p</i> = 0.0009), with variations between <i>C</i>. <i>albicans</i> monospecies and multi-species groups, and similarities amid the latter. (B) Number of epithelial changes and medians observed in candidosis microscopic lesions on the tongue dorsum of mice inoculated with <i>Candida</i> monospecies and multi-species suspensions. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (<i>p</i> = 0.0013), with variations between <i>C</i>. <i>albicans</i> monospecies and multi-species groups, and similarities amid the latter.</p

Epithelial changes caused by candidiasis lesions.

<p>(A) Sagittal incision of the tongue dorsum of mice infected with <i>C</i>. <i>albicans</i> monospecies inoculum, displaying the presence of intraepithelial microabscesses (↓), exocytosis and spongiosis. Moderate inflammatory infiltrate is seen in lamina propria. HE; magnification: 200X. (B) Sagittal incision of the tongue dorsum of mice infected with <i>C</i>. <i>krusei</i> monospecies inoculum, displaying normal tissue appearance. HE; magnification: 200X. (C) Sagittal incision of the tongue dorsum of mice in the <i>C</i>. <i>glabrata</i> control group, displaying normal tissue appearance. HE; magnification: 100X. (D) Sagittal incision of the tongue dorsum of mice in the <i>C</i>. <i>albicans</i> and <i>C</i>. <i>krusei</i> interaction group, displaying the presence of spongiosis, basal layer duplication, hyperplasia and epithelial desquamation. Moderate inflammatory infiltrate is seen in lamina propria (↓). HE; magnification: 200X.</p

<i>Candida</i> hyphae and yeast on the tongue dorsum of mice.

<p>(A) Sagittal incision of the tongue dorsum of mice infected with <i>C</i>. <i>albicans</i> monospecies inoculum, displaying the presence of yeast and hyphae (↓) in keratin. PAS; original magnification: 200X. (B) Sagittal incision of the tongue dorsum of mice infected with <i>C</i>. <i>albicans</i> monospecies inoculum, displaying the presence of yeast and hyphae (↓) in keratin, and polymorphonuclear leukocytes forming intraepithelial microabscesses. PAS; magnification: 630X. C) Sagittal incision of the tongue dorsum of mice infected with <i>C</i>. <i>albicans</i> and <i>C</i>. <i>krusei</i> multi-species inoculum, displaying the presence of yeast and hyphae (↓) in keratin, and spongiosis in the epithelium. PAS; magnification: 200X. D) Sagittal incision of the tongue dorsum of mice infected with <i>C</i>. <i>albicans</i> and <i>C</i>. <i>glabrata</i> multi-species inoculum, displaying the presence of yeast and hyphae (↓) in keratin and in the epithelium. PAS; magnification: 400X.</p

Survival curve of <i>G</i>. <i>mellonella</i> larvae infected with <i>Candida</i> strains.

<p>(A) The larvae were infected with <i>C</i>. <i>albicans</i>, <i>C</i>. <i>krusei</i> or <i>C</i>. <i>glabrata</i> (single infections); (B) The larvae were infected with <i>C</i>. <i>albicans</i> (single infection) and compared with larvae infected by <i>C</i>. <i>albicans</i> and <i>C</i>. <i>glabrata</i> (mixed infection); (C) The larvae were infected with <i>C</i>. <i>albicans</i> (single infection) and compared with larvae infected by <i>C</i>. <i>albicans</i> and <i>C</i>. <i>krusei</i> (mixed infection). Comparison of survival curves was made by Log rank test. These experiments were repeated at least twice and representative experiments are presented.</p

Quantification of <i>Candida</i> cells adhered to the swab.

<p>Mean and standard deviation of the CFU/mL (Log) of <i>C</i>. <i>albicans</i> in monotypic suspension, <i>C</i>. <i>krusei</i> in monotypic suspension, <i>C</i>. <i>glabrata</i> in monotypic suspension, <i>C</i>. <i>albicans</i> in heteroptypic suspension with <i>C</i>. <i>krusei</i>, <i>C</i>. <i>albicans</i> in heteroptypic suspension with <i>C</i>. <i>glabrata</i>, <i>C</i>. <i>krusei</i> in heteroptypic suspension with <i>C</i>. <i>albicans</i>, and <i>C</i>. <i>glabrata</i> in heteroptypic suspension with <i>C</i>. <i>albicans</i>. ANOVA test: there are no statistically significant differences between the groups.</p