Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence

Abstract

C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host's immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host's environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality.

Fig 1. Infection in G.mellonella by sensitive and resistant C.albicans strains.

Comparison of sensitive and resistant strains of C.albicans virulence in G. mellonella infected with 2x10³ fungal cells/larvae and incubated at 37°C up to 5 days. (A) CO23_S,(B) CO23R_FLC, (C) CO23_RFK, (D) CO23_RR, (E) CI1 (F) CI2. Results are expressed as % survival. Experiments with samples untreated or injected with PBS are reported as controls. Each experiment was repeated three times.

Fig 2. Expression levels of C. albicans CDR1 and CDR2 genes determined by quantitative Real-Time PCR.

The expression level of the CDR1 (A) and CDR2 (B) genes in the wild-type (CO23), resistant (CO23RFK, CO23RFLC and CO23RR) strains and Clinical Isolates (CI1 and CI2) are represented as n-fold increase or decrease relative to the level of the control strain (CO23). Data are shown as mean±SEM from three independent experiments performed in triplicate. *p< 0.05, **p< 0.01, ***p< 0.001, ****p<0.0001 vs CO23_S and ^#p< 0.05, ^##p< 0.01, ^###p< 0.001, ^####p< 0.0001 vs CO23_RR.

Fig 3. Expression levels of C. albicans MDR1 gene determined by real-time PCR.

The expression level of the MDR1 gene in the wild-type (CO23), resistant (CO23RFK, CO23RFLC and CO23RR) strains and Clinical Isolates (CI1 and CI2) are represented as n-fold increase or decrease relative to the level of the control strain (CO23). Data are shown as mean±SEM from three independent experiments performed in triplicate. *p< 0,05, **p< 0,01, ***p< 0.001, ****p< 0,0001 vs CO23_S and ^#p< 0.05, ^##p< 0,01,^###p< 0.001, ^####p< 0,0001 vs CO23_RR.

Fig 4. Expression levels of C. albicans ALS1 and HWP1 genes determined by real-time PCR.

The expression level of the ALS1 (A) and HWP1 (B) genes in the wild-type (CO23), resistant (CO23RFK, CO23RFLC and CO23RR) strains and Clinical Isolates (CI1 and CI2) are represented as n-fold increase or decrease relative to the level of the control strain (CO23). Data are shown as mean±SEM from three independent experiments performed in triplicate. *p< 0.05, **p< 0.01, ***p< 0.001, ****p<0.0001 vs CO23_S and ^#p < 0.05, ^##p< 0,01, ^###p< 0.001, ^####p< 0,0001 vs CO23_RR.

Fig 5. Filamentation in C.albicans.

Hyphal growth of C. albicans strains CO23_S, CO23_RFK, CO23_RFLC, CO23_RR, CI1 and CI2. Cells were induced to form hyphae by 24 h incubation at 37°C in RPMI 1640 medium containing 10% fetal calf medium. Photomicrographs were taken by a phase contrast microscope using a 40x objective and they are representative of 50% random fields observed.