HacA Governs Virulence Traits and Adaptive Stress Responses in Trichophyton rubrum

Abstract

The ability of fungi to sense environmental stressors and appropriately respond is linked to secretory system functions. The dermatophyte infection process depends on an orchestrated signaling regulation that triggers the transcription of genes responsible for adherence and penetration of the pathogen into host-tissue. A high secretion system is activated to support the host-pathogen interaction and assures maintenance of the dermatophyte infection. The gateway of secretion machinery is the endoplasmic reticulum (ER), which is the primary site for protein folding and transport. Current studies have shown that ER stress that affects adaptive responses is primarily regulated by UPR and supports fungal pathogenicity; this has been assessed for yeasts and Aspergillus fumigatus, in regard to how these fungi cope with host environmental stressors. Fungal UPR consists of a transmembrane kinase sensor (Ire1/IreA) and a downstream target Hac1/HacA. The active form of Hac is achieved via non-spliceosomal intron removal promoted by endonuclease activity of Ire1/IreA. Here, we assessed features of HacA and its involvement in virulence and susceptibility in Trichophyton rubrum. Our results showed that exposure to antifungals and ER-stressing agents initiated the activation of HacA from T. rubrum. Interestingly, the activation occurs when a 20 nt fragment is removed from part of the exon-2 and part of intron-2, which in turn promotes the arisen of the DNA binding site motif and a dimer interface domain. Further, we found changes in the cell wall and cellular membrane composition in the ΔhacA mutant as well as an increase in susceptibility toward azole and cell wall disturbing agents. Moreover, the ΔhacA mutant presented significant defects in important virulence traits like thermotolerance and growth on keratin substrates. For instance, the development of the ΔhacA mutant was impaired in co-culture with keratinocytes or human nail fragments. Changes in the pro-inflammatory cytokine release were verified for the ΔhacA mutant during the co-culture assay, which might be related to differences in pathogen-associated molecular patterns (PAMPs) in the cell wall. Together, these results suggested that HacA is an integral part of T. rubrum physiology and virulence, implying that it is an important molecular target for antidermatophytic therapy.

Keywords: dermatophytes; endoplasmic reticulum; host-pathogen interaction; mycoses; secretory system; unfolded protein response.

FIGURE 1

The hacA from T. rubrum is processed under antifungals exposure and ER stress-inducing agents. (A) RT-PCR analysis of hacA mRNA was used to detect the processing under antifungals and chemical exposure. (B) Schematic representation of an unconventional fragment removal of parts of the exon-2 and intron-2 from hacA mRNA, primers used for RT-PCR are indicated by gray boxes, the position of the excised fragment is shown by the color contoured box. (C) Prediction of the twin stem-loop structure of an unconventional splicing of T. rubrum and fungi from hacA mRNA carried out by mFOLD (Zuker, 2003) and the drawing of structures with VARNA (Darty et al., 2009). (D) Schematic representation of HacA induced isoform (hacAⁱ) with 395 aa and uninduced isoform (hacA^u) with 402 aa from T. rubrum. (E) Schematic representation of HacA induced (with 350 aa) and non-induced (with 441 aa) isoforms from A. nidulans. The introns in the hacA gene are represented by green boxes, and directly below both of the HacA isoforms are shown. The bZIP domain is shown in a pink box, the coil domain is shown in a blue rectangle, the mobiDB-lite domains are shown as dark green cylinders, a red triangle shows the DNA binding site, and the dimer interface residues are represented by a purple arrow. The transmembrane domain is shown in an orange lozenge and in a yellow lozenge is shown the non-cytoplasmatic domain, and the cytoplasmatic domain is shown as a gray box.

FIGURE 2

hacA plays a role in T. rubrum susceptibility toward antifungals and DTT. Susceptibility of T. rubrum strains to ketoconazole (KTC), in concentrations of 0.98, 1.95, and 3.90 μg/mL; DTT (5 and 10 mM); griseofulvin (GRS) in concentration of 0.98 μg/mL, and terbinafine (TRB), in a concentration of 0.005 μg/mL. Plates were inoculated, from left to right in each panel, with 10⁶, 10⁵, 10⁴, 10³, and 10² conidia/mL and incubated at 28°C for 7 days.

FIGURE 3

hacA affects T. rubrum susceptibility to cell wall inhibitor agents. Susceptibility of T. rubrum strains to CASP (0, 12.5, 25, 50, 100, and 200 μg/mL), and CFW (0, 5, 10, 20, 40, and 80 μg/mL). Plates were inoculated with approximately 1 × 10⁵ cells, and the drugs were added in corresponding concentrations from left to right, respectively, and incubated for 7 days at 28°C.

FIGURE 4

hacA affects thermotolerant growth. Equal concentrations of conidia from each strain were exposed to different temperatures (37 and 42°C) for 30 and 60 min. The control consisted of conidia from both strains without thermal exposition. Thereafter, conidia were inoculated in Sabouraud medium. The percentage reduction for the number of colonies is depicted in this graph. Significantly different values are shown by asterisks, and were determined using ANOVA followed by Tukey’s ad hoc test (**P < 0.01; ***P < 0.001).

FIGURE 5

hacA supports T. rubrum growth on keratin sources. An equal plug from each indicated strain was spotted onto plates with a different culture medium. The growth rate (diameter in cm) was calculated for each culture, and each strain after 9 days at 28°C. MM, minimal medium; MMK, minimal medium contained keratin; Sab, Sabouraud; PDA, Potato dextrose agar; MEA, agar malt extract. Statistical significance was determined using Two-Way RM ANOVA followed by Bonferroni’s post-test (**P < 0.01).

FIGURE 6

hacA supports hyphae directionality in T. rubrum. A microculture from both strains after 6 days at 28°C under microscopy light at magnification ×200.

FIGURE 7

hacA contributes to ergosterol biosynthesis in T. rubrum. Ergosterol content in wild type and mutant strains assessed per gram of mycelium dry weight.

FIGURE 8

hacA contributes to host-fungi interaction. Effect of the hacA gene deletion on the growth of T. rubrum on human molecules. (A) Conidia from wild type and mutant strain were incubated on human nails for 72 h at 28°C. Fungal growth was observed by light microscopy. The black objects seen on the left side are related to nail fragments. (B) Coculture of T. rubrum conidia from both strains with keratinocyte cell type HaCaT for 24 h at 37°C.

FIGURE 9

hacA supports keratinolytic feature of T. rubrum. (A) Dry weight mycelium from wild type and mutant strains expressed per gram of dry weight mycelium. (B) Keratinolytic activity from both strains was determined as mycelium-specific activities in units per gram of dry weight mycelium. Statistical significance determined using Unpaired t-test (**P < 0.01).

FIGURE 10

hacA participates in immune modulation in keratinocyte cells. Levels of pro-inflammatory cytokines output by HaCaT after coculture with conidia from the wild type and mutant strain for 24 h. Comparison of IL-8, IL-1β, and TNF-α secretion by HaCaT. Significantly different values are shown by asterisks, and were determined using ANOVA followed by Tukey’s ad hoc test (*P < 0.05; **P < 0.01).

FIGURE 11

hacA regulates genes belonging to the different metabolic processes. Transcriptional levels of encoding genes of chsD, fks1, hsp75-like, hsp90, erg1, and pkP evaluated by qPCR for 12 h of both T. rubrum strains (wild type and ΔhacA) growth on Sabouraud (SDA), or SDA with DTT (10 mM), or Terbinafine (TRB in 0.014 μg/mL) compared to 0 h (control). Significantly different values are shown by asterisks, and were determined using ANOVA followed by Tukey’s ad hoc test (*P < 0.05; **P < 0.01; ***P < 0.001). Asterisks close to the bars are related to the comparison with the calibrator (0 h WT).

FIGURE 12

hacA regulates genes coding for mannosyltransferase in T. rubrum. Transcriptional levels of N-mann, O-mann, and α-mann from T. rubrum conidia cocultured with human keratinocyte cell line HaCaT for 24 h. Statistical significance was determined using Unpaired t-test (*P < 0.05).

FIGURE 13

Putative HacA target genes in T. rubrum genome. Functional enrichment of genes in T. rubrum genome with a recognition site for UPRE-1 or UPRE-2 or UPR-3 motif consensus.