Differences in cell morphometry, cell wall topography and gp70 expression correlate with the virulence of Sporothrix brasiliensis clinical isolates

Abstract

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.

Figure 1. Virulence profile in a murine model of cutaneous sporotrichosis.

BALB/c mice were subcutaneously infected with 10⁷ conidia of clinical isolates of S. schenckii or S. brasiliensis. (A) Evolution of primary lesions in the dorsal sacral region of mice infected with S. brasiliensis and S. schenckii isolates. (B) Fungal load of the primary cutaneous lesions determined 40 days post-infection. ^a p<0.05 relative to 1099-18 isolate; ^b p<0.05 relative to IPEC 15383 isolate; ^c p<0.05 relative to IPEC 17943 isolate; ^dp<0.05 relative to Ss54 isolate; ^e p<0.05 relative to HUPE 114158 isolate.

Figure 2. Morphology and morphometry of the mycelial phase of S. schenckii and S. brasiliensis clinical isolates.

(A, B) Scanning electron microscopy of 1099-18 and IPEC 15383 strains of S. schenckii, respectively; (C, D) Scanning electron microscopy of IPEC 17943 and 5110 strains of S. brasiliensis, respectively. (E) Morphometric analysis of S. schenckii and S. brasiliensis conidia showing the cell mean size (+/- standard deviation). Forty five conidial cells of each strain were analyzed. *p< 0.05 relative to the S. brasiliensis isolates; ** p< 0.05 relative to the 5110 isolate.

Figure 3. Morphology and morphometry of the yeast phase of S. schenckii and S. brasiliensis clinical isolates.

(A, B) Scanning electron microscopy of 1099-18 and IPEC 15383 of S. schenckii, respectively; (C, D) IPEC 17943 and (D) 5110, S. brasiliensis Scanning electron microscopy of IPEC 17943 and 5110 strains of S. brasiliensis, respectively. (E) Morphometric analysis of yeast cells of S. schenckii and S. brasiliensis isolates showing the cell mean size (+ standard deviation). Fifty eight yeast cells of each strain were analyzed. * p< 0.05 relative to all other isolates.

Figure 4. Scanning electron microscopy (SEM) of the cell surface of S. schenckii and S. brasiliensis yeast cells.

(A, E) 1099-18; (B, F) IPEC 15383; (C, G) IPEC 17943 and (D, H) 5110. For the 1099-18, IPEC 15383 and 5110 strains the outer cell layer shows a dense amorphous fibrillar material. The white arrow indicates a cell wall fracture observed in the IPEC 17943 strain. (A-D) Scale bar -1.0 µm.

Figure 5. Gp70 expression on the cell wall of S. schenckii and S. brasiliensis clinical isolates.

The expression of gp70 in cell wall extracts (see Methods) was verified by western blot analysis of S. schenckii 1099-18 (A) and IPEC 15383 (B); and (C) of S. brasiliensis IPEC 17943 (C) and 5110 (D). The amount of protein loaded was 5 µg (A-C) and 15µg (D). (E-M) Scanning electron microscopy showing the backscattered electron imaging (I-M) of yeast cells of S. schenckii 1099-18 (E, I), IPEC 15383 (F, J) and of S. brasiliensis IPEC 17943 (G, L) and 5110 (H, M) which were incubated with a monoclonal antibody anti-gp70 followed by a mouse anti-IgG gold-conjugate. E-H, Scale bar 1.0 µm.

Figure 6. Western blot analysis of the cell surface gp70 of S. brasiliensis.

The gp70 antigen was revealed on cell surface extracts of the yeast parasitic phase of two S. schenckii reference strains (lanes 1 and 2) and several clinical isolates of S. brasiliensis (lanes 3 to 8) by a monoclonal antibody anti-gp70, mAb P6E7. A purified gp70 was used as a positive control and in C is shown the negative control of the mAb P6E7. Correspondent strains in lanes (1) 1099-18; (2) IPEC 15383; (3) IPEC 17943; (4) 5110; (5) Ss 54; (6) UFTM 01; (7) HUPE 114500 and (8) HUPE 114158. The amount of protein loaded in lanes 1 to 8 was 5 µg..

Figure 7. Distribution and subcellular localization of gp70 on yeast cells of Sporothrix clinical isolates.

Transmission electron micrograph of S. schenckii or S. brasiliensis yeast cells incubated with the monoclonal anti-gp 70 antibody followed by a mouse anti-IgG gold-conjugated antibody. Is shown in (A) S. schenckii 1099-18, (B) S. schenckii IPEC 15383, (C) S. brasiliensis IPEC 17943 and (D) S. brasiliensis 5110. Arrows indicate localization of gp70 on the cell wall (black arrows), in the cytoplasm (white arrows) and at the extracellular compartment (arrowheads). Images featured were digitally magnified.

Figure 8. In silico characterization of gp70 in S. schenckii (1099-18) and S. brasiliensis (5110).

Identification of important domains and putative glycosylation sitesin the gp70 protein sequence using InterPro and EnsembleGly. The gene and protein sequences of gp70 were deposited in the GenBank and EMBL-EBI, respectively.