Silencing of OCH1 unveils the role of Sporothrix schenckii N-linked glycans during the host–fungus interaction

Abstract

Background

Sporothrix schenckii is a neglected fungal pathogen for the human being and other mammals. In several fungal systems, Och1 is a Golgi α1,6-mannosyltransferase with a key function in the synthesis of N-linked glycans; which are important elements during the host-fungus interplay. The role of OCH1 in fungal virulence seems to be species-specific, being an essential component for Candida albicans virulence and dispensable during the interaction of Aspergillus fumigatus with the host.

Methods

Here, we silenced S. schenckii OCH1 and characterized the phenotype of the mutant strains.

Results

The mutant strains did not show defects in the cell or colony morphology, the growth rate or the ability to undergo dimorphism; but the cell wall changed in both composition and exposure of inner components at the surface. When interacting with human monocytes, the silenced strains had a reduced ability to stimulate TNFα and IL-6 but stimulated higher levels of IL-10. The interaction with human macrophages was also altered, with reduced numbers of silenced cells phagocytosed. These strains showed virulence attenuation in both Galleria mellonella and in the mouse model of sporotrichosis. Nonetheless, the cytokine levels in infected organs did not vary significantly when compared with the wild-type strain.

Conclusion

Our data demonstrate that OCH1 silencing affects different aspects of the S. schenckii-host interaction.

Keywords:

• cell wall
• protein glycosylation
• sporotrichosis
• gene silencing
• host–fungus interplay
• mannosyltransferase

Supplementary materials

Figure S1 Comparison of the proteins encoded by members of the Sporothrix schenckii OCH1 gene family.

Notes: (A) Multiple sequence alignment generated with the CLC Sequence Viewer 7.8 using the following protein sequences retrieved from www.ncbi.nlm.nih.gov: ERS99987 (SsOch1), XP_016584588 (SsOch2), XP_016588949 (SsOch3), and XP_016587054 (SsOch4). The predicted consensus sequence is provided below each alignment. (B) Unrooted cladogram generated using the Neighbor-joining tree without distance correction algorithm. The same software and sequences defined in (A) were used to generate the tree. The SsOch1 is the most distant protein among the proteins encoded by members of the OCH1 gene family.

Figure S2 Colony morphology of OCH1-silenced and WT strains.

Notes: Cells were grown on plates containing YPD, pH 4.5, for 6 days at 28°C before the images were obtained. WT, strain 1099-18 ATCC MYA 4821. Scale bars=1 cm.

Abbreviation: WT, wild type.

Figure S3 Doubling time of OCH1-silenced and control strains.

Notes: Growth curves of mycelia in YPD, pH 4.5, were generated by determining the culture dry weight every 120 minutes, and from this, the doubling time was calculated (closed bars). Yeast-like cells were propagated in YPD, pH 7.8, and cell density was estimated by counting in a Neubauer hemocytometer. From the generated growth curve, the doubling time was estimated (open bars). The strains used are 1099-18 ATCC MYA 4821 (WT), HSS12 (WT^H1), and HSS13 (WT^H2).

Abbreviations: h, hours; WT, wild type.

Table S1 Comparison of members of the Sporothrix schenckii OCH1 family to α-1,6-mannosyltransferases belonging to family 32 found in Aspergillus fumigatus, Candida albicans, and Saccharomyces cerevisiae

Table S2 Comparison among members of the Sporothrix schenckii OCH1 family

Acknowledgments

We thank Prof Gordon Brown from the University of Aberdeen for the donation of the IgG Fc-Dectin-1 chimera. This work was supported by Consejo Nacional de Ciencia y Tecnología (ref. PDCPN2014-247109 and FC 2015-02-834), Universidad de Guanajuato (ref. 1025/2016; CIIC 95/2018), and Red Temática Glicociencia en Salud (CONACYT-México). LML-B is a research fellow of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 307169/2015-4) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP 2017/13722-5).

Disclosure

The authors report no conflicts of interest in this work.