The Sporothrix schenckii Gene Encoding for the Ribosomal Protein L6 Has Constitutive and Stable Expression and Works as an Endogenous Control in Gene Expression Analysis

Abstract

Sporothrix schenckii is one of the causative agents of sporotrichosis, a worldwide-distributed mycosis that affects humans and other mammals. The interest in basic and clinical features of this organism has significantly increased in the last years, yet little progress in molecular aspects has been reported. Gene expression analysis is a set of powerful tools that helps to assess the cell response to changes in the extracellular environment, the genetic networks controlling metabolic pathways, and the adaptation to different growth conditions. Most of the quantitative methodologies used nowadays require data normalization, and this is achieved measuring the expression of endogenous control genes. Reference genes, whose expression is assumed to suffer minimal changes regardless the cell morphology, the stage of the cell cycle or the presence of harsh extracellular conditions are commonly used as controls in Northern blotting assays, microarrays, and semi-quantitative or quantitative RT-PCR. Since the biology of the organisms is usually species specific, it is difficult to find a reliable group of universal genes that can be used as controls for data normalization in experiments addressing the gene expression, regardless the taxonomic classification of the organism under study. Here, we compared the transcriptional stability of the genes encoding for elongation factor 1A, Tfc1, a protein involved in transcription initiation on Pol III promoters, ribosomal protein L6, histone H2A, β-actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase, UAF30, the upstream activating factor 30, and the transcription initiation factor TFIID subunit 10, during the fungal growth in different culture media and cell morphologies. Our results indicated that only the gene encoding for the ribosomal protein L6 showed a stable and constant expression. Furthermore, it displayed not transcriptional changes when S. schenckii infected larvae of Galleria mellonella or interacted with immune cells. Therefore, this gene could be used as control for data normalization in expression assays. As a proof of concept, this gene was used to assess the expression of genes encoding for glycosidases involved in the protein N-linked glycosylation pathway, a histidine kinase whose expression is regulated during the fungal dimorphism, and a glycosidase that participates in sucrose assimilation.

Influences of the Culturing Media in the Virulence and Cell Wall of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa

“…Instead, we included YPD, another rich medium used to preserve and stimulate Sporothrix spp. dimorphism [ 14 , 33 , 34 , 83 ]. Moreover, most of the composition and structure analyses of S. schenckii and S. brasiliensis cell walls have been performed in YPD-grown cells [ 14 , 48 ], and it has been reported that the culture media and growth conditions influence fungal biology, including the cell wall composition [ 43 , 45 ].…”

Silencing of <em>OCH1 </em>unveils the role of <em>Sporothrix schenckii</em> <em>N</em>-linked glycans during the host&ndash;fungus interaction

“…Both the OCH1 expression level and the copy number of insertional events of pBGgHg- OCH1 were estimated with the StepOne software V 2.2 (Thermo Fisher Scientific) by calculating 2 −∆∆Ct 34. The encoding gene for the ribosomal protein L6 was used as an endogenous control, whereas the WT strain was defined as the reference condition 35. The primer pair 5′-ATTGCGACAT-CAGAGAAGG-3′ and 5′-TCGACCTTCTTGATGTTGG-3′ was used for amplification of the endogenous control.…”

The Search for Cryptic L-Rhamnosyltransferases on the Sporothrix schenckii Genome

“…To assess whether these sequences are indeed true genes, we first analyzed expression in different growth conditions. The gene encoding for ribosomal protein L6 and the filament growth in YPD broth were used as the reference gene and condition, respectively, for gene expression analysis [ 45 ]. The SPSK_05538 and SPSK_01110 sequences were found to be expressed in yeast-like cells growing in YPD broth, indicating that these are indeed genes ( Figure 1 ).…”

“…Hyphae were propagated at 28 °C in YPD, pH 4.5 medium (1% [ w / v ] yeast extract, 2% [ w / v ] gelatin peptone, and 3% [ w / v ] dextrose); whereas yeast-like cells were obtained in YPD, pH 7.8, as described elsewhere [ 14 ]. Fungal growth in brain-heart infusion (BHI), potato dextrose broth (PDB), Vogel media, and Galleria mellonella larva was carried out at 37 °C, as previously described [ 45 ]. For heterologous gene expression, Escherichia coli BL21 (DE3) from Invitrogen was used.…”

“…Relative gene expression was calculated with the StepOne software V 2.2 (Life Technologies) using the 2 − ∆∆Ct method [ 48 ]. The encoding gene for the ribosomal protein L6 was used as an endogenous control [ 45 ], while the filament growth in YPD at 28 °C was defined as the reference condition. The following primer pairs were used in the RT-qPCR reactions: 5′-ATTGCGACATCAGAGAAGG-3′ and 5′-TCGACCTTCTTGATGTTGG-3′ for the endogenous control; 5′- AACAGATTGAGGCTCTGGGC-3′ and 5′-AGTAAGTCTGGGTTCGCCAC-3′ for SPSK_05538; 5′-CAGAACGGAAAGACGGGACA-3′ and 5′-ACCACCGTTGTTGTTGACCT-3′ for SPSK_05928; 5′-AGGAATGACCATCAGCGCAA-3′ and 5′-AAACGCTCGGGAAAGTCCAT-3′ for SPSK_01368; 5′-AGCCACGAACGTCTCTTCAG-3′ and 5′-CTTCAAACGTGCCTGATGGC-3′ for SPSK_04821; and 5′-GTTTGTCCCCGACGTTTTGG-3′ and 5′-GCCGCGAGATGAAAAAGACG-3′ for SPSK_01110.…”