Blood is a complex milieu composed of several types of immunoactive cells and molecules. As we have previously observed, several clinical strains of S. cerevisiae adapt to blood environment and are able to survive and spread to other tissues in murine models. To better understand the response of S. cerevisiae to this complex environment and to search for special features of virulent strains we have analysed the transcriptome of S. cerevisiae cells after contact with human blood. The results showed that they express several groups of genes in order to rapidly accommodate to this special environment. As expected, a high mRNA level of genes encoding factors involved in protein synthesis were detected in every strain after 15 min inoculation in blood, except for the laboratory strain W303. The strong induction of these set of genes is probably a consequence of the yeast cells transfer from nutrient poor medium (PBS) to a relatively nutrient-rich medium (blood). The downregulation of PCK1, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, and other genes supports that yeast cells are exposed to a carbohydrate-rich environment, even at the later stages of incubation in blood, when most of the yeast cells are phagocytized. In C. albicans, a high level of different transcripts involved in protein synthesis was also detected at the beginning of the incubation in blood, but the mRNA level of these genes decreased during the incubation , indicating that growth initiation occurred only in the early stages. This suggests that C. albicans has a faster response to adapt to blood environment than S. cerevisiae.
We observed that the induction of genes involved in aminoacid biosynthetic pathway is present during the middle and the later time course in the virulent strains, while in non-virulent strains they appear later or don’t appear. Also, following exposure to human neutrophils or cultured macrophages, C. albicans populations upregulate amino acid biosynthetic genes [20, 26]. Rubin-Bejerano et al.  observed an induction of these pathways after yeast cells were ingested by neutrophils, but it was not present when yeast cells were ingested by human monocytes. It may suggest that the microenvironment in the phagosome inside the neutrophil is deficient in amino acids and it generates a rapid response from the virulent S. cerevisiae strains. However, non-virulent strains are slower or less efficient in the adaptation to this microenvironment, decreasing their possibilities to survive. In addition, the methionine and arginine biosynthetic genes are not induced when S. cerevisiae is phagocytized by the murine macrophage-like cell line J774A . Additionally, Kingsbury et al.  revealed the relevance of amino acid biosynthesis for yeast survival in murine host; however processes important for sensing and responding to quality and concentration of nitrogen compounds were not required for yeast survival in vivo, indicating that yeast can use a variety of nitrogen sources in these conditions. The pyridoxine metabolic process genes were downregulated in the virulent strains with regards to non-virulent strains. Padilla et al.  observed that these genes were expressed under nutrient limitation, so it may reflect that strains 60 and D14 were not exposed to a limitation of specific nutrients, such as nitrogen.
The glyoxylate cycle is induced upon phagocytes ingestion of the bacteria Mycobacterium tuberculosis and other fungi such as C. neoformans, C. albicans, Leptosphaeria maculans and S. cerevisiae. This shift in metabolism has been interpreted as a response to the glucose-poor environment of the macrophage, and the ability to make that shift appears to contribute to the virulence of some pathogens. However, in our condition using complex blood medium, the genes encoding the principal enzymes of the glyoxylate cycle, isocitrate lyase (ICL1) and malate synthase (MLS1), were downregulated during the time course, although this repression was less strong at the later stage in strains D14 and 60. This suggests that yeast cells have access to glucose during almost all the experiment. The fact that icl1Δ mutants were only slightly deficient in vivo suggests that the glyoxylate cycle has a minor contribution to S. cerevisiae fitness in vivo. The ICL1 gene of both S. cerevisiae and C. albicans has recently been shown to be substantially induced upon exposure to macrophages in vitro[27, 34]. However, these experiments were performed with murine macrophages in cell culture medium, where glucose concentration may be different from blood. Furthermore, a C. albicans icl1Δ/icl1Δ mutant showed a substantial reduction in virulence , while the same mutant was not attenuated in survival in blood , suggesting that the ICL1 gene may play a general role when C. albicans has left the bloodstream. All these data suggest that the ICL1 gene may play also a general role in S. cerevisiae in human infections but after yeast cells has left the bloodstream.
When we compared the transcriptomes of virulent and control strains, we observed several specific groups of genes that may explain the pathogenic nature of the virulent strains. An interesting functional group of up-regulated genes during blood incubation was Cell redox homeostasis. We confirmed that this increased oxidative stress response correlates with phenotypical advantage of virulent strains in pro-oxidant environments since they have significantly much higher survival in the presence of high concentrations of H2O2. Furthermore, they survive to oxidative burst attack from blood cells significantly better than control strain W303 and at similar levels that pathogenic C. albicans. The decreased survival of YAP1 mutant strain in human blood incubations highlights the importance of the genes included in the Yap1p regulon in determining the virulence of S. cerevisiae strains.
Since we have used a molecular transcriptomic approach and human blood media, it was difficult to use several virulent and non-virulent strains, in order to obtain a broad comparative view. However, Diezmann and Dietrich  compared hundreds of clinical isolates due to the use of easy tractable phenotypical assays. In concordance with our results, Diezmann and Dietrich  showed that S. cerevisiae clinical isolates were more resistant to oxidative stress. This data suggest a correlation between survival in oxidative stress and yeast pathogenicity and strongly supports our data. Macrophages, neutrophils and other phagocytic cells generate potent reactive oxygen and nitrogen species (ROS and RNS), which are toxic to most fungal pathogens, causing damage to DNA, proteins and lipids . Fungal pathogens display different degrees of resistance to the reactive oxygen and nitrogen species used by human cells to counteract infection . Fungal resistance to ROS offers protection from oxidative host defenses and is undoubtedly an advantageous pathobiological property [38, 39]. It is worth mentioning that several genes belonging to the thioredoxin system were upregulated, which suggests its implication in yeast defense against blood defenses. Indeed, the thioredoxin system of C. albicans has been shown to be expressed during growth in human blood or mucosal tissue [19, 20, 40], indicating that ability to respond to oxidative stress might be crucial in the early stages of systemic C. albicans infections. Also, TRX1 is necessary for survival of C. neoformans in the oxidative environment of macrophages and important for virulence of this fungal pathogen . TSA2 and GPX2 genes have been shown to be induced in S. cerevisiae strains when exposed to neutrophils , and a clear antioxidant response has been observed. Fradin et al.  demonstrated that neutrophils play a key role in bloodstream infections with C. albicans. This observation is in line with the high susceptibility of neutropenic patients (deficient in these immune cells) to disseminated candidiasis [42, 43].
In conclusion, this work supports the view that oxidative stress response of S. cerevisiae strains has a special importance for survival in blood, having a high impact in determining their virulence. This characteristic can help these yeasts to prevail in immunocompromised patients and cause systemic infections.