BACKGROUND
s. cerevisiae is a ubiquitous organism that can be found in nature on plants, fruits and in soil.
this species is involved in fermentative processes in beer, bread and wine, and is even consumed as a nutritional supplement, always being traditionally regarded as absolutely safe.
s. cerevisiae and its commercially available preparations known as saccharomyces boulardii, that are used to treat antibiotic-related diarrhea, have recently been shown to have the potential to cause a wide variety of infections, ranging from cutaneous infections and vaginitis in healthy patients to systemic infections of the bloodstream and vital organs in immunocompromised and critically ill patients  <cit> .
the patients infected are mainly premature children, elderly people or patients suffering from immunosuppression due to aids, treatment with immunosuppressive agents, and other conditions associated with an insufficient immune response.
moreover, severe infections by s. cerevisiae have been occasionally reported in patients with no obvious predisposing factors  <cit> .
some s. cerevisiae clinical strains have been isolated from blood  <cit> .
the main routes for bloodstream infections are probably translocation of ingested yeast from the gut or direct contamination of the central venous catheter insertion site  <cit> .
similarly to other opportunistic fungal pathogens such as c. glabrata, the ability to colonize and cause disease in the host depends on the immune status of the host and the expression of certain virulence factors by the pathogen  <cit> .
the majority of s. cerevisiae clinical isolates secrete high levels of proteases and phospholipases, can grow at 42°c, exhibit multiple colony phenotypes, have ability to adhere to epithelial tissue and show invasive pseudohyphal growth on solid agar  <cit> .
these studies showed that clinical isolates differed phenotypically from laboratory and wine strains in vitro, but it remains unclear whether these traits also play a role during infection.
comparison of s. cerevisiae clinical and non-clinical strains by molecular typing and determination of putative virulence traits has not revealed a specific virulence factor that clearly separates the strains of clinical origin from the strains of non-clinical origin  <cit> .
in addition, little is known about the interactions between s. cerevisiae and host defence cells or non-cellular components  <cit> .
mrna level profiles of clinical strains of s. cerevisiae following exposure to whole blood rather than serum may closely reflect the in vivo response of these yeasts during bloodstream infections, since c. albicans expression profiles following intravenous infection of mice closely resembled profiles following incubation in whole blood  <cit> , which in turn strongly correlated with the expression profile of c. albicans exposed to neutrophils  <cit> .
in these studies, the authors identified differentially expressed c. albicans genes involved in general stress response, antioxidative response, the glyoxylate cycle as well as putative virulence genes expressed in response to human blood cells  <cit> .
to better understand the physiology of virulent s. cerevisiae cells during infection, we performed a transcriptional analysis to investigate which genes and pathways are required for survival in blood as a model of human bloodstream infection.
we compare two control non-virulent strains with two virulent isolates .
these strains showed the highest levels in virulence factors, were able to colonize immunocompetent mice brain after blood infections and cause mice death, showing increased adherence ability whereas other clinical isolates showed low virulence levels  <cit> .
comparison of control and virulent strains revealed that amino acid biosynthesis and elevated oxidative stress response can be of special relevance for survival of s. cerevisiae virulent strains to host defences.
phenotypic analysis of virulent strains and a yap <dig> mutant, unable to respond to oxidative stress, confirmed the importance of the increased oxidative stress response for yeast survival in human blood incubations.
RESULTS
transcription profile of yeasts in human blood
to obtain a global view of the s. cerevisiae response to blood environment and to investigate which set of genes are expressed in the different yeast, we analyzed the transcription profile of avirulent  and virulent  strains exposed to blood.
we incubated yeast cells in human blood using the model previously established by fradin et al.
<cit>  and searched for significantly over-represented functional groups in up-regulated  or in the down-regulated  set of genes during the time course of the experiment .
a complete microarray dataset is presented in aditional file 2: table s <dig>
afunctional groups were  or were not  significantly overrepresented .
afunctional groups were  or were not  significantly overrepresented .
ypd precultured cells were preadapted in pbs buffer before inoculation in human blood  <cit> .
preadapted cell suspension was inoculated into fresh whole human blood and incubated for  <dig>   <dig>   <dig> and  <dig> min at 37°c.
mrna levels was determined by microarray hybridization.
the arrays were hybridized with each time point and the time point  <dig> min for each strain, thus, mrna level data for  <dig>   <dig> and  <dig> min reflects the induction respect time  <dig>  data were ratio-based normalized and a fdr of 5% was used to identify statistically significant data.
the microarray transcriptomic data presented corresponds to the average of three biological replicates including a dye swap.
a complete set of data for all genes is included in additional file 2: table s <dig>  data was validated measuring mrna levels of  <dig> genes by qrt-pcr in different strains and conditions  showing that inductions or repressions differ less than 20% between the two methods.
first, we searched in the data for significantly represented functional groups in up-regulated  or in the down-regulated  set of genes .
after  <dig> min of blood incubation, three functional groups  were found in the induced set of genes in all strains except for the laboratory strain w <dig>  these significantly represented functional groups that comprise genes related with normal vegetative growth, appeared in the up-regulated genes during all the experiment, except time point  <dig> min, indicating a continuous adaptation of these strains to the blood environment.
for strain w <dig>  the functional group ribosome biogenesis was significant only after  <dig> min of incubation, revealing a delayed adaptation to the new environment.
strains cect  <dig>   <dig> and d <dig> showed no differences in this set of functional groups.
genes belonging to these functional groups include ribosomal protein genes such as rpl <dig> and rps <dig>  translational elongation factor genes such as tef <dig> and efb <dig>  and trna synthetase genes such as ths <dig>
the functional group amino acid biosynthetic process was observed in strain  <dig> at time point  <dig> min and in strains  <dig>  d <dig> and cect  <dig> after  <dig> and  <dig> min.
furthermore, the functional groups aspartate family amino acid biosynthetic process and arginine biosynthetic process were significant for strain  <dig>  d <dig> and cect  <dig> at  <dig> min and for strain  <dig> at the last time point .
some functional groups were found to be significantly repressed .
protein folding was observed in cect  <dig> after  <dig> min of incubation and during the entire time course in w <dig>  but not in the virulent strains  <dig> and  <dig>  the main function of the genes included in this group, such as ssa <dig>  is to encode molecular chaperones, which bind newly-translated proteins to assist proper folding and to prevent aggregation/misfolding  <cit> .
the down regulation of genes of this functional group in w <dig> is consistent with the observed delay in the functional groups related to preparation for growth like ribosome biogenesis.
in the down-regulated set of genes, the functional group oxidative phosphorylation was observed in strain cect  <dig> after  <dig> and  <dig> min and the functional group biosynthesis of glutamate was detected at  <dig> min.
no significant functional group was observed in the down-regulated genes in virulent strains  <dig> and d <dig>
on the other hand, we found significant changes in the mrna levels of specific genes implicated in important biological process .
at the early stage , several genes encoding glycolytic enzymes were up-regulated.
the genes encoding glucose-6-phosphate isomerase , phosphofructokinase  and aldolase  were up-regulated at early stages of incubation, but the mrna levels were even higher after  <dig> min and  <dig> min for wine strain cect  <dig> and d <dig> respectively.
for strain  <dig>  the maximal mrna level was monitored after  <dig> min.
for the laboratory strain w <dig>  the mrna level of all three genes was down-regulated at  <dig> min, pgi <dig> was weakly induced at  <dig> min, and pfk <dig> mrna level was up-regulated at  <dig> min.
in strain  <dig>  other genes that encode glycolytic enzymes were mostly down-regulated in the early stages compared to  <dig> min time point, but transcripts were considerably elevated at the later stages of the incubation, such as, glyceraldehyde 3-phosphate dehydrogenase , phosphoglycerate kinase  and enolase .
also, the mrna level of gluconeogenic enzyme phosphoenolpyruvate carboxykinase  was strongly down-regulated in this strain.
the genes encoding the key enzymes of the glyoxylate cycle, isocitrate lyase  and malate synthase  were down-regulated in all strains during the time course of the experiment, although this repression was less strong at the later stage in strains d <dig> and  <dig>  these genes are subjected to catabolite repression which suggests that yeast cells have access to glucose during all the experiment.
comparison of mrna levels between virulent and non-virulent strains in human blood
to identify genes specifically expressed in s. cerevisiae virulent isolates in blood, we analyzed the mrna levels at different incubation times and we identified statistically significant data when comparing average of virulent strains group  data with average of non-virulent group  data.
then we searched for functional groups of genes whose mrna level was specifically associated with virulent strains of s. cerevisiae .
a functional groups were  or were not  significantly overrepresented .
after  <dig> min of blood incubation, three functional groups were significantly overrepresented in the up-regulated set of genes in virulent strains compared to non-virulent strains: vacuole organization, xenobiotic transporter and base-excision repair.
the functional group vacuole organization, induced again after  <dig> min, includes genes related with biogenesis, protein-vacuolar targeting and vacuole fusion .
the base-excision repair pathway removes lesions that result from exposure to endogenous or exogenous reactive oxygen species   <cit>  and includes genes such as pol <dig> , rad <dig> , apn <dig>  and ogg <dig> .
the level of mrna of these genes decreased along the experiment in virulent strains.
seven functional groups more were enhanced at time points  <dig> or  <dig> min in virulent strains.
four closely related groups  include genes that take part in the biosynthesis of some aminoacids, such as aromatic aminoacids , histidine , arginine , tryptophan , lysine , leucine , and serine .
we wanted to know if the presence of several auxotrophies in the laboratory strain w <dig> could indirectly affect the appearance of functional groups related to aminoacid metabolism in this analysis.
we repeated the data analysis comparing the two virulent strains  against the wine strain cect  <dig>  excluding w <dig> strain.
the results showed once again the appearance of aminoacid metabolism functional groups as cellular aminoacid metabolic process, containing similar genes as .
then we concluded that w <dig> auxotrophies are not biasing the results, probably because the short time sampling, and that aminoacid metabolism related genes are highly activated in the clinical isolates.
the fifth functional group, phosphatase activity, includes genes such as pho <dig>  pho <dig> and pho <dig> that are expressed under low-phosphate conditions.
it may indicate a response to phosphate starvation during blood incubation.
also, pho <dig> is able to hydrolyze thiamin phosphates in the periplasmic space, increasing cellular thiamine uptake.
another functional group is transmembrane transporter activity which includes genes that take part in the mitochondrial transport , zinc transport  and amino acid transport .
a key functional group, cell redox homeostasis, was found to be enhanced during the incubation in blood in virulent strains.
the genes belonging to this group help to maintain a reduced environment in the cell and are associated with virulence in bacteria  <cit> .
these genes play a significant role in defending the cell against oxidative stress exerted by reactive oxygen species , integral effectors of mammalian host defenses.
stimulated leucocytes can be a source of ros, which may cause oxidative stress.
defense against this stress seems to be enhanced at  <dig> min of incubation of blood.
indeed, when we observed oxidative stress related genes, most of them  show statistically significant higher levels in virulent strains compared to non-virulent strains .
on average, a very significant  increase  is observed in the virulent strains respect to non-virulent strains.
interestingly, all the  <dig> genes are regulated by yap1p, the main regulator of oxidative stress in yeasts .
several components of the thioredoxin system are included in this set of genes.
a thioredoxin peroxidase  and two thioredoxin genes  were strongly upregulated in virulent strains at time  <dig> min.
also mrna levels of trr <dig>  a thioredoxin-disulfide reductase gene, increase during the incubation in blood in strains  <dig> and d <dig>
only the pyridoxine metabolic process functional group was downregulated with regards to non-virulent strains at time point  <dig> min.
this group includes genes  involved in pyridoxine metabolism and thiamine biosynthesis.
resistance to oxidative stress of virulent and non-virulent strains
to confirm the relevance of the oxidative stress response on the virulent nature of yeast strains we measured the percentage of survival of these four strains after one hour in the presence of elevated levels  of h2o <dig> .
virulent isolates exhibited higher survival rates whereas w <dig> and cect  <dig> showed very low survival.
statistical analysis confirmed significant differences in oxidative stress between virulent and non-virulent strains  whereas no significant differences between cect  <dig> and w <dig> or  <dig> and d <dig> were found .
survival of yeast strains in human blood
the aim of this experiment was to study the survival of control and pathogenic s. cerevisiae strains comparing them with c. albicans and also to know which blood cell types are affecting more yeast self-preservation.
in this experiment s. boulardii strain ultralevure, a bio-therapeutic agent which has been described as a potential pathogenic  <cit> , was included.
to investigate the survival of the different strains, we incubated the cells  <dig> min and  <dig> hour at 37°c in human blood .
a survival is expressed as% of cfu respect time  <dig>  *strains individually compared to c. albicans sc <dig> strain presented p <  <dig> .
we observed that after  <dig> min the virulent strains d <dig> and  <dig> showed survival levels similar to the pathogenic c. albicans strain sc <dig>  in contrast, control strain w <dig> presented significantly lower survival.
after  <dig> min of incubation the survival of all strains decreased.
however we observed again a similar pattern since strains  <dig> and d <dig> showed comparable values to virulent sc <dig> whereas w <dig> presented significantly lower survival.
s. boulardii strain showed similar values to the c. albicans strain in this assay.
the wine strain cect <dig> also presented survival levels lower than c. albicans and virulent s. cerevisiae strains but data was not significant.
a virulent strain mutant in yap1p showed decreased survival in human blood
to confirm that oxidative stress response of virulent strains is important to survive in human blood we decided to study performance of a strain unable to grow in oxidative stress conditions.
since all genes with increased mrna levels in virulent strains observed in figure  <dig> are regulated by the transcription factor yap1p, we decided to study survival in human blood comparing the wild type strain d <dig> with a homozygous for a Δ yap <dig> mutation.
first we confirmed that d14Δyap <dig> was unable to survive in the presence of oxidative stress conditions  whereas the wild type strain presented no growth defects .
reincorporation of yap <dig> in d14yap <dig> strain reverted this phenotype.
this experiment confirmed that yap1p is essential to perform a proper oxidative stress response in virulent strains.
then we evaluated the behavior of the d14Δyap <dig> strain in human blood incubation assay.
using the same conditions described above, yeast cells were added to blood and survival was determined after  <dig>   <dig>   <dig> and  <dig> min .
the results showed that d14Δyap <dig> survival decreased significantly  reaching a level comparable to that of the laboratory strain w <dig> at  <dig> min , confirming the relevance of a proper oxidative stress response to survive in human blood.
again, d14yap <dig> showed similar phenotype than the wild type, confirming the involvement of yap1p in blood survival.
discussion
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  <dig> min inoculation in blood, except for the laboratory strain w <dig>  the strong induction of these set of genes is probably a consequence of the yeast cells transfer from nutrient poor medium  to a relatively nutrient-rich medium .
the downregulation of pck <dig>  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  <cit> , 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  <cit> .
rubin-bejerano et al.
<cit>  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  <cit> .
additionally, kingsbury et al.
<cit>  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.
<cit>  observed that these genes were expressed under nutrient limitation, so it may reflect that strains  <dig> and d <dig> 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 <cit>  and other fungi such as c. neoformans <cit> , c. albicans <cit> , leptosphaeria maculans <cit>  and s. cerevisiae <cit> .
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  and malate synthase , were downregulated during the time course, although this repression was less strong at the later stage in strains d <dig> and  <dig>  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 <cit>  suggests that the glyoxylate cycle has a minor contribution to s. cerevisiae fitness in vivo.
the icl <dig> gene of both s. cerevisiae and c. albicans has recently been shown to be substantially induced upon exposure to macrophages in vitro <cit> .
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  <cit> , while the same mutant was not attenuated in survival in blood  <cit> , suggesting that the icl <dig> gene may play a general role when c. albicans has left the bloodstream.
all these data suggest that the icl <dig> 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 h2o <dig>  furthermore, they survive to oxidative burst attack from blood cells significantly better than control strain w <dig> and at similar levels that pathogenic c. albicans.
the decreased survival of yap <dig> 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  <cit>  compared hundreds of clinical isolates due to the use of easy tractable phenotypical assays.
in concordance with our results, diezmann and dietrich  <cit>  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 , which are toxic to most fungal pathogens, causing damage to dna, proteins and lipids  <cit> .
fungal pathogens display different degrees of resistance to the reactive oxygen and nitrogen species used by human cells to counteract infection  <cit> .
fungal resistance to ros offers protection from oxidative host defenses and is undoubtedly an advantageous pathobiological property  <cit> .
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  <cit> , indicating that ability to respond to oxidative stress might be crucial in the early stages of systemic c. albicans infections.
also, trx <dig> is necessary for survival of c. neoformans in the oxidative environment of macrophages and important for virulence of this fungal pathogen  <cit> .
tsa <dig> and gpx <dig> genes have been shown to be induced in s. cerevisiae strains when exposed to neutrophils  <cit> , and a clear antioxidant response has been observed.
fradin et al.
<cit>  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  to disseminated candidiasis  <cit> .
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.
CONCLUSIONS
in the present work we have found significant differences at molecular level between s. cerevisiae non-virulent strains and virulent clinical strains with remarkable capabilities of infect and even produce mice death.
transcription analysis in blood incubations pointed out an enhanced oxidative stress response of virulent strains that is reflected in an increased ability to survive in the presence of high concentrations of the oxidant h2o <dig>  indeed, they are able to survive to a similar extent than pathogenic c. albicans strain in blood incubations.
these data suggest that s. cerevisiae virulent strains are able to extend the survival after the macrophage oxidative burst attack, having more chances to persist and perform systemic infections.

 s. cerevisiae is a ubiquitous organism that can be found in nature on plants, fruits and in soil. this species is involved in fermentative processes in beer, bread and wine, and is even consumed as a nutritional supplement, always being traditionally regarded as absolutely safe. s. cerevisiae and its commercially available preparations known as saccharomyces boulardii, that are used to treat antibiotic-related diarrhea, have recently been shown to have the potential to cause a wide variety of infections, ranging from cutaneous infections and vaginitis in healthy patients to systemic infections of the bloodstream and vital organs in immunocompromised and critically ill patients. the patients infected are mainly premature children, elderly people or patients suffering from immunosuppression due to aids, treatment with immunosuppressive agents, and other conditions associated with an insufficient immune response. moreover, severe infections by s. cerevisiae have been occasionally reported in patients with no obvious predisposing factors. some s. cerevisiae clinical strains have been isolated from blood. the main routes for bloodstream infections are probably translocation of ingested yeast from the gut or direct contamination of the central venous catheter insertion site. similarly to other opportunistic fungal pathogens such as c. glabrata, the ability to colonize and cause disease in the host depends on the immune status of the host and the expression of certain virulence factors by the pathogen. the majority of s. cerevisiae clinical isolates secrete high levels of proteases and phospholipases, can grow at 42°c, exhibit multiple colony phenotypes, have ability to adhere to epithelial tissue and show invasive pseudohyphal growth on solid agar. these studies showed that clinical isolates differed phenotypically from laboratory and wine strains in vitro, but it remains unclear whether these traits also play a role during infection. comparison of s. cerevisiae clinical and non-clinical strains by molecular typing and determination of putative virulence traits has not revealed a specific virulence factor that clearly separates the strains of clinical origin from the strains of non-clinical origin. in addition, little is known about the interactions between s. cerevisiae and host defence cells or non-cellular components. mrna level profiles of clinical strains of s. cerevisiae following exposure to whole blood rather than serum may closely reflect the in vivo response of these yeasts during bloodstream infections, since c. albicans expression profiles following intravenous infection of mice closely resembled profiles following incubation in whole blood, which in turn strongly correlated with the expression profile of c. albicans exposed to neutrophils. in these studies, the authors identified differentially expressed c. albicans genes involved in general stress response, antioxidative response, the glyoxylate cycle as well as putative virulence genes expressed in response to human blood cells. to better understand the physiology of virulent s. cerevisiae cells during infection, we performed a transcriptional analysis to investigate which genes and pathways are required for survival in blood as a model of human bloodstream infection. we compare two control non-virulent strains with two virulent isolates. these strains showed the highest levels in virulence factors, were able to colonize immunocompetent mice brain after blood infections and cause mice death, showing increased adherence ability whereas other clinical isolates showed low virulence levels. comparison of control and virulent strains revealed that amino acid biosynthesis and elevated oxidative stress response can be of special relevance for survival of s. cerevisiae virulent strains to host defences. phenotypic analysis of virulent strains and a yap mutant, unable to respond to oxidative stress, confirmed the importance of the increased oxidative stress response for yeast survival in human blood incubations.  transcription profile of yeasts in human blood to obtain a global view of the s. cerevisiae response to blood environment and to investigate which set of genes are expressed in the different yeast, we analyzed the transcription profile of avirulent and virulent strains exposed to blood. we incubated yeast cells in human blood using the model previously established by fradin et al. and searched for significantly over-represented functional groups in up-regulated or in the down-regulated set of genes during the time course of the experiment. a complete microarray dataset is presented in aditional file 2: table s afunctional groups were or were not significantly overrepresented. afunctional groups were or were not significantly overrepresented. ypd precultured cells were preadapted in pbs buffer before inoculation in human blood. preadapted cell suspension was inoculated into fresh whole human blood and incubated for and min at 37°c. mrna levels was determined by microarray hybridization. the arrays were hybridized with each time point and the time point min for each strain, thus, mrna level data for and min reflects the induction respect time data were ratio-based normalized and a fdr of 5% was used to identify statistically significant data. the microarray transcriptomic data presented corresponds to the average of three biological replicates including a dye swap. a complete set of data for all genes is included in additional file 2: table s data was validated measuring mrna levels of genes by qrt-pcr in different strains and conditions showing that inductions or repressions differ less than 20% between the two methods. first, we searched in the data for significantly represented functional groups in up-regulated or in the down-regulated set of genes. after min of blood incubation, three functional groups were found in the induced set of genes in all strains except for the laboratory strain w these significantly represented functional groups that comprise genes related with normal vegetative growth, appeared in the up-regulated genes during all the experiment, except time point min, indicating a continuous adaptation of these strains to the blood environment. for strain w the functional group ribosome biogenesis was significant only after min of incubation, revealing a delayed adaptation to the new environment. strains cect and d showed no differences in this set of functional groups. genes belonging to these functional groups include ribosomal protein genes such as rpl and rps translational elongation factor genes such as tef and efb and trna synthetase genes such as ths the functional group amino acid biosynthetic process was observed in strain at time point min and in strains d and cect after and min. furthermore, the functional groups aspartate family amino acid biosynthetic process and arginine biosynthetic process were significant for strain d and cect at min and for strain at the last time point. some functional groups were found to be significantly repressed. protein folding was observed in cect after min of incubation and during the entire time course in w but not in the virulent strains and the main function of the genes included in this group, such as ssa is to encode molecular chaperones, which bind newly-translated proteins to assist proper folding and to prevent aggregation/misfolding. the down regulation of genes of this functional group in w is consistent with the observed delay in the functional groups related to preparation for growth like ribosome biogenesis. in the down-regulated set of genes, the functional group oxidative phosphorylation was observed in strain cect after and min and the functional group biosynthesis of glutamate was detected at min. no significant functional group was observed in the down-regulated genes in virulent strains and d on the other hand, we found significant changes in the mrna levels of specific genes implicated in important biological process. at the early stage, several genes encoding glycolytic enzymes were up-regulated. the genes encoding glucose-6-phosphate isomerase, phosphofructokinase and aldolase were up-regulated at early stages of incubation, but the mrna levels were even higher after min and min for wine strain cect and d respectively. for strain the maximal mrna level was monitored after min. for the laboratory strain w the mrna level of all three genes was down-regulated at min, pgi was weakly induced at min, and pfk mrna level was up-regulated at min. in strain other genes that encode glycolytic enzymes were mostly down-regulated in the early stages compared to min time point, but transcripts were considerably elevated at the later stages of the incubation, such as, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase and enolase. also, the mrna level of gluconeogenic enzyme phosphoenolpyruvate carboxykinase was strongly down-regulated in this strain. the genes encoding the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase were down-regulated in all strains during the time course of the experiment, although this repression was less strong at the later stage in strains d and these genes are subjected to catabolite repression which suggests that yeast cells have access to glucose during all the experiment. comparison of mrna levels between virulent and non-virulent strains in human blood to identify genes specifically expressed in s. cerevisiae virulent isolates in blood, we analyzed the mrna levels at different incubation times and we identified statistically significant data when comparing average of virulent strains group data with average of non-virulent group data. then we searched for functional groups of genes whose mrna level was specifically associated with virulent strains of s. cerevisiae. a functional groups were or were not significantly overrepresented. after min of blood incubation, three functional groups were significantly overrepresented in the up-regulated set of genes in virulent strains compared to non-virulent strains: vacuole organization, xenobiotic transporter and base-excision repair. the functional group vacuole organization, induced again after min, includes genes related with biogenesis, protein-vacuolar targeting and vacuole fusion. the base-excision repair pathway removes lesions that result from exposure to endogenous or exogenous reactive oxygen species and includes genes such as pol, rad, apn and ogg. the level of mrna of these genes decreased along the experiment in virulent strains. seven functional groups more were enhanced at time points or min in virulent strains. four closely related groups include genes that take part in the biosynthesis of some aminoacids, such as aromatic aminoacids, histidine, arginine, tryptophan, lysine, leucine, and serine. we wanted to know if the presence of several auxotrophies in the laboratory strain w could indirectly affect the appearance of functional groups related to aminoacid metabolism in this analysis. we repeated the data analysis comparing the two virulent strains against the wine strain cect excluding w strain. the results showed once again the appearance of aminoacid metabolism functional groups as cellular aminoacid metabolic process, containing similar genes as. then we concluded that w auxotrophies are not biasing the results, probably because the short time sampling, and that aminoacid metabolism related genes are highly activated in the clinical isolates. the fifth functional group, phosphatase activity, includes genes such as pho pho and pho that are expressed under low-phosphate conditions. it may indicate a response to phosphate starvation during blood incubation. also, pho is able to hydrolyze thiamin phosphates in the periplasmic space, increasing cellular thiamine uptake. another functional group is transmembrane transporter activity which includes genes that take part in the mitochondrial transport, zinc transport and amino acid transport. a key functional group, cell redox homeostasis, was found to be enhanced during the incubation in blood in virulent strains. the genes belonging to this group help to maintain a reduced environment in the cell and are associated with virulence in bacteria. these genes play a significant role in defending the cell against oxidative stress exerted by reactive oxygen species, integral effectors of mammalian host defenses. stimulated leucocytes can be a source of ros, which may cause oxidative stress. defense against this stress seems to be enhanced at min of incubation of blood. indeed, when we observed oxidative stress related genes, most of them show statistically significant higher levels in virulent strains compared to non-virulent strains. on average, a very significant increase is observed in the virulent strains respect to non-virulent strains. interestingly, all the genes are regulated by yap1p, the main regulator of oxidative stress in yeasts. several components of the thioredoxin system are included in this set of genes. a thioredoxin peroxidase and two thioredoxin genes were strongly upregulated in virulent strains at time min. also mrna levels of trr a thioredoxin-disulfide reductase gene, increase during the incubation in blood in strains and d only the pyridoxine metabolic process functional group was downregulated with regards to non-virulent strains at time point min. this group includes genes involved in pyridoxine metabolism and thiamine biosynthesis. resistance to oxidative stress of virulent and non-virulent strains to confirm the relevance of the oxidative stress response on the virulent nature of yeast strains we measured the percentage of survival of these four strains after one hour in the presence of elevated levels of h2o. virulent isolates exhibited higher survival rates whereas w and cect showed very low survival. statistical analysis confirmed significant differences in oxidative stress between virulent and non-virulent strains whereas no significant differences between cect and w or and d were found. survival of yeast strains in human blood the aim of this experiment was to study the survival of control and pathogenic s. cerevisiae strains comparing them with c. albicans and also to know which blood cell types are affecting more yeast self-preservation. in this experiment s. boulardii strain ultralevure, a bio-therapeutic agent which has been described as a potential pathogenic, was included. to investigate the survival of the different strains, we incubated the cells min and hour at 37°c in human blood. a survival is expressed as% of cfu respect time *strains individually compared to c. albicans sc strain presented p < . we observed that after min the virulent strains d and showed survival levels similar to the pathogenic c. albicans strain sc in contrast, control strain w presented significantly lower survival. after min of incubation the survival of all strains decreased. however we observed again a similar pattern since strains and d showed comparable values to virulent sc whereas w presented significantly lower survival. s. boulardii strain showed similar values to the c. albicans strain in this assay. the wine strain cect also presented survival levels lower than c. albicans and virulent s. cerevisiae strains but data was not significant. a virulent strain mutant in yap1p showed decreased survival in human blood to confirm that oxidative stress response of virulent strains is important to survive in human blood we decided to study performance of a strain unable to grow in oxidative stress conditions. since all genes with increased mrna levels in virulent strains observed in figure are regulated by the transcription factor yap1p, we decided to study survival in human blood comparing the wild type strain d with a homozygous for a Δ yap mutation. first we confirmed that d14Δyap was unable to survive in the presence of oxidative stress conditions whereas the wild type strain presented no growth defects. reincorporation of yap in d14yap strain reverted this phenotype. this experiment confirmed that yap1p is essential to perform a proper oxidative stress response in virulent strains. then we evaluated the behavior of the d14Δyap strain in human blood incubation assay. using the same conditions described above, yeast cells were added to blood and survival was determined after and min. the results showed that d14Δyap survival decreased significantly reaching a level comparable to that of the laboratory strain w at min, confirming the relevance of a proper oxidative stress response to survive in human blood. again, d14yap showed similar phenotype than the wild type, confirming the involvement of yap1p in blood survival. discussion 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 min inoculation in blood, except for the laboratory strain w the strong induction of these set of genes is probably a consequence of the yeast cells transfer from nutrient poor medium to a relatively nutrient-rich medium. the downregulation of pck 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. 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 and d 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 and malate synthase, were downregulated during the time course, although this repression was less strong at the later stage in strains d and 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 icl gene of both s. cerevisiae and c. albicans has recently been shown to be substantially induced upon exposure to macrophages in vitro. 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 icl gene may play a general role when c. albicans has left the bloodstream. all these data suggest that the icl 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 h2o furthermore, they survive to oxidative burst attack from blood cells significantly better than control strain w and at similar levels that pathogenic c. albicans. the decreased survival of yap 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, 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. 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, indicating that ability to respond to oxidative stress might be crucial in the early stages of systemic c. albicans infections. also, trx is necessary for survival of c. neoformans in the oxidative environment of macrophages and important for virulence of this fungal pathogen. tsa and gpx 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 to disseminated candidiasis. 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.  in the present work we have found significant differences at molecular level between s. cerevisiae non-virulent strains and virulent clinical strains with remarkable capabilities of infect and even produce mice death. transcription analysis in blood incubations pointed out an enhanced oxidative stress response of virulent strains that is reflected in an increased ability to survive in the presence of high concentrations of the oxidant h2o indeed, they are able to survive to a similar extent than pathogenic c. albicans strain in blood incubations. these data suggest that s. cerevisiae virulent strains are able to extend the survival after the macrophage oxidative burst attack, having more chances to persist and perform systemic infections. 
furthermore, a virulent strain mutant in the transcription factor yap1p, unable to grow in oxidative stress conditions, presented decreased survival levels in human blood compared with the wild type or yap reconstituted strain. some clinical strains showed in vivo and in vitro virulence traits and were able to cause death in mice whereas other clinical strains were avirulent. in this work, we studied the transcriptional profiles of two s. cerevisiae clinical strains showing virulent traits and two control non-virulent strains during a blood incubation model and detected a specific transcriptional response of clinical strains. we observed that the clinical strains were more resistant to reactive oxygen species in vitro. our data suggest that this enhanced oxidative stress response in virulent clinical isolates, presumably induced in response to oxidative burst from host defense cells, is important to increase survival in human blood and can help to infect and even produce death in mice models. in addition, blood survival of clinical isolates was high, reaching similar levels to pathogenic candida albicans strain. this response involves an mrna levels increase of amino acid biosynthesis genes and especially oxidative stress related genes. in recent years an increasing number of yeast infections in humans have been related to certain clinical isolates of saccharomyces cerevisiae.
 in recent years an increasing number of yeast infections in humans have been related to certain clinical isolates of saccharomyces cerevisiae. some clinical strains showed in vivo and in vitro virulence traits and were able to cause death in mice whereas other clinical strains were avirulent.  in this work, we studied the transcriptional profiles of two s. cerevisiae clinical strains showing virulent traits and two control non-virulent strains during a blood incubation model and detected a specific transcriptional response of clinical strains. this response involves an mrna levels increase of amino acid biosynthesis genes and especially oxidative stress related genes. we observed that the clinical strains were more resistant to reactive oxygen species in vitro. in addition, blood survival of clinical isolates was high, reaching similar levels to pathogenic candida albicans strain. furthermore, a virulent strain mutant in the transcription factor yap1p, unable to grow in oxidative stress conditions, presented decreased survival levels in human blood compared with the wild type or yap reconstituted strain.  our data suggest that this enhanced oxidative stress response in virulent clinical isolates, presumably induced in response to oxidative burst from host defense cells, is important to increase survival in human blood and can help to infect and even produce death in mice models. saccharomyces cerevisiaeclinical strainstranscriptomicsbloodoxidative stress 