Cryptococcus at Work: Gene Expression during Human Infection

Meningitis is a frequent manifestation of infection due to Cryptococcus neoformans and a major cause of increased morbidity in patients with AIDS. Numerous in vitro gene expression and genetic studies of the fungus have predicted a myriad of genes, pathways, and biological processes that may be critical for pathogenesis, and many studies using animal models have supported the role of these processes during infection. However, the relevance of these hypotheses based on in vitro and animal models has often been questioned. A recent study by Chen et al. (Y. Chen, D. L. Toffaletti, J. L. Tenor, A. P. Litvintseva, C. Fang, T. G. Mitchell, T. R. McDonald, K. Nielsen, D. R. Boulware, T. Bicanic, and J. R. Perfect, mBio 5(1):e01087-13, 2014) represents an important step in understanding the cryptococcal response during human infection. C ryptococcusneoformansisdistributedworldwide,growsinsoil, fends off a variety of phagocytic predators, and can infect a wide variety of animals and plants. The presence and survival of the fungus in these diverse environmental and host conditions implythatthefungushasevolvedawell-orchestratedgeneexpres- sionprogramthatenablesittoadaptandproliferateundervarious conditions. The data from testing a multitude of genetic variants under defined in vitro growth conditions and in multiple animal infection models for C. neoformans have been used to formulate and test hypotheses about pathogenesis in humans. Examination of gene expression profiles of C. neoformans un- der specific in vitro stress conditions that are thought to mimic conditions encountered by the fungus within the human host has been widely used to explore the molecular response of C. neofor- manstopredictedhoststressconditions.Amajoradvantageofthe in vitro studies is that many variables can be carefully controlled and sufficient biological and technical replicates can be generated to obtain highly reproducible and significant data. These in vitro- derived data sets have suggested roles for different biological pro- cessesduringpathogenesis.However,datafromonenarrowlyde- fined set of experiments may not be generalizable to animal or human infection. Even within one animal host, the fungus has to survive under diverse tissue and immunologic stress conditions. Our understanding of how the fungus adapts to these changing conditions within the host is limited, and in vitro experiments are unlikely to fully explain these adaptative responses. However, ob- taining sufficient statistical power in studies that are limited by having few biological samples makes it more difficult to generate firm conclusions. Therefore, one way to ascertain the involvement of mecha- nisms of pathogenesis gleaned from experiments carried out un- der laboratory conditions is to directly examine the genomic re- sponse of the fungus growing inside an infected human patient. A major technical hurdle to examining gene expression during hu- man infection has been obtaining sufficient high-quality material to generate the RNA for analysis, due to the limited number of fungal cells and the presence of large amounts of polysaccharide capsule, which interferes with the purity of RNA. More impor- tantly, these experiments are particularly challenging to interpret due to the difficulty of controlling for multiple variables, includ- ing stage of infection, host responses, complications of other in- fections, and the strain of Cryptococcus that is causing the infec- tion. The variability introduced by different infecting genotypes was well demonstrated in a study by Wiesner et al. (1) in which they analyzed 140 Cryptococcus strains isolated from 111 human AIDS patients presenting with Cryptococcus meningitis. The foundasignificantcorrelationbetweenthegenotypeandthephe- notypic response elicited by the fungal strain in the human host. Chen and colleagues in John Perfect's laboratory at Duke Uni- versity (2) have pioneered the examination of C. neoformans gene expression during human infections. They have succeeded in ex- tracting sufficient amounts of good-quality RNA from C. neofor- mans growing in the cerebrospinal fluid (CSF) of two AIDS pa- tients. They subjected the RNA samples to whole-genome transcriptome analysis employing transcriptome sequencing (RNA-Seq). In addition, they purified RNA from the same yeast strains after culturing them in yeast extract-peptone-dextrose (YPD)andinhumanCSFunderlaboratoryconditions.Theywere abletoproducehigh-qualitysequencedatafromtheRNAisolated fromtheyeastcellscollectedfromhumanpatients,with95to98% of the reads mapping to the predicted genes of the H99 genome. Oneofthesurprisingrevelationsofthisstudyisthehighsimilarity of the gene expression profiles between the samples from cells growingintheCSFofthehumanpatientandcellsgrowinginYPD under laboratory conditions. This is intriguing because there are substantial differences in the growth conditions, including CO2 levels, stress, exposure to light, and nutrient composition. The ontology analysis of the differentially expressed genes in the pa- tient's CSF and in YPD in comparison to cells grown in CSF in vitro indicated that the expression of genes in the cellular metab- olism category increased, suggesting that yeast cells are actively growing in CSF inside the human patient. Active metabolism was alsoobservedinC.neoformansintheCSFofinfectedrabbitsbythe use of serial analysis of gene expression (SAGE) (3), along with induction of various other biological processes. The environment in the lung may be very different from the environmentintheCSF.ComparativeSAGEanalysisofyeastcells isolatedfrominfectedmouselungsandthosefrominfectedrabbit