Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host
[['Candida glabrata infections are common in immunocompromised patients and associated with prolonged treatment [1 , 2] , extended length of hospital stay , high costs and high mortality rates [3 , 4] .', 'Over the last decade , the incidence of C . glabrata infections has increased considerably due to higher numbers of immunocompromised patients , as well as broad empiric antifungal prophylaxis , which promotes colonization with azole-resistant C . glabrata [1] .', 'C . glabrata is a very successful human pathogen because it has a high intrinsic stress tolerance , enabling it to withstand oxidative stress [5] .', 'The yeast disseminates and attaches to host cells and indwelling devices , where it forms biofilms [6] .', 'In vitro phagocytosed C . glabrata cells are able to survive and replicate inside human and murine macrophages [7 , 8] .', 'Imaging studies demonstrate that primary human neutrophils can kill or release phagocytosed C . glabrata [9] .', 'Consequently , neutropenia constitutes a major risk factor for disseminated candidiasis in colonized patients [10 , 11] .', 'Most fungal infections are subacute or chronic [1 , 10 , 12] .', 'A major barrier to improved antifungal drug therapy is a lack of understanding of how fungal populations change during chronic infection , and how this microevolution affects in vivo phenotypes and pathogenesis of the respective fungus [13 , 14] .', 'The pathogenic yeasts , Cryptococcus neoformans , Candida albicans and C . glabrata expand clonally in the host even though some of them retain the ability for mating and sexual reproduction [15] .', 'Similar to other unicellular yeasts , C . glabrata is expected to undergo asymmetric mitotic divisions where original mother cells progressively age in a process referred to as replicative aging [16] .', 'Saccharomyces cerevisiae has been the predominant model organism for investigating this process in yeasts .', 'However , this fungus is rarely a pathogen in humans , and therefore research on it has historically focused on elucidating the molecular mechanisms that regulate aging [17] .', 'Recent work in C . neoformans suggests that replicative aging can affect the pathogenesis of fungal populations [18 , 19] .', 'Specifically it was shown that old C . neoformans cells , referring to those with a higher replicative age , accumulate in spinal fluid during chronic infection , indicating the presence of a demographic shift within the infecting fungal population .', 'Older cells manifested a thicker cell wall that rendered them more resistant to host response .', 'This suggested that replicative age might be relevant in a clonally expanding C . neoformans population allowing it to gain resilience in the host environment .', 'We proposed that older C . neoformans cells constitute the persister cells and contribute to the high treatment failure rate despite lack of antifungal resistance .', 'The intent of this study was to characterize the lifespan features of another important fungal pathogen , C . glabrata .', 'Opposed to C . neoformans , this yeast can be a commensal , and it has been proposed that its resilience and high stress tolerance has evolved as a result of its intimate association with the mammalian host ( for review [20] ) .', 'The host is especially vulnerable for invasive disease , when the host becomes neutropenic and the major antifungal defenses are compromised .', 'Then C . glabrata can shift from a commensal to an invasive pathogen .', 'Therefore , we were particularly interested in studying the interplay between replicative aging of a C . glabrata population and the in vivo selection pressures due to neutrophil-mediated killing , which can be done by controlled depletion studies in the murine host .', 'Our data demonstrate that aging leads to remodeling of the cell wall and that selection by neutrophils controls generational distribution within the C . glabrata population .', 'We show that younger C . glabrata cells with more potential to adhere emerge in a neutropenic host , whereas older C . glabrata cells , which exhibit a thicker cell wall with an altered sterol composition mediate enhanced azole resistance and resilience and dominate in the immunocompetent host .'], ['To investigate aging and its effects on various virulence-related phenotypes , we determined replicative lifespans ( RLS ) at 37°C on 14 clinical C . glabrata strains of different multilocus sequence types ( MLST ) and variable chronological lifespans ( CLS ) ( Fig 1A and Table 1 ) .', 'The median RLS of C . glabrata strains varied from 15 . 0 ( strain 43 ) to 61 generations ( strain 143 ) .', 'Median RLS of cells across all strains was 29 generations and ranged from 3–127 generations , confirming the previous observation in C . neoformans [18 , 19] that RLS is highly variable among strains , but a distinct and reproducible trait that defines the individual strain .', 'High variability of RLS within a C . glabrata population was noted , where some cells exhibited a very short , and others a very long RLS .', 'Neither prolonged nor shortened RLS was associated with a specific MLST type .', 'In accordance with other publications [21] , we found calcofluor reliably stains budscars , allowing us to identify the respective replicative age of older C . glabrata cells ( Fig 1B and S1 Video ) .', 'C . glabrata cells , regardless of their MLST , doubled every 45–50 min until they reached an advanced age when their doubling time slowed considerably from 90 to 240 min .', 'Accordingly , the lifespan of C . glabrata strains could be divided into a young , middle , and advanced age phase ( Fig 1C ) .', 'Notably , at the end of their RLS , senescent C . glabrata cells arrested either as pseudohyphae , in a budded state , or as a single mother cell ( unbudded ) ( Fig 1D ) .', 'With every replication , the cell size and cell wall thickness increased ( Fig 1E ) .', 'Although considerable variability in relative cell body volume increase among strains was observed , there was no correlation between maximal volume and median RLS ( Spearman r = -0 . 14 , p = 0 . 78 ) .', 'Also despite variability , no correlation between virulence in the Galleria mellonella model and median RLS ( Spearman r = -0 . 01 , p = 0 . 96 ) , or median CLS ( Spearman r = -0 . 46 , p = 0 . 10 ) was found .', 'C . glabrata strains killed Galleria at markedly different rates ( range 1–14 d , p < 0 . 0001 ) ( Fig 1F ) .', 'C . glabrata strains ( 14 , 43 , 47 ) that formed pseudohyphae were more virulent ( median survival: 2 d ) .', 'Similar to other yeast species , we hypothesized that replicative aging would augment C . glabrata’s resilience in vivo ( Table 2 ) .', 'Old C . glabrata cells were isolated by biotin labeling a virgin population , allowing it ample time to divide and age , and then magnetically labeling it with streptavidin beads before sorting through a magnetic column .', 'These cells , which henceforth we will label as “old , ” are 10–14 generations , or for some experiments , 28 generations old .', 'These are not young cells , but at this age are also far from senescence and thus have comparable growth dynamics to young cells ( Fig 1C ) .', 'The higher the replicative age of a cell , the rarer it becomes without stress: in a clonally expanding population a 14 generation old cell is rare and has a frequency of roughly 1:16 , 000 compared to other ages; this becomes 1:>108 for a 28 generation old cell .', 'We compared the virulence of these old cells to young ones of the standard laboratory strain BG2 using the Galleria model .', 'These experiments demonstrated that old cells ( 14 generations , average diameter 6–8 μm ) were significantly ( p < 0 . 05 ) more virulent compared to young cells ( 0–3 generations , average diameter 4 . 1 μm ) ( Fig 2A ) .', 'It was determined that younger cells were cleared quicker than older cells , while older cells tended to accumulate in the first few hours during infection ( Fig 2B ) .', 'Consistent with that finding , older cells were phagocytosed at a lower rate by Galleria haemocytes ( S1A Fig ) and human neutrophils ( S1B Fig ) , and more resistant to neutrophil-mediated killing ( Fig 2C ) .', 'Although older C . glabrata cells induce more neutrophil extracellular traps ( NETs ) than young C . glabrata cells ( Fig 2D–2F ) , relative to C . albicans hyphae the overall NET induction is considerably lower , and it is unclear if NETs truly kill these C . glabrata cells .', 'Similar enhancement of virulence ( S2A and S2B Fig ) , and resistance to neutrophil-mediated phagocytosis and killing ( S2C and S2D Fig ) was also documented in older cells of C . glabrata strains 89 and 117 .', 'Consistent with augmented resistance to neutrophil killing , older cells exposed to H2O2 exhibited smaller zones of no growth in a disc diffusion assay ( Fig 2G and S2E Fig ) .', 'Interestingly , older cells exhibited decreased adhesion to epithelial cells compared with younger cells ( Fig 2H ) suggesting that they would more successfully invade after dissemination .', 'Lastly , the effect of aging on switching was investigated , in C . glabrata , where the phenotypic switch from the parental smooth ( S ) variant to the dark brown ( DB ) variant , is associated with enhanced persistence in mice ( [22] and S2F Fig ) .', 'These experiments demonstrate that replicative aging of BG2 cells from 2 to 35 generations consistently resulted in higher phenotypic switching rate ( Fig 3A ) , a finding confirmed also in strain 89 ( S2G Fig ) .', 'Interestingly , in both strains , S to DB switching ( Fig 3B and S2H Fig ) resulted in over 50% loss of median RLS , which was reversible and regained in the revertant colony .', 'These findings further support the conception that RLS is a highly regulated trait in a fungus .', 'Given the above described striking phenotypes of resilience we pursued a transcriptome analysis comparing genes transcribed in young and 14-generation-old C . glabrata cells by RNA sequencing .', 'Fungal cells were grown in YPD .', 'RNAseq results were consistent with the observed distinct cell wall morphology on EM images of old cells and indicated that fundamental remodeling of the cell wall occurs throughout the process of aging .', 'Specifically , several genes involved in ergosterol biosynthesis and sterol uptake ( CAGL0I04246g , ERG1 , ERG3 , ERG5 , ERG6 , ERG11 , ) [23–27] were upregulated .', 'ERG19 and ERG20 upstream in the “mevalonate pathway , ” as well as CAGL0L09493g downstream in the “sito steryl glucoside synthesis” of ergosterol biosynthesis were also upregulated .', 'Also CAGL0C04026g , a mannosyl transferase , which aids in protein glycosylation and uses farnesyl pyrophosphate , the endproduct of the mevalonate pathway was upregulated .', 'In addition , several genes involved in cell wall remodeling ( KNH1 , SUN4 ) [28 , 29] , as well as the Hog pathway ( CDC42 , STE20 , MSB2 , CAGL0F03003g ) [30–34] were significantly upregulated .', 'Cell wall adhesins ( PWP3 and PWP5 ) [21 , 35–38] and adhesion proteins ( AWP1 , CAGL0J02530g , CAGL0L06424g ) [21 , 36 , 39] were significantly downregulated ( virulence-associated genes are included in Table 3 , complete list is available at GSE85985 ) , which was predicted by our functional adhesion assays .', 'Interestingly EPA6 , a cell wall adhesin [40–45] , and FKS1 , a β1 , 3-glucan synthase [46 , 47] , both implicated in azole resistance were upregulated in old cells .', 'Given these results we predicted that old cells would be more resilient under the selection pressure of azole treatment .', 'Older ( 5–7 generations ) and young ( 0–2 generations ) C . glabrata cells were grown and aged for 15 h under sub-therapeutic levels of fluconazole ( 32 μg/mL ) , and then subjected 4 h to increasing fluconazole concentrations ( 0–512 μg/mL ) before plating on YPD plates .', 'At fluconazole concentrations of 16–256 μg/mL , the younger cells were inhibited/killed at significantly higher rates than the older cells ( Fig 4A ) .', 'Specifically , failure of young cells to grow on fluconazole containing plates ( 16 to 64 μg/mL ) ranged from 75–79% , respectively .', 'Whereas failure of old cells to grow ranged from 26% ( 32 μg/mL ) to 59% ( 512 μg/mL ) .', 'Inhibition was relatively stable across various fluconazole levels for the young cells , whereas growth inhibition of older cells tended to slightly increase as the amount of fluconazole increased .', 'These data suggest that older cells grown under selection pressure of sub-therapeutic fluconazole are significantly more resistant to subsequent fluconazole exposure compared to young cells exposed to the same fluconazole concentrations .', 'Transcriptome analysis ( GSE85985 ) identified upregulation of several β1 , 3-glucan enzymes: a synthase ( FKS1 ) that is functionally redundant with Fks2 , and in which “hot spot” mutations confer resistance [48] , a exo-1 , 3 , beta-glucosidase ( CAGL0G09515g ) , a transglycosidase ( GAS5 ) with an elongation role [21] , and an ortholog with osmosensor and biosynthetic activity ( CAGL0F03003g ) .', 'Also , KNH1 [28] , which is involved in 1 , 6-glucan synthesis , was upregulated , while a 1 , 4-alpha-glucosidase ( CAGL0G02717g ) was downregulated .', 'In addition to the glucan signature , CHS3B , a Class IV chitin synthase that has a calcineurin pathway- and Tor1p-dependent role in cell wall integrity [49 , 50] was upregulated .', 'Nevertheless , major quantitative or qualitative differences for content of chitin , β1 , 3- , and β1 , 6-glucans could not be established by employed biochemical methods .', 'Old C . glabrata cell walls contained both glucans , and only a slightly higher quantity of β1 , 3-glucans was noted , whereas chitin and β1 , 6-glucan content did not differ ( Fig 4B ) .', 'Detailed transcriptome ( Table', '3 ) and gene ontology analysis ( Table', '4 ) identified enrichment of steroid metabolic and biosynthetic processes .', 'Therefore , we analyzed the lipid composition of older C . glabrata cells to investigate if the upregulation of the ergosterol pathway leads to accumulation of sterol intermediates .', 'The ergosterol content of old and young cells was quantified by GC-MS and normalized both to cell number as well as to lipid content .', 'This quantitative analysis demonstrated approximately 10 fold lower ergosterol levels in old compared to young cells ( Fig 4C ) , whereas analysis of the downstream sito steryl glucosides revealed slightly increased accumulation ( 10 . 5 vs 6 . 6 μg/ml ) .', 'This latter quantification was done by LC-MS in pooled samples of 14-generation-old compared to young cells ( 0–3 generations old ) .', 'Taken together , the transcriptome data show that cell wall remodeling genes , several of which are already implicated in C . glabrata virulence and antifungal resistance are differentially regulated in older cells .', 'Older cells exhibit markedly upregulated ergosterol pathway associated genes and greater resistance under increasing fluconazole selection .', 'Furthermore , biochemical analysis of older C . glabrata cells show differences in ergosterol content and cell wall composition .', 'In a clonally expanding pathogen population , old cells statistically cannot dominate within the population unless forceful selection pressures are operative to promote their emergence and kill off younger cells .', 'Based on the aforementioned in vitro assays , which suggest that old cells have a biological advantage under certain selection pressures , we hypothesized that the immune response of the host controls the generational distribution of a pathogen population by differential mortality .', 'In support of this , larger C . glabrata cells with high budscar count were observed in human patients ( n = 6 ) with urinary tract infections .', 'Those patients were not neutropenic , and violin plots of the budscar counts indicate higher accumulation of old cells from in vivo derived yeast cells when compared to the same cells grown up without selection pressure with in vitro cultures ( Fig 5A shows data from patient #42 , and S3A Fig shows data from the remaining patients ) .', 'Disseminated C . glabrata infections commonly occur in neutropenic patients , and thus we predicted that neutrophils constitute the selective force in mice , as they are the key effector cells that contain C . glabrata infection [51] .', 'To test this hypothesis in vivo , the effect of neutrophil depletion was examined on the generational distribution of an infecting C . glabrata population with different strains ( BG2 and 89 ) injected intravenously into wildtype ( WT ) or neutropenic mice .', 'The latter immune status was achieved by injecting antibody RB6-8C5 , which transiently depletes neutrophils in the mouse .', 'Subsequently , C . glabrata was harvested from homogenized kidneys every 2 days , and yeast cells were directly stained with calcofluor to assess budscar count as a measure of replicative age .', 'In addition , cell size was measured as increase in size correlates with older age .', 'The violin plots demonstrate shift in generational distribution .', 'Specifically , a significantly higher percentage of older cells , as demonstrated by increased budscars and cell size were found in WT mice when compared to neutropenic mice for both strains BG2 ( Fig 5A and 5B , and S1 Table ) and 89 ( S3B Fig , and S1 Table ) .', 'This supported the hypothesis that old cells are selected due to increased killing of cells by host neutrophils .', 'Consequently , in neutropenic mice the pathogen population is younger , since there were fewer neutrophils to kill them , and the generational distribution was more similar to a population that had grown in vitro without exposure to selection forces .', 'To confirm the experimental results theoretically and gain additional insights , the selection pressure on cells of various ages was determined based on the distribution of replicative ages from the mouse experiment ( Fig 5A ) .', 'A system of ordinary differential equations was developed representing the time-dependent population sizes of five age classes ( S4 Fig ) to evaluate the age distributions resulting from various mortality rates on each age class ( Fig 5C ) .', 'Many initial mortality profiles ( sets of mortality rates ) were systematically explored , then local optimization was used to vary each mortality rate separately ( S5 Fig ) .', 'This returned the corresponding best-fit age distribution given the initial profile .', 'Chosen from all local optima , the global-optimum mortality profile was robust to the initial profile , with the local optimizer converging on it for 38% , 52% and 63% ( out of 3125 initial profiles ) of the search space for WT , neutropenic , and in vitro populations , respectively; S6 Fig ) .', 'The experimental age distribution superimposed with the results from the model were parametrized with the most optimal mortality rates , as given by the global optimization ( Fig 5D ) .', 'The model fit the experimental age distribution closely for all three experimental populations .', 'It was also able to capture the overall dynamics of the C . glabrata infections in vivo ( S4 and S5 Figs ) .', 'The estimated mortality rates took different forms for each condition ( Fig 5D ) : high on the first age class and lower on the rest for WT mice; high on the middle age class and lower on the rest for neutropenic mice; very low on all age classes except the second and third for the in vitro population .', 'Comparing the mortality rates with each other , the mortality profile of neutropenic mice was similar to that of in vitro cultures ( Pearson r = 0 . 54 , p = 0 . 35 ) .', 'Interestingly , mortalities in WT mice showed an opposite pattern; they were highest for young cells ( r = -0 . 41 , p = 0 . 49 between WT and neutropenic; r = -0 . 71 , p = 0 . 18 between WT and in vitro ) .', 'The lack of significance was a result of the comparison between two sets of five values ( the number of age classes ) .', 'Overall , the computational results showed that in order to generate the replicative age distributions observed experimentally , a competent host must cause selection pressures on each age class that are different from those of a neutropenic host ( as well as an in vitro population ) .', 'This served as further confirmation of the hypothesis that neutrophils constitute the main selection pressure in a competent host , since the mortality profile with respect to age of a neutropenic host was more similar to that of an in vitro population than a competent host .'], ['To our knowledge , this is the first study to investigate replicative aging in clinical C . glabrata populations .', 'Our data link replicative aging to pathogenesis in this important fungal pathogen .', 'Specifically , we demonstrate that the natural process of aging results in a remodeling of the fungal cell wall leading to enhanced resilience .', 'Furthermore , we show that neutrophil-mediated killing comprises the host’s selection pressure , which shifts the generational distribution of a C . glabrata population in vivo .', 'Thus , this study supports a conceptually novel understanding of pathogenesis , where replicative aging in a yeast population facilitates demographic changes in the presence of a selective host response .', 'Hence , the natural process of replication can be exploited as a unique mechanism of adaptation that contributes to phenotypic variation , resilience , and virulence .', 'Since the phenotype of aging is not genetically inherited , but only seen through shifting selection pressures that promote persistence of older cells , the high risk of random permanent mutations is avoided .', 'Instead constant rejuvenation through budding provides an assurance that all adaptive changes can be rapidly reversed as the daughter inherits asymmetrically [52 , 53] and allows the emergence of a new generation .', 'Our original study indicated accumulation of old C . neoformans cells in chronic cryptococcosis [19 , 54] .', 'That work led to the hypothesis that aging is associated with pathogenesis and that the high number of C . neoformans cells with decreased remaining RLS , which we recovered from spinal fluid of infected rats or humans , represent old cells , which are statistically unlikely to occur in a clonally expanding population unless selected .', 'To examine this hypothesis further and in depth , we chose C . glabrata , a commensal that can become pathogenic to cause invasive disease predominantly in neutropenic patients .', 'Using this yeast we could study directly the association of aging and fungal pathogenesis , because opposed to C . neoformans , it maintains budscars on its cell surface when it continues to replicate , and therefore the actual generational age can be determined in real time .', 'In addition , neutrophils , which are key host defense cells in several human and murine Candida spp .', 'infections [12 , 51 , 55] , can be studied in vitro and depleted in vivo .', 'However , given the lack of prior studies on replicative aging in C . glabrata , baseline assumptions about replicative aging had to first be validated for this species .', 'We found considerable variability of RLS among clinical C . glabrata strains similar to that observed in other fungi [18 , 19 , 56 , 57] .', 'C . glabrata cell size increased consistently with each replication .', 'In contrast to S . cerevisiae [58] , maximal cell volume did not predict RLS , and future imaging studies will need to clarify whether time to first bud emergence correlates with length of RLS as recently demonstrated for S . cerevisiae [59] .', 'Variable length of exposure to the intracellular environment of phagocytic cells has been associated with change in the virulence of several Candida spp .', '[13 , 14] .', 'Thus , it is conceivable that the intracellular environment of other phagocytic cells , especially macrophages , also contributes to variability in replicative age among the clinical strains .', 'C . glabrata can colonize the human host , and cause disease in humans only when host defenses fail and unchecked growth of the C . glabrata population ensues .', 'We were therefore most interested in comparing resilience-associated phenotypes in young and old cells .', 'Consequently , in vivo resilience of old and young C . glabrata cells was compared using a well-established infection model of G . mellonella [60–62] .', 'These data demonstrate that aging indeed enhances resilience , and consequently virulence of this pathogen .', 'Specifically , we found that older cells of both short and long-lived strains killed Galleria significantly faster than younger cells of the same C . glabrata strain .', 'It is important to emphasize that 14-generation-old cells are far from senescence , and therefore fitness would not be significantly decreased .', 'Accordingly , we show that 14-generation-old cells exhibit comparable doubling rates .', 'Several important phenotypic differences between young and old cells explain the observed resilience of the older cells .', 'We demonstrated resistance of older cells to killing and phagocytic uptake .', 'Young cells do not evade the basic immune response of Galleria , and they are therefore killed more rapidly and cannot establish infection as rapidly as the older C . glabrata cells .', 'Galleria has an innate immune system composed of six types of phagocytic cells [60] .', 'Consistent with our finding is the observation that larger C . neoformans cells are also more resistant to uptake by Galleria’s phagocytic cells [63] .', 'Furthermore , our data shows that age confers resistance to intracellular killing after phagocytic uptake .', 'In mammals , one contributing facilitator of killing in neutrophils are reactive oxygen species [55 , 64] .', 'Although C . glabrata is closely related to S . cerevisiae , higher resistance to ROS has evolved in this yeast as an adaptation to the intracellular environment of phagocytic cells , enabling the fungus to persist in the host [5] .', 'Our data now indicate that aging augments this resistance , explaining the resistance of older C . glabrata cells to neutrophil-mediated killing .', 'Transcriptome data indicate that genes in the Hog pathway , including CDC42 , STE20 , MSB2 , were significantly upregulated in older cells , and this pathway is a well-known regulator of cell wall damage imparted by various stresses [65] .', 'Hence , in order to be protected , older cells make a thicker cell wall through a concerted mechanism involving gene regulation , protein reorganization , and lipid remodeling .', 'The observation that glucan is much more abundant in old cells compared to young cells is supported by recent studies in Candida tropicalis , where a thicker cell wall rendered strains less susceptible to amphotericin B and more resistant to stress responses [66] .', 'Also in this instance , the authors found more beta-glucans in strains with thicker cell wall with no change in chitin content , similar to our findings .', 'Our studies extend these observations to another fungus .', 'We also documented a significant decrease in ergosterol in older cells , which could lead to a re-organization of the plasma membrane .', 'In addition , sito steryl glucosides , a downstream product of ergosterol synthesis is known to accumulate in the plasma membrane [67 , 68] .', 'Data from Saccharomyces indicate that mutations of the ergosterol pathway affect the cell wall integrity [67] .', 'Furthermore , vesicular trafficking and release of extracellular vesicles may be affected by ergosterol level , which may also modulate the immune response [69] .', 'The increased azole resistance could be attributed to increased expression of the azole target , Erg11p .', 'It is noteworthy that although ERG11 and several other ergosterol pathway genes are markedly upregulated in old cells , ergosterol levels were found to be significantly lower in old cells .', 'Several factors could contribute to this finding .', 'First the overexpression of upstream mevalonate pathway genes , ERG19 and ERG20 , may redirect the sterol intermediates to the side pathway; as such we found a mannosyl transferase ( CAGL0C04026g ) upregulated .', 'This enzyme aids in protein glycosylation [70] .', 'Second , the accumulation of sito steryl glucosidase , which is downstream of ergosterol may lower the ergosterol levels .', 'This may also promote overexpression of increased uptake .', 'Future studies examining the azole influx and efflux in young and old cells may shed more light on how azole resistance is mediated [67] .', 'Third , the transcriptional upregulation in older cells may be due to a compensatory response to ergosterol depletion .', 'If older cells have mutations in the enzymes involved in ergosterol biosynthesis , it may explain the low ergosterol level and the resistance to fluconazole .', 'Further studies are clearly needed to test these possibilities .', 'The cell wall constitutes the key defense against all external attacks .', 'In addition it is also recognized by the host immune system , which can make both effective and ineffective harmful immune responses as a result of host-pathogen interaction [71 , 72] .', 'Lastly because cell walls of fungi are fundamentally different from the bi-lipid membranes of human cells , they are very attractive targets for antifungal therapy .', 'The concept that cell walls are dynamic and that structural realignment occurs as a result of stress induced upregulation of signaling pathways is not novel and has been described for other yeast as well [73–75] .', 'In our case , however , cell wall reorganization is not a response to an environmental change per se , but a process that is merely driven by an internal clock , namely the natural process of aging .', 'This process is controlled by evolutionarily conserved signaling pathways similar to those of environmentally induced stress ( Hog1 ) , cell integrity ( Mkc1 , Cek1 ) and ergosterol metabolism ( Erg11 , Erg3 , Erg5 , Erg6 , Sur2 ) [76] .', 'A less obvious mechanism of how aging can affect pathogenesis is by augmenting phenotypic switching rates to more persistent or virulent variants .', 'Older C . glabrata cells switch at a significantly higher rate to dark brown colony variants , which colonize the host more efficiently [22] .', 'Interestingly , both the hypervirulent mucoid ( C . neoformans ) [19] , and DB ( C . glabrata ) switch variants exhibit more than 30 generations shortened median RLS , a loss which is instantly recovered with reversal to the parent phenotype .', 'This finding further supports the notion that isogenic strains with shortened RLS may be more virulent because of rapid aging as the more resilient “old age-associated” phenotypes are acquired earlier .', 'In addition , it underscores the fact that lifespan , although variable among C . glabrata strains , is a highly regulated trait in the individual pathogenic yeast cell .', 'Lastly , it should be emphasized that staying young also has some benefits .', 'Our results suggest that adherence to epithelial cells is enhanced in young cells .', 'Adherence of C . glabrata to host cells is mediated in part by the Epa family of adhesins , which are encoded largely at subtelomeric loci and subject to transcriptional silencing of SIR genes [77] .', 'EPA6 for instance is derepressed in response to limiting levels of nicotinamide adenine dinucleotide , which results in reduction of Sir2-mediated histone deacetylation and loss of silencing [41] .', 'In S . cerevisiae , Sir2 levels decrease in old cells , and therefore one would predict higher adherence as a result of derepressed EPA expression [78] .', 'However , it is conceivable that the thicker cell wall of older C . glabrata cells may affect surface exposure of Epa proteins , some of which reside in the C . glabrata cell wall [79] .', 'Nonetheless in C . glabrata , adhesion induces actin-mediated phagocytosis and an increased inflammatory cytokine response [6] .', 'Thus , the decreased adherence observed in the older cells in our studies is consistent with their decreased phagocytosis and ultimately decreased killing .', 'In C . glabrata , stainable budscars permitted us to assess in real-time the generational age distribution of an in vivo expanding C . glabrata population .', 'This allowed us to design experiments that directly assessed the effect of host response on the emergence of old cells .', 'First , we demonstrate by directly counting budscars that the in vivo derived C . glabrata population includes a large percentage of cells with many budscars , the hallmark of 
['Introduction', 'Results', 'Discussion', 'Methods']
['Similar to other yeasts , the human pathogen Candida glabrata ages when it undergoes asymmetric , finite cell divisions , which determines its replicative lifespan .', 'We sought to investigate if and how aging changes resilience of C . glabrata populations in the host environment .', 'Our data demonstrate that old C . glabrata are more resistant to hydrogen peroxide and neutrophil killing , whereas young cells adhere better to epithelial cell layers .', 'Consequently , virulence of old compared to younger C . glabrata cells is enhanced in the Galleria mellonella infection model .', 'Electron microscopy images of old C . glabrata cells indicate a marked increase in cell wall thickness .', 'Comparison of transcriptomes of old and young C . glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins , and suggests that aging is accompanied by remodeling of the fungal cell wall .', 'Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition , leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure .', 'Older C . glabrata cells accumulate during murine and human infection , which is statistically unlikely without very strong selection .', 'Therefore , we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo .', 'When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils , we observed a significantly younger pathogen population in mice .', 'Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population .', 'Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C . glabrata population in a host .', 'We conclude that replicative aging constitutes an emerging trait , which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state .']
['This study shows that aging makes Candida glabrata more resilient and virulent in vivo .', 'Aging results in a thicker cell wall that is also qualitatively different with respect to sterol composition , which conveys enhanced resistance to azoles and cellular stresses .', 'We observed greater accumulation of old cells in competent mice , indicating that selection pressures in the host preferentially kill younger cells .', 'Depletion experiments and mathematical modeling confirm that neutrophils represent the predominant selection pressure , and their removal dramatically alters selection pressures , resulting in a younger pathogen population .', 'We propose that replicative age is an unanticipated virulence trait that is selected in the host environment and may play an important role in the switch from a commensal to a pathogen state .']
['blood cells', 'death rates', 'cell walls', 'medicine and health sciences', 'immune cells', 'body fluids', 'pathology and laboratory medicine', 'demography', 'pathogens', 'immunology', 'age distribution', 'microbiology', 'urine', 'physiological processes', 'developmental biology', 'organism development', 'pharmacology', 'cellular structures and organelles', 'fungal pathogens', 'neutrophils', 'mycology', 'white blood cells', 'antimicrobial resistance', 'animal cells', 'medical microbiology', 'microbial pathogens', 'aging', 'people and places', 'cell biology', 'anatomy', 'physiology', 'microbial control', 'biology and life sciences', 'cellular types']