Xenosiderophore transporter gene expression and clade-specific filamentation in Candida auris killifish infection

Xenosiderophore transporter gene expression and clade-specific filamentation in Candida auris killifish infection | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Xenosiderophore transporter gene expression and clade-specific filamentation in Candida auris killifish infection View ORCID Profile Hugh Gifford , View ORCID Profile Tina Bedekovic , View ORCID Profile Nicolas Helmstetter , View ORCID Profile Jack Gregory , View ORCID Profile Qinxi Ma , View ORCID Profile Alexandra C. Brand , View ORCID Profile Duncan Wilson , View ORCID Profile Johanna Rhodes , View ORCID Profile Mark Ramsdale , View ORCID Profile Tetsuhiro Kudoh , View ORCID Profile Rhys A. Farrer doi: https://doi.org/10.1101/2024.10.22.619744 Hugh Gifford a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hugh Gifford Tina Bedekovic a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tina Bedekovic Nicolas Helmstetter a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicolas Helmstetter Jack Gregory a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jack Gregory Qinxi Ma a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Qinxi Ma Alexandra C. Brand a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexandra C. Brand Duncan Wilson a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Duncan Wilson Johanna Rhodes b Department of Medical Microbiology , Radboudumc, Geert Grooteplein Zuid 10, Nijmegen, Netherlands, 6525 GA c MRC Centre for Global Infectious Disease Analysis, Imperial College London South Kensington Campus , London, United Kingdom , SW7 2AZ Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Johanna Rhodes Mark Ramsdale a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mark Ramsdale Tetsuhiro Kudoh d Biosciences, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tetsuhiro Kudoh Rhys A. Farrer a MRC Center for Medical Mycology, University of Exeter, Geoffrey Pope Building , Stocker Road, Exeter, United Kingdom , EX4 4QD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rhys A. Farrer For correspondence: R.Farrer{at}exeter.ac.uk Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Candida auris is a critical priority fungal pathogen (World Health Organization). Clinical management is challenging due to a high mortality rate, rapidly increasing antifungal resistance, and frequent nosocomial outbreaks. A critical bottleneck in understanding virulence is the lack of gene expression profiling models during infection. We developed a fish embryo yolk-sac microinjection model using Aphanius dispar (Arabian killifish; AK) at human body temperature. This enabled interrogation of infection dynamics via dual host-pathogen RNA-seq across five major clades of C. auris (I-V). Host responses included heat shock, complement activation, and nutritional immunity, notably haem oxygenase ( HMOX ) expression during clade IV infection. We identified a pathogen transcriptional signature across all five clades of C. auris strongly enriched for putative xenosiderophore transmembrane transporters. We describe this novel family and a sub-clade of five putative haem transport-related ( HTR ) genes. Only the basal clade V isolate formed filaments, associated with canonical and atypical regulators of morphogenesis. Clades I and IV demonstrated increased virulence, accompanied by up-regulation of three HTR genes in clade IV, and the non-mating mating-type locus ( MTL ) gene PIKA in both clades. Our study provides new insight into C. auris pathogenesis, highlighting species-wide in vivo up-regulation of XTC genes during host tissue infection. Significance statement Candida auris is an emerging human fungal pathogen and global public health threat, yet in vivo transcriptomic analysis of tissue infection has remained elusive. Using yolk-sac infection in Arabian killifish, we profiled gene expression across five major C. auris clades. We found that the basal clade V uniquely undergoes filamentation during infection, while all clades upregulate members of a large, expanded family of xenosiderophore transporter candidate genes. These findings highlight the important roles for iron acquisition and morphological switching in pathogenesis, revealing potential mechanisms of immune evasion and fungal persistence, and identifying candidate targets for antifungal therapy. Introduction Candida auris is an ascomycetous yeast that has rapidly progressed from a recently identified species to a World Health Organization (WHO) critical priority human fungal pathogen and global public health threat 1 , 2 . The earliest known isolate was retrospectively identified from a bloodstream infection dating to 1996 3 , following its initial description as a novel species in 2008 4 . Over the past decade, C. auris has caused nosocomial outbreaks globally, leading to intensive care unit (ICU) closures and substantial infection control challenges and costs 5 – 7 . Alarmingly, C. auris is now a leading cause of invasive candidiasis in several hospital settings globally 8 , 9 . C. auris bloodstream infections carry an associated mortality rate of approximately 45% 10 and are often resistant to azole antifungals, with an increasing number of pan-drug resistant isolates reported globally 11 . Phylogenetic analysis has revealed five major clades of C. auris , and a minor sixth clade recently found 12 , 13 , suggesting multiple populations of C. auris have simultaneously emerged from unknown environmental reservoirs that have facilitated its rapid adaptation to human infection, drug resistance, and healthcare-associated persistence 14 , 15 . Clade-specific differences have been noted: clades I, III, and IV are more commonly associated with invasive disease, whereas clades II and V have been linked to otomycosis 16 . Mortality from candidaemia may be higher with clades I and IV than clade III, but this difference has not been shown to be statistically significant 17 . These differences in clinical presentation and virulence may reflect underlying genotypic variation across clades. Gene expression profiling has become a standard approach for assessing host and pathogen responses to infection. However, no experiment has described the gene expression of C. auris during in vivo infection of living tissue to date. Studies have assessed the pathogen transcriptome in an in vivo murine catheter model 18 , human whole blood ex vivo 19 , co-incubation with human dermal fibroblasts 20 or murine cell line-derived macrophages 21 . Additionally, host responses have been assessed in murine peripheral skin 22 , human peripheral blood-derived monocytes 23 , and murine bone marrow-derived macrophages 24 . C. auris has been shown to reproduce as budding yeasts in mammalian tissues 25 , 26 and possess multiple adhesins associated with an aggregation phenotype 27 , 28 . There has been no evidence of toxin synthesis unlike distantly related C. albicans 29 , 30 . C. auris digests of host tissue through secreted aspartyl proteases 31 and evades host immune responses through cell surface masking 32 and intra-phagocytic survival with cell lysis 33 . However, these studies have not interrogated in vivo in-host pathogen gene expression, except for the dual RNA-seq study using the ex vivo whole blood model, for which it was possible to compare gene expression profiles between C. auris and other Candida species 19 . The absence of in vivo tissue infection profiling for C. auris may be due to the lack of adequate RNA recovery from current models. Most murine models, such as BALB/c 25 , 34 – 36 and C57BL/6 mice 24 , have rarely demonstrated lethality through C. auris infection 23 , 37 , 38 without major immunosuppression with cyclophosphamide/cortisone 39 – 50 , monoclonal antibody neutrophil depletion 26 , 51 , or other immunodeficiency 27 , 52 , 53 . Additionally, mouse models are also expensive and associated with concerns over environmental pathogen shedding 54 . Alternative host models such as Galleria mellonella 34 , 44 , 55 – 61 , Drosophila melanogaster 62 and Caenorhabditis elegans 60 have been employed, but these may lack key elements of mammalian-relevant immune systems, thus limiting their scope of application. In patients, immune control of C. auris is thought to depend on myeloid cell activity via C-type lectin receptors such as complement receptor 3 and mannose receptor MMR, followed by interleukin (IL-17) pathway activation 63 , 64 . The zebrafish ( Danio rerio ) model has allowed hindbrain 32 , 65 and swim bladder 66 microinjection modelling of C. auris infection, but rarely at temperatures above 30 °C, which zebrafish are broadly intolerant of. Fungal transcriptional programmes can vary greatly across temperatures and are critical for mammalian pathogenesis 67 – 69 . Despite these successes in profiling host responses, none of these models have enabled the profiling of C. auris RNA during infection. A marine model could also shed light not only on human infection but also on a possible evolutionarily adaptive niche. The environmental origins of C. auris have not been conclusively demonstrated; however, a marine origin has been hypothesised and supported by successful isolation of C. auris in coastal waters of both Indian and Pacific oceans 70 , 71 . Notably, a close relative of C. auris in the Metschnikowiaceae clade, C. haemulonii , was first isolated in 1961 from the gut of blue-striped grunt (genera name “ Haemulon ” for their blood-red mouth interior) 72 . Fish larvae must survive hostile and varied ocean environments, demonstrating very early innate immune responses such as antimicrobial peptide production and the complement cascade 73 , 74 . Only in adulthood are teleost fish expected to possess functional IL-17 subtypes (A and F) that lead to T-helper-17 subset differentiation and adaptive immunity in response to fungal infection 75 . A thermotolerant teleost fish embryo model such as the Aphanius dispar , also known as the Arabian killifish (AK), which can acclimatise to temperatures up to and including 40 °C 76 – 78 , offers a balance between considerations such as suitability for experimental manipulation and applicability to human fungal infection at mammalian temperatures 79 , 80 . Additionally, fish embryo models align with the principles of replacement, refinement and reduction of animals in laboratory research and offer an opportunity to develop novel animal models with ethical benefits over models such as adult mice 81 . In this paper, we present the first study exploring in-host virulence of C. auris clades I-V in the AK yolk-sac microinjection model at 37 °C. We supplemented our validation of the model with histology, fungal burden recovery, and dual host-pathogen RNA-seq. By comparing in vivo and in vitro expression profiles of C. auris , we detected a cross-clade expression signature that was significantly enriched for siderophore transporter SIT1 paralogues, which mediate uptake of siderophores produced by other microbes (xenosiderophores). We designated this expanded gene family as xenosiderophore transporter candidates ( XTC ), which included a haem transport-related ( HTR ) sub-clade. We also identified in vivo filamentation by clade V during infection, which was associated with orthologues of key hypha-associated genes from C. albicans . Together, these findings substantially enhance our knowledge of the processes employed by C. auris during infection. Results AK yolk-sac microinjections with each of the five C. auris clades demonstrated lethality (defined by cessation of heartbeat) at 37 °C (92-100% vs 6% for sham injections; Figure 1A-B ). Earlier lethality was significantly higher for clade I compared to clades II, III or V ( p < 0.001) and for clade IV compared to clades II or III ( p < 0.04; Figure 1B , Table S1 ). To investigate morphological outcomes of infection, we compared histological sections of infected yolk-sacs at 48 hours post infection (HPI). All clades exhibited budding yeast morphologies both in vitro and in vivo , except for the clade V strain, which demonstrated predominantly filamentous forms when grown in vivo ( Figure 1C ). Growth kinetics showed only minor inter-clade variation. Clade II grew more slowly in YPD-broth at 37 °C ( Figure S1A ), although this did not translate into lower CFU recovery from infected embryos ( Figure 1D ). On average (X), 673 CFUs were recovered per infected embryo post injection, which increased 166-fold at 24 HPI (X = 1.12 ×10 5 ) and by an additional ∼60% at 48 HPI (X = 1.78 ×10 5 ). Filamentous clade V isolates formed rough ( vs smooth) colonies on YPD-agar in approximately 1-2% of cases ( Figure S1B ), composed of elongated yeast cells, filamentous and pseudohyphal structures measuring >10-50 μm ( Figure S1C ). These results confirm that AK yolk-sac microinjection supports active C. auris infection, leading to embryo mortality by 7 days post-injection. This system therefore provides a robust and informative in vivo model for studying the pathogenicity of human fungal pathogens such as C. auris . Download figure Open in new tab Figure 1: Modelling C. auris infection in the Arabian killifish (A) Illustrative time-lapse microscopy of embryo death including yolk-sac collapse during infection (clade I infection) or survival (sham injection) using Acquifer live imaging. (B) Survival curves for embryos injected with each C. auris clade or sham injection. Significance testing between groups is given in Table S1 ; hours post injection (HPI). (C) H&E Staining of AK yolk-sac at 48 HPI for each clade or sham injection; morphology is indicated by blue arrows, with insets indicating typical yeast (clade III) and filamentous morphology (clade V). The yolk-sac edge is oriented at the top. (D) Colony forming units (CFUs) per clade from embryos homogenised after injection as an indicator of injection dose and in vivo growth. To characterise host responses to C. auris during infection, we identified host differentially expressed genes (DEGs) in AK yolk-sacs between infection by each clade and sham injections ( Figure 2 ). Across all infections, 685 host DEGs were detected (representing 2.49% of potential genes), including 183 up-regulated and 508 down-regulated genes in response to at least one clade. Notably, there were no DEGs common to infection by all five clades. Only five DEGs (four up-regulated genes and one down-regulated gene) were shared across infections with four clades ( Table S2 ). Up-regulated genes included Laminin subunit alpha-1 ( LAMA1 , not significant in response to clade III) and three genes that were not significant in response to clade II, Glucose-6-phosphatase ( G6PC ), DN117939 (hypothetical protein) and Tryptophan aminotransferase protein 1 ( TAR1 ); immunoglobulin-like fibronectin type III domain-containing protein 1 ( IGFN1 ) was down-regulated. These findings suggest host transcriptional responses to C. auris are modest and clade-specific at these early time points, possibly reflecting variation in virulence and stages of infection. Download figure Open in new tab Figure 2: Indicative pipeline diagram for methods used. At three days post fertilization (DPF), A. dispar were microinjected with C. auris from each of five clades I-V. Transcriptional comparisons were made between host and pathogen conditions using either a shared or clade-specific reference. Functional annotations were generated to ascertain enriched features. Illustrations © the authors. Although we did not observe a shared host transcriptional response across all C. auris clades, we identified distinct, clade-specific host responses to infection. At 24 HPI, clade II elicited the largest host response, with 41 up-regulated genes and 210 down-regulated genes ( Figure 3A ). Minimal differences were detected between sham injection and uninjected controls, confirming the specificity of the infection-induced response ( Figure 3B , SI Appendix). By 48 HPI, clade IV induced the strongest transcriptional response, with 49 up-regulated and 104 down-regulated host genes. Notably, we detected differential expression several key immune-related genes across clades, with the most pronounced changes in clades I and IV. These included heat shock proteins ( HSP30 and HSP70 ), complement cascade elements, pattern recognition receptors (PRRs), inflammatory transcription regulators, and genes involved in nutritional immunity ( Figure 3C ). Among genes associated with pathogen recognition, CARD14 was most prominently up-regulated during clade I and IV infection at 48 HPI ( Figure 3D-E ), while NLRP3 (a key inflammasome component) was expressed during clade III infection at 24 HPI. Download figure Open in new tab Figure 3: AK host responses to C. auris infection: (A) Number of differentially expressed genes (DEGs) for each of two comparisons, sham injection vs no injection (left panel), and C. auris injection vs sham injection (right panel), at either 24 or 48 h post injection (HPI). (B) Volcano plot showing log-fold change Functional enrichment analysis of host DEGs revealed several significantly enriched Gene Ontology (GO) terms (and additional annotations), particularly in response to the more virulent clades I and IV at 48 HPI. Clade I infection was associated with 15 enriched GO terms, driven in part by up-regulation of LAMA1 and LAMA2 (orthologues of Laminin subunit-α, Figure 3D ). In clade IV-infected yolk-sacs, enriched terms were related to haem catabolism, haem binding, and haem oxygenase ( HMOX ) activity. Two HMOX orthologues were major contributors to this response, such as haem oxidation (log-fold change X = 3.96, FDR < 0.0003, Figure 3E , Figure S3 ). Quantitative PCR confirmed increased expression of HMOX genes in host; embryos infected by clade IV demonstrated late up-regulation at 48 HPI with a fold change of 3.26 and 9.21 for two HMOX genes DN109585 and DN112160 respectively (compared to uninfected embryos; Figure S2A ). Another host up-regulated gene from infection by clade IV was ferroxidase hephaestin ( HPHL1 ), which is also involved in iron nutritional immunity. Together, these findings suggest that heat shock proteins, nutritional immunity (particularly iron), and the complement effect system are important elements of the AK larval response to C. auris microinjection at 37° C. We compared the in-host transcriptome to expression induced by growth in a standard rich laboratory media (YPD). Principle components and sample correlation clustering from gene expression data indicated a predominant separate clustering of in vivo vs in vitro conditions, indicating that the gene expression profile of C. auris during infection is substantially different to that when it is grown in nutrient-rich laboratory media ( Figure S1G and S1H ). We identified 960 DEGs between in vivo AK infection at 24 HPI or 48 HPI vs in vitro culture in YPD over 24 h (499 up-regulated, 523 down-regulated, 17.7% of potential genes, Figure 4A ). We identified few DEGs between the two time-points 24 HPI to 48 HPI, with zero DEGs for clade IV and V between those time points ( Figure 5A ). The highest numbers of up-regulated DEGs were identified in the filamentous clade V at 24 HPI and in the more virulent clades I and IV at 48 HPI ( Figure 5B-C ). Six functional annotation terms were enriched across all in-host infections at 48 HPI, related to major superfacilitator family carbohydrate and siderophore-iron transmembrane transport ( Figure 6 ). Quantitative PCR confirmed increased expression of SIT genes that had been significantly up-regulated by C. auris at 48 HPI ( Figure S2B ): during embryo infection, clade IV demonstrated up-regulation with a fold change of 2.55 and 8.16 at 24 and 48 HPI respectively for XTC3 (B9J08_003921) compared to culture in YPD alone. Furthermore, XTC7 (B9J08_001487) was up-regulated with a fold change of 39.5 and 130.8 at 24 and 48 HPI respectively. Together, this analysis reveals that during infection, which is a nutrient-limited environment, C. auris is responding by significantly increasing expression of genes involved in sugar uptake, as well as members of the C. auris expanded gene family of siderophore transporters. Download figure Open in new tab Figure 4: C. auris gene expression in vivo vs in vitro (A) Total DEGs per clade for each comparison between 24 h post injection (HPI), 48 HPI, and growth in YPD alone. (B) Combined set of significant DEGs common to all five clades between in vivo infection and in vivo growth in YPD. Genes are coloured by manually annotated category ( Table S3 ). (C) Volcano plots demonstrate mean log-fold change for DEGs identified for any clade. Genes with infinite false discovery rate (FDR) are indicated with an arrow. Genes included in the combined set are coloured by category as per panel B . Download figure Open in new tab Figure 5: C. auris gene expression in vivo vs in vitro . (A-C) Upset plots for each in vivo vs in vitro comparison between 24 h post injection (HPI), 48 HPI, and growth in YPD alone, indicating DEGs that were significant across all five clades (far left) and for each individual clade (far right) and each set in between. Download figure Open in new tab Figure 6: C. auris gene set, pathway and domain enrichment: Transcripts from C. auris reference Thirteen DEGs common to all clades of in-host transcription were transmembrane transporters, including up-regulation of transporters for xenosiderophores ( SIT1 genes 3921 and 1487), sugar transporters ( HGT2 , HGT12 ), an ammonia transporter ( ATO2 ), and a nicotinic acid transporter ( TNA1 ). We also found down-regulation of several drug-resistance-related efflux pumps ( MDR2 and DTR1 ) and peptide transporters ( TPO3 , PTR22 , Figure 4B , Table S3 ). Surprisingly, several putative virulence factors such as lipase LIP1 and cell wall-related KRE6 and SOD6 were also down-regulated across all five clades, as were ALS4 and MDR1 in at least one clade ( Figure 4C ). Several members of the SIT1 family were differentially expressed by <5 of the clades including 0002, 1457, 1458, 1542,2110, 2241, 2465, 3908 and 4097. Meanwhile, two SIT1 genes were down-regulated across 3 of the clades (1519 and 1499). Several SIT1 genes were upregulated in ≥1 clade and down-regulated in ≥1 clade (3908 and 2465). The strong signal of differential expression in the SIT1 expanded gene family highlights the potential importance during in-host survival. The more virulent clades (I and IV) are a -type mating type locus ( MTL ), while the less virulent clades (II, III and V) contain the α-type MTL . Surprisingly, we found differential expression in several non-mating genes between more virulent and less virulent clades, including poly-A polymerase ( PAP1 ), oxysterol binding protein ( OBPA ) and phosphatidylinositol 4-kinase ( PIKA , Figure 7B ). Within the set of 10 DEGs shared across all comparisons, several genes outside the MTL were up-regulated, including vacuolar protein sorting gene, VPS70 , and filamentous growth regulator, FGR14 . Additionally, the drug efflux pump, MDR1 , was down-regulated across all comparisons, supporting the idea of a fitness trade-off between resistance and virulence. Download figure Open in new tab Figure 7: Interclade comparisons for virulence and filamentation: (A): The set of differentially expressed genes (DEGs) shared across all comparisons between clade I and IV (more virulent) and clade II and III (less virulent), coloured by manually annotated category, at either 24 or 48 h post infection (HPI). (B) Volcano plots demonstrate mean log-fold change for each DEG found in any comparison; genes included in the combined set are coloured as per A . (C) The set of DEGs shared across all comparisons between filamenting clade V all other clades (I-IV) in vivo , coloured by manually annotated category. (D): Volcano plots demonstrating mean log-fold change for all comparisons between clade V and clades I-IV at 24 HPI and 48 HPI for each DEG found in any comparison; genes included in the combined set are coloured as per C . To explore drivers of filamentation by the clade V strain, we identified 1,046 DEGs between clade V and each of the other clades (I-IV), comprising 601 up-regulated and 526 down-regulated genes across all comparisons. We identified a set of 57 genes shared across all comparisons for in vivo conditions only, since we did not observe filamentation in nutrient-rich YPD. Twenty-two genes were up-regulated at 24 HPI, seven at 48 HPI, and 18 at both time-points ( Figure 7C ). Most of the up-regulated genes were metabolic ( n = 13), including two cytochrome P450 ERG5 orthologues, though ERG11 was down-regulated. Transmembrane transporters for sugar (including two HGT13 orthologues), amino acids and siderophores ( SIT1 1487) were also up-regulated ( n = 10). Cell wall genes up-regulated in filamentous clade V ( n = 8) included the novel adhesin SCF1 , GPI-anchored genes PGA31 and IFF4 , and orthologues of classical C. albicans hyphal adhesins ALS3 and RBR3 . We identified differentially expressed transcription factors and related regulators, such as HAP41 and the pheromone receptor STE2 , which could play a governing role on these responses. We also examined significant DEGs between clade V and at least one other clade using mean-log fold change ( Figure 7D ). Several genes that play critical roles in C. albicans hypha formation were highly up-regulated, such as transcription factor UME6 and hypha-specific G1 cyclin-dependent protein serine/threonine kinase HGC1 . Enrichment analysis indicated up-regulated gene sets involving CFEM domain, cytochrome P450/oxidoreductase activity, hypha-regulated cell wall GPI-anchored proteins and siderophore transport, particularly at 24 HPI and comparing clade V to clade II ( Figure 7D ). Significantly up-regulated iron-related transport genes in clade V included FRE3 , CFL4 , FRP1 , and SIT1 genes 1487, 1499, 1519, 1542, and 4097. This is consistent with the preservation of a major filamentation programme in C. auris , demonstrated in the basal clade V. Given the prominence of SIT1 orthologues across the transcriptomic analyses of C. auris during in vivo gene expression, we set out to characterise the structural and evolutionary conservation of the expanded gene family across orthogroups, including outgroup species C. haemulonii and C. albicans . We performed phylogenetic analysis on proteins with sequence similarity across seven orthologue clusters, of which two main groupings of transporters with 14 transmembrane domains (TMRs) emerged ( Figure S5 ). The first group included genes with functional annotation relating to siderophore import (including 12 genes in clade I). The second group included haem transmembrane import genes (including 5 genes in clade I). Most of the orthologues retained 14 TMRs, and few had evidence of additional annotations except for the trichothecene mycotoxin efflux pump PFAM domain across multiple groupings. The orthologue SIT1 1547 contained 12 TMRs and was up-regulated with SIT1 1548 containing 2 TMRs, which was not identified as a SIT1 orthologue in our pipeline but has previously been described as such 19 , 82 . To explore the evolutionary history of SIT1 -related genes in fungi, we performed Blastp searches for orthologues of C. albicans SIT1 using an e-value cut-off of 1e - 20 across 226 fungal species’ reference genomes ( n = 304). We identified 1,689 hits, which clustered into two branches: a smaller branch ( n = 397) annotated for siderophore transport including the 12 C. auris groupings which we have designated “xenosiderophore transporter candidates” ( XTC ), and a larger branch ( n = 1,292) featuring genes predicted (by GO term) to encode haem transporters, including the 5 C. auris groupings, which we have additionally designated “haem transport-related” ( HTR , Figure 8A ). There were no putative haem transporters or members of the larger branch for C. albicans , which only contained the single SIT1 gene. We calculated the mean number of orthogroups per genome per species ( Figure 8B ). This showed the most prominent expansion of the XTC branch in the Metschnikowiaceae clade, including C. auris (12 orthologues) and even more so in C. haemulonii , with 15 orthologues. No other fungal species or group of species contained so many. XTC orthologues were not present in any members of the basal Blasocladiomycota , Chytridiomycota and Mucoromycota , or in several Ascomycota (e.g. Pichia kudriavzevii , Schizosaccharomyces pombe and Pyricularia oryzae ) and Basidiomycota (e.g. Ustilago maydis ). Common human fungal pathogens such as Aspergillus fumigatus , C. albicans and Nakaseomyces glabratus contained only one XTC orthologue ( Figure 8B ). The HTR clade, by contrast, was more prominent across fungal phyla, with as many as nine in Mucor species, 16 in A. flavus and 22 in F. solani . We therefore propose a naming of these two expanded gene families as XTC1-12 and HTR1-5 in C. auris . Download figure Open in new tab We note the absence of XTC5 in clade V, XTC10 in clade IV, and in addition to these, XTC4 , XTC9 and XTC11 in clade II ( Figure 8C ). Clade II, which exhibits the greatest degree of XTC gene loss, is also considered by some to be the least virulent strain. It is also noteworthy that the AK embryo expressed HMOX genes during clade IV infection, while clade IV C. auris up-regulated HTR1 , HTR2 and HTR5 . This suggests a specific host-pathogen interaction axis in nutritional immunity related to haem scavenging. No other clades of infection were associated with such iron-related functional enrichment in the host, and no other clades expressed more than one HTR gene. In summary, XTC1 , XTC2 , XTC3 , XTC4 , XTC6 , XTC7 , XTC11 and XTC12 were up-regulated in-host while XTC8 , XTC9 and XTC12 were down-regulated in at least one clade, while up-regulation of XTC3 and XTC7 was significant across all clades. Additionally, the more virulent clades were associated with up-regulation of XTC4 , XTC5 , and XTC8 . The filamentous clade V up-regulated XTC7 across comparisons with all other clades, and up-regulated XTC1 , XTC4 , XTC8 and XTC9 compared to at least one other clade. Discussion We describe the use of an AK yolk-sac microinjection model for comparative functional genomics of C. auris during in vivo in-host infection. All five clades demonstrated lethality, with an increase in virulence during clades I and IV infections, consistent with clinical and laboratory findings 17 . In-host growth was similar across all clades and high enough to indicate a large enough in vivo fungal burden for robust bulk transcriptome interrogation across the species. The contained nature of the yolk-sac provides a host tissue growth medium that enables the assessment of pathogen survival and virulence strategies in vivo and a straightforward experimental unit of host tissue without the challenges of pathogen RNA isolation such as locating foci of infection in larger animals. Thus, an AK yolk-sac microinjection model has the potential to fill a substantial need for in vivo gene expression studies in general. We investigated the host transcriptional response to C. auris infection, which featured an innate immune response, as expected. The most highly up-regulated genes included two predicted haem oxygenase ( HMOX ) genes. HMOX genes have been shown to mask iron from invading pathogens as a feature of anti- Candida nutritional immunity 83 , so presumably have a similar function against C. auris . This was particularly notable given HMOX genes were up-regulated in response to clade IV infection, and the clade IV isolate had a corresponding significant up-regulation of three haem transport-related HTR genes. In addition to nutritional immunity genes, we found up-regulated heat shock and complement cascade genes during infection, confirming the expected immune response to fungal infection 63 , 64 . Genes with complex roles in immunity were up-regulated, such as NLRP3 , for which C. auris may be less activating than e.g. C. albicans 33 , 84 . It is notable that the yolk-sac model was not associated with histological or transcriptional evidence of host neutrophil recruitment and activation, which may relate to the hypothesis that C. auris is immunoevasive 22 . Reference genome bias resulting from the available adult gill transcriptome assembly 85 may limit detection of host gene expression features, so future long and short read assembly from gene expression data across a range of larval stages could further understanding of AK immunity. Additional time-points could be an informative future area of study to develop fine-resolution strain-specific transcription networks. Furthermore, the potential of a translucent and genetically tractable fish embryo model could enable future visualisation of cellular dynamics and genetic pathway manipulation 77 . We provided a thorough investigation of differential expression by C. auris during infection of host tissue compared with in vitro conditions for the first time. The lack of a major distinction across time-points (24 vs 48 HPI) indicates that the transcriptional activity of C. auris in-host was highly similar at these stages. Six functional annotation terms (GO, KEGG pathway and PFAM domain) were significantly enriched among genes that were up-regulated across all five clades, including sugar and siderophore transporters. The enrichment of sugar transporters outside of nutrient-rich YPD-broth is unsurprising, such as high-affinity glucose transporter HGT12 , with evidence of redundancy in C. albicans 86 – 89 , and HGT2 , part of an eight-gene “core filamentation response network” in C. albicans 90 . More surprising was the down-regulation of putative virulence factors such as Secreted Aspartyl Protease 3 ( SAP3 ), thought to be the most important SAP in C. auris , and required for virulence in a mouse model 31 . Copper Superoxide Dismutase SOD6 was also down-regulated, previously shown to be down-regulated in iron starvation 91 . Multi-drug efflux pumps such as MDR2 (down-regulated in vivo ) and MDR1 (down-regulated in vivo and in clades I and IV vs clades II and III) are understood to drive drug resistance through multiple but unclear mechanisms in C. auris 92 , 93 ; in-host down-regulation may indicate a fitness trade-off for virulence vs drug resistance. A total of twelve siderophore transporters were up-regulated during infection, and four were down-regulated, with three family members up-regulated in more virulent clades and five in the filamentous clade V. The prominent finding of these xenosiderophore transport candidate ( XTC ) genes necessitates further experimental work to address their potential for anti-virulence therapeutic targeting. We also identified up-regulation of several genes at the mating type locus ( MTL ) in more virulent strains; MTL genes may contribute to virulence in fungal species including Cryptococcus neoformans 94 through uncertain mechanisms, such as via the expression of non-mating genes contained within the locus. C. auris does not clearly undergo meiotic recombination 95 and it is not clear what role MTL -associated genes play in pathogenesis and morphology. Two of the three non-mating genes ( PAP1 and PIKA ) in the MTL were up-regulated by more virulent strains (I and IV) compared to the less virulent strains (II and III). Importantly, the two more virulent clades (I and IV) contain MTL a rather than MTL α (clades II & III) 29 , and clades V and VI contain MTL a and MTL α respectively 13 . Thus, it is unclear if the up-regulation of non-mating genes is a MTL specific transcriptional nuance, or a feature of more highly virulent C. auris clades. The MTL also did not feature prominently in the filamentation response observed in clade V, providing no evidence for an obvious role for the MTL in such morphological changes. We also observed in vivo filamentation by a representative strain of C. auris clade V, the basal/ancestral clade 96 , accompanied by a genera-typical filamentous gene expression signature. Specifically, we measured the up-regulation of HGC1 and UME6 , which are two of the most important regulators of filamentation in C. albicans , and have recently been described as drivers of C. auris filamentation and biofilm formation 97 . The expression of adhesin SCF1 in filamenting clade V is noteworthy because its cationic adhesion mechanism has been postulated to be consistent with adhesion mechanisms common to marine organisms including bivalves 27 . The generalist marine-isolated pathogen C. haemulonii has also displayed filamentation competence at lower temperatures 98 , consistent with an ancestral aquatic state. Filamentation is an important pathogenicity trait in fungi, especially Candida species, although its roles have been more poorly described in aquatic infections 99 . Human histopathology reports of C. auris infection are rarely published and have not demonstrated classical in-host filamentation, to our knowledge. An ex vivo human epithelial model using a clade I isolate did demonstrate filamentous C. auris at 37 °C after injection into deeper dermis 100 . Clade III C. auris has also been observed to form filamentous/hyphal forms in Galleria mellonella injection 58 . Fan and colleagues have observed filamentous forms of C. auris across all four clades, even demonstrating a higher virulence in the G. mellonella model 101 , though the phenotype required passage through a murine host with additional prolonged growth at 25 °C for at least 5 d before and after injection 25 . Other stimuli have induced filamentation in clade I isolates, such as hydroxyurea genotoxic stress 102 and Hsp90 inhibition 103 , where several up-regulated genes in C. auris filamentation ( SCF1 , IFF4 and PGA31 ) matched those in our study. Constitutive filamentous growth has also been demonstrated with a transposon-mediated disruption of a long non-coding RNA 104 , which sit adjacent to chitin deacetylase CDA2 , which was down-regulated at 24 HPI in our experiment during filamentation. These findings indicate that hyphal formation is possible across C. auris clades. We underscored how the family of predicted xenosiderophore transporter genes is significantly expanded in several members of the Metschnikowiaceae clade compared to other Saccharomycetae 29 , raising questions as to the roles of siderophore transporters in C. auris biology and evolution 105 . Siderophore synthesis may be unlikely in C. auris , given the lack of evidence in other Candida species 106 . As a result, C. albicans Sit1, which is required for virulence 107 , is thought to be a xenosiderophore transporter for iron-chelators produced by other microbes and specific to the ferrioxamine type common in iron-poor ocean environments 108 , 109 . Siderophore transporters can also act intracellularly for compartments such as the mitochondrion 110 , 111 , or act as efflux pumps for various molecules 112 including trichothecene mycotoxins as identified by our functional annotations. Given that XTC gene up-regulation by C. auris has also been detected during ex vivo whole blood infection ( XTC2A-B , B9J08_1547-8 ), this suggests that our findings may be clinically relevant 19 . C. auris encodes a large expanded gene family of 17 siderophore transporters that we named xenosiderophore transporter candidates ( XTC ) 1-12 and haem transport-related ( HTR ) genes 1-5, the latter sharing sequence similarity with the genomic sequence for S. pombe low-affinity haem transporter Str3 113 . In other Candida species such as C. albicans , iron assimilation is understood to be central for in-host survival 114 , 115 , where deprivation triggers various pathogenic programmes such as alterations in cell surface beta-glucan 116 . Haem is acquired by C. albicans through vesicular endocytosis and CFEM-domain containing proteins 117 , 118 and does not contain HTR genes, suggesting a possible loss of transmembrane haem transporters. The ubiquity of HTR across the fungal kingdom and lack of XTC genes in the three basal fungal phyla suggest that SIT1 / HTC could have arisen from a duplication of an HTR -related gene in the Dikarya (the Ascomycota and Basidiomycota ) with subsequent independent loss of both XTC and HTC genes in multiple fungal lineages. These findings together underline the importance of iron metabolism as a clinical target with existing therapies, such as iron chelators deferiprone/deferoxamine with evidence of echinocandin antifungal synergy 119 or low toxicity antihelminthic pyrvinium pamoate as a disruptor of iron homeostasis 120 , 121 . We present the first in vivo host tissue infection transcriptional profile for C. auris , a World Health Organization critical priority fungal pathogen. By examining representative strains from all five major clades implicated in its recent, near-simultaneous global emergence, we uncover clade-specific differences in morphology, virulence and gene expression. Our findings demonstrate the utility of a thermotolerant yolk-sac injection model, and support the hypothesis of a marine origin for C. auris . Notably, we observed natural filamentation in clade V and elevated virulence in clades I and IV, each linked to distinct transcriptional programs. We identify the expanded XTC and HTR families as species-wide, infection-induced features, underscoring their potential as targets for studying fungal biology, pathogenesis, and antifungal development. Together, our results shed light on the mechanisms by which C. auris adapts to and persists in host tissue, offering new insights into its evolution from a likely marine yeast ancestor and advancing our understanding of this emerging fungal pathogen and global public health threat. Materials and methods Full details are given in SI Appendix. Briefly, wild-type adult AK were kept at the Aquatic Resources Facility, Exeter, under Project Licence PP4402521. AK were kept in two 28 °C incubation tanks, with 12-hour day and night light cycles and artificial enrichment foliage. Embryos were collected using three plastic netted collection chambers per tank. Injection needles were prepared using borosilicate glass capillaries (Harvard apparatus, USA) and a heated pulling system (heater level >62, PC-10, Narishige, UK). We used tweezers to snap the tip to an estimated 1 mm graticule-measured aperture of 10-20 μm, and sterilised needles before use with 253.7 nm UV light over 15 min (Environmental Validation Solutions, UK). Injection bays were made with microwaved 1% agarose (Sigma, UK) in 1 g per 100 mL ASW for up to 24 embryos in 100 mm petri dishes using custom-made molds in an inverted 60 mm petri dish. Embryos were randomly chosen from each dish and the sequence of injection for each biological repeat was randomly allocated by computer. C. auris yeasts were counted by haemocytometer and added to 25 μL sterile filtered phenol red solution 0.5% (Sigma, UK) to a concentration of 0.5×10 8 per mL in 100 μL. Injection needles were loaded with 5 μL injection solution using 20 μL Microloader EpT.I.P.S. (Eppendorf, UK) immediately before injection of up to 30 embryos before re-loading. Injections were performed with 400-500 hPa injection pressure and 7-40 hPa compensation pressure over 100-200 ms with the FemtoJet 4i (Eppendorf UK) using a micromanipulator (World Precision Instruments, UK) and a Microtec HM-3 stereo dissection microscope (Optech, UK). To deliver a dose of approx. 500 yeasts in 10 nL solution, an injection bolus was administered to an estimated diameter of 0.2 – 0.3 mm (10-15% of total embryo diameter) via a single chorion puncture. Any embryos that failed to meet these criteria for injection were excluded. Control in vitro inoculations were microinjected as above into 1 mL YPD in 1.5 mL Eppendorf tubes before incubation without shaking at 37 °C for 24 h contemporaneously with embryos as below. We used an unblinded prospective virulence study comparing six groups of AK embryo yolk-sac microinjection, including clades I-V of C. auris , and sham injections as the negative control. We used ≥23 embryos per condition based on a power calculation of (J = 0.25, significance = 0.05, power = 0.9, and o effect size of 25% mortality (two-sided t-test). After excluding embryos that were unhealthy or did not complete successful microinjection, we included n = 10-13 embryos per group per experiment (total n = 34-37 embryos per group). RNA was extracted from three embryos per condition per time-point (0, 24 and 48 HPI, n = 54) plus three embryos per time-point without any injection ( n = 9, total n = 63) as a control for the impact of microinjection. We selected five C. auris strains to represent each major clade ( Table S1 ). Culture stocks were kept in −80 °C in Yeast extract Peptone Dextrose (YPD) broth (Sigma-Aldrich, UK) containing 25% glycerol (ThermoFisher, UK) and plated onto YPD-agar (Sigma-Aldrich) for 48 h before inoculation and overnight growth in 10 mL YPD broth at 30 °C in a shaking incubator at 200 RPM (Multitron, Infors HT, UK). Five mL from each overnight culture was then centrifuged at 5000 g for 2 min before triple washing in 1 mL autoclaved deionised water (DIW, Milli-Q, Merck, USA) to prevent aggregation 28 , then re-suspended again in 200 μL DIW. Individual embryos were kept in 1 mL sterile filtered ASW per embryo in a 48 well flat bottom plate (Corning, USA) sealed with Parafilm “M” (Bemis Company, USA) at 37 °C with passive humidification using a 500 mL open glass beaker of water. Embryos were monitored for presence of heart rate at 24-hourly intervals up to 7 d by stereo dissection microscope. Initial time of injection was subtracted from each time-point to minimise bias between groups. Kaplan-Meier survival curves were plotted with Log-Rank testing and Benjamini-Hochberg (BH) correction 122 using survival v.3.5.8 and survminer v.0.4.9 packages in R v.4.4.0. Colony forming units (CFUs) were calculated by homogenisation of embryos at 0, 24, and 48 HPI by autoclaved plastic micropestle in a 1.5 mL conical tube (Eppendorf, UK) in 100 μL 10,000 U/mL penicillin/streptomycin (P/S) solution (Gibco, UK). Dilutions were made with P/S solution and DIW in 1/10-10,000 and counts were adjusted according to dilution after plating onto YPD agar plates and read after 72 h incubation at 30 °C. Statistical testing between CFU numbers was performed with Wilcoxon testing and BH correction. Total RNA was extracted using the Monarch® Total RNA Miniprep Kit (NEB, USA). Embryos were flash frozen in liquid nitrogen, re-suspended in DNA/RNA Protection Reagent and disrupted with an autoclaved plastic micropestle. Bead beating was performed in a FastPrep 24 homogeniser (MP Biomedicals) with 0.5 mm zirconia/silica beads for 8 cycles of 35 s at 6 m/s plus 45 s on ice. Lysate was recovered and incubated for 5 min. at 55 °C with proteinase K before addition of lysis buffer and RNA purification following manufacturer’s instructions. Library preparation and sequencing were performed by the Exeter Sequencing Service. Samples were normalised and prepared using the NEBNext Ultra II Directional RNA Library Prep - Poly(A) mRNA Magnetic Isolation Module following the standard protocol. Libraries were sequenced on a Novaseq 6000 sequencer with 150bp paired-end reads. Reads were trimmed with fastp 123 v.0.23.1 to remove reads <75 bases and trim based from the 3’ end with q-score <22. Quality control was performed with FastQC ( https://github.com/s-andrews/FastQC ) v.0.11.9 and MultiQC 124 v.1.6. We aligned sequences (X = 52.9 m reads per replicate, range 41.1-112.2 m) using the Trinity pipeline 125 v.2.15.1 to the C. auris clade I B8441 reference genome (GCA_002759435.2) 29 downloaded from GenBank (mean aligned reads in infected embryos 3.83 m, 0.81-13.1%). We further aligned clade-specific reference genomes for each strain ( Table S1 ), including clade II (GCA_003013715.2) 4 , III (GCA_002775015.1) 14 , IV (GCA_008275145.1) 14 and V (GCA_016809505.1) 96 , 126 , 127 . We aligned conditions containing A. dispar to the de novo GJEY01 adult gill transcriptome assembly 85 downloaded from GenBank, for which we detected expression over 27,481 potential genes. Alignment was performed with Bowtie2 128 v.2.5.1, transcript estimation with RNA Seq by Expectation Maximisation (RSEM) 129 v.1.3.3, differential expression with EdgeR 130 v.3.40.0, Samtools 131 v.1.17 and limma 132 v.3.54.0 using a false discovery rate (FDR) p-value cut-off of 0.001 and a log fold change cut-off of +/-2.0. Principal components were calculated and plotted using the prcomp function in Rv4.4.0 and ggfortify v.0.4.17 R package for fragments per kilobase per million, and correlations plots from counts per million as per the default output of the Trinity pipeline. Log fold change was plotted with ComplexHeatmap 133 . Gene ontology terms 134 , 135 were assigned using DIAMOND 136 v.2.1.8 via the DIAMOND2GO pipeline 137 for each of the C. auris reference genomes. KEGG terms 138 were assigned using the BlastKOALA online platform using the eukaryote database 139 . Protein FAMily (PFAM) domains 140 were identified using the PFAM-A.hmm database ( https://ftp.ebi.ac.uk/pub/databases/Pfam/current_release/Pfam-A.full.gz ) and HMMER and HMM Scan 141 with a cut-off of 1e - 5 . Further domain-specific identification was performed with SignalP 5.0 142 , DeepTMHMM 1.0 143 and NetGPI 1.1 144 . To identify accessory genes across C. auris clades, we used the Synima pipeline 145 with Orthofinder v2.5.5 146 with additional reference genomes for C. albicans (SC5314_GCA_000182965.3) and C. haemulonii (B11899_ASM1933202v1). Multiple alignment of single copy orthologues as defined by Orthofinder was performed with Muscle v5.1 147 and a phylogeny was made with FastTree v2.1.11 148 . We visualised the structure using Alphafold v3 149 and ChimeraX v1.8 150 . Clade I-V C. auris and C. haemulonii SIT1 genes were aligned using Muscle as above before using RAXML-NG v1.2.2 with 1000 bootstraps and the LG4X model 151 , which was visualised using ggtree v3.12.0. Author contributions HG: designed and conducted experiments, performed analysis, drafted and wrote the paper. NH, JG, QM, TB: optimised, designed or performed experiments. RF, TK, TB, MR, JR, DW, AB: designed experiments, obtained funding. All authors edited the manuscript. Competing interest statement Authors declare no competing interests. Data availability Raw reads have been deposited via NCBI/GEO via accession GSE277854. All datasets and code required to make figures are available via Github ( https://github.com/hughgifford/Arabian_Killifish_C_auris_2024 ). View this table: View inline View popup Download powerpoint Table 1: Summary of XTC and HTR genes and their chromosomal locations. Acknowledgements We acknowledge funding from the MRC Centre for Medical Mycology at the University of Exeter (MR/N006364/2, MR/V033417/1), MRC Doctoral Training Grants MR/P501955/2 and MR/W502649/1, Wellcome Trust (206412/A/17/Z), Wellcome Trust Career Development Award (215239/Z/19/Z) and Wellcome Trust Fellowship (219551/Z/19/Z), National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (NC/X001121/1), and the NIHR Exeter Biomedical Research Centre. TB also was supported via Elizabeth Ballou and a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (211241/Z/18/Z) and the Lister Institute. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. We thank staff in the Aquatic Resource Center at the University of Exeter for husbandry of the fish and the support for developing protocols for feeding, spawning, and raising new generations of this relatively new model species, including Sam Worthington, Chloe Flint and Richard Silcox. We also thank the Exeter Sequencing Service facility and support from Wellcome Trust Institutional Strategic Support Fund (WT097835MF), Wellcome Trust Multi User Equipment Awards (WT101650MA and 218247/Z/19/Z), Medical Research Council Clinical Infrastructure Funding (MR/M008924/1) and BBSRC LOLA award (BB/K003240/1). We also thank the University of Exeter High-Performance Computing (HPC) facility funded by the UK MRC Clinical Research Infrastructure Initiative (award number MR/M008924/1). We thank Tanmoy Chakraborty, Dhara Malavia and Neil Gow for providing C. auris strains, Teigan Veale, Sumita Roy, Jonathan Ball and Darren Thomson for assistance in microscopy and Chloe Pelletier for advice on aggregation. We also thank Microtechnical Services, Exeter, UK, for assistance with histological sectioning. Footnotes ↵ 1 Co-Senior Author Extended revisions to text, several validatory PCR experiments, an increase in the number of figures in the main manuscript, and careful description of the clinical relevance and experimental limitations. https://github.com/hughgifford/Arabian_Killifish_C_auris_2024 References 1. ↵ Meis , J. F. & Chowdhary , A . Candida auris : A global fungal public health threat . The Lancet Infectious Diseases 18 , 1298 – 1299 ( 2018 ). OpenUrl CrossRef PubMed 2. ↵ WHO . World Health Organisation (WHO) fungal priority pathogens list to guide research, development and public health action . ( 2022 ) doi: https://www.who.int/publications/i/item/9789240060241 . 3. ↵ Lee , W. G. et al. First three reported cases of nosocomial fungemia caused by Candida auris . Journal of Clinical Microbiology 49 , 4 ( 2011 ). OpenUrl 4. ↵ Satoh , K. et al. Candida auris sp. Nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital . Microbiology and Immunology 53 , 41 – 44 ( 2009 ). OpenUrl CrossRef PubMed 5. ↵ Eyre , D. W. et al. A Candida auris outbreak and its control in an intensive care setting . New England Journal of Medicine 379 , 1322 – 1331 ( 2018 ). OpenUrl CrossRef PubMed 6. Taori , S. K. , et al. Candida auris outbreak: Mortality, interventions and cost of sustaining control . Journal of Infection 79 , 601 – 611 ( 2019 ). OpenUrl CrossRef PubMed 7. ↵ Shuping , L. et al. High prevalence of Candida auris colonization during protracted neonatal unit outbreak, South Africa . Emerging Infectious Diseases ( 2023 ) doi: 10.3201/eid2909.230393 . OpenUrl CrossRef 8. ↵ Adam , R. D. et al. Analysis of Candida auris fungemia at a single facility in Kenya . International Journal of Infectious Diseases 85 , 182 – 187 ( 2019 ). OpenUrl CrossRef PubMed 9. ↵ Prayag , P. S. , Patwardhan , S. , Panchakshari , S. , Rajhans , P. A. & Prayag , A . The dominance of Candida auris : A single-center experience of 79 episodes of candidemia from western India . Indian Journal of Critical Care Medicine 26 , 560 – 563 ( 2022 ). OpenUrl CrossRef PubMed 10. ↵ Chen , J. et al. Is the superbug fungus really so scary? A systematic review and meta-analysis of global epidemiology and mortality of Candida auris . BMC Infectious Diseases 20 , 827 ( 2020 ). OpenUrl CrossRef PubMed 11. ↵ Lyman , M. Notes from the field: Transmission of pan-resistant and echinocandin-resistant Candida auris in health care facilities - Texas and the District of Columbia, January-April 2021 . Morbidity and Mortality Weekly Report 70 , ( 2021 ). 12. ↵ Khan , T. et al. Emergence of the novel sixth Candida auris clade VI in Bangladesh . Microbiology Spectrum 12 , e03540 – 23 ( 2024 ). OpenUrl 13. ↵ Suphavilai , C. et al. Detection and characterisation of a sixth Candida auris clade in Singapore: A genomic and phenotypic study . Lancet Microbe 0 , ( 2024 ). 14. ↵ Lockhart , S. R. et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses . Clinical Infectious Diseases 64 , 134 – 140 ( 2017 ). OpenUrl CrossRef PubMed 15. ↵ Gifford , H. , Rhodes , J. & Farrer , R. A . The diverse genomes of Candida auris . The Lancet Microbe 0 , ( 2024 ). 16. ↵ Byun , S. A. et al. Virulence traits and azole resistance in Korean Candida auris Isolates . Journal of Fungi 9 , 979 ( 2023 ). OpenUrl PubMed 17. ↵ Santana , D. J. , Zhao , G. & O’Meara , T. R . The many faces of Candida auris : Phenotypic and strain variation in an emerging pathogen . PLOS Pathogens 20 , e1012011 ( 2024 ). OpenUrl CrossRef PubMed 18. ↵ Wang , T. W. et al. Functional redundancy in Candida auris cell surface adhesins crucial for cell-cell interaction and aggregation . Nature Communications 15 , 9212 ( 2024 ). OpenUrl PubMed 19. ↵ Allert , S. et al. From environmental adaptation to host survival: Attributes that mediate pathogenicity of Candida auris . Virulence 13 , 191 – 214 ( 2022 ). OpenUrl CrossRef PubMed 20. ↵ Fayed , B. et al. Transcriptome analysis of human dermal cells infected with Candida auris identified unique pathogenesis/defensive mechanisms particularly ferroptosis . Mycopathologia 189 , 65 ( 2024 ). OpenUrl CrossRef PubMed 21. ↵ Yang , B. et al. A correlative study of the genomic underpinning of virulence traits and drug tolerance of Candida auris . Infection and Immunity 0 , e00103 – 24 ( 2024 ). OpenUrl 22. ↵ Balakumar , A. et al. Single-cell transcriptomics unveils skin cell specific antifungal immune responses and IL-1Ra-IL-1R immune evasion strategies of emerging fungal pathogen Candida auris . PLOS Pathogens 20 , e1012699 ( 2024 ). OpenUrl CrossRef PubMed 23. ↵ Bruno , M. et al. Transcriptional and functional insights into the host immune response against the emerging fungal pathogen Candida auris . Nature Microbiology 5 , 1516 – 1531 ( 2020 ). OpenUrl PubMed 24. ↵ Wang , Y. et al. Innate immune responses against the fungal pathogen Candida auris . Nature Communications 13 , 3553 ( 2022 ). OpenUrl PubMed 25. ↵ Yue , H. et al. Filamentation in Candida auris , an emerging fungal pathogen of humans: Passage through the mammalian body induces a heritable phenotypic switch . Emerging Microbes & Infections 7 , 1 – 13 ( 2018 ). OpenUrl PubMed 26. ↵ Chadwick , C. , Jesus , M. D. , Ginty , F. & Martinez , J. S . Pathobiology of Candida auris infection analyzed by multiplexed imaging and single cell analysis . PLOS ONE 19 , e0293011 ( 2024 ). OpenUrl CrossRef PubMed 27. ↵ Santana , D. J. et al. A Candida auris -specific adhesin, Scf1, governs surface association, colonization, and virulence . Science 381 , 1461 – 1467 ( 2023 ). OpenUrl CrossRef PubMed 28. ↵ Pelletier , C. , Shaw , S. , Alsayegh , S. , Brown , A. J. P. & Lorenz , A . Candida auris undergoes adhesin-dependent and -independent cellular aggregation . PLOS Pathogens 20 , e1012076 ( 2024 ). OpenUrl CrossRef PubMed 29. ↵ Muñoz , J. F. et al. Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species . Nature Communications 9 , 5346 ( 2018 ). OpenUrl PubMed 30. ↵ Richardson , J. P. et al. Candidalysins are a new family of cytolytic fungal peptide toxins . mBio 13 , e03510 – 21 ( 2022 ). OpenUrl PubMed 31. ↵ Kim , J.-S. , Lee , K.-T. & Bahn , Y.-S . Secreted aspartyl protease 3 regulated by the Ras/cAMP/PKA pathway promotes the virulence of Candida auris . Frontiers in Cellular and Infection Microbiology 13 , 1257897 ( 2023 ). OpenUrl 32. ↵ Horton , M. V. , et al. Candida auris cell wall mannosylation contributes to neutrophil evasion through pathways divergent from Candida albicans and Candida glabrata . mSphere 6 , e00406 – 21 ( 2021 ). OpenUrl PubMed 33. ↵ Weerasinghe , H. , et al. Candida auris uses metabolic strategies to escape and kill macrophages while avoiding robust activation of the NLRP3 inflammasome response . Cell Reports 42 , 112522 ( 2023 ). OpenUrl PubMed 34. ↵ Wang , X. et al. The first isolate of Candida auris in China: Clinical and biological aspects . Emerging Microbes & Infections 7 , 1 – 9 ( 2018 ). OpenUrl PubMed 35. Fan , S. , Li , C. , Bing , J. , Huang , G. & Du , H . Discovery of the diploid form of the emerging fungal pathogen Candida auris . ACS Infectious Diseases 6 , 2641 – 2646 ( 2020 ). OpenUrl PubMed 36. ↵ Fan , S. et al. A biological and genomic comparison of a drug-resistant and a drug-susceptible strain of Candida auris isolated from Beijing, China . Virulence 12 , 1388 – 1399 ( 2021 ). OpenUrl CrossRef PubMed 37. ↵ Wurster , S. , Albert , N. D. & Kontoyiannis , D. P . Candida auris bloodstream infection induces upregulation of the PD-1/PD-L1 immune checkpoint pathway in an immunocompetent mouse model . mSphere 7 , e00817 – 21 ( 2022 ). OpenUrl PubMed 38. ↵ Bing , J. et al. Rapid evolution of an adaptive multicellular morphology of Candida auris during systemic infection . Nature Communications 15 , 2381 ( 2024 ). OpenUrl PubMed 39. ↵ Ben-Ami , R. et al. Multidrug-resistant Candida haemulonii and C. auris , Tel Aviv, Israel . Emerging Infectious Diseases 23 , ( 2017 ). 40. Hager , C. L. , Larkin , E. L. , Long , L. A. & Ghannoum , M. A . Evaluation of the efficacy of rezafungin, a novel echinocandin, in the treatment of disseminated Candida auris infection using an immunocompromised mouse model . Journal of Antimicrobial Chemotherapy 73 , 2085 – 2088 ( 2018 ). OpenUrl CrossRef PubMed 41. Zhao , M. , et al. In vivo pharmacokinetics and pharmacodynamics of APX001 against Candida spp. In a neutropenic disseminated candidiasis mouse model . Antimicrobial Agents and Chemotherapy 62 , e02542 – 17 ( 2018 ). OpenUrl PubMed 42. Singh , S. et al. The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection . PLOS Pathogens 15 , e1007460 ( 2019 ). OpenUrl CrossRef PubMed 43. Zhang , F. et al. A marine microbiome antifungal targets urgent-threat drug-resistant fungi . Science 370 , 974 – 978 ( 2020 ). OpenUrl Abstract / FREE Full Text 44. ↵ Muñoz , J. E. et al. Pathogenicity levels of Colombian strains of Candida auris and Brazilian strains of Candida haemulonii species complex in both murine and Galleria mellonella experimental models . Journal of Fungi 6 , 104 ( 2020 ). OpenUrl PubMed 45. Abe , M. et al. Potency of gastrointestinal colonization and virulence of Candida auris in a murine endogenous candidiasis . PLoS One 15 , e0243223 ( 2020 ). OpenUrl CrossRef PubMed 46. Forgács , L. et al. Comparison of in vivo pathogenicity of four Candida auris clades in a neutropenic bloodstream infection murine model . Emerging Microbes & Infections 9 , 1160 – 1169 ( 2020 ). OpenUrl PubMed 47. Nagy , F. et al. In vitro and in vivo interaction of caspofungin with isavuconazole against Candida auris planktonic cells and biofilms . Medical Mycology 59 , 1015 – 1023 ( 2021 ). OpenUrl CrossRef PubMed 48. Forgács , L. et al. In vivo efficacy of amphotericin B against four Candida auris clades . Journal of Fungi (Basel, Switzerland) 8 , 499 ( 2022 ). OpenUrl PubMed 49. Singh , S. et al. Protective efficacy of anti-Hyr1p monoclonal antibody against systemic candidiasis due to multi-drug-resistant Candida auris . Journal of Fungi 9 , 103 ( 2023 ). OpenUrl PubMed 50. ↵ Salama , E. A. et al. Lansoprazole interferes with fungal respiration and acts synergistically with amphotericin B against multidrug-resistant Candida auris . Emerging Microbes & Infections 13 , 2322649 ( 2024 ). OpenUrl PubMed 51. ↵ Torres , S. R. et al. Impact of Candida auris infection in a neutropenic murine model . Antimicrobial Agents and Chemotherapy 64 , e01625 – 19 ( 2020 ). OpenUrl PubMed 52. ↵ Xin , H. , Mohiuddin , F. , Tran , J. , Adams , A. & Eberle , K . Experimental mouse models of disseminated Candida auris infection . mSphere 4 , e00339 – 19 ( 2019 ). OpenUrl PubMed 53. ↵ Rosario-Colon , J. , Eberle , K. , Adams , A. , Courville , E. & Xin , H . Candida cell-surface-specific monoclonal antibodies protect mice against Candida auris invasive infection . International Journal of Molecular Sciences 22 , 6162 ( 2021 ). OpenUrl PubMed 54. ↵ Torres , S. R. et al. Assessment of environmental and occupational exposure while working with multidrug resistant (MDR) fungus Candida auris in an animal facility . Journal of Occupational and Environmental Hygiene 16 , 507 – 518 ( 2019 ). OpenUrl CrossRef PubMed 55. ↵ Borman , A. M . Of mice and men and larvae: Galleria mellonella to model the early host-pathogen interactions after fungal infection . Virulence 9 , 9 – 12 ( 2017 ). OpenUrl PubMed 56. Romera , D. et al. The Galleria mellonella infection model as a system to investigate the virulence of Candida auris strains . Pathogens and Disease 78 , ftaa067 ( 2020 ). OpenUrl CrossRef PubMed 57. García-Carnero , L. C. et al. Early virulence predictors during the Candida species– Galleria mellonella interaction . Journal of Fungi 6 , 152 ( 2020 ). OpenUrl PubMed 58. ↵ Garcia-Bustos , V. et al. Characterization of the differential pathogenicity of Candida auris in a Galleria mellonella infection model . Microbiology Spectrum 9 , e00013 – 21 ( 2021 ). OpenUrl 59. Carvajal , S. K. et al. Pathogenicity assessment of Colombian strains of Candida auris in the Galleria mellonella invertebrate model . Journal of Fungi 7 , 401 ( 2021 ). OpenUrl PubMed 60. ↵ Hernando-Ortiz , A. et al. Virulence of Candida auris from different clinical origins in Caenorhabditis elegans and Galleria mellonella host models . Virulence 12 , 1063 – 1075 ( 2021 ). OpenUrl CrossRef PubMed 61. ↵ Spettel , K. , et al. Candida auris in Austria—what is new and what is different . Journal of Fungi 9 , 129 ( 2023 ). OpenUrl PubMed 62. ↵ Wurster , S. , et al. Drosophila melanogaster as a model to study virulence and azole treatment of the emerging pathogen Candida auris . Journal of Antimicrobial Chemotherapy 74 , 1904 – 1910 ( 2019 ). OpenUrl CrossRef PubMed 63. ↵ Harpf , V. , Rambach , G. , Würzner , R. , Lass-Flörl , C. & Speth , C . Candida and complement: New aspects in an old battle . Frontiers in Immunology 11 , 1471 ( 2020 ). OpenUrl PubMed 64. ↵ Pechacek , J. & Lionakis , M. S . Host defense mechanisms against Candida auris . Expert Review of Anti-infective Therapy 21 , 1087 – 1096 ( 2023 ). OpenUrl CrossRef PubMed 65. ↵ Johnson , C. J. , Davis , J. M. , Huttenlocher , A. , Kernien , J. F. & Nett , J. E . Emerging fungal pathogen Candida auris evades neutrophil attack . mBio 9 , e01403 – 18 ( 2018 ). OpenUrl PubMed 66. ↵ Pharkjaksu , S. , Boonmee , N. , Mitrpant , C. & Ngamskulrungroj , P . Immunopathogenesis of emerging Candida auris and Candida haemulonii strains . Journal of Fungi 7 , 725 ( 2021 ). OpenUrl PubMed 67. ↵ Shapiro , R. S. & Cowen , L. E . Uncovering cellular circuitry controlling temperature-dependent fungal morphogenesis . Virulence 3 , 400 – 404 ( 2012 ). OpenUrl CrossRef PubMed 68. Leach , M. D. et al. Hsf1 and Hsp90 orchestrate temperature-dependent global transcriptional remodelling and chromatin architecture in Candida albicans . Nature Communications 7 , 11704 ( 2016 ). OpenUrl PubMed 69. ↵ Xiao , W. et al. Response and regulatory mechanisms of heat resistance in pathogenic fungi . Applied Microbiology and Biotechnology 106 , 5415 – 5431 ( 2022 ). OpenUrl CrossRef 70. ↵ Arora , P. et al. Environmental isolation of Candida auris from the coastal wetlands of Andaman Islands, India . mBio 12 , e03181 – 20 ( 2021 ). OpenUrl CrossRef PubMed 71. ↵ Escandón , P . Novel environmental niches for Candida auris : Isolation from a coastal habitat in Colombia . Journal of Fungi 8 , 748 ( 2022 ). OpenUrl PubMed 72. ↵ Uden , V. & Kolipinski , M. C . Torulopsis haemulonii nov. spec. , A yeast from the Atlantic Ocean . Antonie Van Leeuwenhoek 28 , 78 – 80 ( 1962 ). OpenUrl CrossRef PubMed 73. ↵ Bavia , L. , Santiesteban-Lores , L. E. , Carneiro , M. C. & Prodocimo , M. M . Advances in the complement system of a teleost fish, Oreochromis niloticus . Fish & Shellfish Immunology 123 , 61 – 74 ( 2022 ). OpenUrl CrossRef PubMed 74. ↵ Buchmann , K. , Karami , A. M. & Duan , Y . The early ontogenetic development of immune cells and organs in teleosts . Fish & Shellfish Immunology 146 , 109371 ( 2024 ). OpenUrl CrossRef PubMed 75. ↵ Tian , H. et al. Cytokine networks provide sufficient evidence for the differentiation of CD4+ T cells in teleost fish . Developmental & Comparative Immunology 141 , 104627 ( 2023 ). OpenUrl PubMed 76. ↵ Akbarzadeh , A. & Leder , E. H . Acclimation of killifish to thermal extremes of hot spring: Transcription of gonadal and liver heat shock genes . Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 191 , 89 – 97 ( 2016 ). OpenUrl CrossRef 77. ↵ Hamied , A. et al. Identification and characterization of highly fluorescent pigment cells in embryos of the Arabian killifish ( Aphanius Dispar ) . iScience 23 , 101674 ( 2020 ). OpenUrl PubMed 78. ↵ Alsakran , A. et al. Stage-by-stage exploration of normal embryonic development in the Arabian killifish , Aphanius dispar. Developmental Dynamics n/a, ( 2024 ). 79. ↵ Minhas , R. et al. The thermotolerant Arabian killifish, Aphanius dispar, as a novel infection model for human fungal pathogens . 2024.10.08.617174 ( 2024 ) doi: 10.1101/2024.10.08.617174 . OpenUrl Abstract / FREE Full Text 80. ↵ Farrer , R. A. Gifford , H. , Wilson , D. , Rhodes , J. & Farrer , R. A. Seaside to bedside: Assembly in research for emerging human fungal pathogen Candida auris . in Genome Assembly (ed. Farrer , R. A. ) vol. 2955 263 – 291 ( Springer US , New York, NY , 2025 ). OpenUrl 81. ↵ Burden , N. , Chapman , K. , Sewell , F. & Robinson , V . Pioneering better science through the 3Rs: An introduction to the national centre for the replacement, refinement, and reduction of animals in research (NC3Rs) . Journal of the American Association for Laboratory Animal Science 54 , 198 – 208 ( 2015 ). OpenUrl PubMed 82. ↵ Skrzypek , M. S. et al. The Candida Genome Database (CGD): Incorporation of Assembly 22, systematic identifiers and visualization of high throughput sequencing data . Nucleic Acids Research 45 , D592 – D596 ( 2017 ). OpenUrl CrossRef PubMed 83. ↵ Potrykus , J. et al. Fungal iron availability during deep seated candidiasis is defined by a complex interplay involving systemic and local events . PLOS pathogens 9 , e1003676 ( 2013 ). OpenUrl CrossRef PubMed 84. ↵ Kasper , L. et al. The fungal peptide toxin candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes . Nature Communications 9 , 4260 ( 2018 ). OpenUrl PubMed 85. ↵ Bonzi , L. C. et al. The time course of molecular acclimation to seawater in a euryhaline fish . Scientific Reports 11 , 18127 ( 2021 ). OpenUrl PubMed 86. ↵ Varma , A. et al. Molecular cloning and functional characterisation of a glucose transporter, CaHGT1, of Candida albicans . FEMS microbiology letters 182 , 15 – 21 ( 2000 ). OpenUrl CrossRef PubMed 87. Luo , L. , Tong , X. & Farley , P. C . The Candida albicans gene HGT12 (Orf19.7094) encodes a hexose transporter . FEMS Immunology & Medical Microbiology 51 , 14 – 17 ( 2007 ). OpenUrl CrossRef PubMed 88. Brown , V. , Sexton , J. A. & Johnston , M . A glucose sensor in Candida albicans . Eukaryotic Cell 5 , 1726 – 1737 ( 2006 ). OpenUrl Abstract / FREE Full Text 89. ↵ Larcombe , D. E. et al. Glucose-enhanced oxidative stress resistance-A protective anticipatory response that enhances the fitness of Candida albicans during systemic infection . PLOS Pathogens 19 , e1011505 ( 2023 ). OpenUrl CrossRef PubMed 90. ↵ Martin , R. et al. A core filamentation response network in Candida albicans is restricted to eight genes . PLOS ONE 8 , e58613 ( 2013 ). OpenUrl CrossRef PubMed 91. ↵ Schatzman , S. S. et al. Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens . The Journal of Biological Chemistry 295 , 570 – 583 ( 2020 ). OpenUrl Abstract / FREE Full Text 92. ↵ Rybak , J. M. et al. Abrogation of triazole resistance upon deletion of CDR1 in a clinical isolate of Candida auris . Antimicrobial Agents and Chemotherapy 63 , ( 2019 ). 93. ↵ Li , J. , Coste , A. T. , Bachmann , D. , Sanglard , D. & Lamoth , F . Deciphering the Mrr1/Mdr1 pathway in azole resistance of Candida auris . Antimicrobial Agents and Chemotherapy 66 , e00067 – 22 ( 2022 ). OpenUrl CrossRef PubMed 94. ↵ Sun , S. , Coelho , M. A. , David-Palma , M. , Priest , S. J. & Heitman , J . The evolution of sexual reproduction and the mating-type locus: Links to pathogenesis of Cryptococcus human pathogenic fungi . Annual Review of Genetics 53 , 417 – 444 ( 2019 ). OpenUrl CrossRef PubMed 95. ↵ Wang , Y. & Xu , J . Population genomic analyses reveal evidence for limited recombination in the superbug Candida auris in nature . Computational and Structural Biotechnology Journal 20 , 3030 – 3040 ( 2022 ). OpenUrl 96. ↵ Chow , N. A. et al. Potential fifth clade of Candida auris , Iran, 2018 . Emerging Infectious Diseases 25 , 1780 – 1781 ( 2019 ). OpenUrl CrossRef PubMed 97. ↵ Louvet , M. et al. Ume6-dependent pathways of morphogenesis and biofilm formation in Candida auris . Microbiology Spectrum 0 , e01531 – 24 ( 2024 ). OpenUrl 98. ↵ Deng , Y. , Li , S. , Bing , J. , Liao , W. & Tao , L . Phenotypic switching and filamentation in Candida haemulonii , an emerging opportunistic pathogen of humans . Microbiology Spectrum 9 , e00779 – 21 ( 2021 ). OpenUrl PubMed 99. ↵ Overy , D. P. , Rämä , T. , Oosterhuis , R. , Walker , A. K. & Pang , K.-L . The neglected marine fungi, Sensu stricto , and their isolation for natural products’ discovery . Marine Drugs 17 , 42 ( 2019 ). OpenUrl PubMed 100. ↵ Seiser , S. et al. Native human and mouse skin infection models to study Candida auris -host interactions . Microbes and Infection 26 , 105234 ( 2024 ). OpenUrl CrossRef PubMed 101. ↵ Fan , S. et al. Filamentous growth is a general feature of Candida auris clinical isolates . Medical Mycology 59 , 734 – 740 ( 2021 ). OpenUrl CrossRef PubMed 102. ↵ Bravo Ruiz , G. , Ross , Z. K. , Gow , N. A. R. & Lorenz , A. Pseudohyphal growth of the emerging pathogen Candida auris is triggered by genotoxic stress through the S phase checkpoint . mSphere 5 , e00151 – 20 ( 2020 ). OpenUrl CrossRef PubMed 103. ↵ Kim , S. H. et al. Genetic analysis of Candida auris implicates Hsp90 in morphogenesis and azole tolerance and Cdr1 in Azole Resistance . mBio 10 , e02529 – 18 ( 2019 ). OpenUrl CrossRef PubMed 104. ↵ Gao , J. et al. LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris . Nature Microbiology 6 , 842 – 851 ( 2021 ). OpenUrl PubMed 105. ↵ Chybowska , A. D. , Childers , D. S. & Farrer , R. A . Nine things genomics can tell us about Candida auris . Frontiers in Genetics 11 , 351 ( 2020 ). OpenUrl PubMed 106. ↵ Ismail , A. , Bedell , G. W. & Lupan , D. M . Siderophore production by the pathogenic yeast, Candida albicans . Biochemical and Biophysical Research Communications 130 , 885 – 891 ( 1985 ). OpenUrl CrossRef PubMed 107. ↵ Heymann , P. et al. The Siderophore iron transporter of Candida albicans (Sit1p/Arn1p) mediates uptake of ferrichrome-type siderophores and Is required for epithelial invasion . Infection and Immunity 70 , 5246 – 5255 ( 2002 ). OpenUrl Abstract / FREE Full Text 108. ↵ Boiteau , R. M. et al. Siderophore-based microbial adaptations to iron scarcity across the eastern Pacific Ocean . Proceedings of the National Academy of Sciences 113 , 14237 – 14242 ( 2016 ). OpenUrl Abstract / FREE Full Text 109. ↵ Park , J. et al. Siderophore production and utilization by marine bacteria in the North Pacific Ocean . Limnology and Oceanography 68 , 1636 – 1653 ( 2023 ). OpenUrl CrossRef 110. ↵ Renshaw , J. C. et al. Fungal siderophores: Structures, functions and applications . Mycological Research 106 , 1123 – 1142 ( 2002 ). OpenUrl CrossRef Web of Science 111. ↵ Johnstone , T. C. & Nolan , E. M . Beyond iron: Non-classical biological functions of bacterial siderophores . Dalton Transactions 44 , 6320 – 6339 ( 2015 ). OpenUrl CrossRef PubMed 112. ↵ Nakajima , Y. et al. Effect of disrupting the trichothecene efflux pump encoded by FgTri12 in the nivalenol chemotype of Fusarium graminearum . The Journal of General and Applied Microbiology 61 , 93 – 96 ( 2015 ). OpenUrl CrossRef 113. ↵ Xue , P. et al. Heme sensing and trafficking in fungi . Fungal Biology Reviews 43 , 100286 ( 2023 ). OpenUrl CrossRef PubMed 114. ↵ Fourie , R. , Kuloyo , O. O. , Mochochoko , B. M. , Albertyn , J. & Pohl , C. H . Iron at the Centre of Candida albicans Interactions . Frontiers in Cellular and Infection Microbiology 8 , 185 ( 2018 ). OpenUrl 115. ↵ Alselami , A. & Drummond , R. A . How metals fuel fungal virulence, yet promote anti-fungal immunity . Disease Models & Mechanisms 16 , dmm050393 ( 2023 ). OpenUrl 116. ↵ Pradhan , A. et al. Non-canonical signalling mediates changes in fungal cell wall PAMPs that drive immune evasion . Nature Communications 10 , 5315 ( 2019 ). OpenUrl PubMed 117. ↵ Weissman , Z. & Kornitzer , D . A family of Candida cell surface haem-binding proteins involved in haemin and haemoglobin-iron utilization . Molecular Microbiology 53 , 1209 – 1220 ( 2004 ). OpenUrl CrossRef PubMed 118. ↵ Ding , C. et al. Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans . PLOS ONE 6 , e28151 ( 2011 ). OpenUrl CrossRef PubMed 119. ↵ Sharma , P. & Pasrija , R . Iron chelators enhance the potency of Echinocandins against Candida auris . ( 2025 ) doi: 10.21203/rs.3.rs-7042978/v1 . OpenUrl CrossRef 120. ↵ Simm , C. et al. Disruption of iron homeostasis and mitochondrial metabolism are promising targets to inhibit Candida auris . Microbiology Spectrum 10 , e00100 – 22 ( 2022 ). OpenUrl PubMed 121. ↵ Zhang , J. et al. Pyrvinium pamoate synergizes with azoles in vitro and in vivo to exert antifungal efficacy against Candida auris and other Candida species . Infection and Drug Resistance 18 , 783 – 789 ( 2025 ). OpenUrl 122. ↵ Benjamini , Y. & Hochberg , Y . Controlling the false discovery rate: A practical and powerful approach to multiple testing . Journal of the Royal Statistical Society Series B: Statistical Methodology 57 , 289 – 300 ( 1995 ). OpenUrl CrossRef PubMed Web of Science 123. ↵ Chen , S. , Zhou , Y. , Chen , Y. & Gu , J . Fastp: An ultra-fast all-in-one FASTQ preprocessor . Bioinformatics 34 , i884 – i890 ( 2018 ). OpenUrl CrossRef PubMed 124. ↵ Ewels , P. , Magnusson , M. , Lundin , S. & Käller , M . MultiQC: Summarize analysis results for multiple tools and samples in a single report . Bioinformatics 32 , 3047 – 3048 ( 2016 ). OpenUrl CrossRef PubMed 125. ↵ Haas , B. J. et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis . Nature Protocols 8 , 1494 – 1512 ( 2013 ). OpenUrl PubMed 126. ↵ Abastabar , M. , et al. Candida auris otomycosis in Iran and review of recent literature . Mycoses 62 , 101 – 105 ( 2019 ). OpenUrl CrossRef PubMed 127. ↵ Muñoz , J. F. et al. Clade-specific chromosomal rearrangements and loss of subtelomeric adhesins in Candida auris . Genetics 218 , iyab029 ( 2021 ). OpenUrl CrossRef PubMed 128. ↵ Langmead , B. & Salzberg , S. L . Fast gapped-read alignment with Bowtie 2 . Nature Methods 9 , 357 – 359 ( 2012 ). OpenUrl PubMed 129. ↵ Li , B. & Dewey , C. N . RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome . BMC Bioinformatics 12 , 323 ( 2011 ). OpenUrl CrossRef PubMed 130. ↵ Robinson , M. D. , McCarthy , D. J. & Smyth , G. K . edgeR: A Bioconductor package for differential expression analysis of digital gene expression data . Bioinformatics 26 , 139 – 140 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 131. ↵ Li , H. et al. The Sequence Alignment/Map format and SAMtools . Bioinformatics 25 , 2078 – 2079 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 132. ↵ Ritchie , M. E. et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies . Nucleic Acids Research 43 , e47 ( 2015 ). OpenUrl CrossRef PubMed 133. ↵ Gu , Z. , Eils , R. & Schlesner , M . Complex heatmaps reveal patterns and correlations in multidimensional genomic data . Bioinformatics 32 , 2847 – 2849 ( 2016 ). OpenUrl CrossRef PubMed 134. ↵ Ashburner , M. et al. Gene Ontology: Tool for the unification of biology . Nature Genetics 25 , 25 – 29 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 135. ↵ The Gene Ontology Consortium et al. The Gene Ontology knowledgebase in 2023 . Genetics 224 , iyad031 ( 2023 ). OpenUrl CrossRef PubMed 136. ↵ Buchfink , B. , Reuter , K. & Drost , H.-G . Sensitive protein alignments at tree-of-life scale using DIAMOND . Nature Methods 18 , 366 – 368 ( 2021 ). OpenUrl PubMed 137. ↵ Golden , C. , Studholme , D. J. & Farrer , R. A. DIAMOND2GO: A rapid Gene Ontology assignment and enrichment tool for functional genomics . 2024.08.19.608700 ( 2024 ) doi: 10.1101/2024.08.19.608700 . OpenUrl Abstract / FREE Full Text 138. ↵ Kanehisa , M. , Furumichi , M. , Sato , Y. , Kawashima , M. & Ishiguro-Watanabe , M . KEGG for taxonomy-based analysis of pathways and genomes . Nucleic Acids Research 51 , D587 – D592 ( 2022 ). OpenUrl 139. ↵ Kanehisa , M. , Sato , Y. & Morishima , K . BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences . Journal of Molecular Biology 428 , 726 – 731 ( 2016 ). OpenUrl CrossRef PubMed 140. ↵ Bateman , A. et al. The Pfam protein families database . Nucleic Acids Research 32 , D138 – D141 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 141. ↵ Eddy , S. R . Accelerated profile HMM searches . PLOS Computational Biology 7 , e1002195 ( 2011 ). OpenUrl 142. ↵ Almagro Armenteros , J. J. , et al. SignalP 5.0 improves signal peptide predictions using deep neural networks . Nature Biotechnology 37 , 420 – 423 ( 2019 ). OpenUrl CrossRef PubMed 143. ↵ Hallgren , J. et al. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks . ( 2022 ) doi: 10.1101/2022.04.08.487609 . OpenUrl Abstract / FREE Full Text 144. ↵ Gíslason , M. H. , Nielsen , H. , Almagro Armenteros , J. J. & Johansen , A. R . Prediction of GPI-anchored proteins with pointer neural networks . Current Research in Biotechnology 3 , 6 – 13 ( 2021 ). OpenUrl 145. ↵ Farrer , R. A . Synima: A synteny imaging tool for annotated genome assemblies . BMC Bioinformatics 18 , 507 ( 2017 ). OpenUrl CrossRef PubMed 146. ↵ Emms , D. M. & Kelly , S . OrthoFinder: Phylogenetic orthology inference for comparative genomics . Genome Biology 20 , 238 ( 2019 ). OpenUrl CrossRef PubMed 147. ↵ Edgar , R. C . MUSCLE: Multiple sequence alignment with high accuracy and high throughput . Nucleic Acids Research 32 , 1792 – 1797 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 148. ↵ Price , M. N. , Dehal , P. S. & Arkin , A. P . FastTree 2 – approximately maximum-likelihood trees for large alignments . PLOS ONE 5 , e9490 ( 2010 ). OpenUrl CrossRef PubMed 149. ↵ Abramson , J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3 . Nature 630 , 493 – 500 ( 2024 ). OpenUrl CrossRef PubMed 150. ↵ Pettersen , E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers . Protein Science 30 , 70 – 82 ( 2021 ). OpenUrl CrossRef PubMed 151. ↵ Stamatakis , A . RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies . Bioinformatics 30 , 1312 – 1313 ( 2014 ). 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Share Xenosiderophore transporter gene expression and clade-specific filamentation in Candida auris killifish infection Hugh Gifford , Tina Bedekovic , Nicolas Helmstetter , Jack Gregory , Qinxi Ma , Alexandra C. Brand , Duncan Wilson , Johanna Rhodes , Mark Ramsdale , Tetsuhiro Kudoh , Rhys A. Farrer bioRxiv 2024.10.22.619744; doi: https://doi.org/10.1101/2024.10.22.619744 Share This Article: Copy Citation Tools Xenosiderophore transporter gene expression and clade-specific filamentation in Candida auris killifish infection Hugh Gifford , Tina Bedekovic , Nicolas Helmstetter , Jack Gregory , Qinxi Ma , Alexandra C. Brand , Duncan Wilson , Johanna Rhodes , Mark Ramsdale , Tetsuhiro Kudoh , Rhys A. 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