crispr-mediated-genome-editing-human-fungal-pathogen-candida

CRISPR-mediated Genome Editing of the Human Fungal Pathogen <em>Candida albicans</em>

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Whitehead Institute for Biomedical Research

Dectin-1&#xFF0C;&#x4E3B;&#x8981;&#x7684; &beta;-&#x8461;&#x805A;&#x7CD6;&#x53D7;&#x4F53;&#x7684;&#x767D;&#x7EC6;&#x80DE;&#xFF0C;&#x89E6;&#x53D1;&#x540E;&#x771F;&#x83CC;&#x8BC6;&#x522B;&#x6709;&#x6548;&#x7684;&#x514D;&#x75AB;&#x53CD;&#x5E94;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x4F7F;&#x7528; sortase &#x4ECB;&#x5BFC;&#x7684; transpeptidation&#xFF0C;&#x4E00;&#x79CD;&#x6280;&#x672F;&#xFF0C;&#x5141;&#x8BB8;&#x653E;&#x7F6E;&#x5404;&#x79CD;&#x63A2;&#x6D4B;&#x5668;&#x4E0A;&#x7684;&#x591A;&#x80BD;&#x9AA8;&#x5E72;&#xFF0C;&#x8981;&#x76D1;&#x89C6;&#x7684;&#x884C;&#x4E3A;&#x6807;&#x8BB0;&#x529F;&#x80FD; dectin 1 &#x5728;&#x6D3B;&#x7EC6;&#x80DE;&#x5185;&#x4E0E;&#x771F;&#x83CC;&#x7684;&#x6311;&#x6218;&#x3002;&#x5BF9; dectin-1 sortagging &#x7684;&#x63A2;&#x6D4B;&#x5668;&#x5B89;&#x88C5;&#x5141;&#x8BB8;&#x9AD8;&#x5EA6;&#x7279;&#x5B9A;&#x53EF;&#x89C6;&#x5316;&#x529F;&#x80FD;&#x86CB;&#x767D;&#x5728;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x548C;&#x914D;&#x4F53;&#x51FA;&#x793A;&#x53CA;&#x5176;&#x968F;&#x540E;&#x7684;&#x5185;&#x5316;&#x3002;Sortagged dectin-1 &#x5728;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x4E2D;&#x8868;&#x8FBE;&#x7684;&#x68C0;&#x7D22;&#x53D1;&#x73B0; dectin-1 &#x548C;&#x534A;&#x4E73;&#x7CD6;&#x51DD;&#x96C6;&#x7D20;-3 &#x7684;&#x72EC;&#x7279;&#x4E92;&#x52A8;&#x8FD9;&#x5BF9;&#x81F4;&#x75C5;&#x6027;&#x771F;&#x83CC;&#x7684;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x708E;&#x6027;&#x53CD;&#x5E94;&#x4E2D;&#x7684;&#x51FD;&#x6570;&#x3002;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x8868;&#x8FBE; dectin 1 &#x66B4;&#x9732;&#x5728;&#x5FF5;&#x73E0;&#x83CC;&#x767D;&#x7A81;&#x53D8;&#x4F53;&#x7684; &beta;-&#x8461;&#x805A;&#x7CD6;&#x589E;&#x52A0;&#x66DD;&#x5149;&#xFF0C;&#x534A;&#x4E73;&#x7CD6;&#x51DD;&#x96C6;&#x7D20;-3 &#x7684;&#x635F;&#x5931;&#x5927;&#x5927;&#x52A0;&#x5267;&#x6545;&#x969C;&#x89E6;&#x53D1;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x7684;&#x9002;&#x5F53;&#x53CD;&#x5E94;&#x3002;

&#x771F;&#x83CC;&#x8BC6;&#x522B;&#x88AB;&#x4ECB;&#x5BFC;&#x7684; dectin-1 &#x548C;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x534A;&#x4E73;&#x7CD6;&#x51DD;&#x96C6;&#x7D20;-3 &#x534F;&#x4F1A;&#x3002;

Dectin-1, the major Î²-glucan receptor in leukocytes, triggers an effective immune response upon fungal recognition. Here we use sortase-mediated transpeptidation, a technique that allows placement of a variety of probes on a polypeptide backbone, to monitor the behavior of labeled functional dectin-1 in live cells with and without fungal challenge. Installation of probes on dectin-1 by sortagging permitted highly specific visualization of functional protein on the cell surface and its subsequent internalization upon ligand presentation. Retrieval of sortagged dectin-1 expressed in macrophages uncovered a unique interaction between dectin-1 and galectin-3 that functions in the proinflammatory response of macrophages to pathogenic fungi. When macrophages expressing dectin-1 are exposed to Candida albicans mutants with increased exposure of Î²-glucan, the loss of galectin-3 dramatically accentuates the failure to trigger an appropriate TNF-Î± response.

Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages.

Dectin-1, le principal &#x3B2;-glucane r&#xE9;cepteur dans les leucocytes, d&#xE9;clenche une r&#xE9;ponse immunitaire efficace lors de la reconnaissance des champignons. Ici, nous utilisons sortase m&#xE9;di&#xE9;e transpeptidation, une technique qui permet le placement d&#x26;#39;une vari&#xE9;t&#xE9; de sondes sur un squelette polypeptidique, pour surveiller le comportement des &#xE9;tiquet&#xE9; fonctionnelle Dectin-1 dans les cellules vivantes avec et sans d&#xE9;fi fongique. Installation des sondes sur Dectin-1 par sortagging autoris&#xE9;e visualisation hautement sp&#xE9;cifique de la prot&#xE9;ine fonctionnelle sur la surface cellulaire et son internalisation ult&#xE9;rieur apr&#xE8;s la pr&#xE9;sentation ligand. R&#xE9;cup&#xE9;ration de sortagged Dectin-1 exprim&#xE9; dans les macrophages d&#xE9;couvert une interaction unique entre Dectin-1 et galectine-3 qui fonctionne dans la r&#xE9;ponse pro-inflammatoire des macrophages &#xE0; des champignons pathog&#xE8;nes. Lorsque les macrophages exprimant Dectin-1 sont expos&#xE9;s &#xE0; Candida albicans mutants avec une exposition accrue de la &#x3B2;-glucane, la perte de la galectine-3 accentue consid&#xE9;rablement l&#x26;#39;&#xE9;chec de d&#xE9;clencher une TNF-&#x3B1; appropri&#xE9;e r&#xE9;ponse.

Reconnaissance fongique est m&#xE9;di&#xE9;e par l&#x26;#39;Association des Dectin-1 et galectine-3 dans les macrophages

Dectin-1, den gro&szlig;en &beta;-Glucan-Rezeptor in Leukozyten, l&ouml;st eine wirksame Immunantwort auf Pilz-Anerkennung. Hier verwenden wir Sortase-vermittelte Transpeptidation, eine Technik, die erlaubt, Platzierung verschiedener Sonden auf einem Polypeptid-Backbone, um das Verhalten zu &uuml;berwachen funktionale Dectin-1 in lebenden Zellen mit und ohne Pilze Herausforderung beschriftet. Installation von Sonden auf Dectin-1 durch Sortagging gestattet hochspezifische Visualisierung von funktionellen Protein auf der Zelloberfl&auml;che und seine anschlie&szlig;ende Internalisierung Ligand-Vorlage. Abruf von Sortagged Dectin-1 ausgedr&uuml;ckt in Makrophagen deckte einer einzigartigen Interaktion zwischen Dectin-1 und Galectin-3, Funktionen der Makrophagen Proinflammatory auf pathogene Pilze. Wenn Makrophagen Ausdr&uuml;cken Dectin-1 Candida Albicans Mutanten mit erh&ouml;htem Risiko von &beta;-Glucan ausgesetzt sind, akzentuiert der Verlust von Galectin-3 dramatisch das Vers&auml;umnis, eine angemessene TNF-&alpha;-Reaktion ausl&ouml;sen.

Pilzartige Anerkennung wird durch die Assoziation von Dectin-1 und Galectin-3 in Makrophagen vermittelt.

Dectin-1, il recettore &beta;-glucano principali nei leucociti, innesca una risposta immunitaria efficacia su riconoscimento fungina. Qui usiamo transpeptidation sortase-mediata, una tecnica che permette il posizionamento di una variet&agrave; di sonde su una dorsale del polipeptide, per monitorare il comportamento di etichettati funzionale dectin-1 in cellule vive con e senza sfida fungina. Installazione di sonde su dectin-1 da sortagging ammessi visualizzazione altamente specifico della proteina funzionale sulla superficie delle cellule e la sua interiorizzazione successive alla presentazione del ligando. Recupero di sortagged dectin-1 espresso nei macrofagi ha scoperto un&#39;interazione unica tra dectin-1 e galectin-3 che funziona nella risposta proinflammatory di macrofagi a funghi patogeni. Quando i macrofagi che esprimono dectin-1 sono esposti ai mutanti di Candida albicans con maggiore esposizione del &beta;-glucano, la perdita di galectin-3 accentua notevolmente l&#39;incapacit&agrave; di innescare una risposta adeguata di TNF-&alpha;.

Riconoscimento fungina &egrave; mediata dall&#39;associazione dectin-1 e galectin-3 in macrofagi.

&#x30C7;&#x30AF;&#x30C1;&#x30F3;-1&#x3001;&#x767D;&#x8840;&#x7403;&#x306E;&#x4E3B;&#x8981;&#x306A;&#x3B2;-&#x30B0;&#x30EB;&#x30AB;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#x306F;&#x3001;&#x771F;&#x83CC;&#x306E;&#x8A8D;&#x8B58;&#x306B;&#x5FDC;&#x3058;&#x3066;&#x52B9;&#x679C;&#x7684;&#x306A;&#x514D;&#x75AB;&#x5FDC;&#x7B54;&#x3092;&#x30C8;&#x30EA;&#x30AC;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x3068;&#x3001;&#x771F;&#x83CC;&#x6311;&#x6226;&#x305B;&#x305A;&#x306B;&#x751F;&#x304D;&#x3066;&#x3044;&#x308B;&#x7D30;&#x80DE;&#x4E2D;&#x306E;&#x6A19;&#x8B58;&#x306E;&#x6A5F;&#x80FD;&#x30C7;&#x30AF;&#x30C1;&#x30F3;-1&#x306E;&#x52D5;&#x4F5C;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;sortase&#x4ECB;&#x5728;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x8EE2;&#x79FB;&#x3001;&#x30DD;&#x30EA;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x4E3B;&#x9396;&#x4E0A;&#x306E;&#x30D7;&#x30ED;&#x30FC;&#x30D6;&#x306E;&#x69D8;&#x3005;&#x306A;&#x914D;&#x7F6E;&#x3092;&#x53EF;&#x80FD;&#x306B;&#x3059;&#x308B;&#x6280;&#x8853;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; sortagging&#x306B;&#x3088;&#x308B;&#x30C7;&#x30AF;&#x30C1;&#x30F3;-1&#x4E0A;&#x306E;&#x30D7;&#x30ED;&#x30FC;&#x30D6;&#x306E;&#x30A4;&#x30F3;&#x30B9;&#x30C8;&#x30FC;&#x30EB;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x4E0A;&#x306E;&#x6A5F;&#x80FD;&#x6027;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3068;&#x30EA;&#x30AC;&#x30F3;&#x30C9;&#x306E;&#x30D7;&#x30EC;&#x30BC;&#x30F3;&#x30C6;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x6642;&#x306B;&#x3001;&#x305D;&#x306E;&#x5F8C;&#x7D9A;&#x306E;&#x5185;&#x5728;&#x5316;&#x306E;&#x7279;&#x7570;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x53EF;&#x8996;&#x5316;&#x3092;&#x53EF;&#x80FD;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306B;&#x767A;&#x73FE;&#x3057;sortagged&#x30C7;&#x30AF;&#x30C1;&#x30F3;-1&#x306E;&#x691C;&#x7D22;&#x306F;&#x30C7;&#x30AF;&#x30C1;&#x30F3;-1&#x3068;&#x30AC;&#x30EC;&#x30AF;&#x30C1;&#x30F3;-3&#x306E;&#x9593;&#x306E;&#x30E6;&#x30CB;&#x30FC;&#x30AF;&#x306A;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x3001;&#x305D;&#x306E;&#x75C5;&#x539F;&#x6027;&#x771F;&#x83CC;&#x306E;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306E;&#x708E;&#x75C7;&#x53CD;&#x5FDC;&#x3067;&#x6A5F;&#x80FD;&#x3057;&#x307E;&#x3059;&#x3002;&#x30C7;&#x30AF;&#x30C1;&#x30F3;-1&#x3092;&#x767A;&#x73FE;&#x3059;&#x308B;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306F;&#x3001;&#x3B2;-&#x30B0;&#x30EB;&#x30AB;&#x30F3;&#x306E;&#x5897;&#x52A0;&#x66B4;&#x9732;&#x3068;Candida albicans&#x306E;&#x5909;&#x7570;&#x306B;&#x3055;&#x3089;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x5834;&#x5408;&#x306F;&#x3001;&#x30AC;&#x30EC;&#x30AF;&#x30C1;&#x30F3;-3&#x306E;&#x640D;&#x5931;&#x306F;&#x5287;&#x7684;&#x306B;&#x9069;&#x5207;&#x306A;TNF-&#x3B1;&#x5FDC;&#x7B54;&#x3092;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x306B;&#x5931;&#x6557;&#x3057;&#x305F;&#x3053;&#x3068;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;

&#x771F;&#x83CC;&#x306E;&#x8A8D;&#x8B58;&#x306F;&#x30C7;&#x30AF;&#x30C1;&#x30F3;-1&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306B;&#x304A;&#x3051;&#x308B;&#x30AC;&#x30EC;&#x30AF;&#x30C1;&#x30F3;-3&#x306E;&#x5354;&#x4F1A;&#x306B;&#x3088;&#x3063;&#x3066;&#x5A92;&#x4ECB;&#x3055;&#x308C;&#x308B;

Dectin-1, &#xBC31;&#xD608;&#xAD6C;&#xC5D0; &#xC8FC;&#xC694; &beta;-&#xAE00; &#xB8E8; &#xCE78; &#xC218;&#xC6A9; &#xCCB4; &#xADE0; &#xC778;&#xC2DD;&#xC5D0; &#xB530;&#xB77C; &#xD6A8;&#xACFC;&#xC801;&#xC778; &#xBA74;&#xC5ED; &#xBC18;&#xC751;&#xC744; &#xD2B8;&#xB9AC;&#xAC70;&#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; sortase &#xC911;&#xC7AC; transpeptidation, &#xB2E4;&#xC591; &#xD55C; &#xD504;&#xB85C;&#xBE0C;&#xC5D0;&#xC758; &#xB3D9;&#xC791;&#xC744; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB824;&#xBA74; polypeptide &#xBC31;&#xBCF8; &#xBC30;&#xCE58; &#xBD84;&#xB958; &#xAE30;&#xB2A5; dectin-1 &#xACF0; &#xD321;&#xC774; &#xB3C4;&#xC804; &#xD558;&#xC9C0; &#xC54A;&#xACE0; &#xB77C;&#xC774;&#xBE0C; &#xC140; &#xC218; &#xC788;&#xB294; &#xAE30;&#xC220;&#xC744; &#xC0AC;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. Dectin-1, sortagging&#xC5D0; &#xC758;&#xD574; &#xD504;&#xB85C;&#xBE0C;&#xB97C; &#xC124;&#xCE58; &#xD5C8;&#xC6A9; &#xC140; &#xD45C;&#xBA74; &#xBC0F; &#xB9AC;&#xAC04;&#xB4DC; &#xD504;&#xB808; &#xC820; &#xD14C;&#xC774; &#xC158;&#xC5D0; &#xB530;&#xB77C; &#xADF8; &#xD6C4;&#xC18D; &#xAD6D;&#xC81C;&#xD654; &#xAE30;&#xB2A5; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xB9E4;&#xC6B0; &#xB192;&#xC740; &#xC2DC;&#xAC01;&#xD654;. Sortagged dectin 1 &#xB300; &#xC2DD; &#xC138;&#xD3EC; &#xB2E8;&#xC704;&#xB85C; &#xAC80;&#xC0C9; &#xBC1C;&#xACAC; dectin-1 galectin-3 &#xC0AC;&#xC774;&#xC758; &#xB3C5;&#xD2B9;&#xD55C; &#xC0C1;&#xD638; &#xC791;&#xC6A9; proinflammatory &#xC751;&#xB2F5; &#xB300; &#xC2DD; &#xC138;&#xD3EC;&#xC758; &#xBCD1;&#xC6D0; &#xC131; &#xADE0;&#xC73C;&#xB85C; &#xADF8; &#xAE30;&#xB2A5;. Dectin-1&#xC744; &#xD45C;&#xD604; &#xD558;&#xB294; &#xB300; &#xC2DD; &#xC138;&#xD3EC;&#xB294; Candida albicans &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &beta;-&#xAE00; &#xB8E8; &#xCE78;&#xC758; &#xB178;&#xCD9C; &#xC99D;&#xAC00;&#xC5D0; &#xB178;&#xCD9C; &#xB418;&#xB294;, galectin-3 &#xC190;&#xC2E4;&#xC740; &#xADF9;&#xC801;&#xC73C;&#xB85C; TNF-&alpha;&#xC5D0; &#xB300; &#xD55C; &#xC801;&#xC808; &#xD55C; &#xC751;&#xB2F5;&#xC744; &#xC2E4;&#xD589; &#xD558;&#xB294; &#xB370; &#xC2E4;&#xD328; accentuates.

&#xACF0; &#xD321;&#xC774; &#xC778;&#xC2DD; dectin 1 &#xD611;&#xD68C; galectin-3, &#xB300; &#xC2DD; &#xC138;&#xD3EC;&#xC5D0; &#xC758;&#xD574; &#xC911;&#xC7AC;&#xC785;&#xB2C8;&#xB2E4;.

Dectin-1, el principal receptor de &#x3B2;-glucano en los leucocitos, desencadena una respuesta inmune efectiva a partir del reconocimiento de hongos. Aqu&#xED; se utiliza sortase mediada por transpeptidaci&#xF3;n, una t&#xE9;cnica que permite la colocaci&#xF3;n de una variedad de sondas en un esqueleto del polip&#xE9;ptido, para vigilar el comportamiento de etiquetado funcional dectin-1 en c&#xE9;lulas vivas con y sin desaf&#xED;o hongos. La instalaci&#xF3;n de las sondas en dectin-1 por sortagging permitido visualizaci&#xF3;n altamente espec&#xED;fico de la prote&#xED;na funcional en la superficie celular y su internalizaci&#xF3;n posterior a la presentaci&#xF3;n ligando. La recuperaci&#xF3;n de sortagged dectin-1 se expresa en los macr&#xF3;fagos al descubierto una interacci&#xF3;n &#xFA;nica entre dectin-1 y galectina-3 que funciona en la respuesta proinflamatoria de los macr&#xF3;fagos a los hongos pat&#xF3;genos. Cuando los macr&#xF3;fagos que expresan dectin-1 est&#xE1;n expuestos a Candida albicans mutantes con mayor exposici&#xF3;n de &#x3B2;-glucano, la p&#xE9;rdida de galectina-3 dram&#xE1;ticamente acent&#xFA;a el fracaso para desencadenar un apropiado de TNF-&#x3B1; respuesta.

Reconocimiento por hongos est&#xE1; mediada por la Asociaci&#xF3;n de Dectin-1 y galectina-3 en los macr&#xF3;fagos

The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii, restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway (CaAGO1) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans, has additional functions: it is required for cleavage of the 3' external transcribed spacer from unprocessed pre-rRNA and for processing the 3' tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts.

Candida albicans Dicer (CaDcr1) is required for efficient ribosomal and spliceosomal RNA maturation.

While Candida albicans is the most significant fungal pathogen for humans, Candida glabrata accounts for an increasing number of infections. Little is known about how C. glabrata interacts with the innate immune system, the first line of defense against such organisms. The C. glabrata adhesin Epa1p was previously shown to bind mammalian epithelial cells. We hypothesized that Epa1p mediates unique, nonopsonic binding to macrophages, leading to induction of immune responses. We found that Epa1p mediated adhesion by both C. glabrata (Cg) and transformed Saccharomyces cerevisiae (Sc(EPA1) ) to human macrophage-like cells, including Thp1 and U937 lines, and donor PBMCs. Adhesion was distinct from described mechanisms such as Dectin-1. Epa1p expression was necessary and sufficient for S. cerevisiae binding and phagocytosis, the latter of which was actin-mediated. Sc(EPA1)  induced inflammatory cytokine production (IL-8 and TNF-α) by human PBMC-derived macrophages. Despite expressing Epa1p and binding to macrophages, Cg avoided phagocytosis and cytokine induction. In contrast to human results, in murine cell models (RAW264.7, J774A.1, and C57BL/6-derived cells), Epa1p-mediated binding was only revealed after blocking the Dectin-1 system. Recognition of Epa1p represents a novel mechanism by which human innate immune cells bind fungi, and for Sc(EPA1)  results in phagocytosis and subsequent cytokine production.

The Candida glabrata adhesin Epa1p causes adhesion, phagocytosis, and cytokine secretion by innate immune cells.

Our recent finding that the Candida albicans RNase III enzyme CaDcr1 is an unusual, multifunctional RNase III coupled with data on the RNase III enzymes from other fungal species prompted us to seek a model that explained the evolution of RNase III's in modern budding yeast species. CaDcr1 has both dicer function (generates small RNA molecules from dsRNA precursors) and Rnt1 function, (catalyzes the maturation of 35S rRNA and U4 snRNA). Some budding yeast species have two distinct genes that encode these functions, a Dicer and RNT1, whereas others have only an RNT1 and no Dicer. As none of the budding yeast species has the canonical Dicer found in many other fungal lineages and most eukaryotes, the extant species must have evolved from an ancestor that lost the canonical Dicer, and evolved a novel Dicer from the essential RNT1 gene. No single, simple model could explain the evolution of RNase III enzymes from this ancestor because existing sequence data are consistent with two equally plausible models. The models share an architecture for RNase III evolution that involves gene duplication, loss, subfunctionalization, and neofunctionalization. This commentary explains our reasoning, and offers the prospect that further genomic data could further resolve the dilemma surrounding the budding yeast RNase III's evolution.

Genes come and go: the evolutionarily plastic path of budding yeast RNase III enzymes.

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

 is a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe a  CRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome of  and provides a new window into the biology of this pathogen.

A  CRISPR system permits genetic engineering of essential genes and gene families.

The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space.

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in , , , and  These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both  and  rely heavily on homology-directed repair, whereas  and  use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens  and , as well as in the related yeasts  and  The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

Valmik K. Vyas

Whitehead Institute for Biomedical Research

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans