generation-of-fluorescent-protein-fusions-in-candida-species

Generation of Fluorescent Protein Fusions in <em>Candida</em> Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

University of Minnesota

Protein O-mannosyltransferases (Pmt proteins) initiate O-mannosylation of secretory proteins. The PMT gene family of the human fungal pathogen Candida albicans consists of PMT1 and PMT6, as well as three additional PMT genes encoding Pmt2, Pmt4 and Pmt5 isoforms described here. Both PMT2 alleles could not be deleted and growth of conditional strains, containing PMT2 controlled by the MET3- or tetOScHOP1-promoters, was blocked in non-permissive conditions, indicating that PMT2 is essential for growth. A homozygous pmt4 mutant was viable, but synthetic lethality of pmt4 was observed in combination with pmt1 mutations. Hyphal morphogenesis of a pmt4 mutant was defective under aerobic induction conditions, yet increased in embedded or hypoxic conditions, suggesting a role of Pmt4p-mediated O-glycosylation for environment-specific morphogenetic signalling. Although a PMT5 transcript was detected, a homozygous pmt5 mutant was phenotypically silent. All other pmt mutants showed variable degrees of supersensitivity to antifungals and to cell wall-destabilizing agents. Cell wall composition was markedly affected in pmt1 and pmt4 mutants, showing a significant decrease in wall mannoproteins. In a mouse model of haematogenously disseminated infection, PMT4 was required for full virulence of C. albicans. Functional analysis of the first complete PMT gene family in a fungal pathogen indicates that Pmt isoforms have variable and specific roles for in vitro and in vivo growth, morphogenesis and antifungal resistance.

PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance.

Candida albicans, the most prevalent fungal pathogen of humans, causes superficial mycoses, invasive mucosal infections, and disseminated systemic disease. Many studies have shown an intriguing association between C. albicans morphogenesis and the pathogenesis process. For example, hyphal cells have been observed to penetrate host epithelial cells at sites of wounds and between cell junctions. Ras- and Rho-type GTPases regulate many morphogenetic processes in eukaryotes, including polarity establishment, cell proliferation, and directed growth in response to extracellular stimuli. We found that the C. albicans Ras-like GTPase Rsr1p and its predicted GTPase-activating protein Bud2p localized to the cell cortex, at sites of incipient daughter cell growth, and provided landmarks for the positioning of daughter yeast cells and hyphal cell branches, similar to the paradigm in the model yeast Saccharomyces cerevisiae. However, in contrast to S. cerevisiae, CaRsr1p and CaBud2p were important for morphogenesis: C. albicans strains lacking Rsr1p or Bud2p had abnormal yeast and hyphal cell shapes and frequent bends and promiscuous branching along the hypha and were unable to invade agar. These defects were associated with abnormal actin patch polarization, unstable polarisome localization at hyphal tips, and mislocalized septin rings, consistent with the idea that GTP cycling of Rsr1p stabilizes the axis of polarity primarily to a single focus, thus ensuring normal cell shape and a focused direction of polarized growth. We conclude that the Rsr1p GTPase functions as a polarity landmark for hyphal guidance and may be an important mediator of extracellular signals during processes such as host invasion.

Hyphal guidance and invasive growth in Candida albicans require the Ras-like GTPase Rsr1p and its GTPase-activating protein Bud2p.

Proteins that enter the secretory pathway play important roles in virulence and pathogenesis in Candida albicans, but our understanding of the trafficking of these proteins is in its early stages. In Saccharomyces cerevisiae, dominant negative alleles of YPT1 and SEC4 interrupt secretory traffic at pre- and post-Golgi steps, respectively. We therefore used a dominant negative genetic approach to examine the intracellular trafficking of several proteins associated with virulence or azole resistance. When the dominant negative ypt1(N121I) allele of C. albicans was overexpressed, yellow-fluorescent protein (YFP) tagged forms of two plasma membrane transporters (Cdrlp and Ftrlp) and the vacuolar membrane ABC transporter Mltlp accumulated in intracellular structures that appeared related to the ER, but localization of Cdc10p and Int1p was unaffected. When the dominant negative sec4(S28N) allele of C. albicans was overexpressed, Cdrlp and Ftrlp accumulated intracellularly, and localization of Mltlp, Cdc10p and Int1p was unaffected. These results imply that (i) Cdrlp and Ftrlp are transported to the plasma membrane by the general secretory pathway, (ii) Mlt1p enters the secretory pathway but is diverted to the vacuole at an early post-Golgi step, and (iii) like Cdc10p, Int1p does not enter the general secretory pathway.

Intracellular trafficking of fluorescently tagged proteins associated with pathogenesis in Candida albicans.

&#x5B9A;&#x5411;&#x751F;&#x957F;&#x662F;&#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x7684;&#x504F;&#x632F;&#x7B49; neurites&#x3001; &#x82B1;&#x7C89;&#x7BA1;&#x4E0E;&#x771F;&#x83CC;&#x83CC;&#x4E1D;&#x3002;&#x6269;&#x5C55;&#x5355;&#x5143;&#x683C;&#x63D0;&#x793A;&#x6B63;&#x786E;&#x7684;&#x65B9;&#x5411;&#x53D6;&#x51B3;&#x4E8E;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x901A;&#x8DEF;&#x548C;&#x8C03;&#x89E3;&#x5BF9;&#x7279;&#x5B9A;&#x73AF;&#x5883;&#x7EBF;&#x7D22;&#x4E0D;&#x5BF9;&#x79F0;&#x53CD;&#x5E94;&#x7684;&#x6548;&#x5E94;&#x5668;&#x3002;&#x767D;&#x8272;&#x5FF5;&#x73E0;&#x83CC;&#x771F;&#x6838;&#x75C5;&#x539F;&#x771F;&#x83CC;&#x7684;&#x83CC;&#x4E1D;&#x5F62;&#x5F0F;&#xFF0C;&#x8FD9;&#x4E9B;&#x53CD;&#x5E94;&#x5305;&#x62EC; thigmotropism &#x548C; galvanotropism (&#x83CC;&#x4E1D;&#x8F6C;&#x886C;&#x5E95;&#x5730;&#x5F62;&#x548C;&#x65BD;&#x52A0;&#x7684;&#x7535;&#x573A;&#x53D8;&#x5316;&#x54CD;&#x5E94;&#x5206;&#x522B;&#xFF09; &#x548C;&#x534A;&#x56FA;&#x6001;&#x57FA;&#x8D28;&#x6E17;&#x900F;&#x3002;C.&#x767D;&#xFF0C;&#x6A21;&#x578B;&#x9175;&#x6BCD;&#x917F;&#x9152;&#x9175;&#x6BCD;&#xFF0C;&#x5982;&#x8425;&#x517B;&#x751F;&#x957F;&#x671F;&#x95F4; Ras &#x6837;&#x86CB;&#x767D; Rsr1 &#x4ECB;&#x5BFC;&#x5185;&#x90E8;&#x7EC6;&#x80DE;&#x7684;&#x63D0;&#x793A;&#xFF0C;&#x4EE5;&#x5B9A;&#x4F4D;&#x4E2D;&#x9884;&#x5148;&#x6307;&#x5B9A;&#x56FE;&#x6848;&#x7684;&#x6BCD;&#x7EC6;&#x80DE;&#x76AE;&#x5C42;&#x4E0A;&#x7684;&#x65B0;&#x82BD;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x660E; Rsr1 &#x4E5F;&#x662F;&#x91CD;&#x8981;&#x7684;&#x54CD;&#x5E94;&#x5916;&#x90E8;&#x73AF;&#x5883;&#x63D0;&#x793A;&#xFF0C;&#x8BF1;&#x4F7F; thigmotropic &#x548C; galvanotropic &#x751F;&#x957F;&#x83CC;&#x4E1D;&#x63D0;&#x793A;&#x65B9;&#x5411;&#x3002;&#x6B64;&#x5916;&#xFF0C;Rsr1 &#x53C2;&#x4E0E;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x4F53;&#x5916;&#x83CC;&#x4E1D;&#x4EA4;&#x4E92;&#xFF0C;&#x5176;&#x5220;&#x9664;&#x51CF;&#x5C11;&#x80BE;&#x810F;&#x7EC4;&#x7EC7;&#x83CC;&#x4E1D;&#x5165;&#x4FB5;&#x671F;&#x95F4;&#x5168;&#x8EAB;&#x611F;&#x67D3;&#x3002;&#x56E0;&#x6B64;&#xFF0C;Rsr1&#xFF0C;&#x9175;&#x6BCD;&#xFF0C;&#x5185;&#x90E8;&#x6781;&#x6027;&#x91CC;&#x7A0B;&#x7891;&#x8FD8;&#x53C2;&#x4E0E;&#x589E;&#x957F;&#x6781;&#x5316;&#x7684;&#x54CD;&#x5E94;&#x975E;&#x5BF9;&#x79F0;&#x73AF;&#x5883;&#x4FE1;&#x53F7;&#xFF0C;&#x662F;&#x5728;&#x917F;&#x9152;&#x9175;&#x6BCD;&#x4E2D;&#x7684;&#x540C;&#x6E90;&#x86CB;&#x767D;&#x8D28;&#x4E2D;&#x6240;&#x63CF;&#x8FF0;&#x7684;&#x4E0D;&#x540C;&#x7684;&#x8303;&#x5F0F;&#x3002;Rsr1 &#x4ECE;&#x800C;&#x53EF;&#x80FD;&#x901A;&#x8FC7;&#x5F71;&#x54CD;&#x83CC;&#x4E1D;&#x63D0;&#x793A;&#x54CD;&#x5E94;&#x89E6;&#x53D1;&#x4E0E;&#x5BBF;&#x4E3B;&#x7EC4;&#x7EC7;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684;&#x8D21;&#x732E; C.&#x83CC;&#x611F;&#x67D3;&#x7684;&#x53D1;&#x75C5;&#x673A;&#x5236;&#x3002;

&#x5185;&#x90E8;&#x6781;&#x6027;&#x7684;&#x4E00;&#x4E2A;&#x91CC;&#x7A0B;&#x7891;&#x662F;&#x91CD;&#x8981;&#x7684;&#x5916;&#x90E8;&#x8BF1;&#x53D1;&#x83CC;&#x4E1D;&#x884C;&#x4E3A;&#x4E2D;&#x767D;&#x8272;&#x5FF5;&#x73E0;&#x83CC;&#x3002;

Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues.

An internal polarity landmark is important for externally induced hyphal behaviors in Candida albicans.

Croissance directionnelle est une fonction des cellules polaris&eacute;es comme neurites, tubes polliniques et les hyphes fongiques. Orientation correcte de la pointe s&#39;&eacute;tendant de cellule d&eacute;pend de la signalisation des voies et des effecteurs qui m&eacute;dient r&eacute;ponses asym&eacute;triques aux signaux environnementaux sp&eacute;cifiques. Sous la forme des hyphes du champignon pathog&egrave;ne Candida albicans eucaryote, ces r&eacute;actions incluent thigmotropisme et Galvanotropisme (hyphes tournant en r&eacute;ponse &agrave; l&#39;&eacute;volution de la topographie du substrat et des champs &eacute;lectriques impos&eacute;s, respectivement) et la p&eacute;n&eacute;tration dans les substrats semi-solide. Durant la croissance v&eacute;g&eacute;tative chez c. albicans, comme dans la mod&egrave;le levure Saccharomyces cerevisiae, la Ras-comme GTPase Rsr1 m&eacute;diatise les signaux cellulaires internes pour positionner les nouveaux bourgeons de fa&ccedil;on pr&eacute;d&eacute;termin&eacute;e sur le cortex de la cellule-m&egrave;re. Ici, nous d&eacute;montrons que Rsr1 est aussi important pour l&#39;orientation des hyphes pointe en r&eacute;ponse &agrave; la signaux environnementaux externes qui induisent la thigmotropique et la croissance de galvanotropic. En outre, Rsr1 est impliqu&eacute; dans des interactions des hyphes avec in vitro des cellules &eacute;pith&eacute;liales et sa suppression diminue l&#39;invasion des hyphes du tissu r&eacute;nal au cours de l&#39;infection syst&eacute;mique. Ainsi, Rsr1, un point de rep&egrave;re de polarit&eacute; interne chez les levures, participe aux r&eacute;ponses de croissance polaris&eacute;e aux signaux environnementaux asym&eacute;triques, un paradigme qui est diff&eacute;rente de celle d&eacute;crite pour les prot&eacute;ines homologues chez S. cerevisiae. Rsr1 peut contribuer ainsi &agrave; la pathog&eacute;nie des infections &agrave; c. albicans en influen&ccedil;ant les r&eacute;ponses de pointe des hyphes d&eacute;clench&eacute;es par l&#39;interaction avec les tissus de l&#39;h&ocirc;te.

Un point de rep&egrave;re de polarit&eacute; interne est important pour les comportements des hyphes ext&eacute;rieurement induites chez Candida albicans.

Gerichtetes Wachstum ist eine Funktion der polarisierten Zellen wie Neurites, Pollen Rohre und Pilz-Hyphen. Korrekte Ausrichtung der erweitern Zelle Spitze h&auml;ngt davon ab, Signalisierung, Wege und Effektoren, die asymmetrische Reaktionen auf bestimmte &ouml;kologische Hinweise zu vermitteln. In geflochtenen Form der eukaryotischen pilzartige Erreger Candida Albicans geh&ouml;ren diese Antworten Thigmotropism und Galvanotropism (geflochtenen drehen in Reaktion auf &Auml;nderungen im Substrat Topographie und auferlegte elektrische Felder, beziehungsweise) und Eindringen in die halbfeste Substrate. W&auml;hrend des vegetativen Wachstums in C. Albicans, wie in dem Modell Hefe Saccharomyces Cerevisiae vermittelt die Ras-&auml;hnlichen GTPase Rsr1 interne zellulare Hinweise, um neue Knospen in einem vorgegebenen Muster auf der Mutterzelle Kortex zu positionieren. Hier zeigen wir, dass Rsr1 ist auch wichtig f&uuml;r die Orientierung der geflochtenen Tipp als Reaktion auf die externe Umwelt Cues, die thigmotropic ausl&ouml;sen und Galvanotropic Wachstum. Dar&uuml;ber hinaus Rsr1 geflochtenen Interaktionen mit epithelialen Zellen in vitro beteiligt ist und seine L&ouml;schung vermindert die geflochtenen Invasion von Nieren-Gewebe w&auml;hrend systemische Infektion. So ist Rsr1, ein Wahrzeichen der internen Polarit&auml;t in der Hefe, auch polarisierte Wachstum Antworten zu asymmetrischen Umweltsignale, ein Paradigma beteiligt, die f&uuml;r die Homologen Protein in S. Cerevisiae beschrieben unterscheidet. Rsr1 kann somit an der Pathogenese von C. Albicans Infektionen beitragen, durch Beeinflussung der geflochtenen Tipp Antworten ausgel&ouml;st durch Interaktion mit Host-Gewebe.

Eine interne Polarit&auml;t Wahrzeichen ist wichtig f&uuml;r extern induzierten geflochtenen Verhaltensweisen in Candida Albicans.

Crescita direzionale &egrave; una funzione delle cellule polarizzate come neuriti, tubi pollinici e ife fungine. Corretto orientamento della punta della cella si estende dipende dalla segnalazione percorsi ed effettori che mediano risposte asimmetriche a specifici stimoli ambientali. Nella forma ifale dell&#39;agente patogeno negli eucarioti fungo Candida albicans, queste risposte includono tigmotropismo e galvanotropism (ifali girando in risposta a cambiamenti nella topografia del substrato e imposti campi elettrici, rispettivamente) e penetrazione nei substrati semisolidi. Durante la crescita vegetativa in c. albicans, come il modello lievito Saccharomyces cerevisiae, il simil-Ras GTPase Rsr1 media interni cellulari spunti per posizionare nuovi germogli in un modello pre sulla corteccia della cellula madre. Qui, ci dimostrano che &egrave; importante per l&#39;orientamento in risposta agli stimoli ambientali esterni che inducono thigmotropic e galvanotropic crescita ifale punta anche Rsr1. Inoltre, Rsr1 &egrave; coinvolto in ifali interazioni con le cellule epiteliali in vitro e sua eliminazione diminuisce l&#39;invasione ifale dei tessuti renali durante l&#39;infezione sistemica. Cos&igrave;, Rsr1, un punto di riferimento interno polarit&agrave; lievito, &egrave; anche coinvolto nelle risposte di crescita polarizzata a segnali ambientali asimmetriche, un paradigma che &egrave; diverso da quello descritto per le proteine omologhe in S. cerevisiae. Rsr1 possono quindi contribuire alla patogenesi delle infezioni da c. albicans influenzando le risposte punta ifali innescate da interazione con i tessuti dell&#39;ospite.

Un punto di riferimento di polarit&agrave; interna &egrave; importante per comportamenti ifali indotti esternamente in Candida albicans.

&#x4F38;&#x9577;&#x65B9;&#x5411;&#x504F;&#x6CE2;&#x306E;&#x7D30;&#x80DE;&#x7A81;&#x8D77;&#x3001;&#x82B1;&#x7C89;&#x7BA1;&#x3001;&#x7CF8;&#x72B6;&#x83CC;&#x306E;&#x83CC;&#x7CF8;&#x306A;&#x3069;&#x306E;&#x6A5F;&#x80FD;&#x3067;&#x3059;&#x3002;&#x62E1;&#x5F35;&#x30BB;&#x30EB;&#x5148;&#x7AEF;&#x306E;&#x6B63;&#x3057;&#x3044;&#x5411;&#x304D;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x7D30;&#x9053;&#x304A;&#x3088;&#x3073;&#x975E;&#x5BFE;&#x79F0;&#x30EC;&#x30B9;&#x30DD;&#x30F3;&#x30B9;&#x7279;&#x5B9A;&#x74B0;&#x5883;&#x624B;&#x304C;&#x304B;&#x308A;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x30A8;&#x30D5;&#x30A7;&#x30AF;&#x30BF;&#x30FC;&#x306B;&#x3088;&#x3063;&#x3066;&#x7570;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x5FDC;&#x7B54;&#x306B;&#x771F;&#x6838;&#x751F;&#x7269;&#x306E;&#x771F;&#x83CC;&#x75C5;&#x539F;&#x4F53;&#x30AB;&#x30F3;&#x30B8;&#x30C0;&#x306E;&#x83CC;&#x7CF8;&#x5F62;&#x3067;&#x3001;thigmotropism &#x3068; galvanotropism &#xFF08;&#x83CC;&#x7CF8;&#x57FA;&#x677F;&#x5730;&#x5F62;&#x3068;&#x8AB2;&#x3055;&#x308C;&#x305F;&#x96FB;&#x6C17;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x3067;&#x306E;&#x5909;&#x66F4;&#x3078;&#x306E;&#x5FDC;&#x7B54;&#x3067;&#x305D;&#x308C;&#x305E;&#x308C;&#x56DE;&#x3059;&#xFF09; &#x534A;&#x56FA;&#x4F53;&#x57FA;&#x677F;&#x4E0A;&#x3078;&#x306E;&#x6D78;&#x900F;&#x304C;&#x3082;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x6804;&#x990A;&#x751F;&#x9577;&#x306B;&#x5BFE;&#x3059;&#x308B; c.albicans &#x30E2;&#x30C7;&#x30EB;&#x51FA;&#x82BD;&#x9175;&#x6BCD;&#x306E;&#x3088;&#x3046;&#x306B;&#x4E2D;&#x306B; Ras &#x306E;&#x3088;&#x3046;&#x306A; gtp &#x30A2;&#x30FC;&#x30BC; Rsr1 &#x6BCD;&#x7D30;&#x80DE;&#x76AE;&#x8CEA;&#x4E0A;&#x306E;&#x4E8B;&#x524D;&#x6307;&#x5B9A;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306E;&#x65B0;&#x3057;&#x3044;&#x82BD;&#x3092;&#x4F4D;&#x7F6E;&#x306B;&#x5185;&#x90E8;&#x306E;&#x7D30;&#x80DE;&#x624B;&#x304C;&#x304B;&#x308A;&#x3092;&#x4EF2;&#x4ECB;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;Rsr1 &#x3082; thigmotropic &#x3092;&#x8A98;&#x5C0E;&#x3059;&#x308B;&#x5916;&#x90E8;&#x74B0;&#x5883;&#x306E;&#x624B;&#x304C;&#x304B;&#x308A;&#x3068; galvanotropic &#x306E;&#x6210;&#x9577;&#x306E;&#x5FDC;&#x7B54;&#x3067;&#x306E;&#x83CC;&#x7CF8;&#x5148;&#x7AEF;&#x65B9;&#x5411;&#x306E;&#x305F;&#x3081;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;Rsr1 &#x3068;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x57F9;&#x990A;&#x83CC;&#x7CF8;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x3001;&#x305D;&#x306E;&#x524A;&#x9664;&#x814E;&#x81D3;&#x7D44;&#x7E54;&#x306E;&#x83CC;&#x7CF8;&#x4FB5;&#x5165;&#x5168;&#x8EAB;&#x611F;&#x67D3;&#x6642;&#x306B;&#x6E1B;&#x5C11;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;Rsr1&#x3001;&#x9175;&#x6BCD;&#x3001;&#x306A;&#x5185;&#x90E8;&#x6975;&#x6027;&#x30E9;&#x30F3;&#x30C9;&#x30DE;&#x30FC;&#x30AF;&#x3082;&#x504F;&#x5149;&#x6210;&#x9577;&#x5FDC;&#x7B54;&#x975E;&#x5BFE;&#x79F0;&#x74B0;&#x5883;&#x4FE1;&#x53F7;&#x306F;&#x3001;&#x76F8;&#x540C;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306B;&#x304A;&#x3051;&#x308B;&#x51FA;&#x82BD;&#x9175;&#x6BCD;&#x306E;&#x8AAC;&#x660E;&#x306F;&#x7570;&#x306A;&#x308B;&#x30D1;&#x30E9;&#x30C0;&#x30A4;&#x30E0;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;Rsr1 &#x306F;&#x305D;&#x308C;&#x306B;&#x3088;&#x308A;&#x5BBF;&#x4E3B;&#x7D44;&#x7E54;&#x3068;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3055;&#x308C;&#x308B;&#x83CC;&#x7CF8;&#x5148;&#x7AEF;&#x306E;&#x30EC;&#x30B9;&#x30DD;&#x30F3;&#x30B9;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x53CA;&#x307C;&#x3059; C. albicans &#x306B;&#x3088;&#x308B;&#x611F;&#x67D3;&#x75C7;&#x306E;&#x75C5;&#x56E0;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002;

&#x5185;&#x90E8;&#x306E;&#x6975;&#x6027;&#x30E9;&#x30F3;&#x30C9;&#x30DE;&#x30FC;&#x30AF;&#x306F;&#x30AB;&#x30F3;&#x30B8;&#x30C0;&#x3067;&#x5916;&#x90E8;&#x8A98;&#x5C0E;&#x306E;&#x83CC;&#x7CF8;&#x306E;&#x884C;&#x52D5;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;

&#xBC29;&#xD5A5; &#xC131;&#xC7A5; &#xD3B8;&#xAD11;&#xB41C; &#xC140; neurites, &#xAF43;&#xAC00;&#xB8E8; &#xAD00;, &#xACF0; &#xD321;&#xC774; &#xADE0; &#xB4F1;&#xC758; &#xAE30;&#xB2A5;&#xC785;&#xB2C8;&#xB2E4;. &#xD655;&#xC7A5; &#xC140; &#xD301;&#xC758; &#xC62C;&#xBC14;&#xB978; &#xBC29;&#xD5A5; &#xC2E0;&#xD638; &#xACBD;&#xB85C; &#xD2B9;&#xC815; &#xD658;&#xACBD; &#xB2E8;&#xC11C; &#xBE44;&#xB300;&#xCE6D; &#xC751;&#xB2F5; &#xC911;&#xC7AC; &#xC774;&#xD399;&#xD130;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. &#xC9C4; &#xD575; &#xADE0; &#xBCD1;&#xC6D0; &#xCCB4; Candida albicans&#xC758; hyphal &#xD615;&#xD0DC;&#xB85C; &#xC774;&#xB7EC;&#xD55C; &#xC751;&#xB2F5;&#xC5D0;&#xB294; thigmotropism &#xBC0F; galvanotropism (hyphal &#xC120;&#xD68C; &#xAE30;&#xD310; &#xC9C0;&#xD615;&#xACFC; &#xBD80;&#xACFC; &#xC804;&#xAE30; &#xD544;&#xB4DC; &#xBCC0;&#xACBD;&#xC5D0; &#xAC01;&#xAC01;) &#xADF8;&#xB9AC;&#xACE0; semisolid &#xAE30;&#xD310;&#xC73C;&#xB85C; &#xCE68;&#xD22C; &#xD3EC;&#xD568; &#xB429;&#xB2C8;&#xB2E4;. &#xBAA8;&#xB378; &#xD6A8; &#xBAA8; Saccharomyces cerevisiae&#xC5D0;&#xC11C; C. albicans&#xC758; &#xC2DD;&#xBB3C; &#xC131;&#xC7A5; &#xD558;&#xB294; &#xB3D9;&#xC548; Ras &#xAC19;&#xC740; GTPase Rsr1 &#xC5B4;&#xBA38;&#xB2C8; &#xC140; &#xD53C; &#xC9C8;&#xC5D0; prespecified &#xD328;&#xD134;&#xC5D0;&#xC11C; &#xC0C8;&#xB85C;&#xC6B4; &#xC2F9;&#xC744; &#xC704;&#xCE58;&#xB97C; &#xB0B4;&#xBD80; &#xC138;&#xD3EC; &#xC2E0;&#xD638;&#xB97C; &#xC911;&#xC7AC;. &#xC5EC;&#xAE30;&#xC11C;, rsr1&#xC740; thigmotropic&#xC744; &#xC720;&#xBC1C; &#xD558;&#xB294; &#xC678;&#xBD80; &#xD658;&#xACBD; &#xB2E8;&#xC11C;&#xC640; galvanotropic &#xC131;&#xC7A5;&#xC5D0; &#xB300; &#xD55C; &#xC751;&#xB2F5;&#xC5D0;&#xC11C; hyphal &#xD301; &#xBC29;&#xD5A5;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xB610;&#xD55C; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, rsr1&#xC740; &#xC0C1;&#xD53C; &#xC138;&#xD3EC; &#xCCB4; &#xC678; hyphal &#xC0C1;&#xD638; &#xAD00;&#xC5EC; &#xBC0F; &#xC0AD;&#xC81C;&#xD560; &#xAC10;&#xC18C; &#xC870;&#xC9C1;&#xC758; &#xAC10;&#xC5FC; &#xC2DC; &#xC2E0;&#xC7A5; &#xC870;&#xC9C1; hyphal &#xCE68;&#xACF5;. &#xB530;&#xB77C;&#xC11C;, Rsr1, &#xD6A8; &#xBAA8;&#xC5D0; &#xB0B4;&#xBD80; &#xADF9;&#xC131; &#xB79C;&#xB4DC;&#xB9C8;&#xD06C;&#xB3C4; &#xBE44;&#xB300;&#xCE6D; &#xD658;&#xACBD; &#xC2E0;&#xD638;&#xB294; S. cerevisiae&#xC5D0; &#xB3D9;&#xC885; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xB300; &#xD55C; &#xC124;&#xBA85; &#xD558;&#xB294; &#xB2E4;&#xB978; &#xD328;&#xB7EC;&#xB2E4;&#xC784;&#xC744; &#xD3B8;&#xAD11;&#xB41C; &#xC131;&#xC7A5; &#xBC18;&#xC751;&#xC5D0; &#xCC38;&#xC5EC; &#xD558;&#xACE0;&#xC788;&#xB2E4;. Rsr1 &#xD568;&#xC73C;&#xB85C;&#xC368; hyphal &#xD301; &#xC751;&#xB2F5; &#xD638;&#xC2A4;&#xD2B8; &#xC870;&#xC9C1;&#xC640;&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0; &#xC758;&#xD574; &#xC2E4;&#xD589;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xC758;&#xD574; C. albicans &#xAC10;&#xC5FC; &#xBCD1;&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xB0B4;&#xBD80; &#xADF9;&#xC131; &#xB79C;&#xB4DC;&#xB9C8;&#xD06C; Candida albicans&#xC5D0; &#xC678;&#xBD80;&#xC5D0;&#xC11C; &#xC720;&#xB3C4; &#xB41C; hyphal &#xB3D9;&#xC791;&#xC744; &#xC704;&#xD574; &#xC911;&#xC694; &#xD558;&#xB2E4;.

Crecimiento direccional es una funci&oacute;n de c&eacute;lulas polarizadas como neuritas, tubos de polen e hifas de los hongos. Orientaci&oacute;n correcta de la punta extensible de la c&eacute;lula depende de se&ntilde;alizaci&oacute;n v&iacute;as y efectores que median respuestas asim&eacute;tricas a se&ntilde;ales ambientales espec&iacute;ficos. En forma hyphal de los eucariota hongo pat&oacute;geno Candida albicans, estas respuestas incluyen Tigmotropismo y galvanotropism (hifal convirtiendo en respuesta a cambios en la topograf&iacute;a de sustrato y campos el&eacute;ctricos impuestos, respectivamente) y penetraci&oacute;n en sustratos semis&oacute;lidos. Durante el crecimiento vegetativo en C. albicans, como en el modelo levadura Saccharomyces cerevisiae, el Ras-como GTPase Rsr1 media se&ntilde;ales celulares internos para colocar nuevos brotes en un patr&oacute;n preespecificado en la corteza de la c&eacute;lula madre. Aqu&iacute;, demostramos que Rsr1 tambi&eacute;n es importante para la orientaci&oacute;n de la punta hifal en respuesta a se&ntilde;ales ambientales externos que inducen thigmotropic los crecimiento de galvanotropic. Adem&aacute;s, Rsr1 participa en hifal interacciones con las c&eacute;lulas epiteliales in vitro y su eliminaci&oacute;n disminuye la invasi&oacute;n hifal de tejido renal durante la infecci&oacute;n sist&eacute;mica. As&iacute;, Rsr1, un hito de la polaridad interna en la levadura, tambi&eacute;n participa en las respuestas de crecimiento polarizado a se&ntilde;ales ambientales asim&eacute;tricas, un paradigma que difiere de lo descrito para la prote&iacute;na hom&oacute;loga en S. cerevisiae. Rsr1 puede contribuir a la patog&eacute;nesis de las infecciones de C. albicans por influir en las respuestas de punta hifal provocadas por la interacci&oacute;n con los tejidos del hu&eacute;sped.

Un hito de la polaridad interna es importante inducidos externamente comportamientos hifal en Candida albicans.

The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Only sla2 strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis of C. albicans infections.

SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae.

Life-threatening gastrointestinal (GI) diseases of prematurity are highly associated with systemic candidiasis. This implicates the premature GI tract as an important site for invasion by Candida. Invasive interactions of Candida spp. with immature enterocytes have heretofore not been analyzed. Using a primary immature human enterocyte line, we compared the ability of multiple isolates of different Candida spp. to penetrate, injure, and induce a cytokine response from host cells. Of all the Candida spp. analyzed, C. albicans had the greatest ability to penetrate and injure immature enterocytes and to elicit IL-8 release (p < 0.01). In addition, C. albicans was the only Candida spp. to form filamentous hyphae when in contact with immature enterocytes. Similarly, a C. albicans mutant with defective hyphal morphogenesis and invasiveness had attenuated cytotoxicity for immature enterocytes (p < 0.003). Thus, hyphal morphogenesis correlates with immature enterocyte penetration, injury, and inflammatory responses. Furthermore, variability in enterocyte injury was observed among hyphal-producing C. albicans strains, suggesting that individual organism genotypes also influence host-pathogen interactions. Overall, the finding that Candida spp. differed in their interactions with immature enterocytes implicates that individual spp. may use different pathogenesis mechanisms.

Candida species differ in their interactions with immature human gastrointestinal epithelial cells.

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 "fine-tunes" the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis.

Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans.

Hyphae of filamentous fungi maintain generally linear growth over long distances. In Candida albicans, hyphae are able to reorient their growth in the direction of certain environmental cues. In previous work, the C. albicans bud-site selection proteins Rsr1 and Bud2 were identified as important for hyphae to maintain linear growth and were necessary for hyphal responses to directional cues in the environment (tropisms). To ask if hyphal directional responses are general functions of all yeast bud-site selection proteins, we studied the role of Rax2, ortholog of the Saccharomyces cerevisiae bud-site selection protein Rax2, in C. albicans hyphal morphogenesis. Rax2-YFP localized to the hyphal cell surface in puncta and at the hyphal tip in a crescent. Strains lacking Rax2 had hyphal morphologies that did not differ from control strains. In non-cued growth conditions, rax2 mutant strains had defects in both yeast (bud) and hyphal (branch) site selection and mutant hyphae exhibited non-linear growth trajectories as compared to control hyphae. In contrast, when encountering a directional environmental cue, hyphae lacking Rax2 retained the ability to reorient growth in response to both topographical (thigmotropism) and electric-field (galvanotropism) stimuli but exhibited a reduced ability to establish hyphal growth in the direction of a cathodal stimulus. In conclusion, these results indicate that C. albicans Rax2 is important for establishing sites of emergence of yeast and hyphal daughters and for maintaining the linearity of hyphal growth. In contrast to Rsr1 and Bud2, Rax2 is not involved in responses that require a reorientation of the direction of already established hyphal growth (tropisms). Thus, it appears that some hyphal directionality responses are separable in that they are mediated by a different set of polarity proteins.

Rax2 is important for directional establishment of growth sites, but not for reorientation of growth axes, during Candida albicans hyphal morphogenesis.

Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca(2+) influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca(2+) influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca(2+). Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca(2+). Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca(2+). The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca(2+) influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca(2+)-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance.

Cdc42 GTPase dynamics control directional growth responses.

Recent studies highlight the importance of intestinal fungal microbiota in the development of human disease. Infants, in particular, are an important population in which to study intestinal microbiomes because microbial community structure and dynamics during this formative window of life have the potential to influence host immunity and metabolism. When compared to bacteria, much less is known about the early development of human fungal communities, owing partly to their lower abundance and the relative lack of established molecular and taxonomic tools for their study. Herein, we describe the development, validation, and use of complementary amplicon-based genomic strategies to characterize infant fungal communities and provide quantitative information about Candida, an important fungal genus with respect to intestinal colonization and human disease. Fungal communities were characterized from 11 infant fecal samples using primers that target the internal transcribed spacer (ITS) 2 locus, a region that provides taxonomic discrimination of medically relevant fungi. Each sample yielded an average of 27,553 fungal sequences and Candida albicans was the most abundant species identified by sequencing and quantitative PCR (qPCR). Low numbers of Candida krusei and Candida parapsilosis sequences were observed in several samples, but their presence was detected by species-specific qPCR in only one sample, highlighting a challenge inherent in the study of low-abundance organisms. Overall, the sequencing results revealed that infant fecal samples had fungal diversity comparable to that of bacterial communities in similar-aged infants, which correlated with the relative abundance of C. albicans. We conclude that targeted sequencing of fungal ITS2 amplicons in conjunction with qPCR analyses of specific fungi provides an informative picture of fungal community structure in the human intestinal tract. Our data suggests that the infant intestine harbors diverse fungal species and is consistent with prior culture-based analyses showing that the predominant fungus in the infant intestine is C. albicans.

Complementary amplicon-based genomic approaches for the study of fungal communities in humans.

Human milk oligosaccharides (HMOs) are a highly abundant, diverse group of unique glycans that are postulated to promote the development of a protective bacterial microbiota in the intestine and prevent adhesive and invasive interactions of pathogenic bacteria with mucosal epithelia. Candida albicans, a prevalent fungal colonizer of the neonatal gut, causes the majority of fungal disease in premature infants and is highly associated with life-threatening intestinal disorders.

Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells.

Candida parapsilosis is a common cause of invasive candidiasis, especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans, relatively few have been developed for C. parapsilosis. In this study, we show that plasmids harbouring green, yellow and mCherry fluorescent protein sequences, previously developed for expression in C. albicans, can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis, which are typically prototrophic, because the plasmids include NAT1, a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers who require the ability to visualize C. parapsilosis directly, e.g. in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models.

PCR-mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis.

The microbes colonizing the infant gastrointestinal tract have been implicated in later-life disease states such as allergies and obesity. Recently, the medical research community has begun to realize that very early colonization events may be most impactful on future health, with the presence of key taxa required for proper immune and metabolic development. However, most studies to date have focused on bacterial colonization events and have left out fungi, a clinically important sub-population of the microbiota. A number of recent findings indicate the importance of host-associated fungi (the mycobiota) in adult and infant disease states, including acute infections, allergies, and metabolism, making characterization of early human mycobiota an important frontier of medical research. This review summarizes the current state of knowledge with a focus on factors influencing infant mycobiota development and associations between early fungal exposures and health outcomes. We also propose next steps for infant fungal mycobiome research, including longitudinal studies of mother-infant pairs while monitoring long-term health outcomes, further exploration of bacterium-fungus interactions, and improved methods and databases for mycobiome quantitation.

Article	Year
PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance. Molecular microbiology\| PubMed ID: 15659169	2005
Hyphal guidance and invasive growth in Candida albicans require the Ras-like GTPase Rsr1p and its GTPase-activating protein Bud2p. Eukaryotic cell\| PubMed ID: 16002653	2005
Intracellular trafficking of fluorescently tagged proteins associated with pathogenesis in Candida albicans. Medical mycology\| PubMed ID: 16178371	2005
An internal polarity landmark is important for externally induced hyphal behaviors in Candida albicans. Eukaryotic cell\| PubMed ID: 18281602	2008
SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae. Microbiology (Reading, England)\| PubMed ID: 19778960	2009
Candida species differ in their interactions with immature human gastrointestinal epithelial cells. Pediatric research\| PubMed ID: 21283049	2011
Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans. Eukaryotic cell\| PubMed ID: 23223038	2013
Rax2 is important for directional establishment of growth sites, but not for reorientation of growth axes, during Candida albicans hyphal morphogenesis. Fungal genetics and biology : FG & B\| PubMed ID: 23608319	2013
Cdc42 GTPase dynamics control directional growth responses. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24385582	2014
Complementary amplicon-based genomic approaches for the study of fungal communities in humans. PloS one\| PubMed ID: 25706290	2015
Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells. The Journal of nutrition\| PubMed ID: 26180242	2015
PCR-mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis. Yeast (Chichester, England)\| PubMed ID: 26551241	2016
Infant fungal communities: current knowledge and research opportunities. BMC medicine\| PubMed ID: 28190400	2017

Cheryl A. Gale

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