Poster Session : Model Organisms and Disease ; Ssk2 MAPKKK governs the unique regulation of the stress-activated hog1 pathway in human fungal pathogen cryptococcus neoformans

( Yong Sun Bahn ),( Young Joon Ko ),( Joseph Heitman ) 한국생화학분자생물학회 (구 한국생화학회) 2007 생화학분자생물학회 춘계학술발표논문집 Vol.2007 No.-

Unique Evolution of the UPR Pathway with a Novel bZIP Transcription Factor, Hxl1, for Controlling Pathogenicity of <i>Cryptococcus neoformans</i>

Cheon, Seon Ah,Jung, Kwang-Woo,Chen, Ying-Lien,Heitman, Joseph,Bahn, Yong-Sun,Kang, Hyun Ah Public Library of Science 2011 PLoS pathogens Vol.7 No.8

<▼1><P>In eukaryotic cells, the unfolded protein response (UPR) pathway plays a crucial role in cellular homeostasis of the endoplasmic reticulum (ER) during exposure to diverse environmental conditions that cause ER stress. Here we report that the human fungal pathogen <I>Cryptococcus neoformans</I> has evolved a unique UPR pathway composed of an evolutionarily conserved Ire1 protein kinase and a novel bZIP transcription factor encoded by <I>HXL1</I> (<I><U>H</U>AC1</I> and <I><U>X</U>BP1</I>-<I><U>L</U></I>ike gene <I>1</I>). <I>C. neoformans HXL1</I> encodes a protein lacking sequence homology to any known fungal or mammalian Hac1/Xbp1 protein yet undergoes the UPR-induced unconventional splicing in an Ire1-dependent manner upon exposure to various stresses. The structural organization of <I>HXL1</I> and its unconventional splicing is widely conserved in <I>C. neoformans</I> strains of divergent serotypes. Notably, both <I>C. neoformans ire1</I> and <I>hxl1</I> mutants exhibited extreme growth defects at 37°C and hypersensitivity to ER stress and cell wall destabilization. All of the growth defects of the <I>ire1</I> mutant were suppressed by the spliced active form of Hxl1, supporting that <I>HXL1</I> mRNA is a downstream target of Ire1. Interestingly, however, the <I>ire1</I> and <I>hxl1</I> mutants showed differences in thermosensitivity, expression patterns for a subset of genes, and capsule synthesis, indicating that Ire1 has both Hxl1-dependent and -independent functions in <I>C. neoformans</I>. Finally, Ire1 and Hxl1 were shown to be critical for virulence of <I>C. neoformans</I>, suggesting UPR signaling as a novel antifungal therapeutic target.</P></▼1><▼2><P><B>Author Summary</B></P><P>The unfolded protein response (UPR) is a widely conserved signaling pathway in eukaryotic cells and protects cells from the ER stress causing accumulation of toxic unfolded or misfolded proteins. Nevertheless, the UPR pathway has been poorly exploited as a therapeutic target for treatment of eukaryotic fungal pathogens, mainly due to its evolutionarily conserved features. The present study reports unique evolution of the UPR pathway in the basidiomycetous human fungal pathogen, <I>Cryptococcus neoformans</I>, which causes life-threatening meningoencephalitis in both immunocompromised and immunocompetent individuals. Here, for the first time we identified and characterized the <I>C. neoformans</I> UPR pathway, which is composed of an evolutionarily conserved and a distinct signaling component, an ER stress sensor Ire1 and its downstream bZIP transcription factor Hxl1, respectively. Intriguingly, <I>Cryptococcus</I> Hxl1 is very divergent from yeast Hac1 and human Xbp1, but subject to Ire1-mediated unconventional splicing. The Ire1-Hxl1-dependent UPR pathway functions not only in the major response to ER stress, but also plays critical roles in controlling cell wall integrity, growth at host physiological temperature, antifungal drug susceptibility, and virulence of <I>C. neoformans</I>. Therefore we propose Hxl1 is an ideal target for antifungal drug development, based on its marked divergence from the host Xbp1 transcription factor.</P></▼2>

Pleiotropic Roles of the Msi1-Like Protein Msl1 in Cryptococcus neoformans

Yang, Dong-Hoon,Maeng, Shinae,Strain, Anna K.,Floyd, Anna,Nielsen, Kirsten,Heitman, Joseph,Bahn, Yong-Sun American Society for Microbiology 2012 EUKARYOTIC CELL Vol.11 No.12

<B>ABSTRACT</B><P> Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans , which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans . Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as <I>HSP12</I> and <I>HSP78</I> . In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans , providing insight into a potential novel antifungal therapeutic target. </P>

Comparative Transcriptome Analysis of the CO₂ Sensing Pathway Via Differential Expression of Carbonic Anhydrase in <i>Cryptococcus neoformans</i>

Kim, Min Su,Ko, Young-Joon,Maeng, Shinae,Floyd, Anna,Heitman, Joseph,Bahn, Yong-Sun The Genetics Society of America 2010 Genetics Vol.185 No.4

Remodeling of Global Transcription Patterns of Cryptococcus neoformans Genes Mediated by the Stress-Activated HOG Signaling Pathways

Ko, Young-Joon,Yu, Yeong Man,Kim, Gyu-Bum,Lee, Gir-Won,Maeng, Pil Jae,Kim, Sangsoo,Floyd, Anna,Heitman, Joseph,Bahn, Yong-Sun American Society for Microbiology 2009 EUKARYOTIC CELL Vol.8 No.8

<B>ABSTRACT</B><P>The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including <I>Cryptococcus neoformans</I>. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in <I>C. neoformans</I>. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in <I>C. neoformans</I>. In this study, we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses but also elucidate novel characteristics of the HOG pathway in <I>C. neoformans</I>. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress defense and HOG-dependent genes, which encode a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system.</P>

A Flucytosine-Responsive Mbp1/Swi4-Like Protein, Mbs1, Plays Pleiotropic Roles in Antifungal Drug Resistance, Stress Response, and Virulence of Cryptococcus neoformans

Song, Min-Hee,Lee, Jang-Won,Kim, Min Su,Yoon, Ja-Kyung,White, Theodore C.,Floyd, Anna,Heitman, Joseph,Strain, Anna K.,Nielsen, Judith N.,Nielsen, Kirsten,Bahn, Yong-Sun American Society for Microbiology 2012 EUKARYOTIC CELL Vol.11 No.1

<B>ABSTRACT</B><P> Cryptococcosis, caused by the basidiomycetous fungus Cryptococcus neoformans , is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale in C. neoformans . In the present study, we performed comparative transcriptome analysis by employing two-component system mutants ( <I>tco1</I> Δ and <I>tco2</I> Δ) exhibiting opposing flucytosine susceptibility. As a result, a total of 177 flucytosine-responsive genes were identified, and many of them were found to be regulated by Tco1 or Tco2. Among these, we discovered an APSES-like transcription factor, Mbs1 ( Mb p1- and S wi4-like protein 1). Expression analysis revealed that <I>MBS1</I> was regulated in response to flucytosine in a Tco2/Hog1-dependent manner. Supporting this, C. neoformans with the deletion of <I>MBS1</I> exhibited increased susceptibility to flucytosine. Intriguingly, Mbs1 played pleiotropic roles in diverse cellular processes of C. neoformans . Mbs1 positively regulated ergosterol biosynthesis and thereby affected polyene and azole drug susceptibility. Mbs1 was also involved in genotoxic and oxidative stress responses. Furthermore, Mbs1 promoted production of melanin and capsule and thereby was required for full virulence of C. neoformans . In conclusion, Mbs1 is considered to be a novel antifungal therapeutic target for treatment of cryptococcosis. </P>

Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans

Maeng, Shinae,Ko, Young-Joon,Kim, Gyu-Bum,Jung, Kwang-Woo,Floyd, Anna,Heitman, Joseph,Bahn, Yong-Sun American Society for Microbiology 2010 EUKARYOTIC CELL Vol.9 No.3

<B>ABSTRACT</B><P>The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the <I>ras1</I>Δ, <I>gpa1</I>Δ, <I>cac1</I>Δ, <I>aca1</I>Δ, and <I>pka1</I>Δ <I>pka2</I>Δ mutants. The <I>aca1</I>Δ, <I>gpa1</I>Δ, <I>cac1</I>Δ, and <I>pka1</I>Δ <I>pka2</I>Δ mutants displayed similar transcriptome patterns, whereas the <I>ras1</I>Δ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the <I>ras1</I>Δ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans.</P>

<i>In Vitro</i> and <i>In Vivo</i> Assessment of FK506 Analogs as Novel Antifungal Drug Candidates

Lee, Yeonseon,Lee, Kyung-Tae,Lee, Soo Jung,Beom, Ji Yoon,Hwangbo, Areum,Jung, Jin A,Song, Myoung Chong,Yoo, Young Ji,Kang, Sang Hyeon,Averette, Anna F.,Heitman, Joseph,Yoon, Yeo Joon,Cheong, Eunji,Bah American Society for Microbiology 2018 Antimicrobial Agents and Chemotherapy Vol.62 No.11

<P>FK506 (tacrolimus) is an FDA-approved immunosuppressant indicated for the prevention of allograft rejections in patients undergoing organ transplants. In mammals, FK506 inhibits the calcineurin-nuclear factor of activated T cells (NFAT) pathway to prevent T-cell proliferation by forming a ternary complex with its binding protein, FKBP12, and calcineurin. FK506 also exerts antifungal activity by inhibiting calcineurin, which is essential for the virulence of human-pathogenic fungi. Nevertheless, FK506 cannot be used directly as an antifungal drug due to its immunosuppressive action. In this study, we analyzed the cytotoxicity, immunosuppressive activity, and antifungal activity of four FK506 analogs, 31-O-demethyl-FK506, 9-deoxo-FK506, 9-deoxo-31-O-demethyl-FK506, and 9-deoxo-prolyl-FK506, in comparison with that of FK506. The four FK506 analogs generally possessed lower cytotoxicity and immunosuppressive activity than FK506. The FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against Ctyptococcus neoformans and Candida albicans, which are two major invasive pathogenic yeasts, due to the inhibition of the calcineurin pathway. Furthermore, the FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against the invasive filamentous fungus Aspergillus fumigatus. Notably, 9-deoxo-31-O-demethyl-FK506 and 31-O-demethyl-FK506 exhibited robust synergistic antifungal activity with fluconazole, similar to FK506. Considering the antifungal efficacy, cytotoxicity, immunosuppressive activity, and synergistic effect with commercial antifungal drugs, we selected 9-deoxo-31-O-demethyl-FK506 for further evaluation of its in vivo antifungal efficacy in a murine model of systemic cryptococcosis. Although 9-deoxo-31-O-demethyl-FK506 alone was not sufficient to treat the cryptococcal infection, when it was used in combination with fluconazole, it significantly extended the survival of C. neoformans-infected mice, confirming the synergistic in vivo antifungal efficacy between these two agents.</P>

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen <i>Cryptococcus neoformans</i>

Yang, Dong-Hoon,Jung, Kwang-Woo,Bang, Soohyun,Lee, Jang-Won,Song, Min-Hee,Floyd-Averette, Anna,Festa, Richard A.,Ianiri, Giuseppe,Idnurm, Alexander,Thiele, Dennis J.,Heitman, Joseph,Bahn, Yong-Sun The Genetics Society of America 2017 Genetics Vol.205 No.1

Gastrointestinal microbiota alteration induced by Mucor circinelloides in a murine model

Katherine D. Mueller,Hao Zhang,Christian R. Serrano,R. Blake Billmyre,Eun Young Huh,Philipp Wiemann,Nancy P. Keller,Yufeng Wang,Joseph Heitman,Soo Chan Lee 한국미생물학회 2019 The journal of microbiology Vol.57 No.6

Mucor circinelloides is a pathogenic fungus and etiologic agent of mucormycosis. In 2013, cases of gastrointestinal illness after yogurt consumption were reported to the US FDA, and the producer found that its products were contaminated with Mucor. A previous study found that the Mucor strain isolated from an open contaminated yogurt exhibited virulence in a murine systemic infection model and showed that this strain is capable of surviving passage through the gastrointestinal tract of mice. In this study, we isolated another Mucor strain from an unopened yogurt that is closely related but distinct from the first Mucor strain and subsequently examined if Mucor alters the gut microbiota in a murine host model. DNA extracted from a ten-day course of stool samples was used to analyze the microbiota in the gastrointestinal tracts of mice exposed via ingestion of Mucor spores. The bacterial 16S rRNA gene and fungal ITS1 sequences obtained were used to identify taxa of each kingdom. Linear regressions revealed that there are changes in bacterial and fungal abundance in the gastrointestinal tracts of mice which ingested Mucor. Furthermore, we found an increased abundance of the bacterial genus Bacteroides and a decreased abundance of the bacteria Akkermansia muciniphila in the gastrointestinal tracts of exposed mice. Measurements of abundances show shifts in relative levels of multiple bacterial and fungal taxa between mouse groups. These findings suggest that exposure of the gastrointestinal tract to Mucor can alter the microbiota and, more importantly, illustrate an interaction between the intestinal mycobiota and bacteriota. In addition, Mucor was able to induce increased permeability in epithelial cell monolayers in vitro, which might be indicative of unstable intestinal barriers. Understanding how the gut microbiota is shaped is important to understand the basis of potential methods of treatment for gastrointestinal illness. How the gut microbiota changes in response to exposure, even by pathogens not considered to be causative agents of food-borne illness, may be important to how commercial food producers prevent and respond to contamination of products aimed at the public. This study provides evidence that the fungal microbiota, though understudied, may play an important role in diseases of the human gut.