Dataset Information

Fungal-bacterial co-infection of macrophages reveals global reprogramming and adaptation during infection

ABSTRACT: In this study, we use mass spectrometry (MS)-based proteomics to define the host response under single and dual infection states of macrophages with C. neoformans and K. pneumoniae to define critical responses of each biological system. This high-resolution comparative analysis illustrates how protein abundance deviates during a transition from an acute to chronic infectious state with exposure to additional microbial stimuli. We reveal global changes upon infection followed by pathogen-specific host response signatures. Additionally, we define regulatory changes within C. neoformans as the fungi adapts to the host environment and stabilizes prior to further disruption in the presence of chronic bacterial infection. We validate our findings with host cytokine detection and phenotypic profiling of the fungi throughout the host and bacterial exposures. Overall, our study provides an in-depth analysis of cross-kingdom protein level changes during macrophage infection. This information provides new insight into fungal modulation of the immune response, including a stabilization and adaptation of the host and fungi during a chronic infection, which is disrupted upon chronic co-infection with a bacterial pathogen.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Cryptococcus Neoformans Var. Grubii Serotype A (strain H99 / Atcc 208821 / Cbs 10515 / Fgsc 9487) (filobasidiella Neoformans Var. Grubii) Klebsiella Pneumoniae Mus Musculus (mouse)

TISSUE(S): Monocyte

SUBMITTER: Jennifer Geddes-McAlister

LAB HEAD: Jennifer Geddes-McAlister

PROVIDER: PXD033393 | Pride | 2022-06-30

REPOSITORIES: Pride

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Project description:The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. 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 C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses, but also elucidated novel characteristics of the HOG pathway in C. neoformans. 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 encodes a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system. There is more than 95% genome homology between JEC21 (Cryptococcus neoformans var. neoformans serotype D) and H99 (Cryptococcus neoformans var. grubii serotype A). Therefore, 100 slides of JEC21 70-mer oligo arrays are used in this analysis. 3 biological replicate experiments are performed. Total RNAs are extracted under 3 conditions (1M NaCl, 20ug/ul Fludioxonil, 2.5mM Hydrogen peroxide) at 3 time points (0time, 30min, 60min) with 4 strains from H99 (wild type, hog1Î , ssk1Î , skn7Î). We use a mix of all the total RNAs from this experiment as the control RNA. We use Cy5 as the sample dye and Cy3 as the control dye. Several samples are dye swapped.

Project description:Carbon dioxide (CO2) sensing, transport, and metabolism play a pivotal role in survival and proliferation of pathogenic microbes infecting human host from natural environments due to the drastic difference of CO2 levels. Carbonic anhydrases (CAs) are not only key CO2-metabolic enzymes catalyzing reversible interconversion between CO2 and bicarbonate (HCO3-), but also important CO2-signaling modulators. Cryptococcus neoformans that causes fatal fungal meningitis contains two functional CAs, Can1 and Can2, among which Can2 plays essential roles for growth and sexual differentiation of the pathogen. However, downstream genes and signaling network regulated by CAs have not been studied thus far. In this study, we constructed a C. neoformans strain where CAN2 expression is controlled by the CTR4 (copper transporter) promoter and elucidated its transcriptome patterns by using DNA microarray analysis to elucidate downstream target genes of Can2. Expectedly, the CAN2 promoter replacement strain showed growth defects in a CO2-dependent manner when CAN2 is repressed. The CA-transcriptome analysis discovered a number of Can2-dependent genes, including those involved in fatty acid biosynthesis (FAS1), sexual differentiation (GPB1), capsule structure organization (CAS3), iron-metabolism (CFO2), and a number of stress-regulated genes, including an oxidative stress-responsive Atf1 transcription factor, although a majority of them do not have any orthologs in other fungi. The present study revealed other roles of Atf1 in capsule and melanin production and diverse stress responses, including thermotolerance and antifungal drug resistance, of C. neoformans. This study provides further insights into the signaling network of CA/CO2-sensing pathway in pathogenic fungi. 17 slides are used in this analysis, 3 or 2 biological replicate experiments are performed, total RNAs are extracted from 2 strains (H99 Wild type strain, CTR4::CAN2) at 2 time points (0hr time, 12hr time) in 2 conditions (BCS, CuSO4). We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye. Several experiment samples are dye swaped.

Project description:The cAMP-pathway plays a central role in regulation of growth, differentiation, and virulence of human pathogenic fungi, including Cryptococcus neoformans. Three major upstream signaling regulators of the adenylyl cyclase (Cac1), Ras, Aca1 (Adenylyl cyclase-associated protein 1) and G-alpha subunit protein (Gpa1), have been identified to control the cAMP-pathway in C. neoformans, but their functional relationship remains elusive. Here we performed genome-wide transcriptome analysis with C. neoformans ras1, gpa1, cac1, aca1, and pka1 pka2 mutants by DNA microarray. The aca1, gpa1, cac1, and pka1 pka2 mutants displayed similar transcriptome patterns to each other whereas the ras1 mutant exhibited distinctive transcriptome patterns compared to WT and the cAMP mutants. Interestingly, a number of environmental stress response genes are differentially modulated in the ras1 and cAMP mutants. In fact, the Ras1-signaling pathway was found to be involved in osmotic and genotoxic stress response, and maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Through this microarray analysis, we have identified a number of cAMP-dependent genes, including GRE2, HSP12, ENA1, TCO2, PKP2, CAT1, in C. neoformans. Notably, a majority of ergosterol biosynthesis genes were found to be upregulated in the cAMP mutants. Interestingly, the gpa1, cac1, and pka1 mutants, but not the aca1 and pka2 mutants were hypersensitive to amphotericin B, but resistant to fluconazole. In conclusion, we demonstrated in this study that the Ras1- and cAMP-signaling pathways are involved in stress response and sterol biosynthesis of C. neoformans. There are more than 95% of genome homology between JEC21 and H99. Therefore 100 slides of JEC21 (cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted with 6 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), ras1Î, aca1Î, gpa1Î, cac1Î, pka1Îpka2Î), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye. several sample are dye swaped.

Project description:The high osmolarity glycerol response (HOG) pathway plays a pivotal role in the stress response, virulence regulation, and differentiation of fungi, including Cryptococcus neoformans that causes fatal meningoencephalitis. Core signaling components of in the HOG pathway, including the Tco-Ypd1-Ssk1 phosphorelay system and the Ssk2-Pbs2-Hog1 MAPK module, have been elucidated but its downstream transcription factors remain unclear. Here we demonstrated that Atf1 with a basic leucine zipper domain is the transcription factor downstream of Hog1 in C. neoformans. We found that ATF1 expression was differentially regulated by oxidative damaging agents, mainly in a Hog1-dependent, but Mpk1-independent, manner. Interestingly, Atf1 not only promoted oxidative stress response and adaptation, but also played an opposing role to Hog1 in the process. Atf1 primarily localized to the nucleus under both unstressed and oxidative stress conditions in a Hog1-independent manner. Our data demonstrated that Atf1 promoted pheromone production and sexual differentiation under negative control by Hog1. Finally, a DNA microarray-based transcriptome analysis of the atf1M-bM-^HM-^F mutant under unstressed and oxidative stress conditions revealed that Atf1 regulated oxidative stress response genes, including a sulfiredoxin gene (SRX1). Intriguing, the array data further demonstrated that Atf1 modulated basal expression of genes involved in DNA repair and genotoxic stress response. Supporting this, we found that the atf1M-bM-^HM-^F mutant was highly sensitive to genotoxic agents. In conclusion, this study provided a further insight into the Hog1-dependent oxidative and genotoxic stress response and differentiation mechanism of C. neoformans. There are more than 95% of genome homology between JEC21 and H99. Therefore 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted 2 conditions (with or without treatment of Hydroten peroxide) from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), and atf1M-NM-^T). We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy3 as Sample dye and Cy5 as a control dye.

Project description:A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, Msa1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of MSA1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the MSA1 gene exhibited increased susceptibility to flucytosine. Intriguingly, Msa1 plays pleiotropic roles in diverse cellular process of C. neoformans. Msa1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. Msa1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation Msa1 represses pheromone production, but promotes cell-cell fusion. Furthermore Msa1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify Msa1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis. There are more than 95% of genome homology between JEC21 and H99. Therefore 24 slides of JEC21 (cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted under 2 conditions (with or without treatment of Flucytosine) with 4 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), tco1Δ , tco2Δ , skn7Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

Project description:WD40 motif-containing Msi1-like (MSIL) proteins play pleiotropic cellular functions as a negative regulator of the Ras/cAMP-pathways and a component of chromatin assembly factor-I (CAF-I), and yet have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which can cause fatal meningoencephalitis in humans. Notably, Msl1 was not a functional ortholog for the yeast Msi1 but played pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners but mainly Ras-independently. Msl1 negatively controlled antioxidant melanin production and sexual differentiation, which can be repressed by inhibiting the cAMP-signaling pathways. In contrast, Msl1 controlled thermotolerance, diverse stress responses, and antifungal drugs resistance in Ras/cAMP-independent manners. Cac2, which is the second CAF-I component, appeared to play both redundant and distinct function with Msl1. Msl1 is required for full virulence of C. neoformans. Transcriptome and proteomic analysis identified a group of Msl1-regulated genes or -interacting proteins, respectively, which mostly include stress-related genes, including HSP12, HSP78, SSA1, SSA4, and STM1. Furthermore, we identified the third putative component of CAF-1, Rlf2, in C. neoformans. In conclusion, this study demonstrated the pleiotropic roles of Msl1 in human fungal pathogen C. neoformans, providing a novel antifungal therapeutic target. There is more than 95% genome homology between JEC21 and H99. Therefore, 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligos are used in this analysis. Total RNAs are extracted from 2 strains from H99 (H99 wild-type strain (Cryptococcus neoformans var. grubii serotype A), msl1M-NM-^T). 3 biological replicate experiments are performed for each strain. We use the mix of all total RNAs from this experiment as the control RNA. We use Cy3 as the test sample dye and Cy5 as the control dye.

Project description:A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, mbs1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of mbs1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the mbs1 gene exhibited increased susceptibility to flucytosine. Intriguingly, mbs1 plays pleiotropic roles in diverse cellular process of C. neoformans. mbs1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. mbs1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation mbs1 represses pheromone production, but promotes cell-cell fusion. Furthermore mbs1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify mbs1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis. total RNAs are extracted 2 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), mbs1Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

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Fungal-bacterial co-infection of macrophages reveals global reprogramming and adaptation during infection

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