Publications by authors named "Norman Pavelka"

41 Publications

Fungal Symbionts Produce Prostaglandin E to Promote Their Intestinal Colonization.

Front Cell Infect Microbiol 2019 18;9:359. Epub 2019 Oct 18.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (ASTAR), Singapore, Singapore.

is a ubiquitous fungal symbiont that resides on diverse human barrier surfaces. Both mammalian and fungal cells can convert arachidonic acid into the lipid mediator, prostaglandin E2 (PGE), but the physiological significance of fungus-derived PGE remains elusive. Here we report that a mutant deficient in PGE production suffered a loss of competitive fitness in the murine gastrointestinal (GI) tract and that PGE supplementation mitigated this fitness defect. Impaired fungal PGE production affected neither the fitness of nor hyphal morphogenesis and virulence in either systemic or mucosal infection models. Instead, fungal production of PGE was associated with enhanced fungal survival within phagocytes. Consequently, ablation of colonic phagocytes abrogated the intra-GI fitness boost conferred by fungal PGE. These observations suggest that has evolved the capacity to produce PGE from arachidonic acid, a host-derived precursor, to promote its own colonization of the host gut. Analogous mechanisms might undergird host-microbe interactions of other symbiont fungi.
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http://dx.doi.org/10.3389/fcimb.2019.00359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813641PMC
June 2020

Selection of Candida albicans trisomy during oropharyngeal infection results in a commensal-like phenotype.

PLoS Genet 2019 05 15;15(5):e1008137. Epub 2019 May 15.

Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, United States of America.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.
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http://dx.doi.org/10.1371/journal.pgen.1008137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538192PMC
May 2019

Aneuploidy Enables Cross-Adaptation to Unrelated Drugs.

Mol Biol Evol 2019 08;36(8):1768-1782

Department of Molecular Microbiology and Biotechnology, School of Molecular Cell Biology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

Aneuploidy is common both in tumor cells responding to chemotherapeutic agents and in fungal cells adapting to antifungal drugs. Because aneuploidy simultaneously affects many genes, it has the potential to confer multiple phenotypes to the same cells. Here, we analyzed the mechanisms by which Candida albicans, the most prevalent human fungal pathogen, acquires the ability to survive both chemotherapeutic agents and antifungal drugs. Strikingly, adaptation to both types of drugs was accompanied by the acquisition of specific whole-chromosome aneuploidies, with some aneuploid karyotypes recovered independently and repeatedly from very different drug conditions. Specifically, strains selected for survival in hydroxyurea, an anticancer drug, acquired cross-adaptation to caspofungin, a first-line antifungal drug, and both acquired traits were attributable to trisomy of the same chromosome: loss of trisomy was accompanied by loss of adaptation to both drugs. Mechanistically, aneuploidy simultaneously altered the copy number of most genes on chromosome 2, yet survival in hydroxyurea or caspofungin required different genes and stress response pathways. Similarly, chromosome 5 monosomy conferred increased tolerance to both fluconazole and to caspofungin, antifungals with different mechanisms of action. Thus, the potential for cross-adaptation is not a feature of aneuploidy per se; rather, it is dependent on specific genes harbored on given aneuploid chromosomes. Furthermore, pre-exposure to hydroxyurea increased the frequency of appearance of caspofungin survivors, and hydroxyurea-adapted C. albicans cells were refractory to antifungal drug treatment in a mouse model of systemic candidiasis. This highlights the potential clinical consequences for the management of cancer chemotherapy patients at risk of fungal infections.
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http://dx.doi.org/10.1093/molbev/msz104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657732PMC
August 2019

Antibody neutralization of microbiota-derived circulating peptidoglycan dampens inflammation and ameliorates autoimmunity.

Nat Microbiol 2019 05 4;4(5):766-773. Epub 2019 Mar 4.

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore.

The human microbiota provides tonic signals that calibrate the host immune response, but their identity is unknown. Bacterial peptidoglycan (PGN) subunits are likely candidates since they are well-known immunity-enhancing adjuvants, released by most bacteria during growth, and have been found in the blood of healthy people. We developed a monoclonal antibody (mAb), 2E7, that targets muramyl-L-alanyl-D-isoglutamine (MDP), a conserved and minimal immunostimulatory structure of PGN. Using 2E7-based assays, we detected PGN ubiquitously in human blood at a broad range of concentrations that is relatively stable in each individual. We also detected PGN in the serum of several warm-blooded animals. However, PGN is barely detectable in the serum of germ-free mice, indicating that its origin is the host microbiota. Neutralization of circulating PGN via intraperitoneal administration of 2E7 suppressed the development of autoimmune arthritis and experimental autoimmune encephalomyelitis in mice. Arthritic NOD2 mice lacking the MDP sensor did not respond to 2E7, indicating that 2E7 dampens inflammation by blocking nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-mediated pathways. We propose that circulating PGN acts as a natural immune potentiator that tunes the host immune response; altering its level is a promising therapeutic strategy for immune-mediated diseases.
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http://dx.doi.org/10.1038/s41564-019-0381-1DOI Listing
May 2019

Experimental evolution of a fungal pathogen into a gut symbiont.

Science 2018 11;362(6414):589-595

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove Immunos #04, Singapore 138648, Singapore.

Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen in the mouse gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of "trained immunity." Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus.
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http://dx.doi.org/10.1126/science.aat0537DOI Listing
November 2018

Environmental stresses induce karyotypic instability in colorectal cancer cells.

Mol Biol Cell 2019 01 31;30(1):42-55. Epub 2018 Oct 31.

Institute of Medical Biology, Singapore 138648, Republic of Singapore.

Understanding how cells acquire genetic mutations is a fundamental biological question with implications for many different areas of biomedical research, ranging from tumor evolution to drug resistance. While karyotypic heterogeneity is a hallmark of cancer cells, few mutations causing chromosome instability have been identified in cancer genomes, suggesting a nongenetic origin of this phenomenon. We found that in vitro exposure of karyotypically stable human colorectal cancer cell lines to environmental stress conditions triggered a wide variety of chromosomal changes and karyotypic heterogeneity. At the molecular level, hyperthermia induced polyploidization by perturbing centrosome function, preventing chromosome segregation, and attenuating the spindle assembly checkpoint. The combination of these effects resulted in mitotic exit without chromosome segregation. Finally, heat-induced tetraploid cells were on the average more resistant to chemotherapeutic agents. Our studies suggest that environmental perturbations promote karyotypic heterogeneity and could contribute to the emergence of drug resistance.
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http://dx.doi.org/10.1091/mbc.E18-10-0626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337910PMC
January 2019

The mammalian mycobiome: A complex system in a dynamic relationship with the host.

Wiley Interdiscip Rev Syst Biol Med 2019 01 25;11(1):e1438. Epub 2018 Sep 25.

Singapore Immunology Network A*STAR, Singapore.

Mammalian barrier surfaces are densely populated by symbiont fungi in much the same way the former are colonized by symbiont bacteria. The fungal microbiota, otherwise known as the mycobiota, is increasingly recognized as a critical player in the maintenance of health and homeostasis of the host. Here we discuss the impact of the mycobiota on host physiology and disease, the factors influencing mycobiota composition, and the current technologies used for identifying symbiont fungal species. Understanding the tripartite interactions among the host, mycobiota, and other members of the microbiota, will help to guide the development of novel prevention and therapeutic strategies for a variety of human diseases. This article is categorized under: Physiology > Mammalian Physiology in Health and Disease Laboratory Methods and Technologies > Genetic/Genomic Methods Models of Systems Properties and Processes > Organismal Models.
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http://dx.doi.org/10.1002/wsbm.1438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586165PMC
January 2019

The Elusive Anti- Vaccine: Lessons From the Past and Opportunities for the Future.

Front Immunol 2018 27;9:897. Epub 2018 Apr 27.

Singapore Immunology Network (SIgN), Agency of Science, Technology and Research (ASTAR), Singapore, Singapore.

Candidemia is a bloodstream fungal infection caused by species and is most commonly observed in hospitalized patients. Even with proper antifungal drug treatment, mortality rates remain high at 40-50%. Therefore, prophylactic or preemptive antifungal medications are currently recommended in order to prevent infections in high-risk patients. Moreover, the majority of women experience at least one episode of vulvovaginal candidiasis (VVC) throughout their lifetime and many of them suffer from recurrent VVC (RVVC) with frequent relapses for the rest of their lives. While there currently exists no definitive cure, the only available treatment for RVVC is again represented by antifungal drug therapy. However, due to the limited number of existing antifungal drugs, their associated side effects and the increasing occurrence of drug resistance, other approaches are greatly needed. An obvious prevention measure for candidemia or RVVC relapse would be to immunize at-risk patients with a vaccine effective against infections. In spite of the advanced and proven techniques successfully applied to the development of antibacterial or antiviral vaccines, however, no antifungal vaccine is still available on the market. In this review, we first summarize various efforts to date in the development of anti- vaccines, highlighting advantages and disadvantages of each strategy. We next unfold and discuss general hurdles encountered along these efforts, such as the existence of large genomic variation and phenotypic plasticity across strains and species, and the difficulty in mounting protective immune responses in immunocompromised or immunosuppressed patients. Lastly, we review the concept of "trained immunity" and discuss how induction of this rapid and nonspecific immune response may potentially open new and alternative preventive strategies against opportunistic infections by species and potentially other pathogens.
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http://dx.doi.org/10.3389/fimmu.2018.00897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934487PMC
July 2019

Modulation of bacterial metabolism by the microenvironment controls MAIT cell stimulation.

Mucosal Immunol 2018 07 9;11(4):1060-1070. Epub 2018 May 9.

Experimental Immunology, Department of Biomedicine, University and University Hospital Basel, 4031, Basel, Switzerland.

Mucosal-associated invariant T (MAIT) cells are abundant innate-like T lymphocytes in mucosal tissues and recognize a variety of riboflavin-related metabolites produced by the microbial flora. Relevant issues are whether MAIT cells are heterogeneous in the colon, and whether the local environment influences microbial metabolism thereby shaping MAIT cell phenotypes and responses. We found discrete MAIT cell populations in human colon, characterized by the diverse expression of transcription factors, cytokines and surface markers, indicative of activated and precisely controlled lymphocyte populations. Similar phenotypes were rare among circulating MAIT cells and appeared when circulating MAIT cells were stimulated with the synthetic antigens 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil, and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil. Furthermore, bacteria grown in colon-resembling conditions with low oxygen tension and harvested at stationary growth phase, potently activated human MAIT cells. The increased activation correlated with accumulation of the above antigenic metabolites as indicated by mass spectrometry. Thus, the colon environment contributes to mucosal immunity by directly affecting bacterial metabolism, and indirectly controlling the stimulation and differentiation of MAIT cells.
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http://dx.doi.org/10.1038/s41385-018-0020-9DOI Listing
July 2018

Advantages of meta-total RNA sequencing (MeTRS) over shotgun metagenomics and amplicon-based sequencing in the profiling of complex microbial communities.

NPJ Biofilms Microbiomes 2018 18;4. Epub 2018 Jan 18.

1Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (ASTAR), 8A Biomedical Grove, Immunos #04, Singapore, 138648 Singapore.

Sequencing-based microbiome profiling aims at detecting and quantifying individual members of a microbial community in a culture-independent manner. While amplicon-based sequencing (ABS) of bacterial or fungal ribosomal DNA is the most widely used technology due to its low cost, it suffers from PCR amplification biases that hinder accurate representation of microbial population structures. Shotgun metagenomics (SMG) conversely allows unbiased microbiome profiling but requires high sequencing depth. Here we report the development of a meta-total RNA sequencing (MeTRS) method based on shotgun sequencing of total RNA and benchmark it on a human stool sample spiked in with known abundances of bacterial and fungal cells. MeTRS displayed the highest overall sensitivity and linearity for both bacteria and fungi, the greatest reproducibility compared to SMG and ABS, while requiring a ~20-fold lower sequencing depth than SMG. We therefore present MeTRS as a valuable alternative to existing technologies for large-scale profiling of complex microbiomes.
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http://dx.doi.org/10.1038/s41522-017-0046-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773663PMC
January 2018

Emerging and evolving concepts in gene essentiality.

Nat Rev Genet 2018 Jan 16;19(1):34-49. Epub 2017 Oct 16.

Singapore Immunology Network (SIgN), A*STAR, 8A Biomedical Grove, Immunos #04, Singapore 138648, Singapore.

Gene essentiality is a founding concept of genetics with important implications in both fundamental and applied research. Multiple screens have been performed over the years in bacteria, yeasts, animals and more recently in human cells to identify essential genes. A mounting body of evidence suggests that gene essentiality, rather than being a static and binary property, is both context dependent and evolvable in all kingdoms of life. This concept of a non-absolute nature of gene essentiality changes our fundamental understanding of essential biological processes and could directly affect future treatment strategies for cancer and infectious diseases.
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http://dx.doi.org/10.1038/nrg.2017.74DOI Listing
January 2018

The Transcriptional Response of to Weak Organic Acids, Carbon Source, and Inactivation Unveils a Role for in Mediating the Fungistatic Effect of Acetic Acid.

G3 (Bethesda) 2017 11 6;7(11):3597-3604. Epub 2017 Nov 6.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore 138648, Singapore

is a resident fungus of the human intestinal microflora. Commonly isolated at low abundance in healthy people, outcompetes local microbiota during candidiasis episodes. Under normal conditions, members of the human gastrointestinal (GI) microbiota were shown to keep colonization under control. By releasing weak organic acids (WOAs), bacteria are able to moderate yeast growth. This mechanism displays a synergistic effect with the absence of glucose in medium of culture, which underlines the complex interactions that faces in its natural environment. Inactivation of the transcriptional regulator in results in a lack of sensitivity to this synergistic outcome. To decipher transcriptional responses to glucose, WOAs, and the role of , we performed RNA sequencing (RNA-seq) on four biological replicates exposed to combinations of these three parameters. We were able to characterize the (i) glucose response, (ii) response to acetic and butyric acid, (iii) regulation of , and (iv) genes responsible for WOA resistance. We identified a group of six genes linked to WOA sensitivity in a glucose--dependent manner and inactivated one of these genes, the putative glucose transporter , in a SC5314 wild-type background. As expected, the mutant displayed a partial complementation to WOA resistance in the absence of glucose. This result points toward a mechanism of WOA sensitivity in involving membrane transporters, which could be exploited to control yeast colonization in human body niches.
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http://dx.doi.org/10.1534/g3.117.300238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5677169PMC
November 2017

Intrahepatic CD206 macrophages contribute to inflammation in advanced viral-related liver disease.

J Hepatol 2017 09 5;67(3):490-500. Epub 2017 May 5.

Program in Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore; Singapore Immunology Network, A*STAR, 8A Biomedical Grove, Immunos Building, Level 4, Singapore 138648, Singapore. Electronic address:

Background & Aims: Liver inflammation is key in the progression of chronic viral hepatitis to cirrhosis and hepatocellular carcinoma. The magnitude of viral replication and the specific anti-viral immune responses should govern the degree of inflammation, but a direct correlation is not consistently found in chronic viral hepatitis patients. We aim to better define the mechanisms that contribute to chronic liver inflammation.

Methods: Intrahepatic CD14 myeloid cells from healthy donors (n=19) and patients with viral-related liver cirrhosis (HBV, HBV/HDV or HCV; n=15) were subjected to detailed phenotypic, molecular and functional characterisation.

Results: Unsupervised analysis of multi-parametric data showed that liver disease was associated with the intrahepatic expansion of activated myeloid cells mainly composed of pro-inflammatory CD14HLA-DRCD206 cells, which spontaneously produced TNFα and GM-CSF. These cells only showed heightened pro-inflammatory responses to bacterial TLR agonists and were more refractory to endotoxin-induced tolerance. A liver-specific enrichment of CD14HLA-DRCD206 cells was also detected in a humanised mouse model of liver inflammation. This accumulation was abrogated following oral antibiotic treatment, suggesting a direct involvement of translocated gut-derived microbial products in liver injury.

Conclusions: Viral-related chronic liver inflammation is driven by the interplay between non-endotoxin-tolerant pro-inflammatory CD14HLA-DRCD206 myeloid cells and translocated bacterial products. Deciphering this mechanism paves the way for the development of therapeutic strategies specifically targeting CD206 myeloid cells in viral-related liver disease patients. Lay summary: Viral-related chronic liver disease is driven by intrahepatic pro-inflammatory myeloid cells accumulating in a gut-derived bacterial product-dependent manner. Our findings support the use of oral antibiotics to ameliorate liver inflammation in these patients.
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http://dx.doi.org/10.1016/j.jhep.2017.04.023DOI Listing
September 2017

β-glucan Exposure on the Fungal Cell Wall Tightly Correlates with Competitive Fitness of Species in the Mouse Gastrointestinal Tract.

Front Cell Infect Microbiol 2016 22;6:186. Epub 2016 Dec 22.

Singapore Immunology Network, Agency for Science, Technology and Research Singapore, Singapore.

is responsible for ~400,000 systemic fungal infections annually, with an associated mortality rate of 46-75%. The human gastrointestinal (GI) tract represents the largest natural reservoir of species and is a major source of systemic fungal infections. However, the factors that control GI colonization by species are not completely understood. We hypothesized that the fungal cell wall would play an important role in determining the competitive fitness of species in the mammalian GI tract. To test this hypothesis, we generated a systematic collection of isogenic cell wall mutants and measured their fitness in the mouse GI tract via quantitative competition assays. Whereas a large variation in competitive fitness was found among mutants, no correlation was observed between GI fitness and total levels of individual cell wall components. Similar results were obtained in a set of distantly-related species, suggesting that total amounts of individual cell wall components do not determine the ability of fungi to colonize the GI tract. We then subjected this collection of strains and species to an extensive quantitative phenotypic profiling in search for features that might be responsible for their differences in GI fitness, but found no association with the ability to grow in GI-mimicking and stressful environments or with and virulence. The most significant association with GI fitness was found to be the strength of signaling through the Dectin-1 receptor. Using a quantitative assay to measure the amount of exposed β-glucan on the surface of fungal cells, we found this parameter, unlike total β-glucan levels, to be strongly predictive of competitive fitness in the mouse GI tract. These data suggest that fungal cell wall architecture, more so than its crude composition, critically determines the ability of fungi to colonize the mammalian GI tract. In particular, recognition of exposed β-glucan by Dectin-1 receptor appears to severely limit GI fitness and hence represents a promising target to reduce fungal colonization in patients at risks of systemic candidiasis.
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http://dx.doi.org/10.3389/fcimb.2016.00186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5177745PMC
August 2017

Host and Environmental Factors Influencing Individual Human Cytokine Responses.

Cell 2016 11;167(4):1111-1124.e13

Department of Internal Medicine and Radboudumc Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Gelderland 6500HB, the Netherlands. Electronic address:

Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP.
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http://dx.doi.org/10.1016/j.cell.2016.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787854PMC
November 2016

How Chemotherapy Increases the Risk of Systemic Candidiasis in Cancer Patients: Current Paradigm and Future Directions.

Pathogens 2016 Jan 15;5(1). Epub 2016 Jan 15.

Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos Building, Singapore 138648, Singapore.

Candida albicans is a fungal commensal and a major colonizer of the human skin, as well as of the gastrointestinal and genitourinary tracts. It is also one of the leading causes of opportunistic microbial infections in cancer patients, often presenting in a life-threatening, systemic form. Increased susceptibility to such infections in cancer patients is attributed primarily to chemotherapy-induced depression of innate immune cells and weakened epithelial barriers, which are the body's first-line defenses against fungal infections. Moreover, classical chemotherapeutic agents also have a detrimental effect on components of the adaptive immune system, which further play important roles in the antifungal response. In this review, we discuss the current paradigm regarding the mechanisms behind the increased risk of systemic candidiasis in cancer patients. We also highlight some recent findings, which suggest that chemotherapy may have more extensive effects beyond the human host, in particular towards C. albicans itself and the bacterial microbiota. The extent to which these additional effects contribute towards the development of candidiasis in chemotherapy-treated patients remains to be investigated.
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http://dx.doi.org/10.3390/pathogens5010006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810127PMC
January 2016

Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability.

Cell 2015 Dec 25;163(6):1388-99. Epub 2015 Nov 25.

Institute of Medical Biology (IMB), Agency for Science, Technology and Research (A(∗)STAR), Singapore 138648, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. Electronic address:

Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance.
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http://dx.doi.org/10.1016/j.cell.2015.10.069DOI Listing
December 2015

MIG1 Regulates Resistance of Candida albicans against the Fungistatic Effect of Weak Organic Acids.

Eukaryot Cell 2015 Oct 21;14(10):1054-61. Epub 2015 Aug 21.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

Candida albicans is the leading cause of fungal infections; but it is also a member of the human microbiome, an ecosystem of thousands of microbial species potentially influencing the outcome of host-fungal interactions. Accordingly, antibacterial therapy raises the risk of candidiasis, yet the underlying mechanism is currently not fully understood. We hypothesize the existence of bacterial metabolites that normally control C. albicans growth and of fungal resistance mechanisms against these metabolites. Among the most abundant microbiota-derived metabolites found on human mucosal surfaces are weak organic acids (WOAs), such as acetic, propionic, butyric, and lactic acid. Here, we used quantitative growth assays to investigate the dose-dependent fungistatic properties of WOAs on C. albicans growth and found inhibition of growth to occur at physiologically relevant concentrations and pH values. This effect was conserved across distantly related fungal species both inside and outside the CTG clade. We next screened a library of transcription factor mutants and identified several genes required for the resistance of C. albicans to one or more WOAs. A single gene, MIG1, previously known for its role in glucose repression, conferred resistance against all four acids tested. Consistent with glucose being an upstream activator of Mig1p, the presence of this carbon source was required for WOA resistance in wild-type C. albicans. Conversely, a MIG1-complemented strain completely restored the glucose-dependent resistance against WOAs. We conclude that Mig1p plays a central role in orchestrating a transcriptional program to fight against the fungistatic effect of this class of highly abundant metabolites produced by the gastrointestinal tract microbiota.
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http://dx.doi.org/10.1128/EC.00129-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588629PMC
October 2015

The transcriptional stress response of Candida albicans to weak organic acids.

G3 (Bethesda) 2015 Jan 29;5(4):497-505. Epub 2015 Jan 29.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore 138648, Singapore School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore

Candida albicans is the most important fungal pathogen of humans, causing severe infections, especially in nosocomial and immunocompromised settings. However, it is also the most prevalent fungus of the normal human microbiome, where it shares its habitat with hundreds of trillions of other microbial cells. Despite weak organic acids (WOAs) being among the most abundant metabolites produced by bacterial microbiota, little is known about their effect on C. albicans. Here we used a sequencing-based profiling strategy to systematically investigate the transcriptional stress response of C. albicans to lactic, acetic, propionic, and butyric acid at several time points after treatment. Our data reveal a complex transcriptional response, with individual WOAs triggering unique gene expression profiles and with important differences between acute and chronic exposure. Despite these dissimilarities, we found significant overlaps between the gene expression changes induced by each WOA, which led us to uncover a core transcriptional response that was largely unrelated to other previously published C. albicans transcriptional stress responses. Genes commonly up-regulated by WOAs were enriched in several iron transporters, which was associated with an overall decrease in intracellular iron concentrations. Moreover, chronic exposure to any WOA lead to down-regulation of RNA synthesis and ribosome biogenesis genes, which resulted in significant reduction of total RNA levels and of ribosomal RNA in particular. In conclusion, this study suggests that gastrointestinal microbiota might directly influence C. albicans physiology via production of WOAs, with possible implications of how this fungus interacts with its host in both health and disease.
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http://dx.doi.org/10.1534/g3.114.015941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390566PMC
January 2015

Karyotypic changes as drivers and catalyzers of cellular evolvability: a perspective from non-pathogenic yeasts.

Semin Cell Dev Biol 2013 Apr 10;24(4):332-8. Epub 2013 Feb 10.

Institute of Medical Biology, Agency of Science, Technology and Research, 8A Biomedical Grove, Singapore 138648, Singapore.

In spite of the existence of multiple cellular mechanisms that ensure genome stability, thanks to the advent of quantitative genomic assays in the last decade, an unforeseen level of plasticity in cellular genomes has begun to emerge in many different fields of cell biology. Eukaryotic cells not only have a remarkable ability to change their karyotypes in response to various perturbations, but also these karyotypic changes impact cellular fitness and in some circumstances enable evolutionary adaptation. In this review, we focus on recent findings in non-pathogenic yeasts indicating that karyotypic changes generate selectable phenotypic variation and alter genomic instability. Based on these findings, we propose that in highly stressful and thus strongly selective environments karyotypic changes could act both as a driver and as a catalyzer of cellular adaptation, i.e. karyotypic changes drive large phenotypic leaps and at the same time catalyze the accumulation of even more genotypic and karyotypic changes.
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http://dx.doi.org/10.1016/j.semcdb.2013.01.009DOI Listing
April 2013

Karyotypic determinants of chromosome instability in aneuploid budding yeast.

PLoS Genet 2012 17;8(5):e1002719. Epub 2012 May 17.

Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution.
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http://dx.doi.org/10.1371/journal.pgen.1002719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3355078PMC
September 2012

Complexity and dynamics of host-fungal interactions.

Immunol Res 2012 Sep;53(1-3):127-35

Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #03 Immunos, Biopolis, Singapore.

Pathologies attributable to fungal infections represent a growing concern in both developed and developing countries. Initially discovered as opportunistic pathogens of immunocompromised hosts, fungi such as Candida albicans are now being placed at the centre of a more complex and dynamic picture in which the outcome of an infection is the result of an intricate network of molecular interactions between the fungus, the host and the commensal microflora co-inhabiting various host niches, and especially the gastrointestinal (GI) tract. The complexity of the host-fungal interaction begins with the numerous pathogen-associated molecular patterns (PAMPs) present on the fungal cell wall that are recognized by multiple pathogen-recognition receptors (PRRs), expressed by several types of host cells. PAMP-PRR interactions elicit a variety of intracellular signalling pathways leading to a wide array of immune responses, some of which promote fungal clearance while others contribute to pathogenesis. The picture is further complicated by the fact that numerous commensal bacteria normally co-inhabiting the host's GI tract produce molecules that either directly modulate the survival and virulence of commensal fungi such as C. albicans or indirectly modulate the host's antifungal immune responses. On top of this complexity, this host-microbiome-fungal interaction exhibits features of a dynamic system, in which the same fungi can easily switch between different morphological forms presenting different PAMPs at different moments of time. Furthermore, fungal pathogens can rapidly accumulate genomic alterations that further modify their recognition by the immune system, their virulence and their resistance to antifungal compounds. Thus, based on available molecular data alone, it is currently difficult to construct a coherent model able to explain the balance between commensalism and virulence and to predict the outcome of a fungal infection. Here, we review current advances in our understanding of this complex and dynamic system and propose new avenues of investigation to assemble a more complete picture of the host-fungal interaction, integrating microbiological and immunological data under the lens of systems biology and evolutionary genomics.
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http://dx.doi.org/10.1007/s12026-012-8265-yDOI Listing
September 2012

When one and one gives more than two: challenges and opportunities of integrative omics.

Front Genet 2011 6;2:105. Epub 2012 Jan 6.

Saw Swee Hock School of Public Health, National University of Singapore Singapore.

Since the dawn of the post-genomic era a myriad of novel high-throughput technologies have been developed that are capable of measuring thousands of biological molecules at once, giving rise to various "omics" platforms. These advances offer the unique opportunity to study how individual parts of a biological system work together to produce emerging phenotypes. Today, many research laboratories are moving toward applying multiple omics platforms to analyze the same biological samples. In addition, network information of interacting molecules is being incorporated more and more into the analysis and interpretation of these multiple omics datasets, which provides novel ways to integrate multiple layers of heterogeneous biological information into a single coherent picture. Here, we provide a perspective on how such recent "integrative omics" efforts are likely going to shift biological paradigms once again, and what challenges lie ahead.
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http://dx.doi.org/10.3389/fgene.2011.00105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3262227PMC
October 2012

The bZIP transcription factor Rca1p is a central regulator of a novel CO₂ sensing pathway in yeast.

PLoS Pathog 2012 Jan 12;8(1):e1002485. Epub 2012 Jan 12.

School of Biosciences, University of Kent, Canterbury, United Kingdom.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.
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http://dx.doi.org/10.1371/journal.ppat.1002485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257301PMC
January 2012

Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast.

Nature 2010 Nov 20;468(7321):321-5. Epub 2010 Oct 20.

Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA.

Aneuploidy, referring here to genome contents characterized by abnormal numbers of chromosomes, has been associated with developmental defects, cancer and adaptive evolution in experimental organisms. However, it remains unresolved how aneuploidy impacts gene expression and whether aneuploidy could directly bring about phenotypic variation and improved fitness over that of euploid counterparts. Here we show, using quantitative mass spectrometry-based proteomics and phenotypic profiling, that levels of protein expression in aneuploid yeast strains largely scale with chromosome copy numbers, following the same trend as that observed for the transcriptome, and that aneuploidy confers diverse phenotypes. We designed a novel scheme to generate, through random meiotic segregation, 38 stable and fully isogenic aneuploid yeast strains with distinct karyotypes and genome contents between 1N and 3N without involving any genetic selection. Through quantitative growth assays under various conditions or in the presence of a panel of chemotherapeutic or antifungal drugs, we found that some aneuploid strains grew significantly better than euploid control strains under conditions suboptimal for the latter. These results provide strong evidence that aneuploidy directly affects gene expression at both the transcriptome and proteome levels and can generate significant phenotypic variation that could bring about fitness gains under diverse conditions. Our findings suggest that the fitness ranking between euploid and aneuploid cells is dependent on context and karyotype, providing the basis for the notion that aneuploidy can directly underlie phenotypic evolution and cellular adaptation.
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http://dx.doi.org/10.1038/nature09529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978756PMC
November 2010

Dr Jekyll and Mr Hyde: role of aneuploidy in cellular adaptation and cancer.

Curr Opin Cell Biol 2010 Dec 23;22(6):809-15. Epub 2010 Jul 23.

Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA.

When cells in our body change their genome and develop into cancer, we blame it on genome instability. When novel species conquer inhospitable environments, we credit it to genome evolution. From a cellular perspective, however, both processes are outcomes of the same fundamental biological properties-genome and pathway plasticity and the natural selection of cells that escape death and acquire growth advantages. Unraveling the consequences of genome plasticity at a cellular level is not only central to the understanding of species evolution but also crucial to deciphering important cell biological problems, such as how cancer cells emerge and how pathogens develop drug resistance. Aside from the well-known role of DNA sequence mutations, recent evidence suggests that changes in DNA copy numbers in the form of segmental or whole-chromosome aneuploidy can bring about large phenotypic variation. Although usually detrimental under conditions suitable for normal proliferation of euploid cells, aneuploidization may be a frequently occurring genetic change that enables pathogens or cancer cells to escape physiological or pharmacological roadblocks.
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http://dx.doi.org/10.1016/j.ceb.2010.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2974767PMC
December 2010

Delayed correlation of mRNA and protein expression in rapamycin-treated cells and a role for Ggc1 in cellular sensitivity to rapamycin.

Mol Cell Proteomics 2010 Feb 10;9(2):271-84. Epub 2009 Nov 10.

Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.

To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Deltaggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression under TOR control.
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http://dx.doi.org/10.1074/mcp.M900415-MCP200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2830839PMC
February 2010

How do human cells react to the absence of mitochondrial DNA?

PLoS One 2009 May 28;4(5):e5713. Epub 2009 May 28.

Unit of Molecular Neurogenetics-Pierfranco and Luisa Mariani Center for the study of Mitochondrial Disorders in Children, IRCCS Foundation Neurological Institute C. Besta, Milan, Italy.

Background: Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). The mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nDNA. Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. To investigate how the cellular transcriptome is modified in response to the absence of mtDNA, we used Affymetrix HG-U133A GeneChip arrays to study the gene expression profile of two human cell lines, 143BTK(-) and A549, which had been entirely depleted of mtDNA (rho(o) cells), and compared it with that of corresponding undepleted parental cells (rho(+) cells).

Results: Our data indicate that absence of mtDNA is associated with: i) a down-regulation of cell cycle control genes and a reduction of cell replication rate, ii) a down-regulation of nuclear-encoded subunits of complex III of the respiratory chain and iii) a down-regulation of a gene described as the human homolog of ELAC2 of E. coli, which encodes a protein that we show to also target to the mitochondrial compartment.

Conclusions: Our results indicate a strong correlation between mitochondrial biogenesis and cell cycle control and suggest that some proteins could have a double role: for instance in controlling both cell cycle progression and mitochondrial functions. In addition, the finding that ELAC2 and maybe other transcripts that are located into mitochondria, are down-regulated in rho(o) cells, make them good candidates for human disorders associated with defective replication and expression of mtDNA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005713PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683933PMC
May 2009

Aneuploidy underlies rapid adaptive evolution of yeast cells deprived of a conserved cytokinesis motor.

Cell 2008 Nov;135(5):879-93

Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA.

The ability to evolve is a fundamental feature of biological systems, but the mechanisms underlying this capacity and the evolutionary dynamics of conserved core processes remain elusive. We show that yeast cells deleted of MYO1, encoding the only myosin II normally required for cytokinesis, rapidly evolved divergent pathways to restore growth and cytokinesis. The evolved cytokinesis phenotypes correlated with specific changes in the transcriptome. Polyploidy and aneuploidy were common genetic alterations in the best evolved strains, and aneuploidy could account for gene expression changes due directly to altered chromosome stoichiometry as well as to downstream effects. The phenotypic effect of aneuploidy could be recapitulated with increased copy numbers of specific regulatory genes in myo1Delta cells. These results demonstrate the evolvability of even a well-conserved process and suggest that changes in chromosome stoichiometry provide a source of heritable variation driving the emergence of adaptive phenotypes when the cell division machinery is strongly perturbed.
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http://dx.doi.org/10.1016/j.cell.2008.09.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776776PMC
November 2008