Publications by year
Child HT, Deeks MJ, Haynes K, Rudd JJ, Bates S (2022). Distinct roles for different autophagy-associated genes in the virulence of the fungal wheat pathogen Zymoseptoria tritici. Fungal Genetics and Biology, 163, 103748-103748.
(2021). Investigating fungal cellular processes involved in early colonisation of wheat by Zymoseptoria tritici.
Investigating fungal cellular processes involved in early colonisation of wheat by Zymoseptoria tritici
The fungal pathogen Zymoseptoria tritici causes the most economically important disease of wheat in Europe. Despite recent advances in our understanding of its molecular host-pathogen interaction, fundamental questions remain about the cellular processes underlying plant colonisation by this fungus. The work presented in this thesis uses both reverse and forward genetic techniques to further understand the molecular determinants of Z. tritici virulence. To address questions about the source of nutrients utilised by Z. tritici to support symptomless colonisation of the leaf, this thesis explores cellular pathways utilised by other plant pathogenic fungi to access stored macromolecules. While autophagy is crucial for the infection-related development of many fungal plant pathogens, this study reveals that autophagy is dispensable for Z. tritici pathogenicity, and points towards a potential autophagy-independent function of ZtATG8 in virulence. The mitochondrial fatty acid β-oxidation pathway was however found to support the switch to hyphal growth on the leaf surface, providing strong evidence that catabolism of stored lipids is required for early host invasion by Z. tritici. Abstract
Forward genetic investigation identified enzymes within the cell wall integrity (CWI) and cyclic adenosine monophosphate (cAMP) signalling pathways as playing a key role in Z. tritici virulence. In planta transcriptomic analysis revealed that the CWI pathway regulates the expression of infection-related secreted proteins, including the characterised LysM effectors required for host defence evasion, suggesting that Z. tritici may co-regulate virulence gene expression with the response to cell wall perturbation. Findings presented here also suggest that cAMP signalling regulates transcription during the switch to necrotrophic growth, providing insights into the elusive mechanisms controlling this infection cycle transition. Finally, genomic and transcriptomic analysis of a spontaneous Z. tritici mutant revealed the potential function of the light responsive transcription factor white collar 1 in controlling Z. tritici morphological development and infection.
These novel findings advance our understanding of the cellular pathways contributing to Z. tritici infection and inform the development of future strategies to control this devastating pathogen.
Duxbury SJN, Bates S, Beardmore RE, Gudelj I
(2020). Evolution of drug-resistant and virulent small colonies in phenotypically diverse populations of the human fungal pathogen. <i>Candida glabrata</i>. Proceedings of the Royal Society B: Biological Sciences
Evolution of drug-resistant and virulent small colonies in phenotypically diverse populations of the human fungal pathogen. Candida glabrata
. Antimicrobial resistance frequently carries a fitness cost to a pathogen, measured as a reduction in growth rate compared to the sensitive wild-type, in the absence of antibiotics. Existing empirical evidence points to the following relationship between cost of resistance and virulence. If a resistant pathogen suffers a fitness cost in terms of reduced growth rate it commonly has lower virulence compared to the sensitive wild-type. If this cost is absent so is the reduction in virulence. Here we show, using experimental evolution of drug resistance in the fungal human pathogen
. Candida glabrata,
. that reduced growth rate of resistant strains need not result in reduced virulence. Phenotypically heterogeneous populations were evolved in parallel containing highly resistant sub-population small colony variants (SCVs) alongside sensitive sub-populations. Despite their low growth rate in the absence of an antifungal drug, the SCVs did not suffer a marked alteration in virulence compared with the wild-type ancestral strain, or their co-isolated sensitive strains. This contrasts with classical theory that assumes growth rate to positively correlate with virulence. Our work thus highlights the complexity of the relationship between resistance, basic life-history traits and virulence.
Bieger BD, Rogers AM, Bates S, Egan MJ
(2020). Long-distance early endosome motility in Aspergillus fumigatus promotes normal hyphal growth behaviors in controlled microenvironments but is dispensable for virulence. Traffic
Long-distance early endosome motility in Aspergillus fumigatus promotes normal hyphal growth behaviors in controlled microenvironments but is dispensable for virulence.
In filamentous fungi, early endosomes are continuously trafficked to, and from, the growing hyphal tip by microtubule-based motor proteins, serving as platforms for the long-distance transport of diverse cargos including mRNA, signaling molecules, and other organelles which hitchhike on them. While the cellular machinery for early endosome motility in filamentous fungi is fairly well characterized, the broader physiological significance of this process remains less well understood. We set out to determine the importance of long-distance early endosome trafficking in Aspergillus fumigatus, an opportunistic human pathogenic fungus that can cause devastating pulmonary infections in immunocompromised individuals. We first characterized normal early endosome motile behavior in A. fumigatus, then generated a mutant in which early endosome motility is severely perturbed through targeted deletion of the gene encoding for FtsA, one of a complex of proteins that links early endosomes to their motor proteins. Using a microfluidics-based approach we show that contact-induced hyphal branching behaviors are impaired in ΔftsA mutants, but that FtsA-mediated early endosome motility is dispensable for virulence in an invertebrate infection model. Overall, our study provides new insight into early endosome motility in an important human pathogenic fungus. Abstract
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Schaefer K, Wagener J, Ames RM, Christou S, MacCallum DM, Bates S, Gow NAR
(2020). Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That is Essential for Growth and Fungal Virulence. mBio
Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That is Essential for Growth and Fungal Virulence
. We show that the
. C. albicans
. ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for
. C. albicans
. growth and survival in microenvironments of the mammalian host.
Schaefer K, Bates S, Ames R, Christou S, Gow N (2019). The Candida albicans arginase family encodes enzymes with diverse catabolic activities that differentially influence host–fungus interactions. Access Microbiology, 1(1A).
(2018). Candida albicans Cdc15 is essential for mitotic exit and cytokinesis. Sci Rep
Candida albicans Cdc15 is essential for mitotic exit and cytokinesis.
Candida albicans displays a variety of morphological forms, and the ability to switch forms must be linked with cell cycle control. In budding yeast the Mitotic Exit Network (MEN) acts to drive mitotic exit and signal for cytokinesis and cell separation. However, previous reports on the MEN in C. albicans have raised questions on its role in this organism, with the components analysed to date demonstrating differing levels of importance in the processes of mitotic exit, cytokinesis and cell separation. This work focuses on the role of the Cdc15 kinase in C. albicans and demonstrates that, similar to Saccharomyces cerevisiae, it plays an essential role in signalling for mitotic exit and cytokinesis. Cells depleted of Cdc15 developed into elongated filaments, a common response to cell cycle arrest in C. albicans. These filaments emerged exclusively from large budded cells, contained two nuclear bodies and exhibited a hyper-extended spindle, all characteristic of these cells failing to exit mitosis. Furthermore these filaments displayed a clear cytokinesis defect, and CDC15 over-expression led to aberrant cell separation following hyphal morphogenesis. Together, these results are consistent with Cdc15 playing an essential role in signalling for mitotic exit, cytokinesis and cell separation in C. albicans. Abstract
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Champion OL, Titball RW, Bates S
(2018). Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens. J Fungi (Basel)
Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens.
In the past decade, Galleria mellonella (wax moth) larvae have become widely used as a non-mammalian infection model. However, the full potential of this infection model has yet to be realised, limited by the variable quality of larvae used and the lack of standardised procedures. Here, we review larvae suitable for research, protocols for dosing larvae, and methods for scoring illness in larvae infected with fungal pathogens. The development of standardised protocols for carrying out our experimental work will allow high throughput screens to be developed, changing the way in which we evaluate panels of mutants and strains. It will also enable the in vivo screening of potential antimicrobials at an earlier stage in the research and development cycle. Abstract
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Ames L, Duxbury S, Pawlowska B, Ho H-L, Haynes K, Bates S (2017). <i>Galleria mellonella</i> as a host model to study <i>Candida glabrata</i> virulence and antifungal efficacy. Virulence, 8(8), 1909-1917.
Milne SW, Cheetham J, Lloyd D, Shaw S, Moore K, Paszkiewicz KH, Aves SJ, Bates S
(2014). Role of Candida albicans Tem1 in mitotic exit and cytokinesis. Fungal Genet Biol
Role of Candida albicans Tem1 in mitotic exit and cytokinesis.
Candida albicans demonstrates three main growth morphologies: yeast, pseudohyphal and true hyphal forms. Cell separation is distinct in these morphological forms and the process of separation is closely linked to the completion of mitosis and cytokinesis. In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation. Here we have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15. Cells depleted of Tem1 displayed highly polarised growth but ultimately failed to both complete cytokinesis and re-enter the cell cycle following nuclear division. Consistent with its role in activating the mitotic exit network Tem1 localises to spindle pole bodies in a cell cycle-dependent manner. Ultimately, the mitotic exit network in C. albicans appears to co-ordinate the sequential processes of mitotic exit, cytokinesis and cell separation. Abstract
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Parham H, Bates S, Xia Y, Zhu Y
(2013). A highly efficient and versatile carbon nanotube/ceramic composite filter. Carbon
A highly efficient and versatile carbon nanotube/ceramic composite filter
Carbon nanotubes were grown in the open pores of a commercial porous ceramic matrix consisting of mainly Al2O3 and SiO 2 with an average pore size of 300 and 500 μm, using a pre-formed nickel catalyst inside the pores and a camphor solution precursor at 780 °C. The resulting composites, ∅27 mm × 10 mm discs containing about 3 wt.% of carbon nanotubes, were then assessed as a filter for the removal of yeast cells and different heavy metal ions from water, and for the removal of particulates from air. The results showed that the carbon nanotube containing composite filter demonstrated a high efficiency of yeast filtration (98%), ca. 100% heavy metal ion removal from water and excellent particulate filtration from air. The composite filter also exhibited good reusability for these applications, owing to the excellent thermal and chemical stability of the carbon nanotubes. © 2012 Elsevier Ltd. All rights reserved. Abstract
Islahudin F, Khozoie C, Bates S, Ting K-N, Pleass RJ, Avery SV
(2013). Cell wall perturbation sensitizes fungi to the antimalarial drug chloroquine. Antimicrob Agents Chemother
Cell wall perturbation sensitizes fungi to the antimalarial drug chloroquine.
Chloroquine (CQ) has been a mainstay of antimalarial drug treatment for several decades. Additional therapeutic actions of CQ have been described, including some reports of fungal inhibition. Here we investigated the action of CQ in fungi, including the yeast model Saccharomyces cerevisiae. A genomewide yeast deletion strain collection was screened against CQ, revealing that bck1Δ and slt2Δ mutants of the cell wall integrity pathway are CQ hypersensitive. This phenotype was rescued with sorbitol, consistent with cell wall involvement. The cell wall-targeting agent caffeine caused hypersensitivity to CQ, as did cell wall perturbation by sonication. The phenotypes were not caused by CQ-induced changes to cell wall components. Instead, CQ accumulated to higher levels in cells with perturbed cell walls: CQ uptake was 2- to 3-fold greater in bck1Δ and slt2Δ mutants than in wild-type yeast. CQ toxicity was synergistic with that of the major cell wall-targeting antifungal drug, caspofungin. The MIC of caspofungin against the yeast pathogen Candida albicans was decreased 2-fold by 250 μM CQ and up to 8-fold at higher CQ concentrations. Similar effects were seen in Candida glabrata and Aspergillus fumigatus. The results show that the cell wall is critical for CQ resistance in fungi and suggest that combination treatments with cell wall-targeting drugs could have potential for antifungal treatment. Abstract
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Bates S, Hall RA, Cheetham J, Netea MG, MacCallum DM, Brown AJP, Odds FC, Gow NAR
(2013). Role of the Candida albicans MNN1 gene family in cell wall structure and virulence. BMC Res Notes
Role of the Candida albicans MNN1 gene family in cell wall structure and virulence.
BACKGROUND: the Candida albicans cell wall is the first point of contact with the host, and its outer surface is heavily enriched in mannoproteins modified through the addition of N- and O-mannan. Previous work, using mutants with gross defects in glycosylation, has clearly identified the importance of mannan in the host-pathogen interaction, immune recognition and virulence. Here we report the first analysis of the MNN1 gene family, which contains six members predicted to act as α-1,3 mannosyltransferases in the terminal stages of glycosylation. FINDINGS: We generated single null mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro and in vivo defects. Null mutants in other members of the family demonstrated no phenotypic defects, suggesting that these members may display functional redundancy. The mnn14Δ null mutant displayed hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting an altered cell wall structure. However, no gross changes in cell wall composition or N-glycosylation were identified in this mutant, although an extension of phosphomannan chain length was apparent. Although the cell wall defects associated with the mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model. CONCLUSION: Mnn14 plays a distinct role from other members of the MNN1 family, demonstrating that specific N-glycan outer chain epitopes are required in the host-pathogen interaction and virulence. Abstract
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Hall RA, Bates S, Lenardon MD, Maccallum DM, Wagener J, Lowman DW, Kruppa MD, Williams DL, Odds FC, Brown AJP, et al
(2013). The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans. PLoS Pathog
The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans.
The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans. Abstract
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Hall RA, Bates S, Wagener J, Odds FC, Alvarez FJ, Brown AJP, Gow NAR
(2012). Mannosylation of Candida albicans and its importance for immune recognition. MYCOSES
, 67-67. Author URL
Milne SW, Cheetham J, Lloyd D, Aves S, Bates S
(2011). Cassettes for PCR-mediated gene tagging in Candida albicans utilizing nourseothricin resistance. Yeast
Cassettes for PCR-mediated gene tagging in Candida albicans utilizing nourseothricin resistance.
In recent years a number of molecular tools have been reported for use in the human fungal pathogen Candida albicans, including PCR-mediated approaches for gene disruption, conditional expression and epitope tagging. Traditionally these methods have utilized auxotrophic markers; however, the availability of auxotrophic markers can be limiting and in some instances their use may also impact on the interpretation of results. As a result, the use of positive selection markers has now become more commonplace. Here we report the development and validation of a set of cassettes for PCR-mediated gene tagging and overexpression studies utilizing the nourseothricin resistance (CaNAT1) positive selection marker. In particular we have produced cassettes containing yeast-enhanced GFP, YFP, CFP, RFP and a combined V5-6xHis epitope tag. The cassettes are engineered for use in PCR-mediated gene tagging strategies where insertion is targeted to the 3' end of the gene of interest. In addition, to facilitate protein functional analysis and genetic suppression studies through the use of overexpression, we have also constructed a promoter replacement cassette containing the ENO1 promoter which is known to be expressed at a high level. These cassettes expand on the range of molecular tools available for working with C. albicans and may also be used in other Candida species that display sensitivity to nourseothricin. Abstract
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Mora-Montes HM, Bates S, Netea MG, Castillo L, Brand A, Buurman ET, Díaz-Jiménez DF, Jan Kullberg B, Brown AJP, Odds FC, et al
(2010). A multifunctional mannosyltransferase family in Candida albicans determines cell wall mannan structure and host-fungus interactions. J Biol Chem
A multifunctional mannosyltransferase family in Candida albicans determines cell wall mannan structure and host-fungus interactions.
The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTDelta null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of beta-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system. Abstract
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Mora-Montes HM, Netea MG, Sheth CC, Brown GD, Bates S, Kullberg BJ, Brown AJP, Odds FC, Gow NAR
(2009). Candida albicans cell wall glycobiology: biosynthesis of N-linked mannans and interaction with the host innate immune system. MYCOSES
, 24-24. Author URL
Butler G, Rasmussen MD, Lin MF, Santos MAS, Sakthikumar S, Munro CA, Rheinbay E, Grabherr M, Forche A, Reedy JL, et al
(2009). Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature
Evolution of pathogenicity and sexual reproduction in eight Candida genomes.
Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes. Abstract
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Chaves GM, Bates S, Maccallum DM, Odds FC
(2007). Candida albicans GRX2, encoding a putative glutaredoxin, is required for virulence in a murine model. Genet Mol Res
Candida albicans GRX2, encoding a putative glutaredoxin, is required for virulence in a murine model.
Resistance of Candida albicans to reactive oxygen species is thought to enhance its virulence in mammalian hosts. Genes such as SOD1, which encodes the anti-oxidant, superoxide dismutase, are known virulence factors. We disrupted the gene GRX2, which encodes a putative glutathione reductase (glutaredoxin) in C. albicans, and we compared the mutant with an sod1Deltamutant. In vitro, the grx2Deltastrain, but not the sod1Delta strain, was defective in hypha formation. The grx2Deltastrain, but not sod1Delta, was significantly more susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, both mutants were susceptible to 1 mM menadione, but grx2Deltanull alone was resistant to diamide. Both mutants were attenuated in a murine intravenous challenge model, and a GRX2 reintegrant regained partial virulence. Emphasis on the putative function of products of genes such as SOD1 and GRX2 in resistance to oxidative stress may oversimplify their functions in the virulence process, since the grx2Deltastrain also gave defective hypha formation. Both mutants were sensitive to menadione and were slow to form germ tubes, though growth rates matched controls once the lag phase was passed. Abstract
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Bates S, de la Rosa JM, MacCallum DM, Brown AJP, Gow NAR, Odds FC
(2007). Candida albicans Iff11, a secreted protein required for cell wall structure and virulence. Infect Immun
Candida albicans Iff11, a secreted protein required for cell wall structure and virulence.
The Candida albicans cell wall is the immediate point of contact with the host and is implicated in the host-fungal interaction and virulence. To date, a number of cell wall proteins have been identified and associated with virulence. Analysis of the C. albicans genome has identified the IFF gene family as encoding the largest family of cell wall-related proteins. This family is also conserved in a range of other Candida species. Iff11 differs from other family members in lacking a GPI anchor, and we have demonstrated it to be O glycosylated and secreted in C. albicans. A null mutant lacking IFF11 was hypersensitive to cell wall-damaging agents, suggesting a role in cell wall organization. In a murine model of systemic infection the null mutant was highly attenuated in virulence, and survival-standardized infections suggest it is required to establish an infection. This work provides the first evidence of the importance of this gene family in the host-fungal interaction and virulence. Abstract
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Mora-Montes HM, Bates S, Netea MG, Díaz-Jiménez DF, López-Romero E, Zinker S, Ponce-Noyola P, Kullberg BJ, Brown AJP, Odds FC, et al
(2007). Endoplasmic reticulum α-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host-fungus interaction. Eukaryotic Cell
Endoplasmic reticulum α-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host-fungus interaction
The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc3Man9GlcNAc 2 N-glycan is processed by α-glucosidases I and II and α1,2-manno.sidase to generate Man8GlcNAc2. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for α-glucosidase I, α-glucosidase II catalytic subunit, and α1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated β-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions. Copyright © 2007, American Society for Microbiology. All Rights Reserved. Abstract
Gow NAR, Netea MG, Munro CA, Ferwerda G, Bates S, Mora-Montes HM, Walker L, Jansen T, Jacobs L, Tsoni V, et al
(2007). Immune recognition of Candida albicans beta-glucan by dectin-1. J Infect Dis
Immune recognition of Candida albicans beta-glucan by dectin-1.
Beta (1,3)-glucans represent 40% of the cell wall of the yeast Candida albicans. The dectin-1 lectin-like receptor has shown to recognize fungal beta (1,3)-glucans and induce innate immune responses. The importance of beta-glucan-dectin-1 pathways for the recognition of C. albicans by human primary blood cells has not been firmly established. In this study we demonstrate that cytokine production by both human peripheral blood mononuclear cells and murine macrophages is dependent on the recognition of beta-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of beta-glucans on the surface of the cell wall and subsequent recognition by dectin-1, whereas live yeasts stimulated monocytes mainly via recognition of cell-surface mannans. Dectin-1 induced cytokine production through the following 2 pathways: Syk-dependent production of the T-helper (Th) 2-type anti-inflammatory cytokine interleukin-10 and Toll-like receptor-Myd88-dependent stimulation of monocyte-derived proinflammatory cytokines, such as tumor necrosis factor-alpha. In contrast, stimulation of Th1-type cytokines, such as interferon-gamma , by C. albicans was independent of the recognition of beta-glucans by dectin-1. In conclusion, C. albicans induces production of monocyte-derived and T cell-derived cytokines through distinct pathways dependent on or independent of dectin-1. Abstract
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Netea MG, Gow NAR, Munro CA, Bates S, Collins C, Ferwerda G, Hobson RP, Bertram G, Hughes HB, Jansen T, et al
(2006). Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors. J Clin Invest
Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors.
The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall. Abstract
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Bates S, Hughes HB, Munro CA, Thomas WPH, MacCallum DM, Bertram G, Atrih A, Ferguson MAJ, Brown AJP, Odds FC, et al
(2006). Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans. J Biol Chem
Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans.
The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the alpha1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the alpha1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1delta null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence. Abstract
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Braun BR, Hoog MV, d'Enfert C, Martchenko M, Dungan J, Kuo A, Inglis DO, Uhl MA, Hogues H, Berriman M, et al
(2005). A human-curated annotation of the Candida albicans genome. PLOS GENETICS
(1), 36-57. Author URL
Bates S, MacCallum DM, Bertram G, Munro CA, Hughes HB, Buurman ET, Brown AJP, Odds FC, Gow NAR
(2005). Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence. J Biol Chem
Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence.
The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn(2+) as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca(2+) and Mn(2+) ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn(2+)-dependent mannosyltransferases. The Capmr1Delta null mutant was viable in vitro and had no growth defect even on media containing low Ca(2+)/Mn(2+) ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans. Abstract
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Munro CA, Bates S, Buurman ET, Hughes HB, Maccallum DM, Bertram G, Atrih A, Ferguson MAJ, Bain JM, Brand A, et al
(2005). Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence. J Biol Chem
Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence.
The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant alpha-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Delta and mnt1Delta single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Deltamnt2Delta mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections. Abstract
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Chapa y Lazo B, Bates S, Sudbery P
(2005). The G1 cyclin Cln3 regulates morphogenesis in Candida albicans. Eukaryot Cell
The G1 cyclin Cln3 regulates morphogenesis in Candida albicans.
In Saccharomyces cerevisiae, the G1 cyclin Cln3 initiates the Start of a mitotic cell cycle in response to size and nutrient inputs. Loss of Cln3 delays but does not prevent Start, due to the eventual Cln3-independent transcription of CLN1 and CLN2. When unbudded cells of the human pathogen Candida albicans were depleted of the G1 cyclin Cln3 they increased in size but did not bud. Thus, unlike S. cerevisiae, Cln3 is essential for budding in C. albicans. However, eventually the large unbudded cells spontaneously produced filamentous forms. The morphology was growth medium dependent; on nutritionally poor medium the polarized outgrowths fulfilled the formal criteria for true hyphae. This state is stable, and continued growth leads to a hyphal mycelium, which invades the agar substratum. Interestingly, it is also required for normal hyphal development, as Cln3-depleted cells develop morphological abnormalities if challenged with hyphal inducing signals such as serum or neutral pH. Taken together, these results show that, in C. albicans, Cln3 has assumed a critical role in coordinating mitotic cell division with differentiation. Abstract
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Wightman R, Bates S, Amornrrattanapan P, Sudbery P
(2004). In Candida albicans, the Nim1 kinases Gin4 and Hsl1 negatively regulate pseudohypha formation and Gin4 also controls septin organization. J Cell Biol
In Candida albicans, the Nim1 kinases Gin4 and Hsl1 negatively regulate pseudohypha formation and Gin4 also controls septin organization.
In the development of hyphal germ tubes of Candida albicans, a band of septin forms at the base of the germ tube (basal septin band). Later, a septin ring forms, which organizes the first septum within the germ tube (septin ring). We have investigated the role of the Nim1 kinases, Gin4 and Hsl1, in the formation of these septin structures. We show that during germ tube formation, Gin4 is required for the organization of the septin ring but not the basal septin band. Hsl1 is not required for the formation of either septin rings or basal bands. Unexpectedly, we found that both gin4Delta and hsl1Delta mutants form pseudohyphae constitutively, in a fashion that in the case of gin4Delta, is partly independent of Swe1. Gin4-depleted pseudohyphae are unable to form hyphae when challenged with serum, but this can be overcome by ectopic expression of Gin4 from the MET3 promoter. Thus, Gin4 may regulate the developmental switch from pseudohyphae to hyphae. Abstract
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Hobson RP, Munro CA, Bates S, MacCallum DM, Cutler JE, Heinsbroek SEM, Brown GD, Odds FC, Gow NAR
(2004). Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition. J Biol Chem
Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition.
The outer layer of the cell wall of the human pathogenic fungus Candida albicans is enriched with heavily mannosylated glycoproteins that are the immediate point of contact between the fungus and cells of the host, including phagocytes. Previous work had identified components of the acid-labile fraction of N-linked mannan, comprising beta-1,2-linked mannose residues attached via a phosphodiester bond, as potential ligands for macrophage receptors and modulators of macrophage function. We therefore isolated and disrupted the CaMNN4 gene, which is required for mannosyl phosphate transfer and hence the attachment of beta-1,2 mannose oligosaccharides to the acid-labile N-mannan side chains. With the mannosylphosphate eliminated, the mnn4Delta null mutant was unable to bind the charged cationic dye Alcian Blue and was devoid of acid-labile beta-1,2-linked oligomannosaccharides. The mnn4Delta mutant was unaffected in cell growth and morphogenesis in vitro and in virulence in a murine model of systemic C. albicans infection. The null mutant was also not affected in its interaction with macrophages. Mannosylphosphate is therefore not required for macrophage interactions or for virulence of C. albicans. Abstract
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Thomson LM, Bates S, Yamazaki S, Arisawa M, Aoki Y, Gow NA
(2000). Functional characterization of the Candida albicans MNT1 mannosyltransferase expressed heterologously in Pichia pastoris. J Biol Chem
Functional characterization of the Candida albicans MNT1 mannosyltransferase expressed heterologously in Pichia pastoris.
The alpha1,2-mannosyltransferase gene MNT1 of the human fungal pathogen Candida albicans has been shown to be important for its adherence to various human surfaces and for virulence (Buurman, E. T. Westwater, C. Hube, B. Brown, A. P. J. Odds, F. C. and Gow, N. A. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7670-7675). The CaMnt1p is a type II membrane protein, which is part of a family of proteins that are important for both O- and N-linked mannosylation in fungi and which represent a distinct subclass of glycosyltransferase enzymes. Here we use heterologous expression of CaMNT1 in the methylotrophic yeast Pichia pastoris to characterize the properties of the CaMnt1p enzyme as an example of this family of enzymes and to identify key amino acid residues required for coordination of the metal co-factor and for the retaining nucleophilic mechanism of the transferase reaction. We show that the enzyme can use both Mn(2+) and Zn(2+) as metal ion co-factors and that the reaction catalyzed is specific for alpha-methyl mannoside and alpha1,2-mannobiose acceptors. The N-terminal cytoplasmic tail, transmembrane domains, and stem regions were shown to be dispensable for activity, whereas truncations to the C-terminal catalytic domain destroyed activity without markedly affecting transcription of the truncated gene. Abstract
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Gow NAR, Bates S, Brown AJP, Buurman ET, Thomson LM, Westwater C (1999). Candida cell wall mannosylation: importance in host-fungus interaction and potential as a target for the development of antifungal drugs. Biochemical Society Transactions, 27(3), a86-a86.
Gow NA, Bates S, Brown AJ, Buurman ET, Thomson LM, Westwater C
(1999). Candida cell wall mannosylation: importance in host-fungus interaction and potential as a target for the development of antifungal drugs. Biochem Soc Trans
(4), 512-516. Author URL
Bates S, Roscoe RA, Althorpe NJ, Brammar WJ, Wilkins BM
(1999). Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription. Microbiology (Reading)
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Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription.
The leading region of a plasmid is the first sector to enter the recipient cell in bacterial conjugation. This sector of IncI1 plasmid ColIb-P9 includes genes that are transcribed in a transient pulse early in the conjugatively infected cell to promote establishment of the immigrant plasmid. Evidence is presented that the burst of gene expression is regulated by a process which is independent of a repressor but dependent on the orientation of the genes on the unique plasmid strand transferred in conjugation. The nucleotide sequence of 11.7 kb of the leading region was determined and found to contain 10 ORFs; all are orientated such that the template strand for transcription corresponds to the transferred strand. The leading region contains three dispersed repeats of a sequence homologous to a novel promoter in ssDNA described by H. Masai & K. Arai (1997, Cell 89, 897-907). It is proposed that the repeats are promoters that form in the transferring strand of ColIb to support transient transcription of genes transferred early in conjugation. Abstract
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Bates S, Cashmore AM, Wilkins BM
(1998). IncP plasmids are unusually effective in mediating conjugation of Escherichia coli and Saccharomyces cerevisiae: involvement of the tra2 mating system. J Bacteriol
IncP plasmids are unusually effective in mediating conjugation of Escherichia coli and Saccharomyces cerevisiae: involvement of the tra2 mating system.
Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Palpha system caused detectable mobilization to yeast, giving peak values of 5 x 10(-5) transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of oriT in cis and the provision in trans of the Palpha Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Palpha conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Palpha system in mediating gene transfer from bacteria to eukaryotes. Abstract
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Wilkins BM, Bates S (1997). Gene transfer by bacterial conjugation: Establishment of the immigrant plasmid in the recipient cell. In Burby SJW (Ed) Molecular Microbiology, Springer-Verlag, 43-64.