Publications by authors named "Robin C May"

88 Publications

Now for something completely different: Prototheca, pathogenic algae.

PLoS Pathog 2021 Apr 1;17(4):e1009362. Epub 2021 Apr 1.

Institute of Microbiology & Infection and School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

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http://dx.doi.org/10.1371/journal.ppat.1009362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016101PMC
April 2021

Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens.

J Am Soc Mass Spectrom 2021 Mar 1. Epub 2021 Mar 1.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.

The ESKAPE pathogens (, , , , , and ) represent clinically important bacterial species that are responsible for most hospital-acquired drug-resistant infections; hence, the need for rapid identification is of high importance. Previous work has demonstrated the suitability of liquid extraction surface analysis mass spectrometry (LESA MS) for the direct analysis of colonies of two of the ESKAPE pathogens ( and ) growing on agar. Here, we apply LESA MS to the remaining four ESKAPE species ( E745, KP257, AYE, and S11) as well as V583 (a close relative of ) and a clinical isolate of AC02 using an optimized solvent sampling system. In each case, top-down LESA MS/MS was employed for protein identification. In total, 24 proteins were identified from 37 MS/MS spectra by searching against protein databases for the individual species. The MS/MS spectra for the identified proteins were subsequently searched against multiple databases from multiple species in an automated data analysis workflow with a view to determining the accuracy of identification of unknowns. Out of 24 proteins, 19 were correctly assigned at the protein and species level, corresponding to an identification success rate of 79%.
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http://dx.doi.org/10.1021/jasms.0c00466DOI Listing
March 2021

Secretes Small Molecules That Inhibit IL-1 Inflammasome-Dependent Secretion.

Mediators Inflamm 2020 3;2020:3412763. Epub 2020 Dec 3.

Laboratory of Applied Immunology, Department of Cellular Biology, Institute of Biological Sciences, University of Brasília, Brazil.

is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by B3501 strain and its acapsular mutant in inflammasome activation in an model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1 secretion and LDH release via pyroptosis) more strongly than conditioned media from , regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501's conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by . These results were confirmed by experiments, where exposure to conditioned media led to higher fungal burdens in culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival and and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.
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http://dx.doi.org/10.1155/2020/3412763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748918PMC
December 2020

Plugging a Leak: How Phagosomes "Stretch" to Accommodate Pathogen Growth.

Cell Host Microbe 2020 12;28(6):774-775

Institute of Microbiology & Infection and School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Electronic address:

Phagocytes engulf pathogens into a membrane bound compartment called a phagosome, but what happens when engulfed pathogens start growing? In this issue of Cell Host & Microbe,Westman et al. (2020) show that lysosomes fuse with phagosomes to maintain phagosomal membrane integrity as the fungal pathogen Candida albicans expands.
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http://dx.doi.org/10.1016/j.chom.2020.11.005DOI Listing
December 2020

Direct identification of bacterial and human proteins from infected wounds in living 3D skin models.

Sci Rep 2020 07 17;10(1):11900. Epub 2020 Jul 17.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

Trauma is one of the leading causes of death in people under the age of 49 and complications due to wound infection are the primary cause of death in the first few days after injury. The ESKAPE pathogens are a group of bacteria that are a leading cause of hospital-acquired infections and a major concern in terms of antibiotic resistance. Here, we demonstrate a novel and highly accurate approach for the rapid identification of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) directly from infected wounds in 3D in vitro skin models. Wounded skin models were inoculated with bacteria and left to incubate. Bacterial proteins were identified within minutes, directly from the wound, by liquid extraction surface analysis mass spectrometry. This approach was able to distinguish closely related strains and, unlike genomic approaches, can be modified to provide dynamic information about pathogen behaviour at the wound site. In addition, since human skin proteins were also identified, this method offers the opportunity to analyse both host and pathogen biomarkers during wound infection in near real-time.
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http://dx.doi.org/10.1038/s41598-020-68233-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368034PMC
July 2020

AIDS-Related Mycoses: Updated Progress and Future Priorities.

Trends Microbiol 2020 06 19;28(6):425-428. Epub 2020 Feb 19.

Infectious Diseases Institute and Department of Medicine, School of Medicine, College of Health Sciences, Makerere University, Kampala, Uganda; Division of Infectious Diseases and International Medicine, Department of Medicine, University of Minnesota, Minneapolis, USA.

Serious fungal infections continue to devastate people living with HIV and remain a leading cause of infection-related deaths in this population, second only to tuberculosis. The third AIDS-related mycoses workshop updated progress in the field over the last 3 years and highlighted six key action points for the future.
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http://dx.doi.org/10.1016/j.tim.2020.01.009DOI Listing
June 2020

Viral infection triggers interferon-induced expulsion of live Cryptococcus neoformans by macrophages.

PLoS Pathog 2020 02 27;16(2):e1008240. Epub 2020 Feb 27.

Institute of Microbiology & Infection and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Cryptococcus neoformans is an opportunistic human pathogen, which causes serious disease in immunocompromised hosts. Infection with this pathogen is particularly relevant in HIV+ patients, where it leads to around 200,000 deaths per annum. A key feature of cryptococcal pathogenesis is the ability of the fungus to survive and replicate within the phagosome of macrophages, as well as its ability to be expelled from host cells via a novel non-lytic mechanism known as vomocytosis. Here we show that cryptococcal vomocytosis from macrophages is strongly enhanced by viral coinfection, without altering phagocytosis or intracellular proliferation of the fungus. This effect occurs with distinct, unrelated human viral pathogens and is recapitulated when macrophages are stimulated with the anti-viral cytokines interferon alpha or beta (IFNα or IFNβ). Importantly, the effect is abrogated when type-I interferon signalling is blocked, thus underscoring the importance of type-I interferons in this phenomenon. Lastly, our data help resolve previous, contradictory animal studies on the impact of type I interferons on cryptococcal pathogenesis and suggest that secondary viral stimuli may alter patterns of cryptococcal dissemination in the host.
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http://dx.doi.org/10.1371/journal.ppat.1008240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046190PMC
February 2020

Electroporation and Mass Spectrometry: A New Paradigm for In Situ Analysis of Intact Proteins Direct from Living Yeast Colonies.

Anal Chem 2020 02 22;92(3):2605-2611. Epub 2020 Jan 22.

School of Biosciences , University of Birmingham , Edgbaston , Birmingham B15 2TT , U.K.

Yeasts constitute an oft-neglected class of pathogens among which the resistance to first-line treatments, attributed in part to mutations in efflux pumps, is rapidly emerging. Their thick, chitin-reinforced cell walls render cell lysis difficult, complicating their analysis and identification by methods routinely used for bacteria, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Liquid extraction surface analysis mass spectrometry (LESA-MS) has previously been applied to the analysis of intact proteins from Gram-positive and Gram-negative bacterial colonies sampled directly on solid nutrient media. To date, a similar analysis of yeast colonies has not proved possible. Here we demonstrate the rapid release of intact yeast proteins for LESA-MS by electroporation using a home-built high-voltage device designed to lyse cells grown in colonies on agar media. Detection and identification of previously inaccessible proteins from baker's yeast , as well as two clinically relevant yeast species ( and ), is shown. The electroporation approach also has the potential to be translated to other mass spectrometric analysis techniques, including MALDI and various ambient ionization methods.
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http://dx.doi.org/10.1021/acs.analchem.9b04365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145282PMC
February 2020

Pyrifenox, an ergosterol inhibitor, differentially affects Cryptococcus neoformans and Cryptococcus gattii.

Med Mycol 2020 Oct;58(7):928-937

Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz (FIOCRUZ), Curitiba, Brazil.

Cryptococcosis is a life-threatening fungal infection. New therapeutic approaches are necessary to combat cryptococcosis, as the currently available therapeutic protocols are expensive and generally result in deleterious side effects. Pyrifenox is an antifungal compound that affects phytopathogens by inhibiting the biosynthesis of ergosterol. In this study, we investigated the effects of pyrifenox on Cryptococcus neoformans and Cryptococcus gattii growth, capsule architecture and export of the major capsule component, glucuroxylomannan (GXM). Pyrifenox inhibited the growth of C. neoformans, but was significantly less effective against C. gattii. The resistance of C. gattii to pyrifenox was associated with the expression of efflux pump genes, particularly AFR1 and AFR2, since mutant cells lacking expression of these genes became sensitive to pyrifenox. Analysis of the cryptococcal capsule by India ink counterstaining, immunofluorescence, and scanning electron microscopy showed that pyrifenox affected capsular dimensions in both species. However, GXM fibers were shorter and uniformly distributed in C. neoformans, whereas in C. gattii the number of fibers was reduced. Pyrifenox-treated C. gattii developed unusually long chains of undivided cells. The secretion of GXM was markedly reduced in both species after treatment with pyrifenox. Altogether, the results indicated that pyrifenox differently affects C. neoformans and C. gattii. In addition, it highlights a potential role for pyrifenox as an inhibitor of GXM export in experimental models involving pathogenic cryptococci.
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http://dx.doi.org/10.1093/mmy/myz132DOI Listing
October 2020

Vomocytosis: What we know so far.

Cell Microbiol 2020 02 20;22(2):e13145. Epub 2019 Nov 20.

Laboratory of Host and Pathogen Interactions, Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.

Vomocytosis, or nonlytic exocytosis, has been reported for Cryptococcus neoformans since 2006. Since then, the repertoire of vomocytosing pathogens and host cells has increased and so have the molecular components linked to vomocytosis occurrence. Nonetheless, the mechanism underlying this phenomenon, whether it is triggered by the host or the pathogen, and how it affects disease progression are still unresolved. This review contains a summary of the main findings regarding vomocytosis and the outstanding questions puzzling scientists to this day.
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http://dx.doi.org/10.1111/cmi.13145DOI Listing
February 2020

Hepatocytes Delete Regulatory T Cells by Enclysis, a CD4 T Cell Engulfment Process.

Cell Rep 2019 11;29(6):1610-1620.e4

Institute of Immunology and Immunotherapy, Centre for Liver and Gastrointestinal Research, University of Birmingham, Birmingham, UK; NIHR Birmingham Liver Biomedical Research Centre, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK. Electronic address:

CD4 T cells play critical roles in directing immunity, both as T helper and as regulatory T (Treg) cells. Here, we demonstrate that hepatocytes can modulate T cell populations through engulfment of live CD4 lymphocytes. We term this phenomenon enclysis to reflect the specific enclosure of CD4 T cells in hepatocytes. Enclysis is selective for CD4 but not CD8 cells, independent of antigen-specific activation, and occurs in human hepatocytes in vitro, ex vivo, and in vivo. Intercellular adhesion molecule 1 (ICAM-1) facilitates T cell early adhesion and internalization, whereas hepatocytes form membrane lamellipodia or blebs to mediate engulfment. T cell internalization is unaffected by wortmannin and Rho kinase inhibition. Hepatocytes engulf Treg cells more efficiently than non-Treg cells, but Treg cell-containing vesicles preferentially acidify overnight. Thus, enclysis is a biological process with potential effects on immunomodulation and opens a new field for research to fully understand CD4 T cell dynamics in liver inflammation.
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http://dx.doi.org/10.1016/j.celrep.2019.09.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057271PMC
November 2019

Extracellular vesicles of human pathogenic fungi.

Curr Opin Microbiol 2019 12 4;52:90-99. Epub 2019 Jul 4.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Electronic address:

Extracellular vesicles play a significant role in many aspects of cellular life including cell-to-cell communication, pathogenesis and cancer progression. However very little is known about their role in fungi and we are just at the beginning of understanding their influence on fungal pathophysiology and host-pathogen interactions. Recent findings have revealed a role for fungal vesicles in triggering anti-microbial activities as well as in modulating virulence strategies, suggesting potential new avenues for antifungal therapies. In this review, we summarize our current understanding of fungal extracellular vesicles, including their biogenesis, secretion and size variation, and discuss how they may influence the human immune response and some key questions that remain unanswered.
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http://dx.doi.org/10.1016/j.mib.2019.05.007DOI Listing
December 2019

Zinc and Iron Homeostasis: Target-Based Drug Screening as New Route for Antifungal Drug Development.

Front Cell Infect Microbiol 2019 29;9:181. Epub 2019 May 29.

School of Biosciences, Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom.

The incidence of fungal diseases is on the rise and the number of fatalities is still unacceptably high. While advances into antifungal drug development have been made there remains an urgent need to develop novel antifungal agents targeting as-yet unexploited pathways, such as metal ion homeostasis. Here we report such an approach by developing a metal sensor screen in the opportunistic human fungal pathogen . Using this reporter strain, we screened a library of 1,200 compounds and discovered several active compounds not previously described as chemical entities with antifungal properties. Two of these, artemisinin and pyrvinium pamoate, have been further characterized and their interference with metal homeostasis and potential as novel antifungal compounds validated. Lastly, we demonstrate that the same strain can be used to report on intracellular conditions within host phagocytes, paving the way toward the development of novel screening platforms that could identify compounds with the potential to perturb ion homeostasis of the pathogen specifically within host cells.
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http://dx.doi.org/10.3389/fcimb.2019.00181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548825PMC
January 2020

15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection.

PLoS Pathog 2019 03 28;15(3):e1007597. Epub 2019 Mar 28.

Bateson Centre, Firth Court, University of Sheffield, Sheffield, South Yorkshire, United Kingdom.

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.
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http://dx.doi.org/10.1371/journal.ppat.1007597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438442PMC
March 2019

Application of High-Field Asymmetric Waveform Ion Mobility Separation to LESA Mass Spectrometry of Bacteria.

Anal Chem 2019 04 14;91(7):4755-4761. Epub 2019 Mar 14.

We have previously demonstrated the analysis of intact proteins directly from bacterial colonies (including Gram-negative and Gram-positive clinical isolates) grown on agar media by liquid extraction surface analysis mass spectrometry (LESA MS). Several challenges were identified in that work, including (1) interference of background signal derived from the nutrient media ( Escherichia coli), (2) a high density of protein peaks leading to the isolation of multiple protein precursor ions in a single window and consequent acquisition of composite tandem mass spectra ( Pseudomonas aeruginosa), and (3) the overabundance of secreted peptides suppressing peaks corresponding to proteins ( Staphylococcus aureus). Here, we present the coupling of high-field asymmetric waveform ion mobility spectrometry (FAIMS) separation into the LESA MS protocol, with the aim of resolving the aforementioned challenges and thus improving the capabilities of LESA MS for bacterial characterization. The results show that inclusion of FAIMS expands the range of detected proteins through separation of background peaks from protein signal, as well as through resolution of overlapping protein peaks which could not previously be isolated by LESA MS alone.
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http://dx.doi.org/10.1021/acs.analchem.9b00307DOI Listing
April 2019

A Glucuronoxylomannan Epitope Exhibits Serotype-Specific Accessibility and Redistributes towards the Capsule Surface during Titanization of the Fungal Pathogen Cryptococcus neoformans.

Infect Immun 2019 04 25;87(4). Epub 2019 Mar 25.

Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, United Kingdom

Disseminated infections with the fungal species or, less frequently, are an important cause of mortality in immunocompromised individuals. Central to the virulence of both species is an elaborate polysaccharide capsule that consists predominantly of glucuronoxylomannan (GXM). Due to its abundance, GXM is an ideal target for host antibodies, and several monoclonal antibodies (mAbs) have previously been derived using purified GXM or whole capsular preparations as antigens. In addition to their application in the diagnosis of cryptococcosis, anti-GXM mAbs are invaluable tools for studying capsule structure. In this study, we report the production and characterization of a novel anti-GXM mAb, Crp127, that unexpectedly reveals a role for GXM remodeling during the process of fungal titanization. We show that Crp127 recognizes a GXM epitope in an -acetylation-dependent, but xylosylation-independent, manner. The epitope is differentially expressed by the four main serotypes of and , is heterogeneously expressed within clonal populations of serotype B strains, and is typically confined to the central region of the enlarged capsule. Uniquely, however, this epitope redistributes to the capsular surface in titan cells, a recently characterized morphotype where haploid 5-μm cells convert to highly polyploid cells of >10 μm with distinct but poorly understood capsular characteristics. Titan cells are produced in the host lung and critical for successful infection. Crp127 therefore advances our understanding of cryptococcal morphological change and may hold significant potential as a tool to differentially identify cryptococcal strains and subtypes.
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http://dx.doi.org/10.1128/IAI.00731-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434129PMC
April 2019

Transcriptional Heterogeneity of VGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways.

mSphere 2018 10 24;3(5). Epub 2018 Oct 24.

Infectious Disease and Microbiome Program, The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

is a pathogenic yeast of humans and other animals which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual-transcriptome sequencing (RNA-seq) experiment for four lineages of (lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons of to gene expression levels indicated that lineage VGII is transcriptionally divergent from non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment, and ergosterol production. Several paralogous genes demonstrated subfunctionalization between lineages, including upregulation of capsule biosynthesis-related gene and downregulation of in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule gene in VGIV, which have lost the gene. Differential expression of one in five genes was detected following coincubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and downregulation of capsule attachment genes. We also found that VGII switches expression of two laccase paralogs (from to ) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages of and the diversity of transcriptional responses at this host-pathogen interface. The transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of in rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccase to expression of Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response to This work highlights the dynamic roles of key virulence genes in response to macrophages.
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http://dx.doi.org/10.1128/mSphere.00445-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200987PMC
October 2018

In Fungal Intracellular Pathogenesis, Form Determines Fate.

mBio 2018 10 23;9(5). Epub 2018 Oct 23.

Department of Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, Baltimore, Maryland, USA

For pathogenic microbes to survive ingestion by macrophages, they must subvert powerful microbicidal mechanisms within the phagolysosome. After ingestion, undergoes a morphological transition producing hyphae, while the surrounding phagosome exhibits a loss of phagosomal acidity. However, how these two events are related has remained enigmatic. Now Westman et al. (mBio 9:e01226-18, 2018, https://doi.org/10.1128/mBio.01226-18) report that phagosomal neutralization results from disruption of phagosomal membrane integrity by the enlarging hyphae, directly implicating the morphological transition in physical damage that promotes intracellular survival. The intracellular strategy shows parallels with another fungal pathogen, , where a morphological changed involving capsular enlargement intracellularly is associated with loss of membrane integrity and death of the host cell. These similarities among distantly related pathogenic fungi suggest that morphological transitions that are common in fungi directly affect the outcome of the fungal cell-macrophage interaction. For this class of organisms, form determines fate in the intracellular environment.
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http://dx.doi.org/10.1128/mBio.02092-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199499PMC
October 2018

Gene flow contributes to diversification of the major fungal pathogen Candida albicans.

Nat Commun 2018 06 8;9(1):2253. Epub 2018 Jun 8.

Department of Mycology, Fungal Biology and Pathogenicity Unit, Institut Pasteur, INRA, 75015, Paris, France.

Elucidating population structure and levels of genetic diversity and recombination is necessary to understand the evolution and adaptation of species. Candida albicans is the second most frequent agent of human fungal infections worldwide, causing high-mortality rates. Here we present the genomic sequences of 182 C. albicans isolates collected worldwide, including commensal isolates, as well as ones responsible for superficial and invasive infections, constituting the largest dataset to date for this major fungal pathogen. Although, C. albicans shows a predominantly clonal population structure, we find evidence of gene flow between previously known and newly identified genetic clusters, supporting the occurrence of (para)sexuality in nature. A highly clonal lineage, which experimentally shows reduced fitness, has undergone pseudogenization in genes required for virulence and morphogenesis, which may explain its niche restriction. Candida albicans thus takes advantage of both clonality and gene flow to diversify.
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http://dx.doi.org/10.1038/s41467-018-04787-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993739PMC
June 2018

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

PLoS Pathog 2018 05 18;14(5):e1006978. Epub 2018 May 18.

Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen, United Kingdom.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.
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http://dx.doi.org/10.1371/journal.ppat.1006978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959070PMC
May 2018

Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii.

Nat Commun 2018 04 19;9(1):1556. Epub 2018 Apr 19.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

The Pacific Northwest outbreak of cryptococcosis, caused by a near-clonal lineage of the fungal pathogen Cryptococcus gattii, represents the most significant cluster of life-threatening fungal infections in otherwise healthy human hosts currently known. The outbreak lineage has a remarkable ability to grow rapidly within human white blood cells, using a unique 'division of labour' mechanism within the pathogen population, where some cells adopt a dormant behaviour to support the growth of neighbouring cells. Here we demonstrate that pathogenic 'division of labour' can be triggered over large cellular distances and is mediated through the release of extracellular vesicles by the fungus. Isolated vesicles released by virulent strains are taken up by infected host macrophages and trafficked to the phagosome, where they trigger the rapid intracellular growth of non-outbreak fungal cells that would otherwise be eliminated by the host. Thus, long distance pathogen-to-pathogen communication via extracellular vesicles represents a novel mechanism to control complex virulence phenotypes in Cryptococcus gattii and, potentially, other infectious species.
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http://dx.doi.org/10.1038/s41467-018-03991-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908794PMC
April 2018

Variability in innate host immune responses to cryptococcosis.

Mem Inst Oswaldo Cruz 2018 16;113(7):e180060. Epub 2018 Apr 16.

School of Biosciences, Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, UK.

Cryptococcosis is an invasive fungal disease caused by Cryptococcus neoformans and the closely related species C. gattii. The severe form of the disease, cryptococcal meningitis (CM), is rapidly fatal without treatment. Although typically a disease of immunocompromised (especially HIV-positive) individuals, there is growing awareness of cryptococcal disease amongst non-immunocompromised patients. Whilst substantial progress has been made in understanding the pathogenicity of C. neoformans in HIV patients, prospective data on cryptococcosis outside the context of HIV remains lacking. Below we review how innate immune responses vary between hosts depending on immunological status, and discuss risk factors and predictors of disease outcome in different groups.
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http://dx.doi.org/10.1590/0074-02760180060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909084PMC
May 2018

Characterizing the Mechanisms of Nonopsonic Uptake of Cryptococci by Macrophages.

J Immunol 2018 05 11;200(10):3539-3546. Epub 2018 Apr 11.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.

The pathogenic fungus enters the human host via inhalation into the lung and is able to reside in a niche environment that is serum- (opsonin) limiting. Little is known about the mechanism by which nonopsonic phagocytosis occurs via phagocytes in such situations. Using a combination of soluble inhibitors of phagocytic receptors and macrophages derived from knockout mice and human volunteers, we show that uptake of nonopsonized and via the mannose receptor is dependent on macrophage activation by cytokines. However, although uptake of is via both dectin-1 and dectin-2, uptake occurs largely via dectin-1. Interestingly, dectin inhibitors also blocked phagocytosis of unopsonized Cryptococci in wax moth () larvae and partially protected the larvae from infection by both fungi, supporting a key role for host phagocytes in augmenting early disease establishment. Finally, we demonstrated that internalization of nonopsonized Cryptococci is not accompanied by the nuclear translocation of NF-κB or its concomitant production of proinflammatory cytokines such as TNF-α. Thus, nonopsonized Cryptococci are recognized by mammalian phagocytes in a manner that minimizes proinflammatory cytokine production and potentially facilitates fungal pathogenesis.
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http://dx.doi.org/10.4049/jimmunol.1700790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937213PMC
May 2018

Quantifying donor-to-donor variation in macrophage responses to the human fungal pathogen Cryptococcus neoformans.

PLoS One 2018 29;13(3):e0194615. Epub 2018 Mar 29.

Institute of Microbiology & Infection and the School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Cryptococcosis remains the leading cause of fungal meningitis worldwide, caused primarily by the pathogen Cryptococcus neoformans. Symptomatic cryptococcal infections typically affect immunocompromised patients. However, environmental exposure to cryptococcal spores is ubiquitous and most healthy individuals are thought to harbor infections from early childhood onwards that are either resolved, or become latent. Since macrophages are a key host cell for cryptococcal infection, we sought to quantify the extent of individual variation in this early phagocyte response within a small cohort of healthy volunteers with no reported immunocompromising conditions. We show that rates of both intracellular fungal proliferation and non-lytic expulsion (vomocytosis) are remarkably variable between individuals. However, we demonstrate that neither gender, in vitro host inflammatory cytokine profiles, nor polymorphisms in several key immune genes are responsible for this variation. Thus the data we present serve to quantify the natural variation in macrophage responses to this important human pathogen and will hopefully provide a useful "benchmark" for the research community.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5875765PMC
July 2018

Editorial overview: Host-microbe interactions: fungi.

Curr Opin Microbiol 2017 12;40:v-vii

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Electronic address:

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http://dx.doi.org/10.1016/j.mib.2017.11.026DOI Listing
December 2017

IgG1 Is Required for Optimal Protection after Immunization with the Purified Porin OmpD from Typhimurium.

J Immunol 2017 12 10;199(12):4103-4109. Epub 2017 Nov 10.

Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, United Kingdom;

In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as Typhimurium, predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.
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http://dx.doi.org/10.4049/jimmunol.1700952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713499PMC
December 2017

Vomocytosis of live pathogens from macrophages is regulated by the atypical MAP kinase ERK5.

Sci Adv 2017 08 16;3(8):e1700898. Epub 2017 Aug 16.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

Vomocytosis, or nonlytic extrusion, is a poorly understood process through which macrophages release live pathogens that they have failed to kill back into the extracellular environment. Vomocytosis is conserved across vertebrates and occurs with a diverse range of pathogens, but to date, the host signaling events that underpin expulsion remain entirely unknown. We use a targeted inhibitor screen to identify the MAP kinase ERK5 as a critical suppressor of vomocytosis. Pharmacological inhibition or genetic manipulation of ERK5 activity significantly raises vomocytosis rates in human macrophages, whereas stimulation of the ERK5 signaling pathway inhibits vomocytosis. Lastly, using a zebrafish model of cryptococcal disease, we show that reducing ERK5 activity in vivo stimulates vomocytosis and results in reduced dissemination of infection. ERK5 therefore represents the first host signaling regulator of vomocytosis to be identified and a potential target for the future development of vomocytosis-modulating therapies.
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http://dx.doi.org/10.1126/sciadv.1700898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559206PMC
August 2017

Top-Down LESA Mass Spectrometry Protein Analysis of Gram-Positive and Gram-Negative Bacteria.

J Am Soc Mass Spectrom 2017 10 5;28(10):2066-2077. Epub 2017 Jul 5.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

We have previously shown that liquid extraction surface analysis (LESA) mass spectrometry (MS) is a technique suitable for the top-down analysis of proteins directly from intact colonies of the Gram-negative bacterium Escherichia coli K-12. Here we extend the application of LESA MS to Gram-negative Pseudomonas aeruginosa PS1054 and Gram-positive Staphylococcus aureus MSSA476, as well as two strains of E. coli (K-12 and BL21 mCherry) and an unknown species of Staphylococcus. Moreover, we demonstrate the discrimination between three species of Gram-positive Streptococcus (Streptococcus pneumoniae D39, and the viridans group Streptococcus oralis ATCC 35037 and Streptococcus gordonii ATCC35105), a recognized challenge for matrix-assisted laser desorption ionization time-of-flight MS. A range of the proteins detected were selected for top-down LESA MS/MS. Thirty-nine proteins were identified by top-down LESA MS/MS, including 16 proteins that have not previously been observed by any other technique. The potential of LESA MS for classification and characterization of novel species is illustrated by the de novo sequencing of a new protein from the unknown species of Staphylococcus. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1718-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594050PMC
October 2017

Species-specific antifungal activity of blue light.

Sci Rep 2017 07 4;7(1):4605. Epub 2017 Jul 4.

Institute of Microbiology and Infection and School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

Fungal pathogens represent a significant threat to immunocompromised patients or individuals with traumatic injury. Strategies to efficiently remove fungal spores from hospital surfaces and, ideally, patient skin thus offer the prospect of dramatically reducing infections in at-risk patients. Photodynamic inactivation of microbial cells using light holds considerable potential as a non-invasive, minimally destructive disinfection strategy. Recent data indicate that high-intensity blue light effectively removes bacteria from surfaces, but its efficacy against fungi has not been fully tested. Here we test a wide range of fungi that are pathogenic to humans and demonstrate that blue light is effective against some, but not all, fungal species. We additionally note that secondary heating effects are a previously unrecognized confounding factor in establishing the antimicrobial activity of blue light. Thus blue light holds promise for the sterilization of clinical surfaces, but requires further optimization prior to widespread use.
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http://dx.doi.org/10.1038/s41598-017-05000-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496878PMC
July 2017