Cell Wall Perturbation Sensitizes Fungi to the Antimalarial Drug Chloroquine

ABSTRACTChloroquine (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 modelSaccharomyces cerevisiae. A genomewide yeast deletion strain collection was screened against CQ, revealing thatbck1Δ andslt2Δ 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 inbck1Δ andslt2Δ 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 pathogenCandida albicanswas decreased 2-fold by 250 μM CQ and up to 8-fold at higher CQ concentrations. Similar effects were seen inCandida glabrataandAspergillus 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.

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Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence

mBio ◽

10.1128/mbio.02458-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

Author(s):

Aílton Pereira da Costa Filho ◽

Guilherme Thomaz Pereira Brancini ◽

Patrícia Alves de Castro ◽

Clara Valero ◽

Jaire Alves Ferreira Filho ◽

...

Keyword(s):

Cell Wall ◽

G Protein ◽

Galleria Mellonella ◽

G Protein Coupled Receptors ◽

Cell Wall Integrity ◽

Fungal Development ◽

Content Type ◽

Cell Wall Integrity Pathway ◽

Mycotoxin Biosynthesis ◽

G Protein Coupled

ABSTRACT G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

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Interface of Candida albicans Biofilm Matrix-Associated Drug Resistance and Cell Wall Integrity Regulation

Eukaryotic Cell ◽

10.1128/ec.05126-11 ◽

2011 ◽

Vol 10 (12) ◽

pp. 1660-1669 ◽

Cited By ~ 94

Author(s):

Jeniel E. Nett ◽

Hiram Sanchez ◽

Michael T. Cain ◽

Kelly M. Ross ◽

David R. Andes

Keyword(s):

Drug Resistance ◽

Cell Wall ◽

Candida Albicans ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Candidate Gene Approach ◽

Content Type ◽

Resistant Phenotype ◽

Cell Wall Integrity Pathway ◽

Biofilm Matrix

ABSTRACTCandida albicansfrequently infects medical devices by growing as a biofilm, i.e., a community of adherent organisms entrenched in an extracellular matrix. During biofilm growth,Candidaspp. acquire the ability to resist high concentrations of antifungal drugs. One recently recognized biofilm resistance mechanism involves drug sequestration by matrix β-1,3 glucan. Using a candidate gene approach, we investigated potentialC. albicansβ-1,3-glucan regulators, based on their homology toSaccharomyces cerevisiae, includingSMI1and protein kinase C (PKC) pathway components. We identified a role for theSMI1in biofilm matrix glucan production and development of the associated drug resistance phenotype. This pathway appears to act through transcription factor Rlmp and glucan synthase Fks1p. The phenotypes of these mutant biofilms mimicked those of thesmi1Δ/smi1Δ biofilm, and overexpression ofFKS1in thesmi1Δ/smi1Δ mutant restored the biofilm resistant phenotype. However, control of this pathway is distinct from that of the upstream PKC pathway because thepkc1Δ/pkc1Δ,bck1Δ/bck1Δ,mkk2Δ/mkk2Δ, andmkc1Δ/mkc1Δ biofilms retained the resistant phenotype of the parent strain. In addition, resistance to cell-perturbing agents and gene expression data do not support a significant role for the cell wall integrity pathway during the biofilm formation. Here we show that Smi1p functions in conjunction with Rlm1p and Fks1p to produce drug-sequestering biofilm β-glucan. Our work provides new insight into how theC. albicansbiofilm matrix production and drug resistance pathways intersect with the planktonic cell wall integrity pathway. This novel connection helps explain how pathogens in a multicellular biofilm community are protected from anti-infective therapy.

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The Cell Wall Integrity Pathway Contributes to the Early Stages of Aspergillus fumigatus Asexual Development

Applied and Environmental Microbiology ◽

10.1128/aem.02347-19 ◽

2020 ◽

Vol 86 (7) ◽

Cited By ~ 1

Author(s):

Marina Campos Rocha ◽

João Henrique Tadini Marilhano Fabri ◽

Isabelle Taira Simões ◽

Rafael Silva-Rocha ◽

Daisuke Hagiwara ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Filamentous Fungi ◽

Cell Wall Integrity ◽

Asexual Development ◽

Remarkable Feature ◽

Susceptible Host ◽

Content Type ◽

Cell Wall Integrity Pathway ◽

Cell Wall Remodeling

ABSTRACT Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus, and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcAG579R, ΔmpkA, and ΔrlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB, flbC, brlA, abaA, and rasB, as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi. IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi.

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Highly Dynamic and Specific Phosphatidylinositol 4,5-Bisphosphate, Septin, and Cell Wall Integrity Pathway Responses Correlate with Caspofungin Activity against Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02711-15 ◽

2016 ◽

Vol 60 (6) ◽

pp. 3591-3600 ◽

Cited By ~ 8

Author(s):

Hassan Badrane ◽

M. Hong Nguyen ◽

Cornelius J. Clancy

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fluorescent Protein ◽

Cell Wall Integrity ◽

Ph Domain ◽

Calcofluor White ◽

Content Type ◽

Pathway Activation ◽

Cell Wall Integrity Pathway ◽

Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] activates the yeast cell wall integrity pathway.Candida albicansexposure to caspofungin results in the rapid redistribution of PI(4,5)P2and septins to plasma membrane foci and subsequent fungicidal effects. We studiedC. albicansPI(4,5)P2and septin dynamics and protein kinase C (PKC)-Mkc1 cell wall integrity pathway activation following exposure to caspofungin and other drugs. PI(4,5)P2and septins were visualized by live imaging ofC. albicanscells coexpressing green fluorescent protein (GFP)-pleckstrin homology (PH) domain and red fluorescent protein-Cdc10p, respectively. PI(4,5)P2was also visualized in GFP-PH domain-expressingC. albicans mkc1mutants. Mkc1p phosphorylation was measured as a marker of PKC-Mkc1 pathway activation. Fungicidal activity was assessed using 20-h time-kill assays. Caspofungin immediately induced PI(4,5)P2and Cdc10p colocalization to aberrant foci, a process that was highly dynamic over 3 h. PI(4,5)P2levels increased in a dose-response manner at caspofungin concentrations of ≤4× MIC and progressively decreased at concentrations of ≥8× MIC. Caspofungin exposure resulted in broad-based mother-daughter bud necks and arrested septum-like structures, in which PI(4,5)P2and Cdc10 colocalized. PKC-Mkc1 pathway activation was maximal within 10 min, peaked in response to caspofungin at 4× MIC, and declined at higher concentrations. The caspofungin-induced PI(4,5)P2redistribution remained apparent inmkc1mutants. Caspofungin exerted dose-dependent killing and paradoxical effects at ≤4× and ≥8× MIC, respectively. Fluconazole, amphotericin B, calcofluor white, and H2O2did not impact the PI(4,5)P2or Cdc10p distribution like caspofungin did. Caspofungin exerts rapid PI(4,5)P2-septin and PKC-Mkc1 responses that correlate with the extent ofC. albicanskilling, and the responses are not induced by other antifungal agents. PI(4,5)P2-septin regulation is crucial in early caspofungin responses and PKC-Mkc1 activation.

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Auxin-induced changes in the cell wall structure: Changes in the sugar compositions, intrinsic viscosity and molecular weight distributions of matrix polysaccharides of the epicotyl cell wall of Vigna angularis

Physiologia Plantarum ◽

10.1111/j.1399-3054.1981.tb02720.x ◽

1981 ◽

Vol 52 (4) ◽

pp. 482-494 ◽

Cited By ~ 114

Author(s):

Kazuhiko Nishitani ◽

Yoshio Masuda

Keyword(s):

Molecular Weight ◽

Cell Wall ◽

Intrinsic Viscosity ◽

Cell Wall Structure ◽

Wall Structure ◽

Vigna Angularis ◽

Molecular Weight Distributions ◽

Structure Changes ◽

Weight Distributions ◽

Induced Changes

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Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

Eukaryotic Cell ◽

10.1128/ec.00187-10 ◽

2010 ◽

Vol 9 (11) ◽

pp. 1650-1660 ◽

Cited By ~ 11

Author(s):

Encarnación Dueñas-Santero ◽

Ana Belén Martín-Cuadrado ◽

Thierry Fontaine ◽

Jean-Paul Latgé ◽

Francisco del Rey ◽

...

Keyword(s):

Cell Wall ◽

Schizosaccharomyces Pombe ◽

Protein Characterization ◽

Wall Material ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Hydrolysis Of ◽

Controlled Hydrolysis ◽

Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

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In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00523-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4470-4480 ◽

Cited By ~ 29

Author(s):

Min Jung Kwun ◽

Gabriela Novotna ◽

Andrew R. Hesketh ◽

Lionel Hill ◽

Hee-Jeon Hong

Keyword(s):

Cell Wall ◽

Transcriptional Activation ◽

Streptomyces Coelicolor ◽

Constitutive Expression ◽

Transcriptional Response ◽

Vancomycin Resistance ◽

Content Type ◽

Expression Of Genes ◽

Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

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Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽

10.1128/mbio.01120-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 31

Author(s):

Peter Mellroth ◽

Tatyana Sandalova ◽

Alexey Kikhney ◽

Francisco Vilaplana ◽

Dusan Hesek ◽

...

Keyword(s):

Cell Wall ◽

Streptococcus Pneumoniae ◽

Solution Structure ◽

Substrate Binding ◽

Substrate Recognition ◽

Lytic Activity ◽

Site Directed Mutagenesis ◽

Binding Motif ◽

Content Type ◽

Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

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Reconstruction ofmreBExpression in Staphylococcus aureus via a Collection of New Integrative Plasmids

Applied and Environmental Microbiology ◽

10.1128/aem.00759-14 ◽

2014 ◽

Vol 80 (13) ◽

pp. 3868-3878 ◽

Cited By ~ 11

Author(s):

Ana Yepes ◽

Gudrun Koch ◽

Andrea Waldvogel ◽

Juan-Carlos Garcia-Betancur ◽

Daniel Lopez

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Biology ◽

Genetic Manipulation ◽

Protein Localization ◽

Antibiotic Resistance Genes ◽

Wall Material ◽

Content Type ◽

Genetic Manipulations ◽

Discrete Structures

ABSTRACTProtein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial modelsEscherichia coliandBacillus subtilishave been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacteriumStaphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of theS. aureuschromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression ofmreBinS. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that inS. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the useS. aureusas a model system in exploring diverse aspects of cellular microbiology.

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