cspAInfluences Biofilm Formation and Drug Resistance in Pathogenic FungusAspergillus fumigatus

The microbial cell wall plays a crucial role in biofilm formation and drug resistance.cspAencodes a repeat-rich glycophosphatidylinositol-anchored cell wall protein in the pathogenic fungusAspergillus fumigatus. To determine whethercspAhas a significant impact on biofilm development and sensitivity to antifungal drugs inA. fumigatus, a ΔcspAmutant was constructed by targeted gene disruption, and we then reconstituted the mutant to wild type by homologous recombination of a functionalcspAgene. Deletion ofcspAresulted in a rougher conidial surface, reduced biofilm formation, decreased resistance to antifungal agents, and increased internalization by A549 human lung epithelial cells, suggesting thatcspAnot only participates in maintaining the integrity of the cell wall, but also affects biofilm establishment, drug response, and invasiveness ofA. fumigatus.

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Rhamnose Binding Protein as an Anti-Bacterial Agent—Targeting Biofilm of Pseudomonas aeruginosa

Marine Drugs ◽

10.3390/md17060355 ◽

2019 ◽

Vol 17 (6) ◽

pp. 355 ◽

Cited By ~ 2

Author(s):

Tse-Kai Fu ◽

Sim-Kun Ng ◽

Yi-En Chen ◽

Yuan-Chuan Lee ◽

Fruzsina Demeter ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Lung Epithelial Cells ◽

Bacterial Agent ◽

Lung Epithelial ◽

Biofilm Development

More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.

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The Role of Staphylococcus aureus YycFG in Gene Regulation, Biofilm Organization and Drug Resistance

Antibiotics ◽

10.3390/antibiotics10121555 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1555

Author(s):

Shizhou Wu ◽

Junqi Zhang ◽

Qi Peng ◽

Yunjie Liu ◽

Lei Lei ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Drug Resistance ◽

Cell Wall ◽

Gene Regulation ◽

Biofilm Formation ◽

Antimicrobial Drug ◽

Health Concern ◽

Antimicrobial Drug Resistance ◽

Biofilm Development

Antibiotic resistance is a serious global health concern that may have significant social and financial consequences. Methicillin-resistant Staphylococcus aureus (MRSA) infection is responsible for substantial morbidity and leads to the death of 21.8% of infected patients annually. A lack of novel antibiotics has prompted the exploration of therapies targeting bacterial virulence mechanisms. The two-component signal transduction system (TCS) enables microbial cells to regulate gene expression and the subsequent metabolic processes that occur due to environmental changes. The YycFG TCS in S. aureus is essential for bacterial viability, the regulation of cell membrane metabolism, cell wall synthesis and biofilm formation. However, the role of YycFG-associated biofilm organization in S. aureus antimicrobial drug resistance and gene regulation has not been discussed in detail. We reviewed the main molecules involved in YycFG-associated cell wall biosynthesis, biofilm development and polysaccharide intercellular adhesin (PIA) accumulation. Two YycFG-associated regulatory mechanisms, accessory gene regulator (agr) and staphylococcal accessory regulator (SarA), were also discussed. We highlighted the importance of biofilm formation in the development of antimicrobial drug resistance in S. aureus infections. Data revealed that inhibition of the YycFG pathway reduced PIA production, biofilm formation and bacterial pathogenicity, which provides a potential target for the management of MRSA-induced infections.

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Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus

mBio ◽

10.1128/mbio.00215-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 17

Author(s):

Adriana Oliveira Manfiolli ◽

Filipe Silva Siqueira ◽

Thaila Fernanda dos Reis ◽

Patrick Van Dijck ◽

Sanne Schrevens ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Cross Talk ◽

Pathogenic Fungus ◽

Antifungal Drugs ◽

Mitogen Activated Protein Kinase ◽

Melanin Production ◽

Content Type ◽

Mitogen Activated Protein ◽

Salvage Pathways

ABSTRACT The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade. IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

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Antibiofilm activity of antifungal drugs, including the novel drug olorofim, against Lomentospora prolificans

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa157 ◽

2020 ◽

Cited By ~ 1

Author(s):

Lisa Kirchhoff ◽

Silke Dittmer ◽

Ann-Kathrin Weisner ◽

Jan Buer ◽

Peter-Michael Rath ◽

...

Keyword(s):

Amphotericin B ◽

Biofilm Formation ◽

Laser Scanning ◽

Antifungal Agents ◽

Fungal Infections ◽

Pathogenic Fungus ◽

Antifungal Drugs ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Antibiofilm Activity

Abstract Objectives Patients with immunodeficiency or cystic fibrosis frequently suffer from respiratory fungal infections. In particular, biofilm-associated fungi cause refractory infection manifestations, linked to increased resistance to anti-infective agents. One emerging filamentous fungus is Lomentospora prolificans. Here, the biofilm-formation capabilities of L. prolificans isolates were investigated and the susceptibility of biofilms to various antifungal agents was analysed. Methods Biofilm formation of L. prolificans (n = 11) was estimated by crystal violet stain and antibiofilm activity was additionally determined via detection of metabolically active biofilm using an XTT assay. Amphotericin B, micafungin, voriconazole and olorofim were compared with regard to their antibiofilm effects when added prior to adhesion, after adhesion and on mature and preformed fungal biofilms. Imaging via confocal laser scanning microscopy was carried out to demonstrate the effect of drug treatment on the fungal biofilm. Results Antibiofilm activities of the tested antifungal agents were shown to be most effective on adherent cells whilst mature biofilm was the most resistant. The most promising antibiofilm effects were detected with voriconazole and olorofim. Olorofim showed an average minimum biofilm eradication concentration (MBEC) of 0.06 mg/L, when added prior to and after adhesion. The MBECs of voriconazole were ≤4 mg/L. On mature biofilm the MBECs of olorofim and voriconazole were higher than the previously determined MICs against planktonic cultures. In contrast, amphotericin B and especially micafungin did not exhibit sufficient antibiofilm activity against L. prolificans. Conclusions To our knowledge, this is the first study demonstrating the antibiofilm potential of olorofim against the human pathogenic fungus L. prolificans.

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Correlation between Biofilm Formation and the Hypoxic Response in Candida parapsilosis

Eukaryotic Cell ◽

10.1128/ec.00350-08 ◽

2009 ◽

Vol 8 (4) ◽

pp. 550-559 ◽

Cited By ~ 57

Author(s):

Tristan Rossignol ◽

Chen Ding ◽

Alessandro Guida ◽

Christophe d'Enfert ◽

Desmond G. Higgins ◽

...

Keyword(s):

Cell Wall ◽

Biofilm Formation ◽

Candida Parapsilosis ◽

Transcriptional Profile ◽

Open Reading Frames ◽

Hypoxic Response ◽

Hypoxic Conditions ◽

Expression Of Genes ◽

Biofilm Development ◽

Reading Frames

ABSTRACT The ability of Candida parapsilosis to form biofilms on indwelling medical devices is correlated with virulence. To identify genes that are important for biofilm formation, we used arrays representing approximately 4,000 open reading frames (ORFs) to compare the transcriptional profile of biofilm cells growing in a microfermentor under continuous flow conditions with that of cells in planktonic culture. The expression of genes involved in fatty acid and ergosterol metabolism and in glycolysis, is upregulated in biofilms. The transcriptional profile of C. parapsilosis biofilm cells resembles that of Candida albicans cells grown under hypoxic conditions. We therefore subsequently used whole-genome arrays (representing 5,900 ORFs) to determine the hypoxic response of C. parapsilosis and showed that the levels of expression of genes involved in the ergosterol and glycolytic pathways, together with several cell wall genes, are increased. Our results indicate that there is substantial overlap between the hypoxic responses of C. parapsilosis and C. albicans and that this may be important for biofilm development. Knocking out an ortholog of the cell wall gene RBT1, whose expression is induced both in biofilms and under conditions of hypoxia in C. parapsilosis, reduces biofilm development.

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The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungusCryptococcus neoformans.

10.1101/356535 ◽

2018 ◽

Author(s):

Mélissa Caza ◽

Guanggan Hu ◽

Eric David Neilson ◽

Minsu Cho ◽

Won Hee Jung ◽

...

Keyword(s):

Cell Wall ◽

Mitochondrial Function ◽

Iron Uptake ◽

Pathogenic Fungus ◽

Antifungal Drugs ◽

Uptake System ◽

Poor Growth ◽

Enhanced Sensitivity ◽

Electron Transport Complexes ◽

Sm Protein

ABSTRACTThe battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus,Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, avps45deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and avps45mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, thevps45mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of thevps45mutant and found increased sensitivity to agents that challenge cell wall integrity and antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.

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Global Screening of Genomic and Transcriptomic Factors Associated with Phenotype Differences between Multidrug-Resistant and -Susceptible Candida haemulonii Strains

mSystems ◽

10.1128/msystems.00459-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 2

Author(s):

Hao Zhang ◽

Yifei Niu ◽

Jingwen Tan ◽

Weixia Liu ◽

Ming-an Sun ◽

...

Keyword(s):

Drug Resistance ◽

Cell Wall ◽

Multidrug Resistance ◽

Multidrug Resistant ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Close Relative ◽

Content Type ◽

Candida Haemulonii ◽

Resistant Strains

ABSTRACT Candida haemulonii, a close relative of Candida auris, is an emerging pathogen which frequently shows multidrug resistance especially to triazoles, the most used antifungal drugs. The mechanisms of drug resistance in C. haemulonii, however, are largely unknown. Here, we sequenced and annotated the genomes of two reference strains from the C. haemulonii complex, compared the phenotypes, genomes, and transcriptomes of a triazole-susceptible and two triazole-resistant C. haemulonii strains, and identified triazole susceptibility, morphology, fitness, and the major genetic and gene expression differences between the strains. A multidrug efflux gene, CDR1, was recurrently found to be upregulated for expression in triazole-resistant strains. Blocking the activity of Cdr1 increased the susceptibility to triazoles strikingly. Comparative transcriptome analysis also demonstrated impaired cell wall integrity, filamentous growth, and iron homeostasis in triazole-resistant strains. Finally, we also identified a zinc-binding MHR family transcription regulator gene that mutated in triazole-resistant strains spontaneously, contributing to the changes of morphology and, possibly, cell wall integrity between the strains. The study provided important clues for future studies exploring the mechanisms of multidrug resistance and related phenotypic differences of C. haemulonii strains. IMPORTANCE A comprehensive, multi-omic survey was performed to disclose the genetic backgrounds and differences between multidrug-resistant and -susceptible C. haemulonii strains. Genes were identified with mutations or significant expression differences in multidrug-resistant compared to multidrug-susceptible strains, which were mainly involved in multidrug resistance, stress fitness, and morphology. The Cdr1-encoding gene, C. haemulonii 2486 (CH_2486), was expressed at a significantly increased level in multidrug-resistant strains. Functional inhibition experiments further implicated potential roles of CH_2486 in drug resistance. A gene spontaneously mutated in resistant strains, CH_4347, was experimentally validated to influence the morphology of spores, possibly by controlling cell wall integrity.

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Susceptibility of Cryptococcus neoformans Biofilms to Antifungal Agents In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.3.1021-1033.2006 ◽

2006 ◽

Vol 50 (3) ◽

pp. 1021-1033 ◽

Cited By ~ 109

Author(s):

Luis R. Martinez ◽

Arturo Casadevall

Keyword(s):

Confocal Microscopy ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Biofilm Formation ◽

Antifungal Agents ◽

Capsular Polysaccharide ◽

Antifungal Drugs ◽

Enzyme Linked Immunosorbent Assay ◽

Sequence Of Events ◽

Biofilm Development

ABSTRACT Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.

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O-Mannosylation in Candida albicans Enables Development of Interkingdom Biofilm Communities

mBio ◽

10.1128/mbio.00911-14 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 41

Author(s):

Lindsay C. Dutton ◽

Angela H. Nobbs ◽

Katy Jepson ◽

Mark A. Jepson ◽

M. Margaret Vickerman ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Biofilm Formation ◽

Early Stage ◽

Growth Conditions ◽

Wild Type ◽

Content Type ◽

Subsequent Performance ◽

Biofilm Development ◽

Mutant Cells

ABSTRACTCandida albicansis a fungus that colonizes oral cavity surfaces, the gut, and the genital tract.Streptococcus gordoniiis a ubiquitous oral bacterium that has been shown to form biofilm communities withC. albicans. Formation of dual-speciesS. gordonii-C. albicansbiofilm communities involves interaction of theS. gordoniiSspB protein with the Als3 protein on the hyphal filament surface ofC. albicans. Mannoproteins comprise a major component of theC. albicanscell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis ofC. albicanswas necessary for hyphal adhesin functions associated with interkingdom biofilm development. AC. albicans mnt1Δmnt2Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective inO-mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized byS. gordonii. Cell wall proteomes of hypha-formingmnt1Δmnt2Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed bymnt1Δmnt2Δ mutant cells, unlike wild-type hyphae, did not interact withC. albicansAls3 or Hwp1 partner cell wall proteins or withS. gordoniiSspB partner adhesin, suggesting defective functionality of adhesins on themnt1Δmnt2Δ mutant. These observations imply that early stageO-mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such asS. gordonii, and microbial community development.IMPORTANCEIn the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present.Candida albicansis a fungus that is often found within these biofilms. We have focused on the mechanisms by whichC. albicansbecomes incorporated into communities containing bacteria, such asStreptococcus. We find that impairment of early stage addition of mannose sugars toC. albicanshyphal filament proteins deleteriously affects their subsequent performance in mediating formation of polymicrobial biofilms. Our analyses provide new understanding of the way that microbial communities develop, and of potential means to controlC. albicansinfections.

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Pneumocystis carinii Interactions with Lung Epithelial Cells and Matrix Proteins Induce Expression and Activity of the PcSte20 Kinase with Subsequent Phosphorylation of the Downstream Cell Wall Biosynthesis Kinase PcCbk1

Infection and Immunity ◽

10.1128/iai.05066-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4157-4164 ◽

Cited By ~ 1

Author(s):

Theodore J. Kottom ◽

Joshua W. Burgess ◽

Andrew H. Limper

Keyword(s):

Cell Wall ◽

Epithelial Cells ◽

Fungal Pathogen ◽

Pneumocystis Carinii ◽

Specific Region ◽

Lung Epithelial Cells ◽

Matrix Proteins ◽

Content Type ◽

Lung Epithelial ◽

Opportunistic Fungal Pathogen

ABSTRACTEukaryotic cell proliferation and phenotype are highly regulated by contact-dependent mechanisms. We have previously shown that the binding and interaction of the opportunistic fungal pathogenPneumocystis cariniito lung epithelial cells and extracellular matrix proteins induces mRNA expression of both the mitogen-activated protein (MAP) kinaseP. cariniiSte20 (PcSte20) and the cell wall-remodeling enzymePcCbk1(16). Herein, we report that in addition toPcSte20mRNA expression being upregulated,PneumocystisPcSte20 kinase activity is increased upon interacting with these same lung targets. This activity is also significantly suppressed byClostridium difficiletoxin B, a pan-specific inhibitor of small GTPases, demonstrating the potential role of a Cdc42-like molecule in this signaling cascade. We further observed that the PcSte20 kinase physically interacts with a specific region of theP. cariniicell wall biosynthesis kinase, PcCbk1, a downstream kinase important for mating projection formation and cell wall remodeling. This direct binding was mapped to a specific region of the PcCbk1 protein. We also demonstrated that PcSte20 obtained from wholeP. cariniilysates has the ability to phosphorylate PcCbk1 after the organism interacts with lung epithelial cells and extracellular matrix components. These observations provide new insights intoP. cariniisignaling induced by interactions of this important opportunistic fungal pathogen with lung epithelial cells and matrix.

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