scholarly journals Biofilm Formation by Pneumocystis spp.

2008 ◽  
Vol 8 (2) ◽  
pp. 197-206 ◽  
Author(s):  
Melanie T. Cushion ◽  
Margaret S. Collins ◽  
Michael J. Linke
Keyword(s):  
Biofilm Formation ◽  
Fungal Infections ◽  
Morphological Changes ◽  
Continuous Cultivation ◽  
Life Cycles ◽  
Mammalian Host ◽  
Intense Staining ◽  
In Vitro System ◽  
Major Surface

ABSTRACT Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse).Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-β-d-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.

scholarly journals Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

2007 ◽  
Vol 6 (6) ◽  
pp. 931-939 ◽  
Author(s):  
Fang Li ◽  
Michael J. Svarovsky ◽  
Amy J. Karlsson ◽  
Joel P. Wagner ◽  
Karen Marchillo ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Adhesion ◽  
Biofilm Formation ◽  
Fungal Infections ◽  
Gene Dosage ◽  
Venous Catheter ◽  
Flow Chamber ◽  
Dependent Manner

ABSTRACT Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.

scholarly journals Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species

2021 ◽  
Vol 22 (7) ◽  
pp. 3666
Author(s):  
Bettina Szerencsés ◽  
Attila Gácser ◽  
Gabriella Endre ◽  
Ildikó Domonkos ◽  
Hilda Tiricz ◽  
...  
Keyword(s):  
Candida Species ◽  
Biofilm Formation ◽  
Therapeutic Potential ◽  
Fungal Infections ◽  
Combined Treatment ◽  
Antimicrobial Activities ◽  
Dilution Method ◽  
Model Legume ◽  
Anticandidal Activity

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Leishmania mexicana Mutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

2000 ◽  
Vol 11 (4) ◽  
pp. 1183-1195 ◽  
Author(s):  
James D. Hilley ◽  
Jody L. Zawadzki ◽  
Malcolm J. McConville ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Normal Growth ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Putative Protein ◽  
Major Class ◽  
Major Surface Proteins ◽  
Major Surface

The major surface proteins of the parasitic protozoonLeishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (ΔGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein–anchor precursors. However, the synthesis and cellular levels of other non–protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ΔGPI8 mutant. Significantly, the ΔGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

scholarly journals Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation

2009 ◽  
Vol 75 (22) ◽  
pp. 7037-7043 ◽  
Author(s):  
Min Zhu ◽  
Dragana Ajdić ◽  
Yuan Liu ◽  
David Lynch ◽  
Justin Merritt ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Growth Conditions ◽  
Parental Background ◽  
A Cell ◽  
Major Surface ◽  
Biofilm Development ◽  
Small Aggregation

ABSTRACT Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

scholarly journals Role of Sialic Acid and Complex Carbohydrate Biosynthesis in Biofilm Formation by Nontypeable Haemophilus influenzae in the Chinchilla Middle Ear

2005 ◽  
Vol 73 (6) ◽  
pp. 3210-3218 ◽  
Author(s):  
Joseph Jurcisek ◽  
Laura Greiner ◽  
Hiroshi Watanabe ◽  
Anthony Zaleski ◽  
Michael A. Apicella ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Biofilm Formation ◽  
Mammalian Host ◽  
Nontypeable Haemophilus Influenzae ◽  
Biofilm Production ◽  
Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.

scholarly journals Major Surface Protein 2 of Anaplasma phagocytophilum Facilitates Adherence to Granulocytes

2003 ◽  
Vol 71 (7) ◽  
pp. 4018-4025 ◽  
Author(s):  
Jinho Park ◽  
Kyoung Seong Choi ◽  
J. Stephen Dumler
Keyword(s):  
Monoclonal Antibody ◽  
Bacterial Adhesion ◽  
Anaplasma Phagocytophilum ◽  
Human Peripheral Blood ◽  
Myeloid Cells ◽  
Surface Protein ◽  
Mammalian Host ◽  
Blood Neutrophils ◽  
Major Surface

ABSTRACT Anaplasma phagocytophilum is an obligate intracellular bacterium that infects myeloid cells in the mammalian host. Msp2 (p44) is the major immunodominant outer-membrane protein of these bacteria. We hypothesized that Msp2 acts as an adhesin for A. phagocytophilum entry into granulocytes. This potential role was investigated by blocking binding with Msp2 monoclonal antibodies and by antagonizing binding and propagation with recombinant Msp2 (rMsp2) in vitro. With HL-60 cells, fresh human peripheral blood neutrophils, and a cell line devoid of the fucosylated platelet selectin glycoprotein ligand 1 (PSGL-1) receptor for A. phagocytophilum or one that was transfected to express this ligand, Msp2 monoclonal antibody and rMsp2 used as the antagonist caused concentration-dependent reductions in bacterial adhesion (P < 0.007 and P < 0.02, respectively) and propagation (P < 0.05 and P < 0.001), although inhibition of adhesion or propagation was moderate and incomplete. Likewise, rMsp2 bound to surfaces of the transfected cell at a level similar to that of extracellular A. phagocytophilum and significantly (P < 0.05) beyond that of nontransfected cells. Moreover, a dose-dependent reduction (P < 0.019) in PSGL-1 monoclonal antibody binding to HL-60 cells was elicited with rMsp2. We conclude that Msp2s of A. phagocytophilum are involved in bacterial adhesion to ligands on host myeloid cells before intracellular infection.

scholarly journals Chromatin condensation during apoptosis requires ATP

1996 ◽  
Vol 318 (3) ◽  
pp. 749-752 ◽  
Author(s):  
George E. N. KASS ◽  
John E. ERIKSSON ◽  
Marianne WEIS ◽  
Sten ORRENIUS ◽  
Sek C. CHOW
Keyword(s):  
Morphological Changes ◽  
Chromatin Condensation ◽  
Nuclear Periphery ◽  
High Molecular Mass ◽  
Jurkat Cells ◽  
Apoptotic Body ◽  
In Vitro System ◽  
Body Formation ◽  
Isolated Rat

The processes leading to morphological changes of the chromatin in cells that undergo apoptosis are presently unclear. We have recently shown that chromatin fragmentation and the nuclear morphological changes typically seen in apoptosis were reproduced in an in vitro system comprised of isolated rat thymocyte nuclei incubated in the presence of a lysate from Fas/APO-1-stimulated JURKAT cells [Chow, Weis, Kass, Holmström, Eriksson and Orrenius (1995) FEBS Lett. 364, 134–138]. Using this in vitro system, we now report that the presence of ATP is necessary for chromatin condensation, its movement to the nuclear periphery and apoptotic body formation. In clear contrast, chromatin cleavage into high-molecular-mass and oligonucleosomal-length DNA fragments induced by lysates derived from Fas/APO-1-activated JURKAT cells did not require the presence of ATP. The induction of these morphological changes by ATP could not be substituted by the analogues, adenosine 5´-[β,γ-methylene]triphosphate and adenosine 5´-[α,β-methylene]-triphosphate, AMP, cAMP and UTP. However, adenosine 5´-[γ-thio]triphosphate, and to a lesser degree GTP and ADP, could partially replace ATP in inducing nuclear apoptotic morphological changes. It is concluded that ATP is essential for the morphological changes occurring in nuclei during apoptosis, but not for DNA fragmentation.

scholarly journals Permeability properties of monolayers of the human trophoblast cell line BeWo

1997 ◽  
Vol 273 (5) ◽  
pp. C1596-C1604 ◽  
Author(s):  
Fei Liu ◽  
Michael J. Soares ◽  
Kenneth L. Audus
Keyword(s):  
Linoleic Acid ◽  
Cell Line ◽  
Morphological Changes ◽  
Molecular Size ◽  
Bewo Cell ◽  
Human Trophoblast ◽  
In Vitro System ◽  
Size Dependent ◽  
Bewo Cell Line

The BeWo cell line (b30 clone) has been examined as a potential in vitro system to study transplacental transport. At the light and electron microscope level, the cells were observed to form confluent monolayers on polycarbonate filters in ∼5 days and morphologically resembled the typical human trophoblast. BeWo monolayers developed a modest transepithelial electrical resistance and a molecular size-dependent permeability to hydrophilic passive diffusion markers, fluorescein, and selected fluorescein-labeled dextrans. Linoleic acid permeation across BeWo monolayers was asymmetric, saturable, and inhibited by low temperature and excess competing fatty acid. Forskolin and 8-bromoadenosine 3′,5′-cyclic monophosphate treatments stimulated morphological changes in BeWo cultures and enhanced the asymmetric passage of linoleic acid across the BeWo monolayers while having minimal effects on passive permeability, affirming that the differentiation state of the cells can influence membrane transporters and transmonolayer permeability. The basic permeability properties of the BeWo monolayers suggest that the cells grown on permeable supports may be examined as a convenient in vitro system to evaluate some transplacental transport mechanisms.

Expression of LPG and GP63 by different developmental stages ofLeishmania majorin the sandflyPhlebotomus papatasi

Parasitology ◽  
1990 ◽  
Vol 101 (3) ◽  
pp. 337-343 ◽  
Author(s):  
C. R. Davies ◽  
A. M. Cooper ◽  
C. Peacock ◽  
R. P. Lane ◽  
J. M. Blackwell
Keyword(s):  
Developmental Stages ◽  
Morphological Changes ◽  
Morphological Transformation ◽  
Developmentally Regulated ◽  
Surface Molecules ◽  
Natural Vector ◽  
Metacyclic Promastigotes ◽  
Major Surface

SUMMARYDevelopment and forward migration ofLeishmaniaparasites in the sandfly gut is accompanied by morphological transformation to highly motile, non-dividing ‘metacyclic’ forms. Previous studiesin vitrohave demonstrated that this metacyclogenesis is associated with developmentally regulated changes in expression of two major surface glycoconjugates ofLeishmania, the lipophosphoglycan (LPG) and the glycoprotein protease GP63. Studies presented here are the first to examinein situthe changes in expression of these two important surface molecules which occur during amastigote-initiated development ofL. majorin its natural vectorPhlebotomus papatasi. Immunocytochemical analysis using a GP63-specific monoclonal (3.8). and others recognizing metacyclic-specific (3F12) and common (WIC79.3) epitopes of LPG on logarithmic and metacyclic promastigotes, demonstrates: (1) clear expression of LPG and GP63 from 2 and 7 days post-bloodfeeding, respectively; (2) developmental modification of the LPG molecule as parasites undergo forward migration and morphological changes associated with metacyclogenesis; and (3) striking deposition of large amounts of parasite-free excreted LPG on/in the epithelial cells of the gut wall.

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