scholarly journals Biofilm Formation by Moraxella catarrhalis In Vitro: Roles of the UspA1 Adhesin and the Hag Hemagglutinin

2006 ◽  
Vol 74 (3) ◽  
pp. 1588-1596 ◽  
Author(s):  
Melanie M. Pearson ◽  
Cassie A. Laurence ◽  
Sarah E. Guinn ◽  
Eric J. Hansen
Keyword(s):  
Crystal Violet ◽  
Moraxella Catarrhalis ◽  
Biofilm Formation ◽  
Transposon Mutagenesis ◽  
Nucleotide Sequence Analysis ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Negative Effect ◽  
Biofilm Development

ABSTRACT Mutant analysis was used to identify Moraxella catarrhalis gene products necessary for biofilm development in a crystal violet-based assay involving 24-well tissue culture plates. The wild-type M. catarrhalis strains that formed the most extensive biofilms in this system proved to be refractory to transposon mutagenesis, so an M. catarrhalis strain was constructed that was both able to form biofilms in vitro and amenable to transposon mutagenesis. Chromosomal DNA from the biofilm-positive strain O46E was used to transform the biofilm-negative strain O35E; transformants able to form biofilms were identified and subjected to transposon-mediated mutagenesis. Biofilm-negative mutants of these transformants were shown to have a transposon insertion in the uspA1 gene. Nucleotide sequence analysis revealed that the biofilm-positive transformant T14 contained a hybrid O46E-O35E uspA1 gene, with the N-terminal 155 amino acids being derived from the O46E UspA1 protein. Transformant T14 was also shown to be unable to express the Hag protein, which normally extends from the surface of the M. catarrhalis cell. Introduction of a wild-type O35E hag gene into T14 eliminated its ability to form a biofilm. When the hybrid O46E-O35E uspA1 gene from T14 was used to replace the uspA1 gene of O35E, this transformant strain did not form a biofilm. However, inactivation of the hag gene did allow biofilm formation by strain O35E expressing the hybrid O46E-O35E uspA1 gene product. The Hag protein was shown to have an inhibitory or negative effect on biofilm formation by these M. catarrhalis strains in the crystal violet-based assay.

scholarly journals Enhanced Biofilm Formation and Loss of Capsule Synthesis: Deletion of a Putative Glycosyltransferase in Porphyromonas gingivalis

2006 ◽  
Vol 188 (15) ◽  
pp. 5510-5523 ◽  
Author(s):  
Mary E. Davey ◽  
Margaret J. Duncan
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Capsular Polysaccharide ◽  
Sequence Similarity ◽  
Opportunistic Pathogen ◽  
Insertion Mutant ◽  
Wild Type ◽  
High Sequence Similarity ◽  
Biofilm Development

ABSTRACT Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity (∼61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased autoaggregation. Our results indicate that we have identified a gene involved in capsular-polysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.

scholarly journals Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens

2009 ◽  
Vol 191 (13) ◽  
pp. 4207-4217 ◽  
Author(s):  
Janet B. Rollefson ◽  
Caleb E. Levar ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Biofilm Formation ◽  
Electrode System ◽  
Transposon Mutagenesis ◽  
Geobacter Sulfurreducens ◽  
Wild Type ◽  
Multiple Loci ◽  
Respiratory Parameters ◽  
Domain Protein ◽  
Biofilm Development

ABSTRACT Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp.

scholarly journals Streptococcus parasanguisFimbria-Associated Adhesin Fap1 Is Required for Biofilm Formation

2001 ◽  
Vol 69 (4) ◽  
pp. 2512-2519 ◽  
Author(s):  
Eunice H. Froeliger ◽  
Paula Fives-Taylor
Keyword(s):  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Single Species ◽  
Tooth Surface ◽  
Prosthetic Heart Valves ◽  
Dense Layer ◽  
Wild Type ◽  
Vitro Assay ◽  
Biofilm Development

ABSTRACT The sanguis streptococci are primary colonizers of the tooth surface and thus form the foundation for the complex multiple species biofilm known as dental plaque. In addition, these bacteria can colonize native and prosthetic heart valves and are a common cause of endocarditis. Little is known about the molecular mechanisms governing multiple or single species biofilm development within this group of organisms. Using an in vitro assay for biofilm formation, we determined that (i) Streptococcus parasanguis FW213 can form biofilms on inert surfaces such as polystyrene and (ii) environmental and nutritional factors, such as glucose, affect S. parasanguisbiofilm formation. Several isogenic mutants of FW213 were tested in the biofilm assay. Strains containing mutations in fap1, a gene encoding a protein required for assembly of fimbriae, were deficient in biofilm formation. Mutants defective in recA, PepO endopeptidase activity, or the production of a fimbriae-associated protein, FimA, were still capable of biofilm formation. Phase-contrast microscopy was used to follow biofilm development by wild-type andfap1 mutant strains on plastic coverslips over time. Wild-type FW213 attached to the surface, formed aggregates of cells, and eventually formed a dense layer of cells that included microcolonies. In contrast, few fap1 mutant cells were observed attached to the surface, and no cell aggregates or microcolonies were formed. These results suggest that the long peritrichous fimbriae of FW213 are critical for the formation of biofilms on solid surfaces.

scholarly journals An AraC-Type Transcriptional Regulator Encoded on the Enterococcus faecalis Pathogenicity Island Contributes to Pathogenesis and Intracellular Macrophage Survival

2008 ◽  
Vol 76 (12) ◽  
pp. 5668-5676 ◽  
Author(s):  
Phillip S. Coburn ◽  
Arto S. Baghdayan ◽  
GT Dolan ◽  
Nathan Shankar
Keyword(s):  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Transcriptional Regulator ◽  
Pathogenicity Island ◽  
Nucleotide Sequence Analysis ◽  
Infection Model ◽  
Wild Type ◽  
Altered Expression

ABSTRACT A gene encoding a putative AraC-type transcriptional regulator was identified on the 153-kb pathogenicity island (PAI) found among virulent Enterococcus faecalis strains. In an effort to understand the function of this regulator, designated PerA (for pathogenicity island-encoded regulator), we first examined the expression of the perA gene in the original PAI strain MMH594 and in an unrelated clinical isolate E99 by reverse transcription-PCR. Interestingly, expression analysis revealed no detectable perA transcript in MMH594, whereas a transcript was observed in strain E99. Nucleotide sequence analysis revealed that this altered expression between the two strains was attributable to the differential location of an IS1191 element within the putative promoter region upstream of the perA gene. In order to determine the role of this putative regulator in E. faecalis pathogenesis, a perA-deficient mutant was created in strain E99, and the wild-type and mutant pair were compared for phenotypic differences. In in vitro biofilm assays, the mutant strain showed a significantly higher level of growth medium-specific biofilm formation compared to the wild type. However, in a murine intraperitoneal infection model, the mutant strain was significantly less pathogenic. The mutant was also attenuated for survival within macrophages in vitro. These findings highlight the importance of PerA as a regulator of biofilm formation and survival within macrophages and is likely a regulator controlling determinants important to pathogenesis.

scholarly journals Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

2011 ◽  
Vol 80 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chen Li ◽  
Kurniyati ◽  
Bo Hu ◽  
Jiang Bian ◽  
Jianlan Sun ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Adult Population ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Multiple Organs ◽  
Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

scholarly journals Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

2010 ◽  
Vol 59 (10) ◽  
pp. 1225-1234 ◽  
Author(s):  
H. M. H. N. Bandara ◽  
O. L. T. Lam ◽  
R. M. Watt ◽  
L. J. Jin ◽  
L. P. Samaranayake
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Significant Inhibition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Bacterial Lipopolysaccharides ◽  
Species Specific ◽  
Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis

2021 ◽  
pp. 002203452199662
Author(s):  
J.T. Chen ◽  
C.H. Lin ◽  
H.W. Huang ◽  
Y.P. Wang ◽  
P.C. Kao ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Genetic Disorder ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Negative Effect ◽  
Localization Signal ◽  
Gingival Fibromatosis

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

scholarly journals An optimised GAS-pharyngeal cell biofilm model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Heema K. N. Vyas ◽  
Jason D. McArthur ◽  
Martina L. Sanderson-Smith
Keyword(s):  
Crystal Violet ◽  
Biofilm Formation ◽  
Cell Model ◽  
Matrix Material ◽  
Three Dimensional ◽  
Growth Period ◽  
Optimal Growth ◽  
Abiotic Surfaces

AbstractGroup A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

scholarly journals Green Tea Polyphenols and Padma Hepaten Inhibit Candida albicans Biofilm Formation

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Yosi Farkash ◽  
Mark Feldman ◽  
Isaac Ginsburg ◽  
Doron Steinberg ◽  
Miriam Shalish
Keyword(s):  
Candida Albicans ◽  
Green Tea ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Host Cells ◽  
Green Tea Polyphenols ◽  
Crystal Violet Staining ◽  
Biofilm Inhibition ◽  
Therapeutic Concept ◽  
Biofilm Development

Candida albicans (C. albicans) is the most prevalent opportunistic human pathogenic fungus and can cause mucosal membrane infections and invade the blood. In the oral cavity, it can ferment dietary sugars, produce organic acids and therefore has a role in caries development. In this study, we examined whether the polyphenol rich extractions Polyphenon from green tea (PPFGT) and Padma Hepaten (PH) can inhibit the caries-inducing properties of C. albicans. Biofilms of C. albicans were grown in the presence of PPFGT and PH. Formation of biofilms was tested spectrophotometrically after crystal violet staining. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy (CSLM). Treated C. albicans morphology was demonstrated using scanning electron microscopy (SEM). Expression of virulence-related genes was tested using qRT-PCR. Development of biofilm was also tested on an orthodontic surface (Essix) to assess biofilm inhibition ability on such appliances. Both PPFGT and PH dose-dependently inhibited biofilm formation, with no inhibition on planktonic growth. The strongest inhibition was obtained using the combination of the substances. Crystal violet staining showed a significant reduction of 45% in biofilm formation using a concentration of 2.5mg/ml PPFGT and 0.16mg/ml PH. A concentration of 1.25 mg/ml PPFGT and 0.16 mg/ml PH inhibited candidal growth by 88% and EPS secretion by 74% according to CSLM. A reduction in biofilm formation and in the transition from yeast to hyphal morphotype was observed using SEM. A strong reduction was found in the expression of hwp1, eap1, and als3 virulence associated genes. These results demonstrate the inhibitory effect of natural PPFGT polyphenolic extraction on C. albicans biofilm formation and EPS secretion, alone and together with PH. In an era of increased drug resistance, the use of phytomedicine to constrain biofilm development, without killing host cells, may pave the way to a novel therapeutic concept, especially in children as orthodontic patients.

scholarly journals Altering the Ratio of Phenazines in Pseudomonas chlororaphis (aureofaciens) Strain 30-84: Effects on Biofilm Formation and Pathogen Inhibition

2008 ◽  
Vol 190 (8) ◽  
pp. 2759-2766 ◽  
Author(s):  
V. S. R. K. Maddula ◽  
E. A. Pierson ◽  
L. S. Pierson
Keyword(s):  
Fungal Pathogen ◽  
Biofilm Formation ◽  
Flow Cell ◽  
Gaeumannomyces Graminis ◽  
Pseudomonas Chlororaphis ◽  
Wild Type ◽  
Initial Attachment ◽  
Pathogen Inhibition ◽  
Biofilm Development ◽  
Derivatives Of

ABSTRACT Pseudomonas chlororaphis strain 30-84 is a plant-beneficial bacterium that is able to control take-all disease of wheat caused by the fungal pathogen Gaeumannomyces graminis var. tritici. The production of phenazines (PZs) by strain 30-84 is the primary mechanism of pathogen inhibition and contributes to the persistence of strain 30-84 in the rhizosphere. PZ production is regulated in part by the PhzR/PhzI quorum-sensing (QS) system. Previous flow cell analyses demonstrated that QS and PZs are involved in biofilm formation in P. chlororaphis (V. S. R. K. Maddula, Z. Zhang, E. A. Pierson, and L. S. Pierson III, Microb. Ecol. 52:289-301, 2006). P. chlororaphis produces mainly two PZs, phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). In the present study, we examined the effect of altering the ratio of PZs produced by P. chlororaphis on biofilm formation and pathogen inhibition. As part of this study, we generated derivatives of strain 30-84 that produced only PCA or overproduced 2-OH-PCA. Using flow cell assays, we found that these PZ-altered derivatives of strain 30-84 differed from the wild type in initial attachment, mature biofilm architecture, and dispersal from biofilms. For example, increased 2-OH-PCA production promoted initial attachment and altered the three-dimensional structure of the mature biofilm relative to the wild type. Additionally, both alterations promoted thicker biofilm development and lowered dispersal rates compared to the wild type. The PZ-altered derivatives of strain 30-84 also differed in their ability to inhibit the fungal pathogen G. graminis var. tritici. Loss of 2-OH-PCA resulted in a significant reduction in the inhibition of G. graminis var. tritici. Our findings suggest that alterations in the ratios of antibiotic secondary metabolites synthesized by an organism may have complex and wide-ranging effects on its biology.

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