scholarly journals Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and preventPseudomonas aeruginosabiofilms

2015 ◽  
Author(s):  
Perrin Baker ◽  
Preston J HIll ◽  
Brendan D Snarr ◽  
Noor Alnabelseya ◽  
Mathew J Pestrak ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Glycoside Hydrolases ◽  
Bacterial Biofilms ◽  
Human Pathogen ◽  
Host Defenses ◽  
Bacterial Exopolysaccharide ◽  
Opportunistic Human Pathogen ◽  
Potential Therapeutics

Bacterial biofilms are a significant medical challenge as they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogenPseudomonas aeruginosais the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases - PelAhand PslGh- encoded in thepelandpslbiosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to targetP. aeruginosabiofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix and that nanomolar concentrations of these enzymes can both prevent biofilm formation as well as rapidly disrupt preexisting biofilms in vitro. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58-94%. These non-cytotoxic enzymes potentiated antibiotics as the addition of either enzyme to a sub-lethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude. Additionally, PelAhwas able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases and synthetic biology to develop novel anti-biofilm therapeutics.

scholarly journals Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

2016 ◽  
Vol 2 (5) ◽  
pp. e1501632 ◽  
Author(s):  
Perrin Baker ◽  
Preston J. Hill ◽  
Brendan D. Snarr ◽  
Noor Alnabelseya ◽  
Matthew J. Pestrak ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Sublethal Concentration ◽  
Glycoside Hydrolases ◽  
Host Defenses ◽  
Half Maximal Effective Concentration ◽  
Bacterial Exopolysaccharide ◽  
Opportunistic Human Pathogen ◽  
Potential Therapeutics

Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.

scholarly journals Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Derek Fleming ◽  
Laura Chahin ◽  
Kendra Rumbaugh
Keyword(s):  
Glycoside Hydrolase ◽  
Bacterial Population ◽  
Chronic Wounds ◽  
Glycoside Hydrolases ◽  
Bacterial Biofilms ◽  
Planktonic Cells ◽  
Content Type ◽  
Biomass Dissolution

ABSTRACT The persistent nature of chronic wounds leaves them highly susceptible to invasion by a variety of pathogens that have the ability to construct an extracellular polymeric substance (EPS). This EPS makes the bacterial population, or biofilm, up to 1,000-fold more antibiotic tolerant than planktonic cells and makes wound healing extremely difficult. Thus, compounds which have the ability to degrade biofilms, but not host tissue components, are highly sought after for clinical applications. In this study, we examined the efficacy of two glycoside hydrolases, α-amylase and cellulase, which break down complex polysaccharides, to effectively disrupt Staphylococcus aureus and Pseudomonas aeruginosa monoculture and coculture biofilms. We hypothesized that glycoside hydrolase therapy would significantly reduce EPS biomass and convert bacteria to their planktonic state, leaving them more susceptible to conventional antimicrobials. Treatment of S. aureus and P. aeruginosa biofilms, grown in vitro and in vivo, with solutions of α-amylase and cellulase resulted in significant reductions in biomass, dissolution of the biofilm, and an increase in the effectiveness of subsequent antibiotic treatments. These data suggest that glycoside hydrolase therapy represents a potential safe, effective, and new avenue of treatment for biofilm-related infections.

scholarly journals A Candida parapsilosis Overexpression Collection Reveals Genes Required for Pathogenesis

2021 ◽  
Vol 7 (2) ◽  
pp. 97
Author(s):  
Sára E. Pál ◽  
Renáta Tóth ◽  
Joshua D. Nosanchuk ◽  
Csaba Vágvölgyi ◽  
Tibor Németh ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Deletion Mutant ◽  
Candida Parapsilosis ◽  
Galleria Mellonella ◽  
Mutant Library ◽  
Human Pathogen ◽  
Opportunistic Human Pathogen ◽  
Mutant Collection

Relative to the vast data regarding the virulence mechanisms of Candida albicans, there is limited knowledge on the emerging opportunistic human pathogen Candida parapsilosis. The aim of this study was to generate and characterize an overexpression mutant collection to identify and explore virulence factors in C. parapsilosis. With the obtained mutants, we investigated stress tolerance, morphology switch, biofilm formation, phagocytosis, and in vivo virulence in Galleria mellonella larvae and mouse models. In order to evaluate the results, we compared the data from the C. parapsilosis overexpression collection analysis to the results derived from previous deletion mutant library characterizations. Of the 37 overexpression C. parapsilosis mutants, we identified eight with altered phenotypes compared to the controls. This work is the first report to identify CPAR2_107240, CPAR2_108840, CPAR2_302400, CPAR2_406400, and CPAR2_602820 as contributors to C. parapsilosis virulence by regulating functions associated with host-pathogen interactions and biofilm formation. Our findings also confirmed the role of CPAR2_109520, CPAR2_200040, and CPAR2_500180 in pathogenesis. This study was the first attempt to use an overexpression strategy to systematically assess gene function in C. parapsilosis, and our results demonstrate that this approach is effective for such investigations.

scholarly journals Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Poushali Chakraborty ◽  
Sapna Bajeli ◽  
Deepak Kaushal ◽  
Bishan Dass Radotra ◽  
Ashwani Kumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune System ◽  
Biofilm Formation ◽  
Antimycobacterial Activity ◽  
Drug Tolerance ◽  
Host Immune System ◽  
Tissue Sections ◽  
Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

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 A Novel Infection Protocol in Zebrafish Embryo to Assess Pseudomonas aeruginosa Virulence and Validate Efficacy of a Quorum Sensing Inhibitor In Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 401
Author(s):  
Pauline Nogaret ◽  
Fatima El El Garah ◽  
Anne-Béatrice Blanc-Potard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Animal Model ◽  
Quorum Sensing ◽  
Drug Testing ◽  
Drug Efficacy ◽  
Embryo Viability ◽  
Immersion Method ◽  
Opportunistic Human Pathogen

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, new therapeutic strategies against P. aeruginosa are urgently needed. In this context, we aimed to develop a simple vertebrate animal model to rapidly assess in vivo drug efficacy against P. aeruginosa. Zebrafish are increasingly considered for modeling human infections caused by bacterial pathogens, which are commonly microinjected in embryos. In the present study, we established a novel protocol for zebrafish infection by P. aeruginosa based on bath immersion in 96-well plates of tail-injured embryos. The immersion method, followed by a 48-hour survey of embryo viability, was first validated to assess the virulence of P. aeruginosa wild-type PAO1 and a known attenuated mutant. We then validated its relevance for antipseudomonal drug testing by first using a clinically used antibiotic, ciprofloxacin. Secondly, we used a novel quorum sensing (QS) inhibitory molecule, N-(2-pyrimidyl)butanamide (C11), the activity of which had been validated in vitro but not previously tested in any animal model. A significant protective effect of C11 was observed on infected embryos, supporting the ability of C11 to attenuate in vivo P. aeruginosa pathogenicity. In conclusion, we present here a new and reliable method to compare the virulence of P. aeruginosa strains in vivo and to rapidly assess the efficacy of clinically relevant drugs against P. aeruginosa, including new antivirulence compounds.

scholarly journals Attenuation of Aeromonas hydrophila Infection in Carassius auratus by YtnP, a N-acyl Homoserine Lactonase from Bacillus licheniformis T-1

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 631
Author(s):  
Mengfan Peng ◽  
Wentao Tong ◽  
Zhen Zhao ◽  
Ling Xiao ◽  
Zhaoyue Wang ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Bacillus Licheniformis ◽  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Carassius Auratus ◽  
Quorum Quenching ◽  
Inhibition Rate ◽  
Sequence Identity

In this experiment, the quorum quenching gene ytnP of Bacillus licheniformis T-1 was cloned and expressed, and the effect against infection of Aeromonas hydrophila ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the ytnP gene of T-1 and its homolog in B.subtilis sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with B.subtilis 3610, which was categorized as the Aidc cluster of the MBL family. The AHL lactonase activity of the purified YtnP was detected as 1.097 ± 0.7 U/mL with C6-HSL as the substrate. Otherwise, purified YtnP protein could significantly inhibit the biofilm formation of A.hydrophila ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against A. hydrophila reduced from 4 μg/mL and 0.5 μg/mL to 1 μg/mL and 0.125 μg/mL, respectively, in the presence of YtnP. In addition, YtnP significantly inhibited the expression of five virulence factors hem, ahyB, ast, ep, aerA of A. hydrophila ATCC 7966 as well (p < 0.05). The results of inhibition on virulence showed a time-dependence tendency, while the strongest anti-virulence effects were within 4–24 h. In vivo, when the YtnP protein was co-injected intraperitoneally with A. hydrophila ATCC 7966, it attenuated the pathogenicity of A. hydrophila and the accumulated mortality was 27 ± 4.14% at 96 h, which was significantly lower than the average mortality of 78 ± 2.57% of the Carassius auratus injected with 108 CFU/mL of A. hydrophila ATCC 7966 only (p < 0.001). In conclusion, the AHL lactonase in B. licheniformis T-1 was proven to be YtnP protein and could be developed into an agent against infection of A. hydrophila in aquaculture.

scholarly journals Newly Developed Self-Assembling Antioxidants as Potential Therapeutics for the Cancers

2021 ◽  
Vol 11 (2) ◽  
pp. 92 ◽  
Author(s):  
Babita Shashni ◽  
Yukio Nagasaki
Keyword(s):  
Cancer Survival ◽  
Self Assembling ◽  
Therapeutic Regime ◽  
Cancer Models ◽  
Secondary Messengers ◽  
Potential Therapeutics ◽  
Target Effects ◽  
Tempo Radical

Elevated reactive oxygen species (ROS) have been implicated as significant for cancer survival by functioning as oncogene activators and secondary messengers. Hence, the attenuation of ROS-signaling pathways in cancer by antioxidants seems a suitable therapeutic regime for targeting cancers. Low molecular weight (LMW) antioxidants such as 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO), although they are catalytically effective in vitro, exerts off-target effects in vivo due to their size, thus, limiting their clinical use. Here, we discuss the superior impacts of our TEMPO radical-conjugated self-assembling antioxidant nanoparticle (RNP) compared to the LMW counterpart in terms of pharmacokinetics, therapeutic effect, and adverse effects in various cancer models.

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.

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