scholarly journals Function Analysis of Choline Binding Domains of LytA, LytC and CbpD in The Biofilm Formation Process of Streptococcus Pneumoniae

2021 ◽  
Author(s):  
Hongsheng Ji ◽  
Yingshun Zhou ◽  
Luhua Zhang ◽  
Ying Wang ◽  
Feiyang Zhang ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Biofilm Formation ◽  
Function Analysis ◽  
Amino Sugar ◽  
Extracellular Dna ◽  
Transport Pathways ◽  
Binding Domains ◽  
Phenotype Analysis ◽  
High Concentrations

Abstract Background: Choline binding proteins (CBPs) are a family of proteins that can interact with pneumococcal cell wall by choline binding domains (CBDs). In this study, we found a modified choline binding repeat (ChBp-I) with a pI of 7.66 can promote the development of biofilm in vitro. Thus, we aim to characterize the function of CBDs of LytA, LytC and CbpD in biofilm formation.Results: By transcriptome analysis, 81 genes were identified as down regulated and 138 genes were up regulated (|log2 fold change|≥1.5) under ChBp-I of 50mg/L. The up regulated genes are well clustered in membrane transport (carbohydrate, lipid, protein, cation and phosphate) and carbohydrate metabolism (fructose, mannose, galactose, starch, sucrose, amino sugar and nucleotide) related pathways. The up-regulated genes are mostly regulated the same under CBD-A, CBD-C and CBD-D. Phenotype analysis reveal high concentrations of CBD-C and CBD-D (>100μg/mL) but not CBD-A (negative charged) can promote the biofilm formation. Meanwhile, the existence of CBD-C and CBD-D promote the growth rate and both CBDs inhibit the autolysis of pneumococcal cell. By component analysis, these three CBDs were proved involved in the regulation of extracellular DNA, protein, cation and phosphate, and promote the forming of insoluble precipitates.Conclusions: The binding of CBPs can influence the membrane transport pathways and react with extracellular DNA and protein to promote biofilm formation in S. pneumoniae.

scholarly journals Copper Supplementation in Watering Solution Reaches the Xylem But Does Not Protect Tobacco Plants Against Xylella fastidiosa Infection

Plant Disease ◽  
2020 ◽  
Vol 104 (3) ◽  
pp. 724-730 ◽  
Author(s):  
Qing Ge ◽  
Paul A. Cobine ◽  
Leonardo De La Fuente
Keyword(s):  
Biofilm Formation ◽  
Inductively Coupled Plasma ◽  
Tap Water ◽  
Xylella Fastidiosa ◽  
In Vitro Study ◽  
Optical Emission ◽  
In Planta ◽  
Inductively Coupled ◽  
High Concentrations

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that causes disease in many crops worldwide. Copper (Cu) is an antimicrobial agent widely used on X. fastidiosa hosts to control other diseases. Although the effects of Cu for control of foliar pathogens are well known, it is less studied on xylem-colonizing pathogens. Previous results from our group showed that low concentrations of CuSO4 increased biofilm formation, whereas high concentrations inhibited biofilm formation and growth in vitro. In this study, we conducted in planta experiments to determine the influence of Cu in X. fastidiosa infection using tobacco as a model. X. fastidiosa-infected and noninfected plants were watered with tap water or with water supplemented with 4 mM or 8 mM of CuSO4. Symptom progression was assessed, and sap and leaf ionome analysis was performed by inductively coupled plasma with optical emission spectroscopy. Cu uptake was confirmed by increased concentrations of Cu in the sap of plants treated with CuSO4-amended water. Leaf scorch symptoms in Cu-supplemented plants showed a trend toward more severe at later time points. Quantification of total and viable X. fastidiosa in planta indicated that CuSO4-amended treatments did not inhibit but slightly increased the growth of X. fastidiosa. Cu in sap was in the range of concentrations that promote X. fastidiosa biofilm formation according to our previous in vitro study. Based on these results, we proposed that the plant Cu homeostasis machinery controls the level of Cu in the xylem, preventing it from becoming elevated to a level that would lead to bacterial inhibition.

scholarly journals The N-Acetylglucosaminidase LytB of Streptococcus pneumoniae Is Involved in the Structure and Formation of Biofilms

2020 ◽  
Vol 86 (10) ◽  
Author(s):  
Mirian Domenech ◽  
Ernesto García
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Single Gene ◽  
Extracellular Polysaccharide ◽  
Extracellular Dna ◽  
Nasopharyngeal Colonization ◽  
Content Type ◽  
Daughter Cells ◽  
Biofilm Matrix

ABSTRACT The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, ΔlytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytBE585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either ΔlytB or producing the inactive LytBE585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytBE585A mutant formed biofilm more rapidly than ΔlytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed. IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

scholarly journals Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Jayajit Das ◽  
Elaine Mokrzan ◽  
Vinal Lakhani ◽  
Lucia Rosas ◽  
Joseph A. Jurcisek ◽  
...  
Keyword(s):  
Middle Ear ◽  
In Silico ◽  
Biofilm Formation ◽  
Rational Design ◽  
Bacterial Biofilm ◽  
Confocal Imaging ◽  
Extracellular Dna ◽  
Fractal Structures ◽  
Biofilm Development

ABSTRACT Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

scholarly journals Biofilm Formation and Interaction with the Surfaces of Gallstones by Salmonella spp.

2002 ◽  
Vol 70 (5) ◽  
pp. 2640-2649 ◽  
Author(s):  
A. M. Prouty ◽  
W. H. Schwesinger ◽  
J. S. Gunn
Keyword(s):  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Asymptomatic Carrier ◽  
Carrier State ◽  
Luria Bertani ◽  
Salmonella Spp ◽  
Human Gallbladder ◽  
Luria Bertani Broth ◽  
High Concentrations

ABSTRACT Salmonellae can exist in an asymptomatic carrier state in the human gallbladder. Individuals with gallstones are more likely to become typhoid carriers, and antibiotic treatments are often ineffectual against Salmonella enterica serovar Typhi in carriers with gallstones. Therefore, we hypothesized that Salmonella spp. form biofilms on the surfaces of gallstones, where the bacteria are protected from high concentrations of bile and antibiotics. A number of methods were utilized to examine biofilm formation on human gallstones and glass coverslips in vitro, including confocal, light, and scanning electron microscopy. In our assays, salmonellae formed full biofilms on the surfaces of gallstones within 14 days and appeared to excrete an exopolysaccharide layer that bound them to the surfaces and to other bacteria. Efficient biofilm formation on gallstones was dependent upon the presence of bile, as a biofilm did not form on gallstones within 14 days in Luria-Bertani broth alone. The biofilms formed by a Salmonella enterica serovar Typhi Vi antigen mutant, as well as strains with mutations in genes that eliminate production of four different fimbriae, were indistinguishable from the biofilms formed by the parents. Mutants with an incomplete O-antigen, mutants that were nonmotile, and mutants deficient in quorum sensing were unable to develop complete biofilms. In addition, there appeared to be selectivity in salmonella binding to the gallstone surface that did not depend on the topology or surface architecture. These studies should aid in the understanding of the Salmonella carrier state, an important but underresearched area of typhoid fever pathogenesis. If the basis of carrier development can be understood, it may be possible to identify effective strategies to prevent or treat this chronic infection.

scholarly journals Cyclic di-GMP Positively Regulates DNA Repair in Vibrio cholerae

2018 ◽  
Vol 200 (15) ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Disha Srivastava ◽  
Amanda L. Ngouajio ◽  
Christopher M. Waters
Keyword(s):  
Dna Repair ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Binding Assays ◽  
Gene Encoding ◽  
Repair Gene ◽  
Content Type ◽  
Dna Repair Gene ◽  
High Concentrations

ABSTRACT In Vibrio cholerae, high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase (tag) was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of tag that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of tag expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. In vitro binding assays and heterologous host expression studies show that VpsT acts directly at the tag promoter in response to c-di-GMP to induce tag expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in V. cholerae extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade. IMPORTANCE Vibrio cholerae is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in V. cholerae between a sessile biofilm-forming state and a motile lifestyle, both of which are important during V. cholerae environmental persistence and human infections. Here, we report that in V. cholerae c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in V. cholerae.

scholarly journals Characterization of eDNA from the Clinical StrainAcinetobacter baumanniiAIIMS 7 and Its Role in Biofilm Formation

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Praveen K. Sahu ◽  
Pavithra S. Iyer ◽  
Amrita M. Oak ◽  
Karishma R. Pardesi ◽  
Balu A. Chopade
Keyword(s):  
Biofilm Formation ◽  
Membrane Vesicles ◽  
Restriction Enzymes ◽  
Dnase I ◽  
Extracellular Dna ◽  
Clinical Strain ◽  
Biofilm Inhibition ◽  
Whole Cell Lysate

Release of extracellular DNA (eDNA) was observed duringin vitrogrowth of a clinical strain ofAcinetobacter baumannii. Membrane vesicles (MV) of varying diameter (20–200 nm) containing DNA were found to be released by transmission electron microscopy (TEM) and atomic force microscopy (AFM). An assessment of the characteristics of the eDNA with respect to size, digestion pattern by DNase I/restriction enzymes, and PCR-sequencing, indicates a high similarity with genomic DNA. Role of eDNA in static biofilm formed on polystyrene surface was evaluated by biofilm augmentation assay using eDNA available in different preparations, for example, whole cell lysate, cell-free supernatant, MV suspension, and purified eDNA. Biofilm augmentation was seen up to 224.64%, whereas biofilm inhibition was 59.41% after DNase I treatment: confirming that eDNA facilitates biofilm formation inA. baumannii. This is the first paper elucidating the characteristics and role of eDNA inA. baumanniibiofilm, which may provide new insights into its pathogenesis.

scholarly journals Retroviral cDNA Integration: Stimulation by HMG I Family Proteins

2000 ◽  
Vol 74 (23) ◽  
pp. 10965-10974 ◽  
Author(s):  
Ling Li ◽  
Kristine Yoder ◽  
Mark S. T. Hansen ◽  
Jennifer Olvera ◽  
Michael D. Miller ◽  
...  
Keyword(s):  
Dna Binding ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Infected Cells ◽  
High Concentrations ◽  
Cdna Integration ◽  
Hiv 1

ABSTRACT To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by stimulation of intermolecular ligation. This reaction, like reconstitution of integration, depends on the presence of multiple DNA binding domains in each HMG I(Y) monomer. These data suggest that binding of multivalent HMG I(Y) monomers to multiple cDNA sites compacts retroviral cDNA, thereby promoting formation of active integrase-cDNA complexes.

scholarly journals In Vitro Effects of Antimicrobial Agents on Planktonic and Biofilm Forms of Staphylococcus lugdunensis Clinical Isolates

2006 ◽  
Vol 51 (3) ◽  
pp. 888-895 ◽  
Author(s):  
Kristi L. Frank ◽  
Emily J. Reichert ◽  
Kerryl E. Piper ◽  
Robin Patel
Keyword(s):  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Joint Infection ◽  
Staphylococcus Lugdunensis ◽  
Native Valve ◽  
Intrinsic Nature ◽  
Prosthetic Joint ◽  
High Concentrations ◽  
Unexpected Outcome

ABSTRACT Staphylococcus lugdunensis is an atypically virulent coagulase-negative staphylococcal species associated with acute and destructive infections that often resemble Staphylococcus aureus infections. Several types of infection caused by S. lugdunensis (e.g., native valve endocarditis, prosthetic joint infection, and intravascular catheter infection) are associated with biofilm formation, which may lead to an inability to eradicate the infection due to the intrinsic nature of biofilms to resist high levels of antibiotics. In this study, planktonic MICs and MBCs and biofilm bactericidal concentrations of 10 antistaphylococcal antimicrobial agents were measured for 15 S. lugdunensis isolates collected from patients with endocarditis, medical device infections, or skin and soft tissue infections. Planktonic isolates were susceptible to all agents studied, but biofilms were resistant to high concentrations of most of the drugs. However, moxifloxacin was able to kill 73% of isolates growing in biofilms at ≤0.5 μg/ml. Relative to the effect on cell density, subinhibitory concentrations of nafcillin substantially stimulated biofilm formation of most isolates, whereas tetracycline and linezolid significantly decreased biofilm formation in 93 and 80% of isolates, respectively. An unexpected outcome of MBC testing was the observation that vancomycin was not bactericidal against 93% of S. lugdunensis isolates, suggesting widespread vancomycin tolerance in this species. These data provide insights into the response of S. lugdunensis isolates when challenged with various levels of antimicrobial agents in clinical use.

scholarly journals Effect of Commonly Prescribed Liquid Medications on Streptococcus mutans Biofilm. An in vitro study

2017 ◽  
Vol 41 (2) ◽  
pp. 141-146 ◽  
Author(s):  
Samantha A Clark ◽  
LaQuia A Vinson ◽  
George Eckert ◽  
Richard L Gregory
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Fungal Infections ◽  
Sugar Content ◽  
In Vitro Study ◽  
Flat Bottom ◽  
Microtiter Plates ◽  
Oral Liquid ◽  
High Concentrations

Objective: This study addressed the effect of pediatric liquid antibiotic medications on Streptococcus mutans UA159. These suspensions commonly contain sugars such as sucrose to make them more palatable for children. The study was designed to evaluate the effects of oral liquid antibiotics on Streptococcus mutans growth and biofilm formation. Study Design: A 24 hour culture of S. mutans was treated with various concentrations of liquid medications commonly prescribed to children for odontogenic or fungal infections– amoxicillin, penicillin VK, clindamycin, and nystatin. The study was conducted in sterile 96-well flat bottom microtiter plates. The minimum inhibitory and biofilm inhibitory concentrations (MIC/MBIC) of S. mutans were determined for each medication. S. mutans was cultured with and without the test drugs, the amount of total growth measured, the biofilms washed, fixed, and stained with crystal violet. The absorbance was determined to evaluate biofilm formation. Results: Higher concentrations of amoxicillin, penicillin VK and clindamycin had decreased biofilm and overall growth than the control. The MICs were 1:2,560 (1.95 ug/ml), 1:2,560 (1.95 ug/ml) and 1:40 (9.375 ug/ml), while the MBIC were 1:640 (7.8 ug/ml), 1:1,280 (3.9 ug/ml) and 1:20 (18.75 ug/ml), respectively. Lower concentrations provided increased biofilm and overall growth. Nystatin induced significantly more biofilm and overall growth than the control at all concentrations. Conclusion: At high concentrations, approximately at the levels expected to be present in the oral cavity of children, amoxicillin, penicillin, and clindamycin inhibited S. mutans biofilm and overall growth due to their antibiotic activity, while at lower concentrations the three antibiotics demonstrated an increase in biofilm and growth. The increase in S. mutans biofilm and overall growth is most likely attributed to the sugar content in the medications. Nystatin provided an increase in biofilm and growth at each concentration tested.

scholarly journals A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

2016 ◽  
Vol 60 (4) ◽  
pp. 2292-2301 ◽  
Author(s):  
Angeles Estellés ◽  
Anne-Kathrin Woischnig ◽  
Keyi Liu ◽  
Robert Stephenson ◽  
Evelene Lomongsod ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Human Monoclonal Antibody ◽  
Integration Host Factor ◽  
Extracellular Dna ◽  
Infection Model ◽  
Antibody Concentration ◽  
Human Antibody

ABSTRACTMany serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observedin vitroat an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus aureus(MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.

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