scholarly journals Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

2011 ◽  
Vol 193 (22) ◽  
pp. 6315-6322 ◽  
Author(s):  
Daniel J. Schu ◽  
Revathy Ramachandran ◽  
Jared S. Geissinger ◽  
Ann M. Stevens
Keyword(s):  
Conformational Changes ◽  
Homoserine Lactone ◽  
Biologically Active ◽  
Acyl Homoserine Lactone ◽  
Content Type ◽  
Pantoea Stewartii ◽  
Cell Densities ◽  
The Impact

The quorum-sensing regulator EsaR fromPantoea stewartiisubsp.stewartiiis a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogenP. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to studyin vitrobecause AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling anin vitroanalysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stablein vivoin the absence or presence of AHL. Limitedin vitroproteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.

scholarly journals Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

2019 ◽  
Vol 202 (8) ◽  
Author(s):  
Courtney E. Price ◽  
Dustin G. Brown ◽  
Dominique H. Limoli ◽  
Vanessa V. Phelan ◽  
George A. O’Toole
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Physical Space ◽  
Late Time ◽  
Protective Effects ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals Intestinal IgA Regulates Expression of a Fructan Polysaccharide Utilization Locus in Colonizing Gut Commensal Bacteroides thetaiotaomicron

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Payal Joglekar ◽  
Hua Ding ◽  
Pablo Canales-Herrerias ◽  
Pankaj Jay Pasricha ◽  
Justin L. Sonnenburg ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Ex Vivo ◽  
Microbial Colonization ◽  
Bacteroides Thetaiotaomicron ◽  
Content Type ◽  
Microbial Utilization ◽  
Polysaccharide Utilization Locus ◽  
The Impact

ABSTRACT Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro. Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism. IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.

scholarly journals Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form.

2005 ◽  
Vol 52 (2) ◽  
pp. 469-476 ◽  
Author(s):  
Adam Sobczak ◽  
Magdalena Blazejczyk ◽  
Grzegorz Piszczek ◽  
Gang Zhao ◽  
Jacek Kuznicki ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Protein Concentration ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Biologically Active ◽  
Cell Extracts ◽  
Sds Page

The EF-hand Ca(2+)-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca(2+) signals. As oligomerization may represent one of Ca(2+)-signal transduction mechanisms, we characterised recombinant calmyrin forms using non-reducing SDS/PAGE, analytical ultracentrifugation and gel filtration. We also aimed at identification of biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca(2+)-bound calmyrin oligomerizes forming stable intermolecular disulfide bridges. Ultracentrifugation indicated that at a 220 microM initial protein concentration apo-calmyrin existed in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer (trimeric or tetrameric species were not detected). The dimerization constant was calculated as Ka = 1.78 x 103 M(-1) at 6 degrees C. Gel filtration of apo- and Ca(2+)-bound calmyrin at a 100 microM protein concentration confirmed an equilibrium of a monomer and a covalent dimer state. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca(2+). However, when calmyrin forms were analyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brain. Moreover, in contrast to recombinant calmyrin, crosslinking did not preserve any dimeric species of calmyrin regardless of Ca(2+) concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.

scholarly journals The Novel β-Lactam Enhancer Zidebactam Augments theIn VivoPharmacodynamic Activity of Cefepime in a Neutropenic Mouse LungAcinetobacter baumanniiInfection Model

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
S. S. Bhagwat ◽  
H. Periasamy ◽  
S. S. Takalkar ◽  
S. R. Palwe ◽  
H. N. Khande ◽  
...  
Keyword(s):  
Bactericidal Effect ◽  
Multidrug Resistant ◽  
Mouse Lung ◽  
Infection Model ◽  
Bactericidal Action ◽  
Content Type ◽  
Time Kill ◽  
The Impact

ABSTRACTWCK 5222 is a combination of cefepime and the high-affinity PBP2-binding β-lactam enhancer zidebactam. The cefepime-zidebactam combination is active against multidrug-resistant Gram-negative bacteria, including carbapenemase-expressingAcinetobacter baumannii. The mechanism of action of the combination involves concurrent multiple penicillin binding protein inhibition, leading to the enhanced bactericidal action of cefepime. The aim of the present study was to assess the impact of the zidebactam-mediated enhancedin vitrobactericidal action in modulating the percentage of the time that the free drug concentration remains above the MIC (percentfT>MIC) for cefepime required for thein vivokilling ofA. baumannii. Cefepime and cefepime-zidebactam MICs were comparable and ranged from 2 to 16 mg/liter for theA. baumanniistrains (n = 5) employed in the study. Time-kill studies revealed the improved killing of these strains by the cefepime-zidebactam combination compared to that by the constituents alone. Employing a neutropenic mouse lung infection model, exposure-response analyses for all theA. baumanniistrains showed that the cefepimefT>MIC required for 1-log10kill was 38.9%. In the presence of a noneffective dose of zidebactam, the cefepimefT>MIC requirement dropped significantly to 15.5%, but it still rendered a 1-log10kill effect. Thus, zidebactam mediated the improvement in cefepime’s bactericidal effect observed in time-kill studies, manifestedin vivothrough the lowering of cefepime’s pharmacodynamic requirement. This is a first-ever study demonstrating a β-lactam enhancer role of zidebactam that helps augment thein vivoactivity of cefepime by reducing the magnitude of its pharmacodynamically relevant exposures againstA. baumannii.

scholarly journals The Impacts of msaABCR on sarA-Associated Phenotypes Are Different in Divergent Clinical Isolates of Staphylococcus aureus

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Joseph S. Rom ◽  
Aura M. Ramirez ◽  
Karen E. Beenken ◽  
Gyan S. Sahukhal ◽  
Mohamed O. Elasri ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Clinical Isolates ◽  
Protease Production ◽  
Relative Impact ◽  
Extracellular Proteases ◽  
Content Type ◽  
The Impact

ABSTRACT The staphylococcal accessory regulator (sarA) plays an important role in Staphylococcus aureus infections, including osteomyelitis, and the msaABCR operon has been implicated as an important factor in modulating expression of sarA. Thus, we investigated the contribution of msaABCR to sarA-associated phenotypes in the S. aureus clinical isolates LAC and UAMS-1. Mutation of msaABCR resulted in reduced production of SarA and a reduced capacity to form a biofilm in both strains. Biofilm formation was enhanced in a LAC msa mutant by restoring the production of SarA, but this was not true in a UAMS-1 msa mutant. Similarly, extracellular protease production was increased in a LAC msa mutant but not a UAMS-1 msa mutant. This difference was reflected in the accumulation and distribution of secreted virulence factors and in the impact of extracellular proteases on biofilm formation in a LAC msa mutant. Most importantly, it was reflected in the relative impact of mutating msa as assessed in a murine osteomyelitis model, which had a significant impact in LAC but not in UAMS-1. In contrast, mutation of sarA had a greater impact on all of these in vitro and in vivo phenotypes than mutation of msaABCR, and it did so in both LAC and UAMS-1. These results suggest that, at least in osteomyelitis, it would be therapeutically preferable to target sarA rather than msaABCR to achieve the desired clinical result, particularly in the context of divergent clinical isolates of S. aureus.

scholarly journals Three Novel Candidate Probiotic Strains with Prophylactic Properties in a Murine Model of Cow's Milk Allergy

2016 ◽  
Vol 82 (6) ◽  
pp. 1722-1733 ◽  
Author(s):  
Elodie Neau ◽  
Johanne Delannoy ◽  
Candice Marion ◽  
Charles-Henry Cottart ◽  
Chantal Labellie ◽  
...  
Keyword(s):  
Murine Model ◽  
Food Allergies ◽  
T Helper ◽  
Murine Splenocytes ◽  
Content Type ◽  
Cellular Models ◽  
Probiotic Strains ◽  
The Impact

ABSTRACTFood allergies can have significant effects on morbidity and on quality of life. Therefore, the development of efficient approaches to reduce the risk of developing food allergies is of considerable interest. The aim of this study was to identify and select probiotic strains with preventive properties against allergies using a combination ofin vitroandin vivoapproaches. To that end, 31 strains of bifidobacteria and lactic acid bacteria were screened for their immunomodulatory properties in two cellular models, namely, human peripheral blood mononuclear cells (PBMCs) and T helper 2 (Th2)-skewed murine splenocytes. Six strains inducing a high interleukin-10 (IL-10)/IL-12p70 ratio and a low secretion of IL-4 on the two cellular models were selected, and their protective impact was testedin vivoin a murine model of food allergy to β-lactoglobulin. Three strains showed a protective impact on sensitization, with a decrease in allergen-specific IgE, and on allergy, with a decrease in mast cell degranulation. Analysis of the impact of these three strains on the T helper balance revealed different mechanisms of action. TheLactobacillus salivariusLA307 strain proved to block Th1 and Th2 responses, while theBifidobacterium longumsubsp.infantisLA308 strain induced a pro-Th1 profile and theLactobacillus rhamnosusLA305 strain induced pro-Th1 and regulatory responses. These results demonstrate that a combination ofin vitroandin vivoscreening is effective in probiotic strain selection and allowed identification of three novel probiotic strains that are active against sensitization in mice.

scholarly journals Impact of Sickle Cell Trait Hemoglobin on the Intraerythrocytic Transcriptional Program of Plasmodium falciparum

mSphere ◽  
2021 ◽  
Author(s):  
Joseph W. Saelens ◽  
Jens E. V. Petersen ◽  
Elizabeth Freedman ◽  
Robert C. Moseley ◽  
Drissa Konaté ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Mechanisms ◽  
Sickle Cell Trait ◽  
Rna Seq ◽  
Transcriptional Program ◽  
Content Type ◽  
Life Threatening ◽  
The Impact

Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we used transcriptome sequencing (RNA-seq) to measure the impact of HbAS on the blood-stage transcriptome of Plasmodium falciparum in in vitro time series experiments and in vivo samples from natural infections.

scholarly journals Quantitative Impact of Neutrophils on Bacterial Clearance in a Murine Pneumonia Model

2011 ◽  
Vol 55 (10) ◽  
pp. 4601-4605 ◽  
Author(s):  
Beining Guo ◽  
Kamilia Abdelraouf ◽  
Kimberly R. Ledesma ◽  
Kai-Tai Chang ◽  
Michael Nikolaou ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Bacterial Infections ◽  
Dynamic Change ◽  
Parameter Estimates ◽  
Bacterial Clearance ◽  
Content Type ◽  
Susceptibility Data ◽  
The Impact

ABSTRACTThe rapid increase in the prevalence of antibiotic-resistant pathogens is a global problem that has challenged our ability to treat serious infections. Currently, clinical decisions on treatment are often based onin vitrosusceptibility data. The role of the immune system in combating bacterial infections is unequivocal, but it is not well captured quantitatively. In this study, the impact of neutrophils on bacterial clearance was quantitatively assessed in a murine pneumonia model.In vitrotime-growth studies were performed to determine the growth rate constants ofAcinetobacter baumanniiATCC BAA 747 andPseudomonas aeruginosaPAO1. The absolute neutrophil count in mice resulting from different cyclophosphamide preparatory regimens was determined. The dynamic change of bacterial (A. baumanniiBAA 747) burden in mice with graded immunosuppression over 24 h was captured by a mathematical model. The fit to the data was satisfactory (r2= 0.945). The best-fit maximal kill rate (Kk) of the bacterial population by neutrophils was 1.743 h−1, the number of neutrophils necessary for 50% maximal killing was 190.8/μl, and the maximal population size was 1.8 × 109CFU/g, respectively. Using these model parameter estimates, the model predictions were subsequently validated by the bacterial burden change ofP. aeruginosaPAO1 at 24 h. A simple mathematical model was proposed to quantify the contribution of neutrophils to bacterial clearance and predict the bacterial growth/suppression in animals. Our results provide a novel framework to linkin vitroandin vivoinformation and may be used to improve clinical treatment of bacterial infections.

Bioactive peptides from egg: a review

2015 ◽  
Vol 45 (2) ◽  
pp. 190-212 ◽  
Author(s):  
Z. F. Bhat ◽  
Sunil Kumar ◽  
Hina Fayaz Bhat
Keyword(s):  
Bioactive Peptides ◽  
Antioxidant Activities ◽  
Positive Impact ◽  
Antimicrobial Properties ◽  
Specific Protein ◽  
Biologically Active ◽  
Content Type ◽  
Treatment And Prevention

Purpose – The aim of the article was to focus on various peptides identified in the egg and their probable application as novel ingredients in the development of functional food products. Bioactive peptides of egg origin have attracted increasing interest as one of the prominent candidates for development of various health-promoting functional and designer foods. Design/methodology/approach – Traditionally known as a source of highly valuable proteins in human nutrition, eggs are nowadays also considered as an important source of many bioactive peptides which may find wide application in medicine and food production. These specific protein fragments from egg proteins which, above and beyond their nutritional capabilities, have a positive impact on the body’s function or condition by affecting the digestive, endocrine, cardiovascular, immune and nervous systems, and may ultimately influence health. Findings – Several peptides that are released in vitro or in vivo from egg proteins have been attributed to different health effects, including antihypertensive effects, antimicrobial properties, antioxidant activities, anticancer activity, immunomodulating activity, antiadhesive properties and enhancement of nutrient absorption and/or bioavailability. Extensive research has been undertaken to identify and characterize these biologically active peptides of egg origin which has changed the image of egg as a new source of biologically active ingredients for the development of functional foods with specific benefits for human health and treatment and prevention of diseases. Originality/value – The paper mainly describes the above-stated properties of bioactive peptides derived from egg proteins.

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