scholarly journals Lactobacilli Antagonize the Growth, Motility, and Adherence of Brachyspira pilosicoli: a Potential Intervention against Avian Intestinal Spirochetosis

2011 ◽  
Vol 77 (15) ◽  
pp. 5402-5411 ◽  
Author(s):  
Luke J. Mappley ◽  
Monika A. Tchórzewska ◽  
William A. Cooley ◽  
Martin J. Woodward ◽  
Roberto M. La Ragione
Keyword(s):  
Lactobacillus Reuteri ◽  
Control Strategies ◽  
Three Dimensional ◽  
Dependent Manner ◽  
Growth Promoters ◽  
Electron Microscopic ◽  
Content Type ◽  
Brachyspira Pilosicoli ◽  
Intestinal Spirochetosis

ABSTRACTAvian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenicBrachyspiraspecies. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance inBrachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology ofBrachyspirathey antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of twoLactobacillusstrains,Lactobacillus reuteriLM1 andLactobacillus salivariusLM2, suppressed the growth ofBrachyspira pilosicoliB2904 in a pH-dependent manner. Inin vitroadherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecalin vitroorgan culture (IVOC) model, the adherence and invasion ofB. pilosicoliin epithelial cells were reduced significantly by the presence of lactobacilli (P< 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility ofB. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli andBrachyspirawas crucial in inhibiting both adherence and motility. These data suggest that motility is essential forB. pilosicolito adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

Cerebrovascular characterization of clazosentan, the first nonpeptide endothelin receptor antagonist clinically effective for the treatment of cerebral vasospasm. Part I: Inhibitory effect on endothelinA receptor—mediated contraction

2005 ◽  
Vol 102 (6) ◽  
pp. 1101-1107 ◽  
Author(s):  
Hartmut Vatter ◽  
Michael Zimmermann ◽  
Veronika Tesanovic ◽  
Andreas Raabe ◽  
Lothar Schilling ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Cerebral Vasospasm ◽  
Isometric Force ◽  
Inhibitory Effect ◽  
Affinity Constant ◽  
Dependent Manner ◽  
Content Type ◽  
The Right ◽  
Fine Print

Object. The central role of endothelin (ET)—1 in the development of cerebral vasospasm after subarachnoid hemorrhage is indicated by the successful treatment of this vasospasm in several animal models by using selective ETA receptor antagonists. Clazosentan is a selective ETA receptor antagonist that provides for the first time clinical proof that ET-1 is involved in the pathogenesis of cerebral vasospasm. The aim of the present investigation was, therefore, to define the pharmacological properties of clazosentan that affect ETA receptor—mediated contraction in the cerebrovasculature. Methods. Isometric force measurements were performed in rat basilar artery (BA) ring segments with (E+) and without (E−) endothelial function. Concentration effect curves (CECs) were constructed by cumulative application of ET-1 or big ET-1 in the absence or presence of clazosentan (10−9, 10−8, and 10−7 M). The inhibitory potency of clazosentan was determined by the value of the affinity constant (pA2). The CECs for contraction induced by ET-1 and big ET-1 were shifted to the right in the presence of clazosentan in a parallel dose-dependent manner, which indicates competitive antagonism. The pA2 values for ET-1 were 7.8 (E+) and 8.6 (E−) and the corresponding values for big ET-1 were 8.6 (E+) and 8.3 (E−). Conclusions. The present data characterize clazosentan as a potent competitive antagonist of ETA receptor—mediated constriction of the cerebrovasculature by ET-1 and its precursor big ET-1. These functional data may also be used to define an in vitro profile of an ET receptor antagonist with a high probability of clinical efficacy.

scholarly journals NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Samuel Fenn ◽  
Jean-Frédéric Dubern ◽  
Cristina Cigana ◽  
Maura De Simone ◽  
James Lazenby ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Disease Models ◽  
Attractive Alternative ◽  
Dependent Manner ◽  
Content Type ◽  
Isogenic Mutant ◽  
Virulence Traits ◽  
Infection Models

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

scholarly journals Three-Dimensional Observations of an Aperiodic Oscillatory Gliding Behavior in Myxococcus xanthus Using Confocal Interference Reflection Microscopy

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Liam M. Rooney ◽  
Lisa S. Kölln ◽  
Ross Scrimgeour ◽  
William B. Amos ◽  
Paul A. Hoskisson ◽  
...  
Keyword(s):  
Myxococcus Xanthus ◽  
Three Dimensional ◽  
Oscillatory Behavior ◽  
Gliding Motility ◽  
Bacterial Cells ◽  
Force Transmission ◽  
Content Type ◽  
Gliding Bacteria ◽  
Microscopy Techniques

ABSTRACT The deltaproteobacterium Myxococcus xanthus is a model for bacterial motility and has provided unprecedented insights into bacterial swarming behaviors. Fluorescence microscopy techniques have been invaluable in defining the mechanisms that are involved in gliding motility, but these have almost entirely been limited to two-dimensional (2D) studies, and there is currently no understanding of gliding motility in a three-dimensional (3D) context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, revealing aperiodic oscillatory behavior with changes in the position of the basal membrane relative to the substrate on the order of 90 nm in vitro. First, we use a model planoconvex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus bacteria and show that cells undergo previously unobserved changes in their adhesion profile as they glide. We compare the wild type with mutants that have reduced motility, which also exhibit the same changes in the adhesion profile during gliding. We find that the general gliding behavior is independent of the proton motive force-generating complex AglRQS and suggest that the novel behavior that we present here may be a result of recoil and force transmission along the length of the cell body following firing of the type IV pili. IMPORTANCE 3D imaging of live bacteria with optical microscopy techniques is a challenge due to the small size of bacterial cells, meaning that previous studies have been limited to observing motility behavior in 2D. We introduce the application of confocal multiwavelength interference reflection microscopy to bacteria, which enables visualization of 3D motility behaviors in a single 2D image. Using the model organism Myxococcus xanthus, we identified novel motility behaviors that are not explained by current motility models, where gliding bacteria exhibit aperiodic changes in their adhesion to an underlying solid surface. We concluded that the 3D behavior was not linked to canonical motility mechanisms and that IRM could be applied to study a range of microbiological specimens with minimal adaptation to a commercial microscope.

scholarly journals Inhibition of Staphylococcus aureus Invasion into Bovine Mammary Epithelial Cells by Contact with Live Lactobacillus casei

2012 ◽  
Vol 79 (3) ◽  
pp. 877-885 ◽  
Author(s):  
Damien S. Bouchard ◽  
Lucie Rault ◽  
Nadia Berkova ◽  
Yves Le Loir ◽  
Sergine Even
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Lactobacillus Casei ◽  
Mammary Epithelial Cells ◽  
Control Strategies ◽  
Mammary Epithelial ◽  
Dairy Herds ◽  
Dependent Manner ◽  
Content Type ◽  
Bovine Mammary Epithelial Cells

ABSTRACTStaphylococcus aureusis a major pathogen that is responsible for mastitis in dairy herds.S. aureusmastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability ofS. aureusto invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability ofLactobacillus caseistrains to prevent invasion of bMEC by twoS. aureusbovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively.L. caseistrains affected adhesion and/or internalization ofS. aureusin a strain-dependent manner. Interestingly,L. caseiCIRM-BIA 667 reducedS. aureusNewbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two otherL. caseistrains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate ofS. aureuswas not affected byL. casei. It should be noted thatL. caseiwas internalized at a low rate but survived in bMEC cells with a better efficiency than that ofS. aureusRF122. Inhibition ofS. aureusadhesion was maintained with heat-killedL. casei, whereas contact between liveL. caseiandS. aureusor bMEC was required to preventS. aureusinternalization. This first study of the antagonism of LAB towardS. aureusin a mammary context opens avenues for the development of novel control strategies against this major pathogen.

scholarly journals ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Hang Yang ◽  
Yujing Gong ◽  
Huaidong Zhang ◽  
Irina Etobayeva ◽  
Paulina Miernikiewicz ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
High Specificity ◽  
Multidrug Resistant ◽  
Penicillin G ◽  
Dependent Manner ◽  
Pneumococcal Infections ◽  
Promising Alternative ◽  
Content Type ◽  
Conventional Antibiotics

ABSTRACT Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae. By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro. In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

scholarly journals Membrane Protein Complex ExbB4-ExbD1-TonB1from Escherichia coli Demonstrates Conformational Plasticity

2015 ◽  
Vol 197 (11) ◽  
pp. 1873-1885 ◽  
Author(s):  
Aleksandr Sverzhinsky ◽  
Jacqueline W. Chung ◽  
Justin C. Deme ◽  
Lucien Fabre ◽  
Kristian T. Levey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Acquisition ◽  
Proton Translocation ◽  
Three Dimensional ◽  
Structural Data ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type

ABSTRACTIron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-taggedexbB-exbDand S-taggedtonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB fromEscherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstratein vitroevidence of ExbB4-ExbD1-TonB1complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

scholarly journals Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

2001 ◽  
Vol 75 (11) ◽  
pp. 5335-5342 ◽  
Author(s):  
Kartik Chandran ◽  
Xing Zhang ◽  
Norman H. Olson ◽  
Stephen B. Walker ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Molecular Genetic ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Dependent Manner ◽  
Viral Attachment ◽  
Structure Assembly

ABSTRACT Mammalian reoviruses, prototype members of theReoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.

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