scholarly journals Adhesive Surface Proteins of Erysipelothrix rhusiopathiae Bind to Polystyrene, Fibronectin, and Type I and IV Collagens

2003 ◽  
Vol 185 (9) ◽  
pp. 2739-2748 ◽  
Author(s):  
Yoshihiro Shimoji ◽  
Yohsuke Ogawa ◽  
Makoto Osaki ◽  
Hidenori Kabeya ◽  
Soichi Maruyama ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Solid Phase ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Type I ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Erysipelothrix Rhusiopathiae ◽  
Gram Positive ◽  
Adhesive Surface

ABSTRACT Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation of biofilm formation through their binding abilities to abiotic and biotic surfaces.

scholarly journals Titanium Dioxide Nanoparticles Induce Inhibitory Effects against Planktonic Cells and Biofilms of Human Oral Cavity Isolates of Rothia mucilaginosa, Georgenia sp. and Staphylococcus saprophyticus

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1564
Author(s):  
Saher Fatima ◽  
Khursheed Ali ◽  
Bilal Ahmed ◽  
Abdulaziz A. Al Kheraif ◽  
Asad Syed ◽  
...  
Keyword(s):  
Titanium Dioxide Nanoparticles ◽  
Anatase Phase ◽  
Spherical Shape ◽  
Rrna Gene ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Gram Positive ◽  
Concentration Dependent Manner ◽  
Staphylococcus Saprophyticus ◽  
Rothia Mucilaginosa

Multi-drug resistant (MDR) bacterial cells embedded in biofilm matrices can lead to the development of chronic cariogenesis. Here, we isolated and identified three Gram-positive MDR oral cocci, (1) SJM-04, (2) SJM-38, and (3) SJM-65, and characterized them morphologically, biochemically, and by 16S rRNA gene-based phylogenetic analysis as Georgenia sp., Staphylococcus saprophyticus, and Rothia mucilaginosa, respectively. These three oral isolates exhibited antibiotic-resistance against nalidixic acid, tetracycline, cefuroxime, methicillin, and ceftazidime. Furthermore, these Gram positive MDR oral cocci showed significant (p < 0.05) variations in their biofilm forming ability under different physicochemical conditions, that is, at temperatures of 28, 30, and 42 °C, pH of 6.4, 7.4, and 8.4, and NaCl concentrations from 200 to 1000 µg/mL. Exposure of oral isolates to TiO2NPs (14.7 nm) significantly (p < 0.05) reduced planktonic cell viability and biofilm formation in a concentration-dependent manner, which was confirmed by observing biofilm architecture by scanning electron microscopy (SEM) and optical microscopy. Overall, these results have important implications for the use of tetragonal anatase phase TiO2NPs (size range 5–25 nm, crystalline size 13.7 nm, and spherical shape) as an oral antibiofilm agent against Gram positive cocci infections. We suggest that TiO2NPs pave the way for further applications in oral mouthwash formulations and antibiofilm dental coatings.

Sequencing and Characterization of the xyl Operon of a Gram-Positive Bacterium, Tetragenococcus halophila

1998 ◽  
Vol 64 (7) ◽  
pp. 2513-2519 ◽  
Author(s):  
Yasuo Takeda ◽  
Kazuma Takase ◽  
Ichiro Yamato ◽  
Keietsu Abe
Keyword(s):  
Consensus Sequence ◽  
Regulatory Gene ◽  
Regulation Of Gene Expression ◽  
Regulatory Region ◽  
Gc Content ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Palindromic Structure

ABSTRACT The xyl operon of a gram-positive bacterium,Tetragenococcus halophila (previously calledPediococcus halophilus), was cloned and sequenced. The DNA was about 7.7 kb long and contained genes for a ribose binding protein and part of a ribose transporter, xylR (a putative regulatory gene), and the xyl operon, along with its regulatory region and transcription termination signal, in this order. The DNA was AT rich, the GC content being 35.8%, consistent with the GC content of this gram-positive bacterium. The xyl operon consisted of three genes, xylA, encoding a xylose isomerase, xylB, encoding a xylulose kinase, andxylE, encoding a xylose transporter, with predicted molecular weights of 49,400, 56,400, and 51,600, respectively. The deduced amino acid sequences of the XylR, XylA, XylB, and XylE proteins were similar to those of the corresponding proteins in other gram-positive and -negative bacteria, the similarities being 37 to 64%. Each polypeptide of XylB and XylE was expressed functionally inEscherichia coli. XylE transported d-xylose in a sodium ion-dependent manner, suggesting that it is the first described xylose/Na+ symporter. The XylR protein contained a consensus sequence for binding catabolites of glucose, such as glucose-6-phosphate, which has been discovered in glucose and fructose kinases in bacteria. Correspondingly, the regulatory region of this operon contained a putative binding site of XylR with a palindromic structure. Furthermore, it contained a consensus sequence, CRE (catabolite-responsive element), for binding CcpA (catabolite control protein A). We speculate that the transcriptional regulation of this operon resembles the regulation of catabolite-repressible operons such as the amy, lev, xyl, andgnt operons in various gram-positive bacteria. We discuss the significance of the regulation of gene expression of this operon inT. halophila.

scholarly journals A Newly Identified Leptospiral Adhesin Mediates Attachment to Laminin

2006 ◽  
Vol 74 (11) ◽  
pp. 6356-6364 ◽  
Author(s):  
Angela S. Barbosa ◽  
Patricia A. E. Abreu ◽  
Fernanda O. Neves ◽  
Marina V. Atzingen ◽  
Mônica M. Watanabe ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Collagen Type ◽  
Outer Membrane Proteins ◽  
Collagen Type I ◽  
Surface Proteins ◽  
Collagen Type Iv ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Sodium Metaperiodate

ABSTRACT Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Several pathogens, including spirochetes, have been shown to express surface proteins that interact with the extracellular matrix (ECM). This adhesin-mediated binding process seems to be a crucial step in the colonization of host tissues. This study examined the interaction of putative leptospiral outer membrane proteins with laminin, collagen type I, collagen type IV, cellular fibronectin, and plasma fibronectin. Six predicted coding sequences selected from the Leptospira interrogans serovar Copenhageni genome were cloned, and proteins were expressed, purified by metal affinity chromatography, and characterized by circular dichroism spectroscopy. Their capacity to mediate attachment to ECM components was evaluated by binding assays. We have identified a leptospiral protein encoded by LIC12906, named Lsa24 (leptospiral surface adhesin; 24 kDa) that binds strongly to laminin. Attachment of Lsa24 to laminin was specific, dose dependent, and saturable. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. Triton X-114-solubilized extract of L. interrogans and phase partitioning showed that Lsa24 was exclusively in the detergent phase, indicating that it is a component of the leptospiral membrane. Moreover, Lsa24 partially inhibited leptospiral adherence to immobilized laminin. This newly identified membrane protein may play a role in mediating adhesion of L. interrogans to the host. To our knowledge, this is the first leptospiral adhesin with laminin-binding properties reported to date.

scholarly journals The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

2014 ◽  
Vol 197 (5) ◽  
pp. 882-892 ◽  
Author(s):  
Jessica R. Galloway-Peña ◽  
Xiaowen Liang ◽  
Kavindra V. Singh ◽  
Puja Yadav ◽  
Chungyu Chang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Bile Salt ◽  
Enterococcus Faecium ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Type I ◽  
Gram Positive ◽  
Content Type

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6Enterococcus faeciumproteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence inE. faeciumand possibly other Gram-positive bacterial species.

scholarly journals Protection from Lethal Gram-Positive Infection by Macrophage Scavenger Receptor–Dependent Phagocytosis

2000 ◽  
Vol 191 (1) ◽  
pp. 147-156 ◽  
Author(s):  
Christian A. Thomas ◽  
Yongmei Li ◽  
Tatsuhiko Kodama ◽  
Hiroshi Suzuki ◽  
Samuel C. Silverstein ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Critical Role ◽  
Scavenger Receptor ◽  
Lipoteichoic Acid ◽  
Peritoneal Macrophages ◽  
Type I ◽  
Dependent Manner ◽  
Gram Positive ◽  
Gram Positive Bacteria

Infections with gram-positive bacteria are a major cause of morbidity and mortality in humans. Opsonin-dependent phagocytosis plays a major role in protection against and recovery from gram-positive infections. Inborn and acquired defects in opsonin generation and/or recognition by phagocytes are associated with an increased susceptibility to bacterial infections. In contrast, the physiological significance of opsonin-independent phagocytosis is unknown. Type I and II class A scavenger receptors (SR-AI/II) recognize a variety of polyanions including bacterial cell wall products such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting a role for SR-AI/II in innate immunity to bacterial infections. Here, we show that SR-AI/II–deficient mice (MSR-A−/−) are more susceptible to intraperitoneal infection with a prototypic gram-positive pathogen, Staphylococcus aureus, than MSR-A+/+ control mice. MSR-A−/− mice display an impaired ability to clear bacteria from the site of infection despite normal killing of S. aureus by neutrophils and die as a result of disseminated infection. Opsonin-independent phagocytosis of gram-positive bacteria by MSR-A−/− macrophages is significantly decreased although their phagocytic machinery is intact. Peritoneal macrophages from control mice phagocytose a variety of gram-positive bacteria in an SR-AI/II–dependent manner. Our findings demonstrate that SR-AI/II mediate opsonin-independent phagocytosis of gram-positive bacteria, and provide the first evidence that opsonin-independent phagocytosis plays a critical role in host defense against bacterial infections in vivo.

scholarly journals Characterization of Streptococcus suis Genes Encoding Proteins Homologous to Sortase of Gram-Positive Bacteria

2002 ◽  
Vol 184 (4) ◽  
pp. 971-982 ◽  
Author(s):  
Makoto Osaki ◽  
Daisuke Takamatsu ◽  
Yoshihiro Shimoji ◽  
Tsutomu Sekizaki
Keyword(s):  
Cell Wall ◽  
Streptococcus Suis ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Gram Positive ◽  
Two Dimensional Gel Electrophoresis ◽  
Gram Positive Bacteria ◽  
Genes Encoding ◽  
Covalently Linked ◽  
Sorting System

ABSTRACT Many surface proteins which are covalently linked to the cell wall of gram-positive bacteria have a consensus C-terminal motif, Leu-Pro-X-Thr-Gly (LPXTG). This sequence is cleaved, and the processed protein is attached to an amino group of a cross-bridge in the peptideglycan by a specific enzyme called sortase. Using the type strain of Streptococcus suis, NCTC 10234, we found five genes encoding proteins that were homologous to sortases of other bacteria and determined the nucleotide sequences of the genetic regions. One gene, designated srtA, was linked to gyrA, as were the sortase and sortase-like genes of other streptococci. Three genes, designated srtB, srtC, and srtD, were tandemly clustered in a different location, where there were three segments of directly repeated sequences of approximately 110 bp in close vicinity. The remaining gene, designated srtE, was located separately on the chromosome with a pseudogene which may encode a transposase. The deduced amino acid sequences of the five Srt proteins showed 18 to 31% identity with the sortases of Streptococcus gordonii and Staphylococcus aureus, except that SrtA of S. suis had 65% identity with that of S. gordonii. Isogenic mutants deficient for srtA, srtBCD, or srtE were generated by allelic exchanges. The protein fraction which was released from partially purified cell walls by digestion with N-acetylmuramidase was profiled by two-dimensional gel electrophoresis. More than 15 of the protein spots were missing in the profile of the srtA mutant compared with that of the parent strain, and this phenotype was completely complemented by srtA cloned from S. suis. Four genes encoding proteins corresponding to such spots were identified and sequenced. The deduced translational products of the four genes possessed the LPXTG motif in their C-terminal regions. On the other hand, the protein spots that were missing in the srtA mutant appeared in the profiles of the srtBCD and srtE mutants. These results provide evidence that the cell wall sorting system involving srtA is also present in S. suis.

scholarly journals MgrA regulates interaction of Staphylococcus aureus with mucin

2020 ◽  
Author(s):  
Connor P. Parker ◽  
Nour Akil ◽  
Cullen R. Shanrock ◽  
Patrick D. Allen ◽  
Anna L. Chaly ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Dose Range ◽  
Microtiter Plate ◽  
Bacterial Attachment ◽  
Surface Proteins ◽  
Respiratory Pathogen ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Wild Type

AbstractBackgroundTo defend the lungs, mucus adheres to bacterial cells and facilitates their removal by ciliary transport. Our goals were to measure the affinity of mucus for the respiratory pathogen Staphylococcus aureus and identify bacterial genes that regulate this interaction.MethodsS. aureus was added to pig tracheas to determine whether it binds mucus or epithelial cells. To quantify its affinity for mucus, we developed a competition assay in microtiter plates. Mucin was added over a dose range as an inhibitor of bacterial attachment. We then examined how transcriptional regulator MgrA and cell wall transpeptidase sortase (SrtA) affect bacterial interaction with mucin.ResultsIn pig tracheas, S. aureus bound mucus strands from submucosal glands more than epithelial cells. In microtiter plate assays, ΔsrtA failed to attach even in the absence of mucin. Mucin blocked wild type S. aureus attachment in a dose-dependent manner. Higher concentrations were needed to inhibit binding of ΔmgrA. Co-deletion of ebh and sraP, which encode surface proteins repressed by MgrA, suppressed the ΔmgrA binding phenotype. No differences between ΔmgrA and wild type were observed when methylcellulose or heparin sulfate were substituted for mucin, indicating specificity.ConclusionsMucin decreases attachment of S. aureus to plastic, consistent with its physiologic role in host defense. S. aureus deficient in MgrA has decreased affinity for mucin. Ebh and SraP, which are normally repressed by MgrA, may function as inhibitors of attachment to mucin. These data show that specific bacterial factors may regulate the interaction of S. aureus with mucus.

scholarly journals Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rowan P. Rimington ◽  
Jacob W. Fleming ◽  
Andrew J. Capel ◽  
Patrick C. Wheeler ◽  
Mark P. Lewis
Keyword(s):  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Unit ◽  
Motor Nerve ◽  
Motor Units ◽  
Extracellular Matrices ◽  
Synaptic Membrane ◽  
Type I ◽  
Dependent Manner ◽  
Human Motor

AbstractInvestigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.

scholarly journals Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro

2021 ◽  
Vol 22 (1) ◽  
pp. 394
Author(s):  
Simone Krueger ◽  
Alexander Riess ◽  
Anika Jonitz-Heincke ◽  
Alina Weizel ◽  
Anika Seyfarth ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Electric Fields ◽  
Cartilage Tissue ◽  
Type I ◽  
Dependent Manner ◽  
New Device ◽  
Alternating Electric Fields ◽  
Cartilage Cells ◽  
Stimulation Of

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.

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