scholarly journals Proteomics of Extracellular Vesicles Produced by Granulicatella Species that cause Infective Endocarditis

2019 ◽  
Author(s):  
Sarah A Alkandari ◽  
Radhika G Bhardwaj ◽  
Arjuna Ellepola ◽  
Maribasappa Karched
Keyword(s):  
Extracellular Vesicles ◽  
Ribosomal Proteins ◽  
Immune Modulation ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Oral Bacteria ◽  
Human Pbmcs ◽  
Bacterial Survival ◽  
Dental Procedures ◽  
Associated Proteins

AbstractWhen oral bacteria accidentally enter the bloodstream due to transient tissue damage during dental procedures, they have the potential to attach to the endocardium or an equivalent surface of an indwelling prosthesis and cause infection. Many bacterial species produce extracellular vesicles (EVs) as part of normal physiology, but also use it as a virulence strategy. In this study, it was hypothesized that Granulicatella species produce EVs that possibly help them in virulence. Therefore, the objectives were to isolate and characterize EVs produced by these species and to investigate their immune-stimulatory effects. The reference strains G. adiacens CCUG 27809 and G. elegans CCUG 38949 were cultured on chocolate blood agar for 2 days. From subsequent broth cultures, the EVs were isolated using differential centrifugation and filtration protocol and then observed using scanning electron microscopy. Proteins in the vesicle preparations were identified by nano LC-ESI-MS/MS. The EVs proteomes were analyzed and characterized using different bioinformatics tools. The immune-stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1β and CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It was revealed that both G. adiacens and G. elegans produced EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 160 proteins, and G. elegans EVs contained 107 proteins. Both proteomes consist of several ribosomal proteins, DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown that these EVs carry putative virulence factors including moonlighting proteins. These EVs were able to induce the production of IL-8, IL-1β and CCL5 from human PBMCs. The diversity in EVs content indicates that these vesicles could have possible roles in bacterial survival, invasion, host immune modulation as well as infection. Further functional characterization of the Granulicatella EVs may provide new insights into virulence mechanisms of these important but less studied oral bacterial species.

scholarly journals Proteomics of extracellular vesicles produced by Granulicatella adiacens, which causes infective endocarditis

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0227657
Author(s):  
Sarah A. Alkandari ◽  
Radhika G. Bhardwaj ◽  
Arjuna Ellepola ◽  
Maribasappa Karched
Keyword(s):  
Extracellular Vesicles ◽  
Ribosomal Proteins ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Oral Bacteria ◽  
Broth Culture ◽  
Human Pbmcs ◽  
Dental Procedures ◽  
Associated Proteins ◽  
Granulicatella Adiacens

When oral bacteria accidentally enter the bloodstream due to transient tissue damage during dental procedures, they have the potential to attach to the endocardium or an equivalent surface of an indwelling prosthesis and cause infection. Many bacterial species produce extracellular vesicles (EVs) as part of normal physiology, but also use it as a virulence strategy. In this study, it was hypothesized that Granulicatella adiacens produce EVs that possibly help it in virulence. Therefore, the objectives were to isolate and characterize EVs produced by G. adiacens and to investigate its immune-stimulatory effects. The reference strain G. adiacens CCUG 27809 was cultured on chocolate blood agar for 2 days. From subsequent broth culture, the EVs were isolated using differential centrifugation and filtration protocol and then observed using scanning electron microscopy. Proteins in the vesicle preparation were identified by nano LC-ESI-MS/MS. The EVs proteome was analyzed and characterized using different bioinformatics tools. The immune-stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1β and CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It was revealed that G. adiacens produced EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 112 proteins. The proteome consists of several ribosomal proteins, DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown that these EVs carry putative virulence factors including moonlighting proteins. These EVs were able to induce the production of IL-8, IL-1β and CCL5 from human PBMCs. Further functional characterization of the G. adiacens EVs may provide new insights into virulence mechanisms of this important but less studied oral bacterial species.

scholarly journals Structure of the virulence-associated protein VapD from the intracellular pathogenRhodococcus equi

2014 ◽  
Vol 70 (8) ◽  
pp. 2139-2151 ◽  
Author(s):  
Jean L. Whittingham ◽  
Elena V. Blagova ◽  
Ciaran E. Finn ◽  
Haixia Luo ◽  
Raúl Miranda-CasoLuengo ◽  
...  
Keyword(s):  
Spectroscopic Analysis ◽  
Laser Light ◽  
Bacterial Species ◽  
Structural Similarity ◽  
Structural Features ◽  
Virulence Plasmid ◽  
Bacterial Survival ◽  
Sequence Comparisons ◽  
Functional Implications ◽  
Associated Proteins

Rhodococcus equiis a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity ofR. equito divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins fromR. equistrains are similar to that of VapD. It is further shown that sequences encoding putativeR. equiVap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

Filifactor alocis: Recent Insights and Advances

2021 ◽  
pp. 002203452110006
Author(s):  
E. Aja ◽  
M. Mangar ◽  
H.M. Fletcher ◽  
A. Mishra
Keyword(s):  
Oxidative Stress ◽  
Periodontal Disease ◽  
Structural Damage ◽  
Community Dynamics ◽  
Functional Characterization ◽  
Oral Bacteria ◽  
Host Cells ◽  
Periodontal Pocket ◽  
Bacterial Survival ◽  
Filifactor Alocis

Filifactor alocis, a fastidious Gram-positive obligate anaerobic bacterium, is a newly appreciated member of the periodontal community that is now proposed to be a diagnostic indicator of periodontal disease. Its pathogenic characteristics are highlighted by its ability to survive in the oxidative stress–rich environment of the periodontal pocket and to significantly alter the microbial community dynamics by forming biofilms and interacting with several oral bacteria. Here, we describe the current understanding of F. alocis virulence attributes, such as its comparative resistance to oxidative stress, production of unique proteases and collagenases that can cause structural damage to host cells, and dysregulation of the immune system, which enable this bacterium to colonize, survive, and outcompete other traditional pathogens in the inflammatory environment of the periodontal pocket. Furthermore, we explore the recent advancements and future directions for F. alocis research, including the potential mechanisms for oxidative stress resistance and our evolving understanding of the interactions and mechanisms of bacterial survival inside neutrophils. We also discuss the current genetic tools and challenges involved in manipulating the F. alocis genome for the functional characterization of the putative virulence genes. Collectively, this information will expedite F. alocis research and should lead to the identification of prime targets for the development of novel therapeutics to aid in the control and prevention of periodontal disease.

scholarly journals Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Matthew R. Olm ◽  
Alexander Crits-Christoph ◽  
Spencer Diamond ◽  
Adi Lavy ◽  
Paula B. Matheus Carnevali ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Ribosomal Proteins ◽  
Large Scale ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Species Discrimination ◽  
Bacterial Genomes ◽  
Discrimination Power

ABSTRACT Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

Development of a Cavity Disinfectant Containing Antibacterial Monomer MDPB

2016 ◽  
Vol 95 (13) ◽  
pp. 1487-1493 ◽  
Author(s):  
N. Hirose ◽  
R. Kitagawa ◽  
H. Kitagawa ◽  
H. Maezono ◽  
A. Mine ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Bond Strength ◽  
Bacterial Species ◽  
Oral Bacteria ◽  
Resin Cement ◽  
Resin Cements ◽  
Adhesive Resin ◽  
Significant Difference ◽  
Adhesive Resin Cement

An experimental cavity disinfectant (ACC) that is intended to be used for various direct and indirect restorations was prepared by adding an antibacterial monomer 12-methacryloyloxydodecylpyridinum bromide (MDPB) at 5% into 80% ethanol. The antibacterial effectiveness of ACC and its influences on the bonding abilities of resin cements were investigated. To examine the antibacterial activity of unpolymerized MDPB, the minimum inhibitory and bactericidal concentrations (MIC and MBC) were determined for Streptococcus mutans, Lactobacillus casei, Actinomyces naeslundii, Parvimonas micra, Enterococcus faecalis, Fusobacterium nucleatum, and Porphyromonas gingivalis. Antibacterial activities of ACC and the commercial cavity disinfectant containing 2% chlorhexidine and ethanol (CPS) were evaluated by agar disk diffusion tests through 7 bacterial species and by MIC and MBC measurement for S. mutans. The effects of ACC and CPS to kill bacteria in dentinal tubules were compared with an S. mutans–infected dentin model. Shear bond strength tests were used to examine the influences of ACC on the dentin-bonding abilities of a self-adhesive resin cement and a dual-cure resin cement used with a primer. Unpolymerized MDPB showed strong antibacterial activity against 7 oral bacteria. ACC produced inhibition zones against all bacterial species similar to CPS. For ACC and CPS, the MIC value for S. mutans was identical, and the MBC was similar with only a 1-step dilution difference (1:2). Treatment of infected dentin with ACC resulted in significantly greater bactericidal effects than CPS ( P < 0.05, analysis of variance and Tukey’s honest significant difference test). ACC showed no negative influences on the bonding abilities to dentin for both resin cements, while CPS reduced the bond strength of the self-adhesive resin cement ( P < 0.05). This study clarified that the experimental cavity disinfectant containing 5% MDPB is more effective in vitro than the commercially available chlorhexidine solution to eradicate bacteria in dentin, without causing any adverse influences on the bonding abilities of resinous luting cements.

Effects of Low-energy Shock Waves on Oral Bacteria

2008 ◽  
Vol 87 (10) ◽  
pp. 928-931 ◽  
Author(s):  
K.F. Novak ◽  
M. Govindaswami ◽  
J.L. Ebersole ◽  
W. Schaden ◽  
N. House ◽  
...  
Keyword(s):  
Alveolar Bone ◽  
Antibacterial Effect ◽  
Bacterial Species ◽  
Extracorporeal Shock Wave Therapy ◽  
Oral Bacteria ◽  
Low Energy ◽  
Shock Wave Therapy ◽  
Extracorporeal Shock Wave ◽  
Bacterial Aggregates ◽  
Viability Determination

We have recently demonstrated that extracorporeal shock-wave therapy (ESWT) is effective in promoting the healing of dermal wounds and in regenerating alveolar bone lost through periodontal disease. The objective of the present study was to determine any antibacterial effect of ESWT on oral bacteria. Monoculture suspensions of 6 bacterial species were treated with 100 to 500 pulses of ESWT at energy flux densities (EFD) of 0.12 mJ/mm2, 0.22 mJ/mm2, and 0.3 mJ/mm2. Following treatment, aliquots were plated for viability determination and compared with untreated controls. ESWT showed a significant microbicidal effect for Streptococcus mutans and an unencapsulated strain of Porphyromonas gingivalis following as few as 100 pulses at 0.3 mJ/mm2 (p ≤ 0.001). In addition, a significant disruption of bacterial aggregates was observed at lower EFDs. No significant reduction in viability was observed for all other bacteria at EFDs and pulses tested (p > 0.05). These findings suggest that low-energy ESWT may be bactericidal for selected oral bacteria.

scholarly journals Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Mario Codemo ◽  
Sandra Muschiol ◽  
Federico Iovino ◽  
Priyanka Nannapaneni ◽  
Laura Plant ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Plasma Membrane ◽  
Streptococcus Pneumoniae ◽  
Extracellular Vesicles ◽  
Factor H ◽  
Host Cells ◽  
Immunomodulatory Effects ◽  
Content Type ◽  
Associated Proteins ◽  
Tract Infections

ABSTRACTGram-positive bacteria, including the major respiratory pathogenStreptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.IMPORTANCEStreptococcus pneumoniaeis a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

scholarly journals Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

2021 ◽  
Vol 22 (14) ◽  
pp. 7521
Author(s):  
Marko Nedeljković ◽  
Diego Emiliano Sastre ◽  
Eric John Sundberg
Keyword(s):  
Immune Responses ◽  
Basal Body ◽  
Vaccine Development ◽  
Bacterial Species ◽  
High Variability ◽  
Bacterial Survival ◽  
Bacterial Flagellum ◽  
Capping Proteins ◽  
Flagellar Filament ◽  
Flagellar Assembly

The bacterial flagellum is a complex and dynamic nanomachine that propels bacteria through liquids. It consists of a basal body, a hook, and a long filament. The flagellar filament is composed of thousands of copies of the protein flagellin (FliC) arranged helically and ending with a filament cap composed of an oligomer of the protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases are even completely absent. Flagellar assembly is a complex and energetically costly process triggered by environmental stimuli and, accordingly, highly regulated on transcriptional, translational and post-translational levels. Apart from its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction and pathogenicity, such as adhesion to surfaces, secretion of virulence factors and formation of biofilms. Additionally, due to its ability to provoke potent immune responses, flagellins have a role as adjuvants in vaccine development. In this review, we summarize the latest knowledge on the structure of flagellins, capping proteins and filaments, as well as their regulation and role during the colonization and infection of the host.

Functional Characterization of Some Ribosomal Proteins from Wheat Germ

1980 ◽  
pp. 195-201
Author(s):  
A. B. Legocki ◽  
C. J. Madrzak ◽  
D. Przybył ◽  
M. Sikorski ◽  
U. Szybiak
Keyword(s):  
Ribosomal Proteins ◽  
Wheat Germ ◽  
Functional Characterization
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