scholarly journals Fibronectin Binding Proteins SpsD and SpsL Both Support Invasion of Canine Epithelial Cells by Staphylococcus pseudintermedius

2015 ◽  
Vol 83 (10) ◽  
pp. 4093-4102 ◽  
Author(s):  
Giampiero Pietrocola ◽  
Valentina Gianotti ◽  
Amy Richards ◽  
Giulia Nobile ◽  
Joan A. Geoghegan ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Kinase Inhibitors ◽  
Epidermal Keratinocytes ◽  
Bacterial Invasion ◽  
Staphylococcus Pseudintermedius ◽  
Content Type ◽  
Gentamicin Protection Assay ◽  
Internalization Process ◽  
Fibronectin Binding ◽  
Recombinant Fragments

In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogenStaphylococcus pseudintermediusfor their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsLmutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants.Lactococcus lactistransformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5β1markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520–846and SpsL538–823regions harboring the major fibronectin-binding sites inhibitedS. pseudintermediusinternalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections.

scholarly journals The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus

2015 ◽  
Vol 197 (23) ◽  
pp. 3720-3730 ◽  
Author(s):  
Jessica L. Danger ◽  
Nishanth Makthal ◽  
Muthiah Kumaraswami ◽  
Paul Sumby
Keyword(s):  
Cell Surface ◽  
Virulence Factors ◽  
Binding Proteins ◽  
Mrna Translation ◽  
Regulatory Rna ◽  
Collagen Binding ◽  
Content Type ◽  
Small Regulatory Rna ◽  
Group A ◽  
Fibronectin Binding

ABSTRACTThe group AStreptococcus(GAS;Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to theprtF1andprtF2mRNAs within their 5′ untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and theprtF1andprtF2mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection.IMPORTANCEMore than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens.

scholarly journals Staphylococcus aureus Fibronectin Binding Proteins Are Essential for Internalization by Osteoblasts but Do Not Account for Differences in Intracellular Levels of Bacteria

2001 ◽  
Vol 69 (5) ◽  
pp. 2872-2877 ◽  
Author(s):  
Saddif Ahmed ◽  
Sajeda Meghji ◽  
Rachel J. Williams ◽  
Brian Henderson ◽  
Jeremy H. Brock ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Proteins ◽  
Laboratory Strain ◽  
Large Numbers ◽  
Short Period ◽  
Internalization Process ◽  
Fibronectin Binding ◽  
Kinetics Of Uptake ◽  
Kinetics Of ◽  
Strain Dependent

ABSTRACT Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains ofS. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

scholarly journals The Increased Accumulation of Staphylococcus aureus Virulence Factors Is Maximized in a purR Mutant by the Increased Production of SarA and Decreased Production of Extracellular Proteases

2021 ◽  
Vol 89 (4) ◽  
Author(s):  
Duah Alkam ◽  
Piroon Jenjaroenpun ◽  
Aura M. Ramirez ◽  
Karen E. Beenken ◽  
Horace J. Spencer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Binding Proteins ◽  
Protease Production ◽  
Alpha Toxin ◽  
Extracellular Proteases ◽  
Content Type ◽  
Enhanced Production ◽  
Fibronectin Binding ◽  
The Impact

ABSTRACT Mutation of purR was previously shown to enhance the virulence of Staphylococcus aureus in a murine sepsis model, and this cannot be fully explained by increased expression of genes within the purine biosynthesis pathway. Rather, the increased production of specific S. aureus virulence factors, including alpha toxin and the fibronectin-binding proteins, was shown to play an important role. Mutation of purR was also shown previously to result in increased abundance of SarA. Here, we demonstrate by transposon sequencing that mutation of purR in the USA300 strain LAC increases fitness in a biofilm while mutation of sarA has the opposite effect. Therefore, we assessed the impact of sarA on reported purR-associated phenotypes by characterizing isogenic purR, sarA, and sarA/purR mutants. The results confirmed that mutation of purR results in increased abundance of alpha toxin, protein A, the fibronectin-binding proteins, and SarA, decreased production of extracellular proteases, an increased capacity to form a biofilm, and increased virulence in an osteomyelitis model. Mutation of sarA had the opposite effects on all of these phenotypes and, other than bacterial burdens in the bone, all of the phenotypes of sarA/purR mutants were comparable to those of sarA mutants. Limiting the production of extracellular proteases reversed all of the phenotypes of sarA mutants and most of those of sarA/purR mutants. We conclude that a critical component defining the virulence of a purR mutant is the enhanced production of SarA, which limits protease production to an extent that promotes the accumulation of critical S. aureus virulence factors.

scholarly journals Identification and Characterization of Staphylococcus delphini Internalization Pathway in Nonprofessional Phagocytic Cells

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Yousef Maali ◽  
Alan Diot ◽  
Patrícia Martins-Simões ◽  
Michele Bes ◽  
Daniel Bouvard ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Surface Protein ◽  
Phagocytic Cells ◽  
Typical Structure ◽  
Staphylococcus Pseudintermedius ◽  
Content Type ◽  
Repeated Pattern ◽  
Fibronectin Binding ◽  
Identification And Characterization

ABSTRACT The intracellular lifestyle of bacteria is widely acknowledged to be an important mechanism in chronic and recurring infection. Among the Staphylococcus genus, only Staphylococcus aureus and Staphylococcus pseudintermedius have been clearly identified as intracellular in nonprofessional phagocytic cells (NPPCs), for which the mechanism is mainly fibronectin-binding dependent. Here, we used bioinformatics tools to search for possible new fibronectin-binding proteins (FnBP-like) in other Staphylococcus species. We found a protein in Staphylococcus delphini called Staphylococcus delphini surface protein Y (SdsY). This protein shares 68% identity with the Staphylococcus pseudintermedius surface protein D (SpsD), 36% identity with S. aureus FnBPA, and 39% identity with S. aureus FnBPB. The SdsY protein possesses the typical structure of FnBP-like proteins, including an N-terminal signal sequence, an A domain, a characteristic repeated pattern, and an LPXTG cell wall anchor motif. The level of adhesion to immobilized fibronectin was significantly higher in all S. delphini strains tested than in the fibronectin-binding-deficient S. aureus DU5883 strain. By using a model of human osteoblast infection, the level of internalization of all strains tested was significantly higher than with the invasive-incompetent S. aureus DU5883. These findings were confirmed by phenotype restoration after transformation of DU5883 by a plasmid expression vector encoding the SdsY repeats. Additionally, using fibronectin-depleted serum and murine osteoblast cell lines deficient for the β1 integrin, the involvement of fibronectin and β1 integrin was demonstrated in S. delphini internalization. The present study demonstrates that additional staphylococcal species are able to invade NPPCs and proposes a method to identify FnBP-like proteins.

Protein M and Fibronectin-Binding Proteins are Not Sufficient to Promote Internalization of Group a Streptococci into Hela Cells

1998 ◽  
Vol 11 (3) ◽  
pp. 163-169 ◽  
Author(s):  
R. Greco ◽  
C. von Hunolstein ◽  
G. Orefici ◽  
G. Donnarumma ◽  
M. Polidoro ◽  
...  
Keyword(s):  
Hela Cells ◽  
Binding Proteins ◽  
Group A Streptococcus ◽  
Protein A ◽  
Group A Streptococci ◽  
Cell Monolayers ◽  
Invasive Diseases ◽  
Internalization Process ◽  
Group A ◽  
Fibronectin Binding

Infection by group A streptococci is often associated with highly invasive diseases resulting in sepsis and shock. Recently, this species has renewed interest in the mechanism by which some strains are better able to invade mucosal epithelia and to penetrate into the bloodstream than are others. In this work we have evaluated the invasive ability of eight group A Streptococcus pyogenes strains isolated either from patients with severe invasive diseases, or with pharyngitis, or from healthy carrier. Five out of the eight strains studied were efficiently internalized within HeLa cells and, of these, four produced the M6 protein. A recombinant S.gordonii strain constitutively expressing the M6 protein failed to invade HeLa cell monolayers, suggesting that the expression of the M6 protein is not sufficient to allow the non-invasive S.gordonii to be internalized within Hela cells. As fibronectin-binding proteins have been implicated as primary adhesins in host-parasite interactions, we assayed the adhesiveness and the invasiveness of five invasive GAS strains in competition experiments where HeLa cells were infected with bacteria in the presence of purified fibronectin. The results obtained indicated that fibronectin moderately inhibits bacterial adhesion, while it does not affect internalization. These results indicate that other factors, together with fibronectin-binding proteins, participate in the adhesion of streptococci, and that fibronectin-mediated adhesion does not seem to be important in the internalization process of streptococci within HeLa cells.

scholarly journals EGFR inhibitors switch keratinocytes from a proliferative to a differentiative phenotype affecting epidermal development and barrier function

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nicolas Joly-Tonetti ◽  
Thomas Ondet ◽  
Mario Monshouwer ◽  
Georgios N. Stamatas
Keyword(s):  
Growth Factor ◽  
Barrier Function ◽  
Kinase Inhibitors ◽  
Treatment Protocol ◽  
Growth Factor Receptor ◽  
Skin Barrier ◽  
Keratinocyte Differentiation ◽  
Skin Barrier Function ◽  
Epidermal Keratinocytes ◽  
Epidermal Structure

Abstract Background Cutaneous adverse drug reactions (CADR) associated with oncology therapy involve 45–100% of patients receiving kinase inhibitors. Such adverse reactions may include skin inflammation, infection, pruritus and dryness, symptoms that can significantly affect the patient’s quality of life. To prevent severe skin damages dose adjustment or drug discontinuation is often required, interfering with the prescribed oncology treatment protocol. This is particularly the case of Epidermal Growth Factor Receptor inhibitors (EGFRi) targeting carcinomas. Since the EGFR pathway is pivotal for epidermal keratinocytes, it is reasonable to hypothesize that EGFRi also affect these cells and therefore interfere with the epidermal structure formation and skin barrier function. Methods To test this hypothesis, the effects of EGFRi and Vascular Endothelial Growth Factor Receptor inhibitors (VEGFRi) at therapeutically relevant concentrations (3, 10, 30, 100 nM) were assessed on proliferation and differentiation markers of human keratinocytes in a novel 3D micro-epidermis tissue culture model. Results EGFRi directly affect basal keratinocyte growth, leading to tissue size reduction and switching keratinocytes from a proliferative to a differentiative phenotype, as evidenced by decreased Ki67 staining and increased filaggrin, desmoglein-1 and involucrin expression compared to control. These effects lead to skin barrier impairment, which can be observed in a reconstructed human epidermis model showing a decrease in trans-epidermal water loss rates. On the other hand, pan-kinase inhibitors mainly targeting VEGFR barely affect keratinocyte differentiation and rather promote a proliferative phenotype. Conclusions This study contributes to the mechanistic understanding of the clinically observed CADR during therapy with EGFRi. These in vitro results suggest a specific mode of action of EGFRi by directly affecting keratinocyte growth and barrier function.

scholarly journals Fibronectin‐Binding Proteins and Fibrinogen‐Binding Clumping Factors Play Distinct Roles in Staphylococcal Arthritis and Systemic Inflammation

2005 ◽  
Vol 191 (5) ◽  
pp. 791-798 ◽  
Author(s):  
Niklas Palmqvist ◽  
Timothy Foster ◽  
J. Ross Fitzgerald ◽  
Elisabet Josefsson ◽  
Andrzej Tarkowski
Keyword(s):  
Systemic Inflammation ◽  
Binding Proteins ◽  
Fibrinogen Binding ◽  
Fibronectin Binding

scholarly journals Dissociation Rate Constants of Human Fibronectin Binding to Fibronectin-binding Proteins on LivingStaphylococcus aureusIsolated from Clinical Patients

2012 ◽  
Vol 287 (9) ◽  
pp. 6693-6701 ◽  
Author(s):  
Nadia N. Casillas-Ituarte ◽  
Brian H. Lower ◽  
Supaporn Lamlertthon ◽  
Vance G. Fowler ◽  
Steven K. Lower
Keyword(s):  
Rate Constants ◽  
Binding Proteins ◽  
Dissociation Rate ◽  
Human Fibronectin ◽  
Fibronectin Binding ◽  
Dissociation Rate Constants

scholarly journals Human Lung Tissue Explants Reveal Novel Interactions during Legionella pneumophila Infections

2013 ◽  
Vol 82 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Jens Jäger ◽  
Sebastian Marwitz ◽  
Jana Tiefenau ◽  
Janine Rasch ◽  
Olga Shevchuk ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Legionella Pneumophila ◽  
Human Lung ◽  
Transcriptional Response ◽  
Human Infection ◽  
Bacterial Invasion ◽  
Human Lung Tissue ◽  
Content Type ◽  
Tissue Explants ◽  
Legionnaires Disease

ABSTRACTHistological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model forLegionella pneumophilainfection comprising living human lung tissue. We stimulated lung explants withL. pneumophilastrains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion ofL. pneumophilato the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA−strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context ofL. pneumophilainfections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.

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