Publications by authors named "Joan A Geoghegan"

59 Publications

Enantioselective effect of cysteine functionalized mesoporous silica nanoparticles in U87 MG and GM08680 human cells and bacteria.

J Mater Chem B 2021 04;9(16):3544-3553

School of Chemistry, CRANN and AMBER Research Centres, Trinity College Dublin, College Green, Dublin 2, Ireland.

Chirality is a fundamental phenomenon in biological systems, since most of the biomolecules and biological components and species are chiral and therefore recognize and respond differently depending on the enantiomer present. With increasing research into the use of nanomaterials for biomedical purposes, it is essential to understand the role that chirality of nanoparticles plays at the cellular level. Here, the chiral cysteine functionalization of mesoporous silica nanoparticles has been shown to broadly affect its interaction with U87 MG human glioblastoma cell, healthy human fibroblast (GM08680) and methicillin-resistant S. aureus bacteria. We believe that this research is important to further advancement of nano-biotechnology.
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http://dx.doi.org/10.1039/d0tb02532aDOI Listing
April 2021

binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Proc Natl Acad Sci U S A 2021 01;118(1)

Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2, Ireland;

colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual cells to corneocytes. Finally, we found that mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of to AD corneocytes.
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http://dx.doi.org/10.1073/pnas.2014444118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817190PMC
January 2021

Dysregulated skin barrier function in Tmem79 mutant mice promotes IL-17A-dependent spontaneous skin and lung inflammation.

Allergy 2020 12 22;75(12):3216-3227. Epub 2020 Jul 22.

Trinity Biomedical Sciences Institute, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Background: Atopic dermatitis (AD) is associated with a dysregulation of the skin barrier and may predispose to the development of secondary allergic conditions, such as asthma. Tmem79 mice harbor a mutation in the gene encoding Transmembrane Protein 79 (or Mattrin), which has previously been associated with AD. As a result of the Tmem79 gene mutation, these mice have a defective skin barrier and develop spontaneous skin inflammation. In this study, Tmem79 mice were assessed for the underlying immunological response in the development of spontaneous skin and lung inflammation.

Methods: Development of spontaneous skin and lung inflammation in Tmem79 mice was analyzed. We further investigated susceptibility to cutaneous Staphylococcus aureus infection. Tmem79 were crossed to IL-17A-deficient mice to address the contribution of IL-17A to spontaneous skin and lung disease.

Results: Tmem79 mice developed IL-17A-dependent spontaneous AD-like inflammation and were refractory to S aureus infection. Mutant mice progressed to airway inflammation subsequent to the occurrence of dermatitis. The progression from skin to lung disease is dependent on adaptive immunity and is facilitated by cutaneous expansion of Th17 and TCRγδ T cells.

Conclusion: Mice lacking Tmem79/Mattrin expression have a defective skin barrier. In adulthood, these mice develop dermatitis with secondary progression to lung inflammation. The development of skin and lung inflammation is IL-17A-dependent and mediated by TCRγδ T cells.
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http://dx.doi.org/10.1111/all.14488DOI Listing
December 2020

Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

Nat Commun 2020 01 9;11(1):140. Epub 2020 Jan 9.

Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, D02 R590, Ireland.

Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.
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http://dx.doi.org/10.1038/s41467-019-13724-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952399PMC
January 2020

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion.

PLoS Pathog 2019 06 19;15(6):e1007816. Epub 2019 Jun 19.

The Roslin Institute and Edinburgh Infectious Diseases, University of Edinburgh, Easter Bush Campus, Edinburgh, Scotland, United Kingdom.

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology.
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http://dx.doi.org/10.1371/journal.ppat.1007816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602291PMC
June 2019

Clumping factor B is an important virulence factor during Staphylococcus aureus skin infection and a promising vaccine target.

PLoS Pathog 2019 04 22;15(4):e1007713. Epub 2019 Apr 22.

Host-Pathogen Interactions Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.

Staphylococcus aureus expresses a number of cell wall-anchored proteins that mediate adhesion and invasion of host cells and tissues and promote immune evasion, consequently contributing to the virulence of this organism. The cell wall-anchored protein clumping factor B (ClfB) has previously been shown to facilitate S. aureus nasal colonization through high affinity interactions with the cornified envelope in the anterior nares. However, the role of ClfB during skin and soft tissue infection (SSTI) has never been investigated. This study reveals a novel role for ClfB during SSTIs. ClfB is crucial in determining the abscess structure and bacterial burden early in infection and this is dependent upon a specific interaction with the ligand loricrin which is expressed within the abscess tissue. Targeting ClfB using a model vaccine that induced both protective humoral and cellular responses, leads to protection during S. aureus skin infection. This study therefore identifies ClfB as an important antigen for future SSTI vaccines.
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http://dx.doi.org/10.1371/journal.ppat.1007713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497315PMC
April 2019

Fibronectin-binding protein B (FnBPB) from protects against the antimicrobial activity of histones.

J Biol Chem 2019 03 8;294(10):3588-3602. Epub 2019 Jan 8.

From the Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, 27100 Pavia, Italy,

is a Gram-positive bacterium that can cause both superficial and deep-seated infections. Histones released by neutrophils kill bacteria by binding to the bacterial cell surface and causing membrane damage. We postulated that cell wall-anchored proteins protect from the bactericidal effects of histones by binding to and sequestering histones away from the cell envelope. Here, we focused on strain LAC and by using an array of biochemical assays, including surface plasmon resonance and ELISA, discovered that fibronectin-binding protein B (FnBPB) is the main histone receptor. FnBPB bound all types of histones, but histone H3 displayed the highest affinity and bactericidal activity and was therefore investigated further. H3 bound specifically to the A domain of recombinant FnBPB with a of 86 nm, ∼20-fold lower than that for fibrinogen. Binding apparently occurred by the same mechanism by which FnBPB binds to fibrinogen, because FnBPB variants defective in fibrinogen binding also did not bind H3. An FnBPB-deletion mutant of LAC bound less H3 and was more susceptible to its bactericidal activity and to neutrophil extracellular traps, whereas an FnBPB-overexpressing mutant bound more H3 and was more resistant than the WT. FnBPB bound simultaneously to H3 and plasminogen, which after activation by tissue plasminogen activator cleaved the bound histone. We conclude that FnBPB provides a dual immune-evasion function that captures histones and prevents them from reaching the bacterial membrane and simultaneously binds plasminogen, thereby promoting its conversion to plasmin to destroy the bound histone.
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http://dx.doi.org/10.1074/jbc.RA118.005707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416437PMC
March 2019

Mobile-Genetic-Element-Encoded Hypertolerance to Copper Protects Staphylococcus aureus from Killing by Host Phagocytes.

mBio 2018 10 16;9(5). Epub 2018 Oct 16.

Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin, Ireland

Pathogens are exposed to toxic levels of copper during infection, and copper tolerance may be a general virulence mechanism used by bacteria to resist host defenses. In support of this, inactivation of copper exporter genes has been found to reduce the virulence of bacterial pathogens Here we investigate the role of copper hypertolerance in methicillin-resistant (MRSA). We show that a copper hypertolerance operon (), carried on a mobile genetic element (MGE), is prevalent in a collection of invasive strains and more widely among clonal complex 22, 30, and 398 strains. The and genes encode a copper efflux pump and a multicopper oxidase, respectively. Isogenic mutants lacking or had impaired growth in subinhibitory concentrations of copper. Transfer of a carrying plasmid to a naive clinical isolate resulted in a gain of copper hypertolerance and enhanced bacterial survival inside primed macrophages. The and genes were upregulated within infected macrophages, and their expression was dependent on the copper-sensitive operon repressor CsoR. Isogenic and mutants were impaired in their ability to persist intracellularly in macrophages and were less resistant to phagocytic killing in human blood than the parent strain. The importance of copper-regulated genes in resistance to phagocytic killing was further elaborated using mutants expressing a copper-insensitive variant of CsoR. Our findings suggest that the gain of mobile genetic elements carrying copper hypertolerance genes contributes to the evolution of virulent strains of that are better equipped to resist killing by host immune cells. Methicillin-resistant (MRSA) poses a substantial threat to human health worldwide and evolves rapidly by acquiring mobile genetic elements, such as plasmids. Here we investigate how the - copper hypertolerance operon carried on a mobile genetic element contributes to the virulence potential of clinical isolates of MRSA. Copper is a key component of innate immune bactericidal defenses. Here we show that copper hypertolerance genes enhance the survival of inside primed macrophages and in whole human blood. The and genes are carried by clinical isolates responsible for invasive infections across Europe, and more broadly among three successful clonal lineages of Our findings show that a gain of copper hypertolerance genes increases the resistance of MRSA to phagocytic killing by host immune cells and imply that acquisition of this mobile genetic element can contribute to the success of MRSA.
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http://dx.doi.org/10.1128/mBio.00550-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191537PMC
October 2018

Adhesion of Staphylococcus aureus to Corneocytes from Atopic Dermatitis Patients Is Controlled by Natural Moisturizing Factor Levels.

mBio 2018 08 14;9(4). Epub 2018 Aug 14.

Institute of Life Sciences, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

The bacterial pathogen plays an important role in atopic dermatitis (AD), a chronic disorder that mostly affects children. Colonization of the skin of AD patients by exacerbates the disease, but the molecular determinants of the bacterium-skin adhesive interactions are poorly understood. Specifically, reduced levels of natural moisturizing factor (NMF) in the stratum corneum have been shown to be associated with more severe AD symptoms, but whether this is directly related to adhesion is still an open question. Here, we demonstrate a novel relationship between NMF expression in AD skin and strength of bacterial adhesion. Low-NMF corneocytes, unlike high-NMF ones, are covered by a dense layer of nanoscale villus protrusions. bacteria isolated from AD skin bind much more strongly to corneocytes when the NMF level is reduced. Strong binding forces originate from a specific interaction between the bacterial adhesion clumping factor B (ClfB) and skin ligands. Remarkably, mechanical tension dramatically strengthens ClfB-mediated adhesion, as observed with catch bonds, demonstrating that physical stress plays a role in promoting colonization of AD skin by Collectively, our findings demonstrate that patient NMF levels regulate the strength of -corneocyte adhesion, the first step in skin colonization, and suggest that the ClfB binding mechanism could represent a potential target for new therapeutic treatments. Bacterium-skin interactions play important roles in skin disorders, yet their molecular details are poorly understood. In this study, we decipher the molecular forces at play during adhesion of to skin corneocytes in the clinically important context of atopic dermatitis (AD), also known as eczema. We identify a unique relationship between the level of natural moisturizing factor (NMF) in the skin and the strength of bacterium-corneocyte adhesion. Bacterial adhesion is primarily mediated by the surface protein clumping factor B (ClfB) and is enhanced by physical stress, highlighting the role of protein mechanobiology in skin colonization. Similar to a catch bond behavior, this mechanism represents a promising target for the development of novel antistaphylococcal agents.
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http://dx.doi.org/10.1128/mBio.01184-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094479PMC
August 2018

Mechanomicrobiology: How Mechanical Forces Activate Staphylococcus aureus Adhesion.

Trends Microbiol 2018 08 1;26(8):645-648. Epub 2018 Jun 1.

Institute of Life Sciences, Université catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06., B-1348 Louvain-la-Neuve, Belgium; Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Belgium. Electronic address:

During colonization of biomaterials and host tissues, surface-attached bacteria are subjected to mechanical stresses, including hydrodynamic flow and cell-surface contacts. Two publications show that mechanical force activates the adhesive function of Staphylococcus aureus surface proteins, thereby providing the pathogen with a means to withstand high shear stress during colonization.
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http://dx.doi.org/10.1016/j.tim.2018.05.004DOI Listing
August 2018

clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion.

Proc Natl Acad Sci U S A 2018 05 7;115(21):5564-5569. Epub 2018 May 7.

Institute of Life Sciences, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium;

Clumping factor A (ClfA), a cell-wall-anchored protein from , is a virulence factor in various infections and facilitates the colonization of protein-coated biomaterials. ClfA promotes bacterial adhesion to the blood plasma protein fibrinogen (Fg) via molecular forces that have not been studied so far. A unique, yet poorly understood, feature of ClfA is its ability to favor adhesion to Fg at high shear stress. Unraveling the strength and dynamics of the ClfA-Fg interaction would help us better understand how colonizes implanted devices and withstands physiological shear stress. By means of single-molecule experiments, we show that ClfA behaves as a force-sensitive molecular switch that potentiates staphylococcal adhesion under mechanical stress. The bond between ClfA and immobilized Fg is weak (∼0.1 nN) at low tensile force, but is dramatically enhanced (∼1.5 nN) by mechanical tension, as observed with catch bonds. Strong bonds, but not weak ones, are inhibited by a peptide mimicking the C-terminal segment of the Fg γ-chain. These results point to a model whereby ClfA interacts with Fg via two distinct binding sites, the adhesive function of which is regulated by mechanical tension. This force-activated mechanism is of biological significance because it explains at the molecular level the ability of ClfA to promote bacterial attachment under high physiological shear stress.
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http://dx.doi.org/10.1073/pnas.1718104115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003445PMC
May 2018

A horizontally gene transferred copper resistance locus confers hyper-resistance to antibacterial copper toxicity and enables survival of community acquired methicillin resistant Staphylococcus aureus USA300 in macrophages.

Environ Microbiol 2018 04 26;20(4):1576-1589. Epub 2018 Mar 26.

Department of Genetics, University of Leicester, University Road, Leicester, LE1 7RH, UK.

Excess copper is highly toxic and forms part of the host innate immune system's antibacterial arsenal, accumulating at sites of infection and acting within macrophages to kill engulfed pathogens. We show for the first time that a novel, horizontally gene transferred copper resistance locus (copXL), uniquely associated with the SCCmec elements of the highly virulent, epidemic, community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) USA300, confers copper hyper-resistance. These genes are additional to existing core genome copper resistance mechanisms, and are not found in typical S. aureus lineages, but are increasingly identified in emerging pathogenic isolates. Our data show that CopX, a putative P -ATPase efflux transporter, and CopL, a novel lipoprotein, confer copper hyper-resistance compared to typical S. aureus strains. The copXL genes form an operon that is tightly repressed in low copper environments by the copper regulator CsoR. Significantly, CopX and CopL are important for S. aureus USA300 intracellular survival within macrophages. Therefore, the emergence of new S. aureus clones with the copXL locus has significant implications for public health because these genes confer increased resistance to antibacterial copper toxicity, enhancing bacterial fitness by altering S. aureus interaction with innate immunity.
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http://dx.doi.org/10.1111/1462-2920.14088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947656PMC
April 2018

Staphylococcus aureus and Atopic Dermatitis: A Complex and Evolving Relationship.

Trends Microbiol 2018 06 9;26(6):484-497. Epub 2017 Dec 9.

Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2, Ireland. Electronic address:

Staphylococcus aureus is frequently isolated from the skin of atopic dermatitis (AD) patients during flares. The normal microbiota is disrupted and the diversity of the microorganisms on the skin is reduced. Many species that produce inhibitors of S. aureus growth decline. Strains from S. aureus clonal complex 1 are enriched among AD sufferers whereas the CC30 strains most frequently isolated from nasal carriers in the normal population are much rarer in AD. S. aureus expresses several molecules that contribute to the intensity of symptoms, including δ-toxin which stimulates mast cells, α-toxin which damages keratinocytes, phenol-soluble modulins which stimulate cytokine release by keratinocytes, protein A which triggers inflammatory responses from keratinocytes, superantigens which trigger B cell expansion and cytokine release, and proinflammatory lipoproteins. Proteases contribute to disruption of the epidermal barrier. S. aureus isolated from AD patients adheres to the deformed corneocytes from AD patients in a clumping factor B-dependent fashion. Novel targeted therapies for AD have recently been introduced to clinical practice with many more in development, including monoclonal antibodies that specifically target cytokines and their receptors, and a bacteriophage lysin that eliminates S. aureus from AD skin.
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http://dx.doi.org/10.1016/j.tim.2017.11.008DOI Listing
June 2018

Force-Induced Strengthening of the Interaction between Clumping Factor B and Loricrin.

mBio 2017 Dec 5;8(6). Epub 2017 Dec 5.

Institute of Life Sciences, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

Bacterial pathogens that colonize host surfaces are subjected to physical stresses such as fluid flow and cell surface contacts. How bacteria respond to such mechanical cues is an important yet poorly understood issue. uses a repertoire of surface proteins to resist shear stress during the colonization of host tissues, but whether their adhesive functions can be modulated by physical forces is not known. Here, we show that the interaction of clumping factor B (ClfB) with the squamous epithelial cell envelope protein loricrin is enhanced by mechanical force. We find that ClfB mediates adhesion to loricrin through weak and strong molecular interactions both in a laboratory strain and in a clinical isolate. Strong forces (~1,500 pN), among the strongest measured for a receptor-ligand bond, are consistent with a high-affinity "dock, lock, and latch" binding mechanism involving dynamic conformational changes in the adhesin. Notably, we demonstrate that the strength of the ClfB-loricrin bond increases as mechanical force is applied. These findings favor a two-state model whereby bacterial adhesion to loricrin is enhanced through force-induced conformational changes in the ClfB molecule, from a weakly binding folded state to a strongly binding extended state. This force-sensitive mechanism may provide with a means to finely tune its adhesive properties during the colonization of host surfaces, helping cells to attach firmly under high shear stress and to detach and spread under low shear stress. colonizes the human skin and the nose and can cause various disorders, including superficial skin lesions and invasive infections. During nasal colonization, the surface protein clumping factor B (ClfB) binds to the squamous epithelial cell envelope protein loricrin, but the molecular interactions involved are poorly understood. Here, we unravel the molecular mechanism guiding the ClfB-loricrin interaction. We show that the ClfB-loricrin bond is remarkably strong, consistent with a high-affinity "dock, lock, and latch" binding mechanism. We discover that the ClfB-loricrin interaction is enhanced under tensile loading, thus providing evidence that the function of an surface protein can be activated by physical stress.
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http://dx.doi.org/10.1128/mBio.01748-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717387PMC
December 2017

The Staphylococcus aureus Cell Wall-Anchored Protein Clumping Factor A Is an Important T Cell Antigen.

Infect Immun 2017 12 17;85(12). Epub 2017 Nov 17.

Host-Pathogen Interactions Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

has become increasingly resistant to antibiotics, and vaccines offer a potential solution to this epidemic of antimicrobial resistance. Targeting of specific T cell subsets is now considered crucial for next-generation anti- vaccines; however, there is a paucity of information regarding T cell antigens of This study highlights the importance of cell wall-anchored proteins as human CD4 T cell activators capable of driving antigen-specific Th1 and Th17 cell activation. Clumping factor A (ClfA), which contains N1, N2, and N3 binding domains, was found to be a potent human T cell activator. We further investigated which subdomains of ClfA were involved in T cell activation and found that the full-length ClfA N123 and N23 were potent Th1 and Th17 activators. Interestingly, the N1 subdomain was capable of exclusively activating Th1 cells. Furthermore, when these subdomains were used in a model vaccine, N23 and N1 offered Th1- and Th17-mediated systemic protection in mice upon intraperitoneal challenge. Overall, however, full-length ClfA N123 is required for maximal protection both locally and systemically.
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http://dx.doi.org/10.1128/IAI.00549-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695125PMC
December 2017

Live-Cell Nanoscopy in Antiadhesion Therapy.

Trends Microbiol 2017 07 8;25(7):512-514. Epub 2017 May 8.

Institute of Life Sciences, Université catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06., B-1348 Louvain-la-Neuve, Belgium; Walloon Excellence in Life sciences and Biotechnology (WELBIO), Belgium. Electronic address:

Live-cell nanoscopy has contributed significantly to assessing the inhibition of adhesion of uropathogenic Escherichia coli and Staphylococcus aureus by glycoconjugates and monoclonal antibodies, respectively, and of S. aureus surface attachment and cell-cell association by a synthetic peptide. This new technology shows promise for the development of antiadhesion therapies against bacterial pathogens.
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http://dx.doi.org/10.1016/j.tim.2017.04.002DOI Listing
July 2017

Conjugate of an IgG Binding Domain with Botulinum Neurotoxin A Lacking the Acceptor Moiety Targets Its SNARE Protease into TrkA-Expressing Cells When Coupled to Anti-TrkA IgG or Fc-βNGF.

Bioconjug Chem 2017 06 22;28(6):1684-1692. Epub 2017 May 22.

International Centre for Neurotherapeutics, Dublin City University , Glasnevin, Dublin 9, Ireland.

Numerous naturally occurring toxins can perturb biological systems when they invade susceptible cells. Coupling of pertinent targeting ligands to the active domains of such proteins provides a strategy for directing these to particular cellular populations implicated in disease. A novel approach described herein involved fusion of one mutated immunoglobulin G (IgG) binding moiety of staphylococcal protein A to the SNARE protease and translocation domain of botulinum neurotoxin A (BoNT/A). This chimera could be monovalently coupled to IgG or via its Fc region to recombinant targeting ligands. The utility of the resulting conjugates is demonstrated by the delivery of a SNARE protease into a cell line expressing tropomyosin receptor kinase A (TrkA) through coupling to anti-TrkA IgG or a fusion of Fc and nerve-growth factor. Thus, this is a versitile and innovative technology for conjugating toxins to diverse ligands for retargeted cell delivery of potential therapeutics.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00157DOI Listing
June 2017

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis.

Infect Immun 2017 06 23;85(6). Epub 2017 May 23.

Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin, Ireland

skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of to AD corneocytes. strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single bacteria to corneocytes from AD patients was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of colonization in AD patients. ClfB is a key adhesion molecule for the interaction of with AD corneocytes and represents a target for intervention.
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http://dx.doi.org/10.1128/IAI.00994-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442637PMC
June 2017

Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC.

Proc Natl Acad Sci U S A 2017 04 20;114(14):3738-3743. Epub 2017 Mar 20.

Institute of Life Sciences, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium;

forms biofilms on indwelling medical devices using a variety of cell-surface proteins. There is growing evidence that specific homophilic interactions between these proteins represent an important mechanism of cell accumulation during biofilm formation, but the underlying molecular mechanisms are still not well-understood. Here we report the direct measurement of homophilic binding forces by the serine-aspartate repeat protein SdrC and their inhibition by a peptide. Using single-cell and single-molecule force measurements, we find that SdrC is engaged in low-affinity homophilic bonds that promote cell-cell adhesion. Low-affinity intercellular adhesion may play a role in favoring biofilm dynamics. We show that SdrC also mediates strong cellular interactions with hydrophobic surfaces, which are likely to be involved in the initial attachment to biomaterials, the first stage of biofilm formation. Furthermore, we demonstrate that a peptide derived from β-neurexin is a powerful competitive inhibitor capable of efficiently blocking surface attachment, homophilic adhesion, and biofilm accumulation. Molecular modeling suggests that this blocking activity may originate from binding of the peptide to a sequence of SdrC involved in homophilic interactions. Our study opens up avenues for understanding the role of homophilic interactions in staphylococcal adhesion, and for the design of new molecules to prevent biofilm formation during infection.
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http://dx.doi.org/10.1073/pnas.1616805114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389287PMC
April 2017

Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody.

EBioMedicine 2016 Nov 1;13:328-338. Epub 2016 Oct 1.

Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A & M University Health Science Center, 2121 W Holcombe Blvd., Houston, TX 77030, USA. Electronic address:

The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the "top" of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.
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http://dx.doi.org/10.1016/j.ebiom.2016.09.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264652PMC
November 2016

Forces between Staphylococcus aureus and human skin.

Nanoscale Horiz 2016 Jul 6;1(4):298-303. Epub 2016 May 6.

Institute of Life Sciences, Université catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06, B-1348 Louvain-la-Neuve, Belgium.

Characterization of the molecular interactions between microbial cells and the human skin is essential to understand the functions of the skin microbiome, and to gain insight into the molecular basis of skin disorders. Although various molecular approaches have been used to study microbe-skin interactions, the underlying molecular forces were not accessible to study. Here we present a novel atomic force microscopy approach to localize and quantify the nanoscale interaction forces between the bacterial pathogen Staphylococcus aureus and human skin. A method combining nanoscale multiparametric imaging with single bacterial probes is developed to map simultaneously the topography and bacterial-binding properties of corneocytes at high spatiotemporal resolution. Further quantification of the forces between bacteria and corneocytes is achieved using single-cell force spectroscopy. The results show that the S. aureus-skin adhesion is strong (∼500 pN) and originates from multiple specific bonds between adhesins on the bacterial cell surface and target ligands on the corneocyte surface. Applicable to a wide variety of microbes and skin cells, our methodology offers exciting prospects for understanding the molecular details of skin colonization and infection.
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http://dx.doi.org/10.1039/c6nh00057fDOI Listing
July 2016

Molecular Interactions of Human Plasminogen with Fibronectin-binding Protein B (FnBPB), a Fibrinogen/Fibronectin-binding Protein from Staphylococcus aureus.

J Biol Chem 2016 08 7;291(35):18148-62. Epub 2016 Jul 7.

From the Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, 27100 Pavia, Italy and

Staphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen- and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureus- or Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a KD of 0.532 μm as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein.
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http://dx.doi.org/10.1074/jbc.M116.731125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000064PMC
August 2016

CRIg Functions as a Macrophage Pattern Recognition Receptor to Directly Bind and Capture Blood-Borne Gram-Positive Bacteria.

Cell Host Microbe 2016 Jul 23;20(1):99-106. Epub 2016 Jun 23.

The Calvin, Phoebe & Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, T2N 4N1, Canada; Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada. Electronic address:

Kupffer cells (KCs), the vast pool of intravascular macrophages in the liver, help to clear blood-borne pathogens. The mechanisms by which KCs capture circulating pathogens remain unknown. Here we use intra-vital imaging of mice infected with Staphylococcus aureus to directly visualize the dynamic process of bacterial capture in the liver. Circulating S. aureus were captured by KCs in a manner dependent on the macrophage complement receptor CRIg, but the process was independent of complement. CRIg bound Staphylococcus aureus specifically through recognition of lipoteichoic acid (LTA), but not cell-wall-anchored surface proteins or peptidoglycan. Blocking the recognition between CRIg and LTA in vivo diminished the bacterial capture in liver and led to systemic bacterial dissemination. All tested Gram-positive, but not Gram-negative, bacteria bound CRIg in a complement-independent manner. These findings reveal a pattern recognition role for CRIg in the direct capture of circulating Gram-positive bacteria from the bloodstream.
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http://dx.doi.org/10.1016/j.chom.2016.06.002DOI Listing
July 2016

The Role of Staphylococcus aureus Virulence Factors in Skin Infection and Their Potential as Vaccine Antigens.

Pathogens 2016 Feb 17;5(1). Epub 2016 Feb 17.

Host Pathogen Interactions Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.

Staphylococcus aureus (S. aureus) causes the vast majority of skin and soft tissue infections (SSTIs) in humans. S. aureus has become increasingly resistant to antibiotics and there is an urgent need for new strategies to tackle S. aureus infections. Vaccines offer a potential solution to this epidemic of antimicrobial resistance. However, the development of next generation efficacious anti-S. aureus vaccines necessitates a greater understanding of the protective immune response against S. aureus infection. In particular, it will be important to ascertain if distinct immune mechanisms are required to confer protection at distinct anatomical sites. Recent discoveries have highlighted that interleukin-17-producing T cells play a particularly important role in the immune response to S. aureus skin infection and suggest that vaccine strategies to specifically target these types of T cells may be beneficial in the treatment of S. aureus SSTIs. S. aureus expresses a large number of cell wall-anchored (CWA) proteins, which are covalently attached to the cell wall peptidoglycan. The virulence potential of many CWA proteins has been demonstrated in infection models; however, there is a paucity of information regarding their roles during SSTIs. In this review, we highlight potential candidate antigens for vaccines targeted at protection against SSTIs.
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http://dx.doi.org/10.3390/pathogens5010022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810143PMC
February 2016

Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG.

Proc Natl Acad Sci U S A 2016 Jan 29;113(2):410-5. Epub 2015 Dec 29.

Institute of Life Sciences, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium; Walloon Excellence in Life Sciences and Biotechnology, B-1300 Wavre, Belgium

Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between β-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.
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http://dx.doi.org/10.1073/pnas.1519265113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720321PMC
January 2016

Cell Wall-Anchored Surface Proteins of Staphylococcus aureus: Many Proteins, Multiple Functions.

Curr Top Microbiol Immunol 2017;409:95-120

Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College Dublin, The University of Dublin, Dublin 2, Ireland.

Staphylococcus aureus persistently colonizes about 20 % of the population and is intermittently associated with the remainder. The organism can cause superficial skin infections and life-threatening invasive diseases. The surface of the bacterial cell displays a variety of proteins that are covalently anchored to peptidoglycan. They perform many functions including adhesion to host cells and tissues, invasion of non-phagocytic cells, and evasion of innate immune responses. The proteins have been categorized into distinct classes based on structural and functional analysis. Many surface proteins are multifunctional. Cell wall-anchored proteins perform essential functions supporting survival and proliferation during the commensal state and during invasive infections. The ability of cell wall-anchored proteins to bind to desquamated epithelial cells is important during colonization, and the binding to fibrinogen is of particular significance in pathogenesis.
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http://dx.doi.org/10.1007/82_2015_5002DOI Listing
May 2019

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection.

PLoS Pathog 2015 5;11(11):e1005226. Epub 2015 Nov 5.

Host-Pathogen Interactions Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.
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http://dx.doi.org/10.1371/journal.ppat.1005226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634925PMC
April 2016

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

Infect Immun 2015 Oct 3;83(10):4093-102. Epub 2015 Aug 3.

Department of Molecular Medicine, Unit of Biochemistry, Pavia, Italy

In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogen Staphylococcus pseudintermedius for 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 ΔspsL mutant 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 lactis transformed 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 α5β1 markedly 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 SpsD520-846 and SpsL538-823 regions harboring the major fibronectin-binding sites inhibited S. pseudintermedius internalization. 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.
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http://dx.doi.org/10.1128/IAI.00542-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567651PMC
October 2015

Staphylokinase Control of Staphylococcus aureus Biofilm Formation and Detachment Through Host Plasminogen Activation.

J Infect Dis 2016 Jan 1;213(1):139-48. Epub 2015 Jul 1.

Department of Rheumatology and Inflammation Research, Institute of Medicine.

Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.
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http://dx.doi.org/10.1093/infdis/jiv360DOI Listing
January 2016

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection.

Infect Immun 2015 Sep 22;83(9):3445-57. Epub 2015 Jun 22.

Host Pathogen Interactions Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.
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http://dx.doi.org/10.1128/IAI.00358-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534639PMC
September 2015
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