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Pier, Gerald

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Gerald

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Pier, Gerald
Author's Publications: search.filters.isAuthorOfPublication.41f8105b-b83d-4ac6-a0bb-2ab0373134a0

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    PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms
    (Public Library of Science, 2018) Little, Dustin J.; Pfoh, Roland; Le Mauff, François; Bamford, Natalie C.; Notte, Christina; Baker, Perrin; Guragain, Manita; Robinson, Howard; Pier, Gerald; Nitz, Mark; Deora, Rajendar; Sheppard, Donald C.; Howell, P. Lynne
    Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.
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    Antibodies to Conserved Surface Polysaccharides Protect Mice Against Bacterial Conjunctivitis
    (The Association for Research in Vision and Ophthalmology, 2018) Zaidi, Tanweer S.; Zaidi, Tauqeer; Pier, Gerald
    Purpose Bacterial conjunctivitis is a major problem in ocular health. Little is known about protective immune effectors in the conjunctiva. We evaluated whether opsonic antibody to the conserved surface/capsular polysaccharide poly-N-acetyl glucosamine (PNAG) expressed by Streptococcus pneumoniae and Staphylococcus aureus was protective against bacterial conjunctivitis, as well as an antibody to the Pseudomonas aeruginosa surface polysaccharide alginate. Methods: Bacteria were injected directly into the conjunctivae of either A/J mice or into conjunctivae of wild type C57Bl/6 mice for comparisons to responses of recombination activating gene 1-knock out (RAG 1 KO) or germ-free mice in the C57Bl/6 genetic background. Human IgG1 monoclonal antibodies (MAb) to either PNAG or alginate were administered as follows: direct injection of 10 μg into the conjunctivae or topical application onto the cornea 4, 24, and 32 hours post infection; or intraperitoneal injection of 200 μg 18 hours prior to and then 4, 24, and 32-hours postinfection. After 48 hours, eyes were scored for pathology, mice were euthanized, and CFU/conjunctiva was determined. Results: All methods of antibody administration reduced S. pneumoniae, S. aureus, or P. aeruginosa pathology and bacterial levels in the conjunctivae. Histopathologic analysis showed severe inflammatory cell infiltrates in conjunctivae of mice treated with control MAb, whereas immune mice showed only very mild cellular infiltration. The protective effect of MAb to PNAG was abolished in RAG 1 KO and germ-free mice. Conclusions: Antibodies to both PNAG and alginate demonstrated therapeutic efficacy in models of S. pneumoniae, S. aureus, and P. aeruginosa conjunctivitis, validating the protective capacity of antibodies to surface polysaccharides in distinct ocular tissues.
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    Production of poly-β-1,6-N-acetylglucosamine by MatAB is required for hyphal aggregation and hydrophilic surface adhesion by Streptomyces
    (Shared Science Publishers OG, 2018) van Dissel, Dino; Willemse, Joost; Zacchetti, Boris; Claessen, Dennis; Pier, Gerald; van Wezel, Gilles P.
    Streptomycetes are multicellular filamentous microorganisms, and major producers of industrial enzymes and bioactive compounds such as antibiotics and anticancer drugs. The mycelial lifestyle plays an important role in the productivity during industrial fermentations. The hyphae of liquid-grown streptomycetes can self-aggregate into pellets, which hampers their industrial exploitation. Here we show that the Mat complex, which is required for pellet formation, catalyzes the synthesis of extracellular poly-β-1,6-N-acetylglucosamine (PNAG) in the model organisms Streptomyces coelicolor and Streptomyces lividans. Extracellular accumulation of PNAG allows Streptomyces to attach to hydrophilic surfaces, while attachment to hydrophobic surfaces requires a cellulase-degradable extracellular polymer (EPS) produced by CslA. Over-expression of matAB was sufficient to restore pellet formation to cslA null mutants of S. lividans. The two EPS systems together increase the robustness of mycelial pellets. These new insights allow better control of liquid-culture morphology of streptomycetes, which may be harnessed to improve growth and industrial exploitation of these highly versatile natural product and enzyme producers.
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    A Comprehensive Analysis of In Vitro and In Vivo Genetic Fitness of Pseudomonas aeruginosa Using High-Throughput Sequencing of Transposon Libraries
    (Public Library of Science, 2013) Skurnik, David; Roux, Damien; Aschard, Hugues; Cattoir, Vincent; Yoder-Himes, Deborah; Lory, Stephen; Pier, Gerald
    High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200–1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.
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    Monoclonal Antibody Raised against PNAG Has Variable Effects on Static S. epidermidis Biofilm Accumulation In Vitro
    (Ivyspring International Publisher, 2013) França, Angela; Vilanova, Manuel; Cerca, Nuno; Pier, Gerald
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    Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa
    (Frontiers Media S.A., 2015) Korneev, Kirill V.; Arbatsky, Nikolay P.; Molinaro, Antonio; Palmigiano, Angelo; Shaikhutdinova, Rima Z.; Shneider, Mikhail M.; Pier, Gerald; Kondakova, Anna N.; Sviriaeva, Ekaterina N.; Sturiale, Luisa; Garozzo, Domenico; Kruglov, Andrey A.; Nedospasov, Sergei A.; Drutskaya, Marina S.; Knirel, Yuriy A.; Kuprash, Dmitry V.
    Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.
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    Staphylococcus epidermidis Biofilm-Released Cells Induce a Prompt and More Marked In vivo Inflammatory-Type Response than Planktonic or Biofilm Cells
    (Frontiers Media S.A., 2016) França, Angela; Pérez-Cabezas, Begoña; Correia, Alexandra; Pier, Gerald; Cerca, Nuno; Vilanova, Manuel
    Staphylococcus epidermidis biofilm formation on indwelling medical devices is frequently associated with the development of chronic infections. Nevertheless, it has been suggested that cells released from these biofilms may induce severe acute infections with bacteraemia as one of its major associated clinical manifestations. However, how biofilm-released cells interact with the host remains unclear. Here, using a murine model of hematogenously disseminated infection, we characterized the interaction of cells released from S. epidermidis biofilms with the immune system. Gene expression analysis of mouse splenocytes suggested that biofilm-released cells might be particularly effective at activating inflammatory and antigen presenting cells and inducing cellular apoptosis. Furthermore, biofilm-released cells induced a higher production of pro-inflammatory cytokines, in contrast to mice infected with planktonic cells, even though these had a similar bacterial load in livers and spleens. Overall, these results not only provide insights into the understanding of the role of biofilm-released cells in S. epidermidis biofilm-related infections and pathogenesis, but may also help explain the relapsing character of these infections.
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    Characterization of an in vitro fed-batch model to obtain cells released from S. epidermidis biofilms
    (Springer Berlin Heidelberg, 2016) França, Angela; Carvalhais, Virgínia; Vilanova, Manuel; Pier, Gerald; Cerca, Nuno
    Both dynamic and fed-batch systems have been used for the study of biofilms. Dynamic systems, whose hallmark is the presence of continuous flow, have been considered the most appropriate for the study of the last stage of the biofilm lifecycle: biofilm disassembly. However, fed-batch is still the most used system in the biofilm research field. Hence, we have used a fed-batch system to collect cells released from Staphylococcus epidermidis biofilms, one of the most important etiological agents of medical device-associated biofilm infections. Herein, we showed that using this model it was possible to collect cells released from biofilms formed by 12 different S. epidermidis clinical and commensal isolates. In addition, our data indicated that biofilm disassembly occurred by both passive and active mechanisms, although the last occurred to a lesser extent. Moreover, it was observed that S. epidermidis biofilm-released cells presented higher tolerance to vancomycin and tetracycline, as well as a particular gene expression phenotype when compared with either biofilm or planktonic cells. Using this model, biofilm-released cells phenotype and their interaction with the host immune system could be studied in more detail, which could help providing significant insights into the pathophysiology of biofilm-related infections.
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    Efficacy of Antibody to PNAG Against Keratitis Caused by Fungal Pathogens
    (The Association for Research in Vision and Ophthalmology, 2016) Zhao, Ge; Zaidi, Tanweer S.; Bozkurt-Guzel, Cagla; Zaidi, Tauqeer H.; Lederer, James; Priebe, Gregory; Pier, Gerald
    Purpose Developing immunotherapies for fungal eye infections is a high priority. We analyzed fungal pathogens for expression of the surface polysaccharide, poly-N-acetyl glucosamine (PNAG), and used a mouse model of ocular keratitis caused by Aspergillus flavus, A. fumigatus, or Fusarium solani to determine if PNAG was an immunotherapy target and requirements for ancillary cellular and molecular immune effectors. Methods: Enzyme-linked immunosorbent assay (ELISA) or immunofluorescence was used to detect PNAG on fungal cells. Keratitis was induced by scratching corneas of C57BL/6, IL-17R KO, RAG-1 KO, or IL-22 KO mice followed by inoculation with fungal pathogens. Goat antibodies to PNAG, a PNAG-specific human IgG1 monoclonal antibody, or control antibodies were injected either prophylactically plus therapeutically or therapeutically only, and corneal pathology and fungal levels determined in infected eyes at 24 or 48 hours after infection. Results: All tested fungal species produced PNAG. Prophylactic or therapeutic treatment by intraperitoneal (IP) injection of antibody to PNAG combined with post-infection topical application of antibody, the latter also used for A. fumigatus, led to reduced fungal levels, corneal pathology, and cytokine expression. Topical administration only of the PNAG monoclonal antibodies (MAb) reduced fungal loads and corneal pathology. There was no antibody protection in IL-17R KO, RAG-1 KO, or IL-22 KO mice. Conclusions: Poly-N-acetyl glucosamine is produced by clinically important fungal ocular pathogens. Antibody to PNAG demonstrated protection against Aspergillus and Fusarium keratitis, requiring T cells producing IL-17 and IL-22. These findings indicate the potential to prevent or treat fungal infections by vaccines and immunotherapeutics to PNAG.