1H, 15N, and 13C resonance assignments of the third domain from the S. aureus innate immune evasion protein Eap

Herrera, Alvaro I.; Nicoleta, Ploscariu,; Geisbrecht, Brian V.; Prakash, Om

doi:10.1007/s12104-018-9804-9

Cited by 6 publications

(4 citation statements)

References 21 publications

(35 reference statements)

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“…2). This prediction is in agreement with the secondary structures observed in EapH1, EapH2, and Eap2, as determined by X-ray crystallography (Geisbrecht et al 2005), and the structural characteristics described by NMR for Eap3 and Eap4 (Herrera et al 2018, Woehl et al 2016. The chemical shift assignments have been deposited in BioMagResBank (http:// www.bmrb.wisc.edu) under the accession number 27540.…”

Section: Extent Of Assignments and Data Depositionsupporting

confidence: 84%

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Backbone resonance assignments of innate immune evasion protein EapH2 from the S. aureus

et al. 2019

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Staphylococcus aureus is a ubiquitous and persistent pathogen of humans and livestock. The bacterium disrupts the host's innate immune system's ability to recognize and clear bacteria with optimal efficiency by expressing a wide variety of virulence proteins. Two single domain protein homologs (EapH1, EapH2) of the extracellular adherence protein (Eap) have been reported. Eap is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement and Neutrophil Serine Proteases (NSPs). EapH1 and EapH2 are also inhibitors of NSPs (Stapels et al. 2014), but lack the ability to inhibit the classical, and lectin pathways of the complement activation system (Woehl et al. 2014).We continue the characterization of Eap domains, here with the experiments on EapH2, we acquired a series of 2D and 3D NMR spectra of EapH2 in solution. We completed 99% of expected non-proline backbone 1 H, 15 N, and 13 C resonance assignments of EapH2 and predicted secondary structure via the TALOS-N server. The assignment data have been deposited in the BMRB data bank under accession number 27540.

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Section: Extent Of Assignments and Data Depositionsupporting

confidence: 84%

“…EapH2 was overexpressed following methods established at our lab and reported earlier (Geisbrecht et al 2006;Herrera et al 2018). A DNA fragment encoding the protein sequence was subcloned into the Sal1 and Not1 sites of the prokaryotic expression vector pT7HMT.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Backbone resonance assignments of innate immune evasion protein EapH2 from the S. aureus

et al. 2019

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“…EAP domain proteins are defined by an eponymous motif of approximately ∼110 residues that adopts a β-grasp fold . Their overall structure consists of a prominent α-helix superimposed on a five-stranded mixed β-sheet, and has been extensively characterized by both X-ray crystallography and solution NMR spectroscopy. , Since the amino and carboxy termini lay on opposite sides of the fold, the structure of the EAP domain allows for tandem repeats of this motif to occur within the same molecule. Such an arrangement is seen in the multidomain Eap protein, , and appears to impart additional innate immune evasion properties to this molecule aside from inhibition of neutrophil serine proteases. − …”

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confidence: 99%

Investigation of Human Neutrophil Elastase Inhibition by Staphylococcus aureus EapH1: The Key Role Played by Arginine 89

Herdendorf

Geisbrecht

2018

Biochemistry

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Staphylococcus aureus secretes a family of potent, non-covalent inhibitory proteins that selectively target the neutrophil serine proteases neutrophil elastase, cathepsin-G, and proteinase-3. A majority of our understanding of these so-called EAP domain proteins has come from structure/function studies on EapH1 and its effects on human neutrophil elastase (hNE). Inspection of the EapH1/hNE co-crystal structure suggested that EapH1 residues R89, E94, and K95 are positioned near the EapH1/hNE interface and might contribute to the potent inhibition of hNE by EapH1. In this study, we used site directed mutagenesis, kinetic evaluation, and surface plasmon resonance to probe the individual contributions of R89, E94, and K95 to EapH1 function. We found that the wild-type EapH1/hNE complex is characterized by a fast association rate (2.0×106 M−1s−1) and a very slow dissociation rate (4.3×10−5 s−1), yielding an apparent inhibition constant of 21 pM. The slow dissociation rate of EapH1 from hNE resulted in a time-dependent inhibition pattern. Although conservative mutants E94Q and K95M, as well as the E94Q/K95M double mutant, had on- and off-rates comparable to wild-type EapH1, mutation of R89 to methionine resulted in a 15,000-fold decrease in inhibition (321 nM) and loss of the time-dependent inhibition characteristic. The double mutants R89M/E94Q and R89M/K95M, as well as the triple mutant R89M/E94Q/K95M were similarly perturbed. Mutation of R89 to lysine restored a portion of the inhibition of hNE (27 nM). Given these observations, we conclude that R89 is a primary contributor to EapH1 function vis-à-vis time-dependent inhibition of hNE.

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“…G. adiacens EVs induced the release of IL-8 and IL-1β to significantly (P < 0.05) higher levels compared to WCP. These observations overemphasize the importance of bacterial vesicle Copper resistance protein CopC [67] gi|1489647414| DUF1307 domain-containing protein [68,69] gi|1489650843| hypothetical protein D8B48_01700 [70] gi|259035675| 3D domain protein [71] gi|491798643|…”

Section: Elisa Quantification Of Selected Cytokines Produced From Stimentioning

confidence: 99%

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

et al. 2020

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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.

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1H, 15N, and 13C resonance assignments of the third domain from the S. aureus innate immune evasion protein Eap

Cited by 6 publications

References 21 publications

Backbone resonance assignments of innate immune evasion protein EapH2 from the S. aureus

Backbone resonance assignments of innate immune evasion protein EapH2 from the S. aureus

Investigation of Human Neutrophil Elastase Inhibition by Staphylococcus aureus EapH1: The Key Role Played by Arginine 89

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

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