Ambrose L Cheung

Dartmouth College, Hanover, New Hampshire, United States

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Publications (162)664.12 Total impact

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    ABSTRACT: The toxin MazFsa in Staphylococcus aureus is a sequence-specific endoribonuclease that cleaves the majority of the mRNAs in vivo, but spares many essential (e.g. secY mRNA) and, surprisingly, an mRNA encoding a regulatory protein (i.e. sarA mRNA). We hypothesize that some mRNAs may be protected by RNA binding protein(s) from degradation by MazFsa. Using heparin-Sepharose enriched fractions that hybridized to sarA mRNA on Northwestern blots, we identified among multiple proteins the DEAD-box RNA helicase CshA (NWMN_1985 or SA1885) by mass spectroscopy. Purified CshA exhibits typical RNA helicase activities as exemplified by RNA-dependent ATPase activity and unwinding of the DNA-RNA duplex. A severe growth defect was observed in the cshA mutant as compared with the parent when grown at 25°C, but not at 37°C. Activation of MazFsa in the cshA mutant resulted in lower CFU/ml accompanied by a precipitous drop in viability (∼40%) as compared to that of the parent and complemented strains. Nanostring analysis reveals diminished expression of a small number of mRNAs and 22 sRNAs in the cshA mutant vs. the parent upon MazFsa induction, thus implying protection of these RNA by CshA. In the case of the sRNA teg049 within the sarA locus, we showed that the protective effect was likely due to transcript stability as revealed by reduced half-life in the cshA mutant vs. the parent. Accordingly, CshA likely stabilizes selective mRNAs and sRNA in vivo and as a result, enhances S. aureus survival upon MazFsa induction during stress.
    No preview · Article · Jan 2016 · Infection and Immunity
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    ABSTRACT: Staphylococcus aureus uses the two-component regulatory system, GraRS, to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS, or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Null mutants in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin, hNP-1, cutaneous β-defensin, hBD-2 or the platelet kinocidin congener, RP-1. Actions studied by flow cytometry included: energetics (ENR); membrane permeabilization (PRM); annexin-V binding (ANX); or cell death protease activation (CDP). Assay conditions simulated bloodstream (7.5) or phagolysosomal (5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 versus pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔEL on HDP resistance were peptide- and pH-dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs, and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔEL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5, and all study HDPs at pH 5.5. An exogenous EL-mimic protected mutant strains from hNP-1 and hBD-2, but not RP-1, indicating GraS and its EL play non-redundant roles in S. aureus survival responses to specific HDPs. These findings suggest GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.
    No preview · Article · Nov 2015 · Infection and Immunity
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    ABSTRACT: Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.
    Full-text · Article · Apr 2015 · PLoS Pathogens
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    ABSTRACT: Methicillin resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen found in hospital and community environments that can cause serious infections. A major barrier to genetic manipulations of clinical isolates has been the considerable difficulty in transforming these strains with foreign plasmids, such as those from E. coli, in part due to the type I and IV Restriction Modification (R-M) barriers. Here we combine a Plasmid Artificial Modification (PAM) system with DC10B E. coli cells (dcm mutants) to bypass the barriers of both type I and IV R-M of S. aureus, thus allowing E. coli plasmid DNA to be transformed directly into clinical MRSA strains MW2, N315 and LAC, representing three of the most common clonal complexes. Successful transformation of clinical S. aureus isolates with E. coli-derived plasmids should greatly increase the ability to genetically modify relevant S. aureus strains and advance our understanding of S. aureus pathogenesis.
    Full-text · Article · Mar 2015 · PLoS ONE
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    ABSTRACT: In a loss of viability screen using small molecules against methicillin resistant S. aureus MRSA USA300 with sub-MIC β-lactam, we found a small molecule, designated as DNAC-1, that potentiated the effect of oxacillin (MIC of oxacillin decreased from 64 to 0.25 μg/ml). Fluorescence microscopy indicated disruption of membrane structures within 15 minutes of exposure to DNAC-1 at 2x MIC. This permeabilization was accompanied by a rapid loss of membrane potential, as monitored by the DiOC2 dye. Macromolecular analysis showed inhibition of staphylococcal cell wall synthesis by DNAC-1. Transmission electron microscopy of treated MRSA USA300 cells revealed a slightly thicker cell wall together with mesosome -like projections into the cytosol. Exposure of USA300 cells to DNAC-1 was associated with mislocalization of FtsZ accompanied by localization of PBP2 and PBP4 away from the septum as well as mild activation of the vraRS-mediated cell-wall stress response. However, DNAC-1 does not have any generalized toxicity towards mammalian host cells. DNAC-1, in combination with ceftriaxone, is also effective against an assortment of Gram negative pathogens. Using a murine subcutaneous co-injection model with 10(8) CFU of USA 300 as a challenge inoculum, DNAC-1 alone or DNAC-1 with sub-MIC oxacillin resulted in a 6-log reduction in bacterial load and decreased abscess formation as compared to the untreated control. We propose that DNAC-1, by exerting a bimodal effect on the cell membrane and the cell wall, is a viable candidate for development of combination therapy against many common bacterial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    No preview · Article · Jan 2015 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ΔgraS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.
    Full-text · Article · Oct 2014 · Infection and Immunity
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    Niles P Donegan · Jonathan S Marvin · Ambrose L Cheung
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    ABSTRACT: Staphylococcus aureus responds to changing extracellular environments in part by adjusting its proteome through alterations of transcriptional priorities and selective degradation of the preexisting pool of proteins. In Bacillus subtilis, the proteolytic adaptor protein MecA has been shown to play a role in assisting with the proteolytic degradation of proteins involved in competence and the oxidative stress response. However, the targets of TrfA, the MecA homolog in S. aureus, have not been well characterized. In this work, we investigated how TrfA assists chaperones and proteases to regulate the proteolysis of several classes of proteins in S. aureus. By fusing the last 3 amino acids of the SsrA degradation tag to Venus, a rapidly folding yellow fluorescent protein, we obtained both fluorescence-based and Western blot assay-based evidence that TrfA and ClpCP are the adaptor and protease, respectively, responsible for the degradation of the SsrA-tagged protein in S. aureus. Notably, the impact of TrfA on degradation was most prominent during late log phase and early stationary phase, due in part to a combination of transcriptional regulation and proteolytic degradation of TrfA by ClpCP. We also characterized the temporal transcriptional regulation governing TrfA activity, wherein Spx, a redox-sensitive transcriptional regulator degraded by ClpXP, activates trfA transcription while repressing its own promoter. Finally, the scope of TrfA-mediated proteolysis was expanded by identifying TrfA as the adaptor that works with ClpCP to degrade antitoxins in S. aureus. Together, these results indicate that the adaptor TrfA adds temporal nuance to protein degradation by ClpCP in S. aureus.
    Preview · Article · Sep 2014 · Journal of Bacteriology
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    Samin Kim · Dindo Reyes · Marie Beaume · Patrice Francois · Ambrose Cheung
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    ABSTRACT: High-throughput RNA sequencing technology has found the 5′ untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA.
    Preview · Article · Aug 2014 · Infection and Immunity
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    ABSTRACT: Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.
    Full-text · Article · Jun 2014 · PLoS Pathogens
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    ABSTRACT: The Staphylococcus aureus genome contains three toxin–antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE.
    Full-text · Article · Apr 2014 · Nucleic Acids Research
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    ABSTRACT: Background. Staphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation. Methods. Two healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub–minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model. Results. All sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model. Conclusions. These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation.
    Full-text · Article · Jan 2014 · The Journal of Infectious Diseases
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    ABSTRACT: In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.
    Full-text · Article · Oct 2013 · Infection and immunity
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    ABSTRACT: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) frequently causes skin and soft-tissue infections, including impetigo, cellulitis, folliculitis, and infected wounds and ulcers. Uncomplicated CA-MRSA skin infections are typically managed in an outpatient setting with oral and topical antibiotics and/or incision and drainage whereas complicated skin infections often require hospitalization, intravenous antibiotics, and sometimes surgery. The aim of this study was to develop a mouse model of CA-MRSA wound infection to compare the efficacy of commonly used systemic and topical antibiotics. A bioluminescent USA300 CA-MRSA strain was inoculated into full-thickness scalpel wounds on the backs of mice and digital photography/image analysis and in vivo bioluminescence imaging were used to measure wound healing and the bacterial burden. Subcutaneous vancomycin, daptomycin and linezolid similarly reduced the lesion sizes and bacterial burden. Oral linezolid, clindamycin and doxycycline all decreased the lesion sizes and bacterial burden. Oral trimethoprim/sulfamethoxazole decreased the bacterial burden but did not decrease the lesion size. Topical mupirocin and retapamulin ointments both reduced the bacterial burden. However, the petrolatum vehicle ointment for retapamulin, but not the polyethylene glycol vehicle ointment for mupirocin, promoted wound healing and initially increased the bacterial burden. Finally, in type 2 diabetic mice, subcutaneous linezolid and daptomycin had the most rapid therapeutic effect compared with vancomycin. Taken together, this mouse model of CA-MRSA wound infection, which utilizes in vivo bioluminescence imaging to monitor the bacterial burden, represents an alternative method to evaluate the preclinical in vivo efficacy of systemic and topical antimicrobial agents.
    Full-text · Article · Dec 2012 · Antimicrobial Agents and Chemotherapy
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    Dataset: Figure S12
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    ABSTRACT: Immunoblotting for detection of pro-IL-1β and cleaved IL-1β. Neutrophils from mouse bone marrow were purified using anti-Ly6G MACS bead separation (Miltenyi Biotec) were infected with live S. aureus (SH1000) or MRSA (USA300 LAC strain) (MOI bacteria∶neutrophils of 5∶1) for a total culture time of 6 hrs and gentamicin was added at 60 min from the start of the infection to prevent bacterial overgrowth. Pro-IL-1β protein (35 kDa) and cleaved IL-1β protein (17 kDa) was detected by immunoblot of cell lysates using a polyclonal antibody against IL-1β. Cleaved IL-1β protein was only detected in S. aureus- or MRSA-infected neutrophils but not in uninfected neutrophils. (TIF)
    Preview · Dataset · Nov 2012
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    Dataset: Figure S4
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    ABSTRACT: Additional representative confocal images of IL-1β expression in neutrophils and monocytes and isotype controls. pIL1-DsRed mice were infected intradermally with S. aureus and lesional skin specimens were collected at 4 and 24 hrs. Representative photomicrographs of sections labeled with anti-DsRed (IL-1β, red) and anti-MOMA2 (monocytes/macrophages, green) (A) or anti-7/4 (neutrophils, green) (C) and sections analyzed by confocal microscopy. Cells expressing both markers appear yellow (merge). High (left) and low (right) magnification images are shown (Scale bars = 50 µm). Dotted line = dermoepidermal junction. (B, D) Representative photomicrographs of sections labeled with isotype control antibodies. Data are representative from 4 mice per group. (TIF)
    Preview · Dataset · Nov 2012
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    Dataset: Figure S16
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    ABSTRACT: Cellular composition in the S. aureus-infected skin at day 10 after infection. TLR2-, NOD2-, FPR1-, and IL-1β-deficient mice as well as wt mice were inoculated intradermally with S. aureus and lesional skin specimens were collected at 10 days after infection. Representative photomicrographs of sections labeled with anti-7/4 (neutrophils) or anti-MOMA2 (monocytes/macrophages) or isotype control mAb (immunoperoxidase method) of frozen sections lesional skin at 10 days after skin inoculation with S. aureus (Scale bars = 100 µm). Data is representative of 3 mice per group. The cellular composition of 7/4+ neutrophils and MOMA2+ monocytes/macrophages on day 10 in TLR2-, NOD2-, FPR1-deficient mice after infection was similar to the cellular composition in wt mice whereas IL-1β-deficient mice had a paucity of 7/4+ neutrophils at this time point. (TIF)
    Preview · Dataset · Nov 2012
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    Dataset: Figure S17
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    ABSTRACT: Localization of EGFP in neutrophils from LysEGFP mice. (A) Neutrophils from LysEGFP mice labeled with Hoescht 33342 counterstain, mounted on microscope slides and imaged with an Olympus BX61 fluorescence microscope (100× objective). Green = EGFP and Blue = nucleus. Scale bar = 10 µm. (B) Mouse neutrophils enriched from bone marrow of LysEGFP mice were left unstimulated or stimulated with fMLF or PMA and EGFP fluorescent signals and CD11b expression (as an positive marker for neutrophil activation) were evaluated using flow cytometry. Neutrophils were first gated on forward and side scatter and mature neutrophils were then identified by high Ly6G+ expression. Mean fluorescence intensity (MFI) of EGFP and CD11b on neutrophils is indicated for unstimulated and stimulated neutrophils. Data are representative from 3 different LysEGFP mice. (TIF)
    Preview · Dataset · Nov 2012
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    Dataset: Figure S13
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    ABSTRACT: Purity of mouse neutrophils for the in vitro infection experiments in Fig. 6. (A) The purity of the of mouse neutrophils obtained from bone marrow cells using anti-Ly6G MACS bead separation [Miltenyi Biotec]) for the in vitro infection experiments in Fig. 6 was determined by labeling the cells with mAbs specific for Ly6G (clone 1A8) and CD11b (clone M1/70) and by flow cytometry analysis. Plots are representative of mouse neutrophil purity from 3 different experiments. There were 99.1% Ly6G+ CD11bhigh neutrophils. There were very few Ly6G− CD11blow monocytes (0.1%) and the remaining cells (0.6%) were Ly6G− CD11b− cells. (TIF)
    Preview · Dataset · Nov 2012
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    Dataset: Figure S3
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    ABSTRACT: Other cell types that produce IL-1β at early time points after S. aureus skin infection. pIL1-DsRed mice were infected intradermally with S. aureus and lesional skin specimens were collected at 4 hrs. Representative photomicrographs of sections labeled with anti-DsRed (IL-1β, red) and anti-CD45 (pan-leukocytes, green) (A) or anti-MHC II (antigen presenting cells, green) (B) and sections analyzed by confocal microscopy. Cells expressing both markers appear yellow (merge). High (left) and low (right) magnification images are shown (Scale bars = 50 mm). Dotted line = dermoepidermal junction. (C) Quantification of co-localization of IL-1β-DsRed fluorescence with CD45+ leukocytes or MHCII+ antigen presenting cells using the Manders' coefficient for a value range of 0 to 1 in which 0 = no pixels co-localize and 1 = all pixels co-localize. Data are representative from 4 mice per group. (TIF)
    Preview · Dataset · Nov 2012
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    Dataset: Figure S11
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    ABSTRACT: Viability of mouse neutrophils for the in vitro infection experiments in Fig. 6C and D. Neutrophils from mouse bone marrow were infected with live S. aureus (SH1000) or MRSA (USA300 LAC strain) (MOI bacteria∶neutrophils of 5∶1) for a total culture time of 6 hrs and gentamicin was added at 60 min from the start of the infection to prevent bacterial overgrowth. (A, B) Cell viability of the neutrophils infected with (A) S. aureus or (B) MRSA in the presence or absence of an NLRP3-inhibitor (glibenclamide), a caspase-1 inhibitor (Z-YVAD-FMK) or anti-staphylococcal α-toxin antibodies was measured using a viability assay kit (Promega, Madison, WI). Data presented as the percent viability (mean ± SEM) compared with the viability of uninfected neutrophils. Data are from 3 mice per group. (TIF)
    Preview · Dataset · Nov 2012

Publication Stats

8k Citations
664.12 Total Impact Points

Institutions

  • 2003-2015
    • Dartmouth College
      • Department of Microbiology and Immunology
      Hanover, New Hampshire, United States
  • 2000-2015
    • Geisel School of Medicine at Dartmouth
      • Department of Microbiology and Immunology
      Hanover, New Hampshire, United States
  • 2010
    • Cornell University
      Ithaca, New York, United States
  • 2007-2010
    • Dartmouth–Hitchcock Medical Center
      Lebanon, New Hampshire, United States
    • University of Tuebingen
      • Institute of Medical Microbiology and Hygiene
      Tübingen, Baden-Wuerttemberg, Germany
    • University of Arkansas for Medical Sciences
      • Department of Microbiology and Immunology
      Little Rock, Arkansas, United States
  • 2003-2010
    • University of California, Los Angeles
      • • Division of Dermatology
      • • Department of Medicine
      • • Division of Infectious Diseases
      Los Angeles, CA, United States
  • 2006-2008
    • University of South Dakota
      • Division of Basic Biomedical Sciences
      Vermillion, South Dakota, United States
    • Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
      • Department of Medicine
      Torrance, California, United States
  • 2005
    • University CEU Cardenal Herrera
      Valenza, Valencia, Spain
  • 2003-2005
    • Utrecht University
      • Division of Microbiology
      Utrecht, Utrecht, Netherlands
  • 2001
    • Saratov State Medical University
      Saratow, Saratov, Russia
  • 1988-2000
    • The Rockefeller University
      • Laboratory of Bacterial Pathogenesis and Immunology
      New York City, New York, United States
  • 1989-1995
    • Harbor-UCLA Medical Center
      • Department of Pediatrics
      Torrance, California, United States