Lack of a functional MntABC system renders <i>S</i>. <i>aureus</i> more sensitive to killing by methyl viologen and human neutrophils but not murine macrophages, unless <i>S</i>. <i>aureus</i> is pre-exposed to oxidative stress.

<p>(A, B) Survival of wild-type and <i>mntC</i> mutant strains within neutrophils harvested from heparin-treated human blood (A) and INF-γ-activated murine macrophages (B). Bacteria were either untreated or pre-exposed to 1 μM methyl viologen for 1 hour. Neutrophils (A) and macrophages (B) were lysed after 90 min and 24 hours of infection, respectively, to enumerate CFU. Bars represent the mean value of triplicate samples and error bars are standard deviation. <i>P-</i>values were determined using one-way ANOVA with multiple comparisons between samples via Tukey’s post-test.</p

The NrdEF proteins are highly induced in the <i>mntC</i> mutant when exposed to oxidative stress <i>in vitro</i>.

<p>(A) MA plot of the log₂ transformed normalized ratio of “light” <i>mntC</i> mutant relative to the wild-type strain against the log₂ transformed signal intensity arising from the “light” <i>mntC</i> mutant. (B) MA plot of the log₂ transformed normalized ratio of “heavy” <i>mntC</i> mutant relative to the wild-type strain against the log₂ transformed signal intensity arising from the “heavy” <i>mntC</i> mutant. Proteins with z-scores for ratios between the <i>mntC</i> mutant and wild-type strains in all replicates greater than 2 and smaller than −2 are shown in green and red dots, respectively. Proteins that were identified both in (A) and (B) that had the strongest mass spectrometric signals are shown in bold.</p

Recovery of the <i>mntC</i> mutant is delayed after phagocytosis.

<p>(A) Wild-type and <i>mntC</i> mutant strains that were harvested from murine macrophages were plated onto agar plates containing 5% defibrinated sheep blood. Images were taken after 16 and 40 hours of incubation at 37°C. (B, C) Growth after phagocytosis by macrophages (B) or neutrophils (C) was measured by BacTiter-Glo (Promega) 22 hours after recovery from phagocytic cells and inoculation into manganese-restricted media. Data are mean values of triplicate samples with standard deviation.</p

The <i>nrdEF</i> genes are highly upregulated in the <i>mntC</i> mutant after exposure to murine macrophages and human neutrophils <i>ex vivo</i>.

<p>Expression of genes involved in oxidative stress response (filled bars) and DNA repair (striped bars) in the <i>mntC</i> mutant strain relative to the wild-type strain that were phagocytosed by murine macrophages for 2 hours (A) or human neutrophils for 45 minutes (B). Expression levels were determined via qRT-PCR. Bars represent the mean value of triplicate samples and error bars are standard deviation. <i>P</i>-values (ns = non-significant, * = <0.05, *** = <0.001, **** = <0.0001) were determined using student’s t-test.</p

Genes involved in oxidative stress response and DNA repair are highly upregulated in the <i>mntC</i> mutant after exposure to a sub-lethal concentration of methyl viologen <i>in vitro</i>.

<p>Expression of genes involved in oxidative stress response (A), DNA repair (B) and the <i>srtA</i> gene (C) was determined via qRT-PCR in wild-type (filled bars) and <i>mntC</i> (open bars) cells grown in RPMI-H media and treated with or without 1 μM methyl viologen (MV) for 1 hour. Bars represent the mean value of triplicate samples and error bars are standard deviation. <i>P</i>-values (* = <0.05, *** = <0.001, **** = <0.0001) were determined using student’s t-test.</p

The <i>Staphylococcus aureus</i> ABC-Type Manganese Transporter MntABC Is Critical for Reinitiation of Bacterial Replication Following Exposure to Phagocytic Oxidative Burst

<div><p>Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for <i>Staphylococcus aureus</i>, a major human pathogen. Here, we show that a <i>mntC</i> mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the <i>mntC</i> mutant was pre-exposed to oxidative stress. Notably, the <i>mntC</i> mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the <i>mntC</i> mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the <i>mntC</i> mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for <i>S</i>. <i>aureus</i> viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for <i>S</i>. <i>aureus</i> pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host.</p></div

MntABC is important for efficient proliferation of <i>S</i>. <i>aureus</i> after oxidative stress.

<p>(A) Percentage of wild-type and <i>mntC</i> mutant cells that lost CFSE fluorescence when inoculated into TSB media after recovery from IFN-γ activated murine macrophages with and without DPI treatment. Bacteria were identified by cell size and further differentiated from macrophage debris by co-staining with an anti-<i>S</i>. <i>aureus</i> antibody. Data are median values from triplicate samples with standard deviation. (B) Wild-type and <i>mntC</i> mutant cells were treated with 1 μM methyl viologen (MV) for 1 hour. Growth after treatment was measured at indicated time points for 12 hours post exposure, via an increase in BacTiter-Glo luminescence values. Once bacterial cultures reached stationary phase growth, growth was no longer measured. Data are mean values of triplicated samples with standard deviation.</p

Recognition of SdgB-dependent epitope by human antibodies.

<p>(<b>A</b>) Four different human IgG preparations were reacted with plate-bound CWP from WT or Δ<i>sdgB</i> USA300 by ELISA. To calculate the specific anti-staphylococcal IgG content, data were normalized using a calibration curve with known IgG concentrations of a mAb against peptidoglycan, which has the same reactivity with both USA300 strains by ELISA. Data are expressed as µg/mL of anti-staphylococcal IgG in the serum. The reduction in reactivity observed for CWP from Δ<i>sdgB</i> (red bars) as compared to wild-type CWP (black bars) reflects IgG specific for SdgB-dependent epitopes. Asterisks indicate significant differences (p < 0.05) from WT CWP. (<b>B</b>) CWP from WT, Δ<i>sdgA</i>, or Δ<i>sdgB</i>, Δ<i>sdgAΔsdgB</i> USA300 were immunoblotted with rF1 and three additional human mAbs (SD2, SD3, and SD4) from different patients. All four mAbs showed similar epitope specificity.</p

SdgB glycosylation protects SDR proteins from cleavage by human neutrophil-derived cathepsin G.

<p>(<b>A</b>) Live, in tact WT or Δ<i>sdgB</i> USA300 bacteria were incubated in the presence or absence of human neutrophil lysosomal extracts (NLE). Culture supernatants were immunoblotted with a mAb against the A-domain of ClfA (9E10) to detect cleaved ClfA fragments released from the bacteria. (<b>B</b>) Live, in tact WT or Δ<i>sdgB</i> cells were incubated in the presence or absence of lysosomal extracts from human THP1 cells or mouse RAW cells and culture supernatants were immunoblotted with anti-ClfA. (<b>C</b>) Live, intact WT or Δ<i>sdgB</i> cells were incubated with a panel of purified human neutrophil serine proteases, ie. neutrophil elastase (NE), cathepsin G (CatG), proteinase-3 (P3), and neutrophil serine protease-4 (NSP4). (<b>D</b>) <b>Δ</b><i>sdgB</i> cells were treated with human neutrophil lysosomal extract in the presence or absence of a biochemical inhibitor of cathepsin G. (<b>E</b>) WT or various Sdg-mutant strains were treated with purified human cathepsin G. (<b>B-E</b>) Culture supernatants were analyzed by immunoblotting as in (A) to detect released ClfA fragments. (<b>F</b>) Live bacteria of WT, <b>Δ</b><i>sdgB</i>, or Δ<i>sdgB</i> complemented with exogenous SdgB (p<i>sdgB</i>) were treated with purified human cathepsin G. Culture supernatants (Sup) or cell wall preparations (CWP) were immunoblotted with mAb against the A-domain of ClfA (S4675), SdrD (17H4), or IsdA (2D3). In addition to S4675, another mAb against the A-domain of ClfA (9E10) showed similar results (not shown). (<b>G</b>) Human cathepsin G inhibits adherence of glycosylation-deficient <i>S. aureus</i> to human fibrinogen. Live WT or Δ<i>sdgB</i> USA300 bacteria were pre-incubated with cathepsin G, and allowed to adhere to fibrinogen-precoated plates. Bacterial adhesion was quantified by measuring the amount of bacterial ATP associated with the plates.</p

SdgB and SdgA sequentially modify the SDR-domain with GlcNAc moieties.

<p>(<b>A</b>) SdgB generates rF1 epitopes on SDR protein. A combination of MBP-SDR-His and SdgA or SdgB was co-expressed in <i>E. coli</i>, and cell lysates were immunoblotted with mAb rF1, or with mAb against unmodified SDR (9G4) or anti-His. (<b>B</b>) Cell-free system to reconstitute SDR glycosylation using purified components. Recombinant MBP-SDR-His was incubated with purified SdgA or SdgB, and in the presence or absence of UDP-GlcNAc; rF1 reactivity was induced only in the presence of SdgB and UDP-GlcNAc. (<b>C</b>) Final model for step-wise glycosylation of SDR proteins by SdgA and SdgB. First, SdgB appends GlcNAc moieties onto the SD-region on SDR proteins, followed by additional GlcNAc modification by SdgA. The epitope for mAb rF1 includes the SdgB-dependent GlcNAc moieties. (<b>D</b>) Mass spectrometry analysis to identify the SDR-sugar moieties using purified MBP-SDR-His expressed in <i>E. coli</i>. (Upper panel) Deconvoluted mass spectrum of purified MBP-SDR-His protein, showing the expected intact mass of 58719 Da. (Middle panel) MBP-SDR-His protein was treated with purified SdgB enzyme in the presence of UDP-GlcNAc for 2 h at 37°C. After incubation, the mass of the MBP-SDR-His protein showed several peaks, each peak being separated from the others by the mass of additional GlcNAc residues. (Bottom panel) The above-mentioned reaction mixture of MBP-SDR-His and SdgB (middle panel) was additionally treated with purified SdgA enzyme. After further incubation for 2 hrs at 37°C, up to an additional 47 GlcNAc groups were found to be added. Thus, most of the serines in the DSD motifs in MBP-SD can be modified with these disaccharide sugar moieties.</p