CcpA Affects Infectivity of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e in a Hyperglycemic Environment

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14), and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤10 mM) were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment

Nck1-Nck2-deficient fibroblasts show diminished CEACAM3-mediated uptake of bacteria.

<p>(A) Nck1/Nck2-deficient mouse fibroblasts (Nck1−/− Nck2−/−) and Nck1 and Nck2-expressing fibroblasts (Nck1+/− Nck2+/−) were transfected with a plasmid encoding GFP-tagged CEACAM3 and infected with biotinylated, Pacific blue-labelled Opa_CEA-expressing <i>N. gonorrhoeae</i>. 60 minutes after infection, extracellular bacteria were stained with streptavidin-Alexafluor647 allowing detection of intracellular bacteria by their selective Pacific blue staining (arrowheads). (B) Total cell-associated and intracellular bacteria were quantified in cells stained as in (A). Bars represent the mean values ± S.E.M of intracellular bacteria (n = 30 cells). (C) Whole cell lysates (WCL) of cells used in (A) were probed with a monoclonal anti-Nck1/Nck2 antibody (top panel) or a monoclonal anti-tubulin antibody (lower panel).</p

The SH2 domain of Nck1 associates with the cytoplasmatic domain of CEACAM3.

<p>(A) 293 cells were transfected with a plasmid encoding GFP- and HA-tagged CEACAM3 (CEACAM3-GFP) or GFP alone and co-transfected or not with v-Src. CEACAM3 was immunoprecipitated (IP) from lysates and the phosphorylation status was assayed by Western blotting with monoclonal antibodies against phospho-tyrosine (pTyr)(upper panel). The expression of the proteins was verified by probing whole cell lysates (WCL) with antibodies against GFP, vSrc, or tubulin (lower panels). (B) The indicated GST-SH2 domains or GST alone were used in pulldown assays with lysates from cells co-expressing CEACAM3-GFP and vSrc as in (A). Precipitates were analysed by Western blot with monoclonal HA-tag antibody to detect SH2 domain-bound CEACAM3 (upper panel). Input shows CEACAM3 expression in 1/10 of the lysate used for pulldown. Membranes were stripped and re-probed with GST antibodies to detect the GST fusion proteins (lower panel). (C) 293 cells were transfected with GFP- and HA-tagged wildtype CEACAM3 (CEACAM3 WT), a CEACAM3 mutant with a deletion of the complete cytoplasmatic domain (CEACAM3 ΔCT), or an empty vector (pcDNA) and co-transfected with v-Src. The expression of the receptor proteins was verified by probing whole cell lysates (WCL) with an antibody against the HA-tag. (D) Lysates from (C) were used in pulldown assays with GST, GST-Grb2-SH2, or GST-Nck1-SH2. Precipitates were analysed by Western blotting as in (B). (E) Lysates from cells co-expressing CEACAM3-GFP and vSrc were precipitated with GST, GST-Nck1-SH2, or a mutant of the Nck1 SH2 domain that is unable to bind phospho-tyrosine (GST-Nck1-R308K). Precipitates were analysed as in (B).</p

Nck1 and Nck2-deficient cells lack CEACAM3-initiated lamellipodia.

<p>Nck1 and Nck2-expressing fibroblasts (Nck1+/−/Nck2+/−, upper panel) and Nck1/Nck2-deficient mouse fibroblasts (Nck1−/−/Nck2−/−, lower panel) were transfected with a plasmid encoding GFP-tagged CEACAM3 and infected with Pacific blue-labelled Opa_CEA-expressing <i>N. gonorrhoeae</i> for 30 minutes. Fixed samples were first analyzed by confocal laser-scanning microscopy and the identical cells were imaged in a second step by scanning electron microscopy (SEM). Confocal images (left) showing CEACAM3-transfected cells (green) with bound gonococci (blue) were overlayed with the SEM image of the same cell (middle) allowing identification of subcellular areas of CEACAM3-mediated bacterial engulfment (white boxes). Higher resolution SEM images of the boxed areas demonstrate extensive lamellipodial protrusions in Nck-expressing cells (small arrows), whereas CEACAM3-engagement in Nck-deficient cells does not elicit lamellipodia in the vicinity of bound bacteria (arrowhead).</p

Association of Nck1 and CEACAM3 occurs in intact cells upon <i>N. gonorrhoeae</i> infection.

<p>(A) 293 cells were transfected with plasmids encoding mKate-tagged CEACAM3 WT (red) and co-transfected with either GFP, GFP-Nck1-SH2, or GFP-Nck1-R308K-SH2 (green). Cells were infected for 30 minutes with PacificBlue-labelled Opa_CEA-expressing <i>N. gonorrhoeae</i> (blue). Fixed samples were analysed by confocal laser scanning microscopy. Bacteria cluster CEACAM3 and induce recruitment of Nck1-SH2 (arrowhead), but not GFP or Nck1-R308K-SH2 (small arrows). Bars indicate 20 µm. (B) 293 cells were transfected with mKate-tagged CEACAM3 WT (red) and co-transfected with full-length GFP-Nck1 (GFP-Nck1; green). Samples were infected for 30 minutes with Opa_CEA-expressing <i>N. gonorrhoeae</i> (blue) and, after fixation, bacteria were stained with polyclonal antibody and Cy5-coupled secondary reagents. Confocal laser scanning microscopy revealed that Nck was strongly enriched at sites of bacterial contact with CEACAM3 (arrowheads). Bars indicate 5 µm. (C) 293 cells were transfected with plasmids encoding vSrc together with pcDNA or HA-tagged CEACAM3 WT. Where indicated, cell were co-transfected with full-length myc-tagged Nck1. After lysis, CEACAM3 WT was immunoprecipitated (IP) with mAb against the HA-epitope. After Western blotting, precipitates were probed with mAb against myc-Nck1 (upper panel) and, after stripping of the membrane against the immunoprecipitated CEACAM3 WT with mAb against the HA-tag (lower panel). The immunoglobulin heavy (Ig-H) and light chain (Ig-L) of the precipitating antibody are indicated.</p

WAVE2 function is required during CEACAM3-mediated uptake of bacteria.

<p>(A) 293 cells were co-transfected with CEACAM3WT-GFP or GFP (green) together with myc-tagged WAVE2 or WAVE ΔVCA (red) and infected for 30 min with PacificBlue-labelled Opa_CEA-expressing <i>N. gonorrhoeae</i> (blue). After fixation, WAVE2 was stained with anti-myc antibodies and samples were analysed by confocal laser scanning microscopy. Recruitment of WAVE2 or WAVE ΔVCA to cell-bound bacteria (arrowheads) in the presence of CEACAM3 was further visualized by line profiles representing the relative fluorescence intensity values in the three detection channels (lower panels). (B) 293 cells were transfected with pcDNA or a plasmid encoding HA-tagged CEACAM3 WT and co-transfected with either GFP alone or GFP-WAVE2 ΔVCA. Cells were infected for 30 minutes with Opa_CEA-expressing <i>N. gonorrhoeae</i>. Parallel samples were analysed by bacterial adhesion assays (left panel) or gentamicin protection assays (middle panel). Bars represent mean values ± S.E.M of three independent experiments done in triplicate. Significance was tested using a paired, two-sided Student's t-test; ***, p<0.001. Expression of constructs was verified by Western blotting of whole cell lysates (WCL) with mAb against GFP, HA-tag, or tubulin as indicated (right panels).</p

Nck recruits the WAVE2 complex to the phosphorylated cytoplasmatic domain of CEACAM3.

<p>(A) 293 cells were transfected with CEACAM3 WT-GFP, CEACAM3 ΔCT-GFP or GFP alone (green) and co-transfected with myc-WAVE2 (red). Samples were infected for 30 minutes with PacificBlue-labelled Opa_CEA-expressing <i>N. gonorrhoeae</i> (blue), and, after fixation, myc-WAVE2 was detected using mAb myc and Cy5-goat-anti-mouse secondary antibody. Confocal laser scanning microscopy revealed the recruitment of WAVE2 to sites of bacteria-induced CEACAM3 WT clustering (arrowheads), whereas CEACAM3 ΔCT or GFP did not induce major relocation of WAVE2 (small arrows). Bars indicate 20 µm. (B) 293 cells were transfected with GFP-WAVE2, myc-Nck1, and vSrc as indicated. GFP-WAVE2 was immunoprecipitated (IP) with rabbit polyclonal GFP-antibody and precipitates (upper panels) as well as WCLs (lower panels) were analysed by Western blot with mAb against the myc-tag or against GFP. (C) 293 cells were co-transfected with GFP-WAVE2, myc-Nck1, CEACAM3 WT-HA and vSrc as indicated. WCLs were analysed by Western blotting with mAb against GFP, the myc-tag, or the HA-tag. (D) GFP-WAVE2 was immunoprecipitated (IP) from lysates generated in (C) and precipitates were analysed as in (C). The immunoglobulin light chain (Ig-L) of the precipitating antibody is indicated.</p

CcpA Affects Infectivity of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e in a Hyperglycemic Environment

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14), and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤10 mM) were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals