Extracellular polysaccharides impair pro-inflammatory response.

<p>(A) <i>S</i>. <i>aureus</i> strain 15981 was grown in TSB ON at 37°C, 130 rpm. The overnight culture was diluted 1/20 in fresh TSB and grown for 2h 30 min. (B) DCs were stimulated with UV-inactivated strain 15981 or its isogenic <i>ica</i> mutant in exponential or stationary phase. After 20h IL-12p70, TNF-α and IL-10 were detected in the supernatant by ELISA. Data represents one experiment out of three. (C) Phagocytosis was inhibited by treatment with cytochalasin D (0.5 μg/ml) for 1h and subsequently DCs were stimulated and analyzed as in B. Means and SD are based on technical replicates. Data show one out of three experiments. (error bars, SD; ns, no significant difference; *, ** and ***p<0.05).</p

<i>S</i>. <i>aureus</i> induces <i>inf-β</i> expression but does not induce increased macropinocytosis.

<p>(A) DCs were stimulated with UV-killed 8325–5 in exponential or stationary phase (MOI 6), <i>L</i>. <i>acidophilus</i> NCFM (10 μg/ml) or <i>E</i>. coli Nissle (10 μg/ml). Expression of i<i>fn-β</i> and i<i>l-10</i> was analyzed by qPCR and normalized to β-actin after 2, 4, 6 and 10h. Means and SD are based on technical replicates. Data represent one out of three experiments. (B) DCs were pre-treated with or without cytochalasin D (0.5 μg/ml) for 1h. Next, cells were added complete DC medium, LPS (1 μg/ml) or 15981 in exponential or stationary phase (MOI 2) for 1h together with FITC-coupled dextran particles (500 μg/ml). Uptake of dextran particles was measured by flow cytometry. Data are based on the count of 10,000 cells and double cells were excluded by FSC-A/FSC-H. Numbers indicate mean and standard deviations of the geometrical mean fluorescence intensity of three technical replicates (upper numbers represents the solid line of cells with no inhibitor, and the lower numbers represent dotted line of cells incubated with cytochalasin D).</p

Only intact <i>S</i>. <i>aureus</i> is able to fully mature dendritic cells.

<p>(A) Intact or lysostaphin lysed UV-inactivated 8325–4 (MOI 6) or LPS (1μg/ml) +/- lysostaphin were subjected to DCs. After 20h of stimulation, IL-12p70, TNF-α and IL-10 levels were detected in the supernatant by ELISA. (B) Mean fluorescence intensity (MFI) of CD40, CD80 and CD86 were detected by flow cytometry after maturation of DCs for 20h with intact or lysed 8325–4 in exponential or stationary phase. Means and SD are based on technical replicates. Data represent one experiment out of three. (C) As in B, combination of three experiments. (error bars, SD).</p

Phagocytosis and acidification is important for <i>S</i>. <i>aureus</i> induced IL-12 production.

<p>(A) DCs were pre-treated for 1h with 0.5 μg/ml cytochalasin D before stimulation with UV-inactivated 8325–4 in exponential or stationary phase. ELISA was used to detect the levels of IL-12p70 and IL-10 in the supernatant after 20h. Means and SD are based on technical replicates. Data represent one out of three experiments. (B) DCs were incubated with cytochalasin D (0.5 μg/ml) for 1h and subsequently stimulated wit PGN (10 μg/ml), LTA (10 μg/ml) or LPS (1 μg/ml). IL-12p70 and IL-10 were measured in the supernatant after 20h. Means and SD are based on technical replicates. Data represents one out of three experiments. (C) DCs pre-incubated with 0.5 μg/ml cytochalasin D were subsequently stimulated for 1h with AlexaFluor647 labeled <i>S</i>. <i>aureus</i> 15981 in exponential phase (MOI 2). The uptake was measured by flow cytometry. Numbers indicate the percentages of AlexaFluor647 positive cells. (D) Bafilomycin A1 (50nM) or chloroquine (10 μM) were added to the DCs for 1h. Subsequently, DCs were stimulated with LPS (1 μg/ml) or <i>S</i>. <i>aureus</i> 15981 in EP. After 20h IL-12p70 and IL-10 were measured in the supernatant. (E) DCs were pre-treated for 1h with bafilomycin A1 (50 nM) or chloroquine (10 μM) followed by 30 min of stimulation with AlexaFlour647 labeled <i>S</i>. <i>aureus</i> 15981 in EP (MOI 2). AlexaFlour647 positive cells were measured by flow cytometry. Data are based on 10,000 cell counts and double cells were excluded by gating FSC-A/FSC-H. (error bars, SD; ns, no significant difference; * p<0.05, ** p< 0.01 and ***p<0.001).</p

Exponentially growing S. aureus strains are strong IL-12 inducers.

<p>(A) DCs were stimulated with live <i>S</i>. <i>aureus</i> 15981 or 8325–4 in exponential or stationary phase with a MOI 10. Production of IL-12p70, TNF-α and IL-10 was detected in the supernatants by ELISA after 20h of stimulation. Data shown represents one representative experiment of three. Means and SD are based on technical replicates. (B) DCs were stimulated for 20h with UV-inactivated 8325–4 and 15981 in exponential or stationary phase with a MOI of 6. At least three experiments were performed. C) DC viability was analyzed by flow cytometry after 1h or 20h of stimulation with UV-inactivated strain 8325 in exponential (EP) or stationary (SP) phase, or 8% supernatant harvested from 8325–4 or RN6911 in EP (SEP) or SP (SSP). The amount of alive, apoptotic, dead or cells double positive for dead and apoptotic cells are shown as percentages based on counting 5000 cells.</p

D-Alanylation of Teichoic Acids and Loss of Poly-N-Acetyl Glucosamine in <i>Staphylococcus aureus</i> during Exponential Growth Phase Enhance IL-12 Production in Murine Dendritic Cells

<div><p><i>Staphylococcus aureus</i> is a major human pathogen that has evolved very efficient immune evading strategies leading to persistent colonization. During different stages of growth, <i>S</i>. <i>aureus</i> express various surface molecules, which may affect the immune stimulating properties, but very little is known about their role in immune stimulation and evasion. Depending on the growth phase, <i>S</i>. <i>aureus</i> may affect antigen presenting cells differently. Here, the impact of growth phases and the surface molecules lipoteichoic acid, peptidoglycan and poly-N-acetyl glucosamine on the induction of IL-12 imperative for an efficient clearance of <i>S</i>. <i>aureus</i> was studied in dendritic cells (DCs). Exponential phase (EP) <i>S</i>. <i>aureus</i> was superior to stationary phase (SP) bacteria in induction of IL-12, which required actin-mediated endocytosis and endosomal acidification. Moreover, addition of staphylococcal cell wall derived peptidoglycan to EP <i>S</i>. <i>aureus</i> stimulated cells increased bacterial uptake but abrogated IL-12 induction, while addition of lipoteichoic acid increased IL-12 production but had no effect on the bacterial uptake. Depletion of the capability to produce poly-N-acetyl glucosamine increased the IL-12 inducing activity of EP bacteria. Furthermore, the mutant <i>dltA</i> unable to produce D-alanylated teichoic acids failed to induce IL-12 but like peptidoglycan and the toll-like receptor (TLR) ligands LPS and Pam3CSK4 the mutant stimulated increased macropinocytosis. In conclusion, the IL-12 response by DCs against <i>S</i>. <i>aureus</i> is highly growth phase dependent, relies on cell wall D-alanylation, endocytosis and subsequent endosomal degradation, and is abrogated by receptor induced macropinocytosis.</p></div

D-alanylation prevents induction of macropinocytosis and is required for IL-12 production.

<p>(A) DCs were stimulated with SA113 or its isogenic <i>dltA</i> mutant in exponential or stationary phase. IL-12p70, TNF-α and IL-10 were measured in the supernatant after 20h. (B) Cytochalasin D (0.5 μg/ml) treated DCs were stimulated with SA113 and its isogenic <i>dltA</i> mutant in exponential phase and the levels of IL-12p70 were measured in the supernatant after 20h. (C) DCs were treated with cytochalasin D for 1h and were subsequently subjected for 30 min to <i>S</i>. <i>aureus</i> SA113 or its isogenic <i>dltA</i> mutant both in EP. Next the cells were chased with FITC-dextran for 10 min, and the uptake of dextran was measured by flow cytometry and plotted against FSC. (D) Combination of three technical replicates of C. Means and SD are based on technical replicates. Data represents one experiment out of three.</p

Staphylococcal PGN, but not LTA induce macropinocytosis and abrogate IL-12 production.

<p>(A) DCs was stimulated with, LPS, Pam2CSK4, or Pam3CSK4 (1 μg/ml) or PGN or LTA (10 μg/ml) for 30 min with or without pre-treatment with cytochalasin D. Then, cells were chased with FITC-coupled dextran (500 μg/ml) for 10 min. Dextran uptake was measured by flow cytometry by counting 10,000 cells. Numbers indicate the percentages of FITC-positive cells. (B) DCs were pre-stimulated with PGN or LTA (1 and 10 μg/ml), LPS, Pam2CSK4, Pam3CSK4 (1 μg/ml), or <i>S</i>. <i>aureus</i> SA113 in stationary phase (MOI for: High 3; Intermediate, 2; Low, 1) for 30 min. Next, the DCs were subjected to <i>S</i>. <i>aureus</i> SA113 in exponential phase (MOI 6), (Black bars), or DC’s were stimulated with the ligands alone (white bars). After 20h, IL-12 and IL-10 levels were measured in the supernatant by ELISA. (C) DCs was pre-treated with cytochalasin D for 1h and subsequently stimulated with DC complete medium, PGN or LTA (10 μg/ml) for 30 min. Next, the cells were stimulated with AlexaFluor647 labeled <i>S</i>. <i>aureus</i> 15981 in exponential phase for 30 min, and chased with FITC-dextran for 10 min. Dot plots are based on 10,000 cells counted on FACS CantoII and single cell gating by the use of FSC-A/FSC-H. Means and SD are based on technical replicates. Data represent one out of three experiments.</p

Table_1_Intrinsic tet(L) sub-class in Bacillus velezensis and Bacillus amyloliquefaciens is associated with a reduced susceptibility toward tetracycline.pdf

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes.</p

Table_1.pdf

<p>Incomplete killing of bacterial pathogens by antibiotics is an underlying cause of treatment failure and accompanying complications. Among those avoiding chemotherapy are persisters being individual cells in a population that for extended periods of time survive high antibiotic concentrations proposedly by being in a quiescent state refractory to antibiotic killing. While investigating the human pathogen Staphylococcus aureus and the influence of growth phase on persister formation, we noted that spent supernatants of stationary phase cultures of S. aureus or S. epidermidis, but not of distantly related bacteria, significantly reduced the persister cell frequency upon ciprofloxacin challenge when added to exponentially growing and stationary phase S. aureus cells. Curiously, the persister reducing activity of S. aureus supernatants was also effective against persisters formed by either S. carnosus or Listeria monocytogenes. The persister reducing component, which resisted heat but not proteases and was produced in the late growth phase in an agr quorum-sensing dependent manner, was identified to be the phenol-soluble modulin (PSM) toxins. S. aureus express several PSMs, each with distinct cytolytic and antimicrobial properties; however, the persister reducing activity was specifically linked to synthesis of the PSMα family. Correspondingly, a high-persister phenotype of a PSMα mutant was observed upon fluoroquinolone or aminoglycoside challenge, demonstrating that the persister reducing activity of PSMs can be endogenously synthesized or extrinsically added. Given that PSMs have been associated with lytic activity against bacterial membranes we propose that PSM toxins increase the susceptibility of persister cells to killing by intracellularly acting antibiotics and that chronic and re-occurring infections with quorum sensing, agr negative mutants may be difficult to treat with antibiotics because of persister cells formed in the absence of PSM toxins.</p