D-Alanylation of Lipoteichoic Acids Confers Resistance to Cationic Peptides in Group B Streptococcus by Increasing the Cell Wall Density

International audienceCationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistanc

The HTLV-1 gp21 fusion peptide inhibits antigen specific T-cell activation in-vitro and in mice.

The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait

Succination inactivates gasdermin D and blocks pyroptosis

Activated macrophages undergo a metabolic switch to aerobic glycolysis accumulating Krebs cycle intermediates that alter transcription of immune response genes. Here we extend these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against LPS shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis (EAE) by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics including DMF used to treat multiple sclerosis

Aggregation and membrane interaction of CAMPs with GBS.

a<p>Percentage of change in signal intensity relatively to the basal signal of the peptides.</p>b<p>Difference in the signal change between <i>dltA</i> strain to the WT strain.</p>c<p>Value is significantly different from that of the WT strain (P<0.05).</p><p>Data are means of triplicate measurements from two independent experiments.</p

Quantitative binding assay.

<p>The binding of cytochrome C (A) and NBD-labeled CAMPs (B) to WT (black) and <i>dltA</i> (gray) mutant strains was studied. Data are means ± SD of triplicate measurements from three independent experiments and are presented as the percentage of the maximum signal (peptide with no bacteria) ± SD.</p

Peptide designation and properties.

a<p>Underlined and bold amino acids are D-enantiomers. All linear peptides are amidated in their C-terminus.</p>b<p>Calculated by dividing the net charge by the total number of amino acids.</p>c<p>The peptides were eluted in 40 min using a linear gradient of acetonitrile (AcN) from 30 to 70% v/v in water containing TFA (0.05% v/v) on a C4 reverse analytical column.</p

Susceptibility of GBS strains to perforation by CAMPs.

<p>Peptide-dependent influx of the vital dye SYTOX green following 30 min exposure of GBS to magainin 2 (A), LL-37 (B), 5D-K₆L₉ (C) and K₅L₇ (D). Solid lines represent the WT strain whereas dashed lines represent the <i>dltA</i> muatnt. All readings were normalized by subtracting the basal fluorescence of the dye. Data are means ± SD of triplicate measurements.</p