A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

New Noncovalent Inhibitors of Penicillin-Binding Proteins from Penicillin-Resistant Bacteria

BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for beta-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.Eur-Intafa

In vitro rumen fermentation of diets with different types of condensed tannins derived from sainfoin (Onobrychis viciifolia Scop.) pellets and hazelnut (Corylus avellana L.) pericarps

The aim of this study was to evaluate the in vitro rumen fermentation parameters of diets including pellets of sainfoin pellets and/or hazelnut pericarps, which are two plant resources that containing different types of condensed tannins (CT) with contrasted structures, using a batch culture system during for 24 h. The treatments were a basal diet (control), the basal diet + pellets of dehydrated sainfoin (PS), the basal diet + freeze-dried hazelnut pericarps (HP), and the basal diet + PS + HP. The diets were adjusted to be isotannic (20 g/kg dry matter (DM), except for the control) and isoproteic (132 g/kg DM). Total gas and methane (CH4) productions were measured after 3.5 h and 24 h of incubation. At the end of incubation, pH, in vitro DM degradability (IVDMD) and the concentration of fermentation end-products in the medium were also measured. The CT structures of CT infrom PS and HP were very different: as PS showed a PD-dominant profilehad mostly prodelphinidins and HP showed a PC-dominant profile mostly procyanidins. After 24 h of incubation, the total gas and methane productions and IVDMD were greater for the basal diet than for the diet + HP and the diet + PS (P<0.05). The CH4 production increased significantly with the diet + HP in the presence of PEG, a compound CT-inactivating CTcompound (P<0.001), and tended to increase for the diet + PS (P<0.1). The volatile fatty acids (VFA) net productions were globally similar among treatments, while the NH3 concentration was lower for the diet + PS (with a significant PEG effect) than for the diets including HP, and was the highest for the basal diet. It was concluded that the inclusion of PS and HP in a basal diet results in lower rumen fermentability and that but their CT decreased CH4 production and protein degradability;, PS being were more efficient effective than HP for the latterreducing protein degradability

A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

Compensatory Evolution of pbp Mutations Restores the Fitness Cost Imposed by β-Lactam Resistance in Streptococcus pneumoniae

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae

Molecular Dynamics Simulation of the Complex PBP-2x with Drug Cefuroxime to Explore the Drug Resistance Mechanism of Streptococcus suis R61

Drug resistance of Streptococcus suis strains is a worldwide problem for both humans and pigs. Previous studies have noted that penicillin-binding protein (PBPs) mutation is one important cause of β-lactam antibiotic resistance. In this study, we used the molecular dynamics (MD) method to study the interaction differences between cefuroxime (CES) and PBP2x within two newly sequenced Streptococcus suis: drug-sensitive strain A7, and drug-resistant strain R61. The MM-PBSA results proved that the drug bound much more tightly to PBP2x in A7 (PBP2x-A7) than to PBP2x in R61 (PBP2x-R61). This is consistent with the evidently different resistances of the two strains to cefuroxime. Hydrogen bond analysis indicated that PBP2x-A7 preferred to bind to cefuroxime rather than to PBP2x-R61. Three stable hydrogen bonds were formed by the drug and PBP2x-A7, while only one unstable bond existed between the drug and PBP2x-R61. Further, we found that the Gln569, Tyr594, and Gly596 residues were the key mutant residues contributing directly to the different binding by pair wise energy decomposition comparison. By investigating the binding mode of the drug, we found that mutant residues Ala320, Gln553, and Thr595 indirectly affected the final phenomenon by topological conformation alteration. Above all, our results revealed some details about the specific interaction between the two PBP2x proteins and the drug cefuroxime. To some degree, this explained the drug resistance mechanism of Streptococcus suis and as a result could be helpful for further drug design or improvement

The fibrinogen-binding M1 protein reduces pharyngeal cell adherence and colonization phenotypes of M1T1 Group A Streptococcus

Background: The group A Streptococcus (GAS) M1 protein binds fibrinogen (Fg) to block phagocytosis and to form a proinflammatory complex. Results: M1 and Fg limit GAS adherence and invasion of pharyngeal keratinocytes in vitro. Conclusion: Protease SpeB modulates M1 expression and GAS host cell interactions differentially during the course of infection. Significance: M1 protein is shown to impede pharyngeal colonization in vivo. Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis ("strep throat") to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-Anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulentWTM1T1GASstrain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surfaceM1protein cleavage by theGAScysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-Associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreasedGAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities