PBP4: A New Perspective on Staphylococcus aureus β-Lactam Resistance.

β-lactam antibiotics are excellent drugs for treatment of staphylococcal infections, due to their superior efficacy and safety compared to other drugs. Effectiveness of β-lactams is severely compromised due to resistance, which is widespread among clinical strains of Staphylococcus aureus. β-lactams inhibit bacterial cells by binding to penicillin binding proteins (PBPs), which perform the penultimate steps of bacterial cell wall synthesis. Among PBPs of S. aureus, PBP2a has received the most attention for the past several decades due to its preeminent role in conferring both high-level and broad-spectrum resistance to the entire class of β-lactam drugs. Studies on PBP2a have thus unraveled incredible details of its mechanism of action. We have recently identified that an uncanonical, low molecular weight PBP of S. aureus, PBP4, can also provide high-level and broad-spectrum resistance to the entire class of β-lactam drugs at a level similar to that of PBP2a. The role of PBP4 has typically been considered not so important for β-lactam resistance of S. aureus, and as a result its mode of action remains largely unknown. In this article, we review our current knowledge of PBP4 mediating β-lactam resistance in S. aureus

Fibronectin binding protein B binds to loricrin and promotes corneocyte adhesion by Staphylococcus aureus.

Colonisation of humans by Staphylococcus aureus is a major risk factor for infection, yet the bacterial and host factors involved are not fully understood. The first step during skin colonisation is adhesion of the bacteria to corneocytes in the stratum corneum where the cornified envelope protein loricrin is the main ligand for S. aureus. Here we report a novel loricrin-binding protein of S. aureus, the cell wall-anchored fibronectin binding protein B (FnBPB). Single-molecule force spectroscopy revealed both weak and ultra-strong (2 nN) binding of FnBPB to loricrin and that mechanical stress enhanced the strength of these bonds. Treatment with a peptide derived from fibrinogen decreased the frequency of strong interactions, suggesting that both ligands bind to overlapping sites within FnBPB. Finally, we show that FnBPB promotes adhesion to human corneocytes by binding strongly to loricrin, highlighting the relevance of this interaction to skin colonisation

Interaction of the Staphylococcus aureus Surface Protein FnBPB with Corneodesmosin Involves Two Distinct, Extremely Strong Bonds

Attachment of Staphylococcus aureus to human skin corneocyte cells plays a critical role in exacerbating the severity of atopic dermatitis (AD). Pathogen-skin adhesion is mediated by bacterial cell-surface proteins called adhesins, including fibronectin-binding protein B (FnBPB). FnBPB binds to corneodesmosin (CDSN), a glycoprotein exposed on AD patient corneocytes. Using single-molecule experiments, we demonstrate that CDSN binding by FnBPB relies on a sophisticated two-site mechanism. Both sites form extremely strong bonds with binding forces of ∼1 and ∼2.5 nN albeit with faster dissociation rates than those reported for homologues of the adhesin. This previously unidentified two-binding site interaction in FnBPB illustrates its remarkable variety of adhesive functions and is of biological significance as the high strength and short bond lifetime will favor efficient skin colonization by the pathogen

PBP4 Mediates β-Lactam Resistance by Altered Function

Penicillin binding protein 4 (PBP4) can provide high-level β-lactam resistance in Staphylococcus aureus A series of missense and promoter mutations associated with pbp4 were detected in strains that displayed high-level resistance. We show here that the missense mutations facilitate the β-lactam resistance mediated by PBP4 and the promoter mutations lead to overexpression of pbp4 Our results also suggest a cooperative interplay among PBPs for β-lactam resistance

PBP4 Mediates β-Lactam Resistance by Altered Function

Penicillin binding protein 4 (PBP4) can provide high-level β-lactam resistance in Staphylococcus aureus A series of missense and promoter mutations associated with pbp4 were detected in strains that displayed high-level resistance. We show here that the missense mutations facilitate the β-lactam resistance mediated by PBP4 and the promoter mutations lead to overexpression of pbp4 Our results also suggest a cooperative interplay among PBPs for β-lactam resistance

Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes

Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes

Computational design of cyclic peptide inhibitors of a bacterial membrane lipoprotein peptidase

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.</p

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance. </p