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Gram-positive bacteria colonize mucosal tissues, withstanding large mechanical perturbations such as coughing, which generate shear forces that exceed the ability of non-covalent bonds to remain attached. To overcome these challenges, the pathogen Streptococcus pyogenes utilizes the protein Cpa, a pilus tip-end adhesin equipped with a Cys–Gln thioester bond. The reactivity of this bond towards host surface ligands enables covalent anchoring; however, colonization also requires cell migration and spreading over surfaces. The molecular mechanisms underlying these seemingly incompatible requirements remain unknown. Here we demonstrate a magnetic tweezers force spectroscopy assay that resolves the dynamics of the Cpa thioester bond under force. When folded at forces <6 pN, the Cpa thioester bond reacts reversibly with amine ligands, which are common in inflammation sites; however, mechanical unfolding and exposure to forces >6 pN block thioester reformation. We hypothesize that this folding-coupled reactivity switch (termed a smart covalent bond) could allow the adhesin to undergo binding and unbinding to surface ligands under low force and remain covalently attached under mechanical stress.Bacteria use thioester-bond-containing proteins to covalently bind to host surfaces and withstand large mechanical shocks. Now, thioester bond reactivity has been shown to be force-dependent: forces >35 pN inhibit bond cleavage by primary amine ligands, whereas forces <6 pN enable reversible reformation. This force-modulated thioester bond reactivity could potentially enable bacterial mobility and a route by which they optimize infection.

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions. Validating crosslinking-mass spectrometry workflows is hampered by the lack of a ground truth to assess the robustness of the crosslink identifications. Here, the authors present a synthetic library of crosslinked peptides, enabling unambiguous discrimination of correct and incorrect crosslink identifications.

Many bacterial pathogens have long, slender pili through which they adhere to host cells. The crystal structure of the major pilin subunit from the Gram-positive human pathogen Streptococcus pyogenes at 2.2 angstroms resolution reveals an extended structure comprising two all-β domains. The molecules associate in columns through the crystal, with each carboxyl terminus adjacent to a conserved lysine of the next molecule. This lysine forms the isopeptide bonds that link the subunits in native pili, validating the relevance of the crystal assembly. Each subunit contains two lysine-asparagine isopeptide bonds generated by an intramolecular reaction, and we find evidence for similar isopeptide bonds in other cell surface proteins of Gram-positive bacteria. The present structure explains the strength and stability of such Gram-positive pili and could facilitate vaccine development.

Streptococcus pyogenes (group A Streptococcus) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a “cap and release” mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr–Hb complex which function to transiently uncap HID1 from the heme bound to Hb’s β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr’s NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum , for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold–sulfur bonds (Au–S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes , which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond. The conjugation of nanoparticles and proteins can require complex optimization for the addition of different proteins. Here, the authors report on the development of a simple isopeptide bond forming method of conjoining gold nanoparticles and fusion proteins.

The pathogenicity of group A Streptococcus (GAS) is mediated by direct bacterial invasivity and toxin-associated damage. Among the extracellular products, the exotoxin streptolysin O (SLO) is produced by almost all GAS strains. SLO is a pore forming toxin (PFT) hemolitically active and extremely toxic in vivo . Recent evidence suggests that human serum albumin (HSA), the most abundant protein in plasma, is a player in the innate immunity “orchestra.” We previously demonstrated that HSA acts as a physiological buffer, partially neutralizing Clostridioides difficile toxins that reach the bloodstream after being produced in the colon. Here, we report the in vitro and ex vivo capability of HSA to neutralize the cytotoxic and hemolytic effects of SLO. HSA binds SLO with high affinity at a non-conventional site located in domain II, which was previously reported to interact also with C. difficile toxins. HSA:SLO recognition protects HEp-2 and A549 cells from cytotoxic effects and cell membrane permeabilization induced by SLO. Moreover, HSA inhibits the SLO-dependent hemolytic effect in red blood cells isolated from healthy human donors. The recognition of SLO by HSA may have a significant protective role in human serum and sustains the emerging hypothesis that HSA is an important constituent of the innate immunity system.

Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and atomic-force microscopy force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a type of highly specific antiadhesive antibiotics to treat gram-positive pathogens.

Group A Streptococci (GAS) are involved in a number of life threatening diseases and biofilm formation by these pathogens are considered as an important virulence determinant as it mediates antibiotic resistance among them. In the present study, we have explored the ability of (+)-usnic acid, a lichen secondary metabolite, as an antibiofilm agent against four serotypes of Streptococcus pyogenes causing pharyngitis. Usnic acid inhibited the biofilms of M serotypes M56, st38, M89 efficiently and the biofilm of M74 to a lesser extent. Confocal imaging of the treated samples showed that usnic acid reduced the biomass of the biofilms when compared to that of the control. Fourier Transfer Infrared (FT-IR) spectroscopy indicated that usnic acid reduced the cellular components (proteins and fatty acids) of the biofilms. Interestingly, the FT-IR spectrum further revealed that usnic acid probably acted upon the fatty acids of the biofilms as evident from the disappearance of a peak at 2,455–2,100 cm⁻¹when compared to the control only in serotypes M56, st38 and M89 but not in M74. The present study shows, for the first time, that usnic acid can act as an effective antibiofilm agent against GAS.

•emm clusters were used as the basis for structure-based design of vaccines.•3-D structures and physicochemical properties identified five M peptides representing 17 emm types.•Pentavalent synthetic peptide vaccine antibodies reacted with all 17M peptides in the cluster.•Vaccine antibodies promoted bactericidal activity against 15/17 streptococcal emm types.•Structure-based design may result in broadly protective group A streptococcal vaccines. A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. M protein sequences (AA 16–50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4GAS. The synthetic vaccine rabbit antisera reacted with all 17 E4M peptides and demonstrated bactericidal activity against 15/17 E4GAS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4M peptides. Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS.

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV–vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.