Crystal Structure of Spy0129, a Streptococcus pyogenes Class B Sortase Involved in Pilus Assembly

Sortase enzymes are cysteine transpeptidases that mediate the covalent attachment of substrate proteins to the cell walls of Gram-positive bacteria, and thereby play a crucial role in virulence, infection and colonisation by pathogens. Many cell-surface proteins are anchored by the housekeeping sortase SrtA but other more specialised sortases exist that attach sub-sets of proteins or function in pilus assembly. The sortase Spy0129, or SrtC1, from the M1 SF370 strain of Streptococcus pyogenes is responsible for generating the covalent linkages between the pilin subunits in the pili of this organism. The crystal structure of Spy0129 has been determined at 2.3 Å resolution (R = 20.4%, Rfree  = 26.0%). The structure shows that Spy0129 is a class B sortase, in contrast to other characterised pilin polymerases, which belong to class C. Spy0129 lacks a flap believed to function in substrate recognition in class C enzymes and instead has an elaborated β6/β7 loop. The two independent Spy0129 molecules in the crystal show differences in the positions and orientations of the catalytic Cys and His residues, Cys221 and His126, correlated with movements of the β7/β8 and β4/β5 loops that respectively follow these residues. Bound zinc ions stabilise these alternative conformations in the crystal. This conformational variability is likely to be important for function although there is no evidence that zinc is involved in vivo

Superantigens and Streptococcal Toxic Shock Syndrome

Superantigens produced by Streptococcus pyogenes have been implicated with streptococcal toxic shock syndrome (STSS). We analyzed 19 acute-phase serum samples for mitogenic activity from patients with severe streptococcal disease. The serum samples from two patients in the acute phase of STSS showed strong proliferative activity. Streptococcal mitogenic exotoxin (SME) Z-1 and streptococcal pyrogenic exotoxin (SPE)-J were identified in one patient with peritonitis who recovered after 2 weeks in intensive care. SMEZ-16 was found in a second patient who died on the day of admission. Sequential serum samples taken on day 3 after admission from patient 1 showed clearance of mitogenic activity but absence of neutralizing anti-SMEZ antibodies. Serum samples taken on day 9 from this patient showed evidence of seroconversion with high levels of anti-SMEZ antibodies that neutralized SMEZ-1 and 12 other SMEZ-variants. These results imply that a high level of SMEZ production by group A streptococcus is a causative event in the onset and subsequent severity of STSS

A new approach to local hardness

The applicability of the local hardness as defined by the derivative of the chemical potential with respect to the electron density is undermined by an essential ambiguity arising from this definition. Further, the local quantity defined in this way does not integrate to the (global) hardness - in contrast with the local softness, which integrates to the softness. It has also been shown recently that with the conventional formulae, the largest values of local hardness do not necessarily correspond to the hardest regions of a molecule. Here, in an attempt to fix these drawbacks, we propose a new approach to define and evaluate the local hardness. We define a local chemical potential, utilizing the fact that the chemical potential emerges as the additive constant term in the number-conserving functional derivative of the energy density functional. Then, differentiation of this local chemical potential with respect to the number of electrons leads to a local hardness that integrates to the hardness, and possesses a favourable property; namely, within any given electron system, it is in a local inverse relation with the Fukui function, which is known to be a proper indicator of local softness in the case of soft systems. Numerical tests for a few selected molecules and a detailed analysis, comparing the new definition of local hardness with the previous ones, show promising results.Comment: 30 pages (including 6 figures, 1 table

Photoactivation of titanium-oxo cluster [Ti 6 O 6 (OR) 6 (O 2 C t Bu) 6 ] : mechanism, photoactivated structures, and onward reactivity with O 2 to a peroxide complex

The molecular titanium-oxo cluster [Ti6O6(OiPr)6(O2CtBu)6] (1) can be photoactivated by UV light, resulting in a deeply coloured mixed valent (photoreduced) Ti (iii/iv) cluster, alongside alcohol and ketone (photooxidised) organic products. Mechanistic studies indicate that a two-electron (not free-radical) mechanism occurs in this process, which utilises the cluster structure to facilitate multielectron reactions. The photoreduced products [Ti6O6(OiPr)4(O2CtBu)6(sol)2], sol = iPrOH (2) or pyridine (3), can be isolated in good yield and are structurally characterized, each with two, uniquely arranged, antiferromagnetically coupled d-electrons. 2 and 3 undergo onward oxidation under air, with 3 cleanly transforming into peroxide complex, [Ti6O6(OiPr)4(O2CtBu)6(py)(O2)] (5). 5 reacts with isopropanol to regenerate the initial cluster (1) completing a closed cycle, and suggesting opportunities for the deployment of these easily made and tuneable clusters for sustainable photocatalytic processes using air and light. The redox reactivity described here is only possible in a cluster with multiple Ti sites, which can perform multi-electron processes and can adjust its shape to accommodate changes in electron density

B Cell Numbers Predict Humoral and Cellular Response Upon SARS–CoV-2 Vaccination Among Patients Treated With Rituximab

Objective: Patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) therapy are at higher risk of poor COVID-19 outcomes and show substantially impaired humoral immune response to anti-SARS-CoV-2 vaccine. However, the complex relationship between antigen-specific B cells and T cells and the level of B cell repopulation necessary to achieve anti-vaccine responses remain largely unknown. Methods: Antibody responses to SARS-CoV-2 vaccines and induction of antigen-specific B and CD4/CD8 T cell subsets were studied in 19 patients with rheumatoid arthritis (RA) or antineutrophil cytoplasmic antibody-associated vasculitis receiving RTX, 12 patients with RA receiving other therapies, and 30 healthy controls after SARS-CoV-2 vaccination with either messenger RNA or vector-based vaccines. Results: A minimum of 10 B cells per microliter (0.4% of lymphocytes) in the peripheral circulation appeared to be required for RTX-treated patients to mount seroconversion to anti-S1 IgG upon SARS-CoV-2 vaccination. RTX-treated patients who lacked IgG seroconversion showed reduced receptor-binding domain-positive B cells (P = 0.0005), a lower frequency of Tfh-like cells (P = 0.0481), as well as fewer activated CD4 (P = 0.0036) and CD8 T cells (P = 0.0308) compared to RTX-treated patients who achieved IgG seroconversion. Functionally relevant B cell depletion resulted in impaired interferon-γ secretion by spike-specific CD4 T cells (P = 0.0112, r = 0.5342). In contrast, antigen-specific CD8 T cells were reduced in both RA patients and RTX-treated patients, independently of IgG formation. Conclusion: In RTX-treated patients, a minimum of 10 B cells per microliter in the peripheral circulation is a candidate biomarker for a high likelihood of an appropriate cellular and humoral response after SARS-CoV-2 vaccination. Mechanistically, the data emphasize the crucial role of costimulatory B cell functions for the proper induction of CD4 responses propagating vaccine-specific B cell and plasma cell differentiation

Innovative solutions to sticky situations: Antiadhesive strategies for treating bacterial infections

ABSTRACT Bacterial adherence to host tissue is an essential process in pathogenesis, necessary for invasion and colonization and often required for the efficient delivery of toxins and other bacterial effectors. As existing treatment options for common bacterial infections dwindle, we find ourselves rapidly approaching a tipping point in our confrontation with antibiotic-resistant strains and in desperate need of new treatment options. Bacterial strains defective in adherence are typically avirulent and unable to cause infection in animal models. The importance of this initial binding event in the pathogenic cascade highlights its potential as a novel therapeutic target. This article seeks to highlight a variety of strategies being employed to treat and prevent infection by targeting the mechanisms of bacterial adhesion. Advancements in this area include the development of novel antivirulence therapies using small molecules, vaccines, and peptides to target a variety of bacterial infections. These therapies target bacterial adhesion through a number of mechanisms, including inhibition of pathogen receptor biogenesis, competition-based strategies with receptor and adhesin analogs, and the inhibition of binding through neutralizing antibodies. While this article is not an exhaustive description of every advancement in the field, we hope it will highlight several promising examples of the therapeutic potential of antiadhesive strategies.</jats:p

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone