Biology subjects

Fischetti, V. A.

Publications and source records attributed to Fischetti, V. A..

5 recordsLinked to original sources

bioRxiv2026

Removing an S-layer enables endolysin-derived probes to detect Paenibacillus thiaminolyticus in mixed bacterial populations

The emerging neonatal bacterial pathogen Paenibacillus thiaminolyticus causes sepsis and postinfectious hydrocephalus, yet few tools are available to study or specifically detect it. We developed fluorescent probes based on cell wall-binding domains (CBDs) from bacteriophage-derived cell-wall hydrolases. CBDs can target structurally conserved cell-wall glycans, making them attractive affinity reagents for bacterial detection. None of the 11 probes we designed labeled intact cells under standard conditions, and we identified a surface layer (S-layer) that prevented access to their cell-wall targets. Brief acidic treatment (pH 2.0) released abundant high-molecular-mass proteins from the cell surface and enabled labeling by five distinct CBD probes, each containing a single C-terminal S-layer homology domain. Mass spectrometry identified the main released protein as an ortholog of the P. alvei SpaA S-layer protein, which we designate SlsA. Across 26 surveyed P. thiaminolyticus genomes, we identified four distinct SlsA variants with 58-79% pairwise amino acid identity, encoded within a conserved cluster homologous to the P. alvei S-layer-associated locus. After either the acidic treatment or a quick flame fixation, the lead probe mGL-BDPt9 labeled six P. thiaminolyticus strains, including three Ugandan clinical isolates, as well as a P. dendritiformis clinical isolate from a US infant, and three additional Paenibacillus species. mGL-BDPt9 also distinguished P. thiaminolyticus from five gram-positive sepsis pathogens in pairwise mixtures and detected it in a five-species suspension. Together, these findings establish the presence of an S-layer in P. thiaminolyticus and provide a rapid fluorescent labeling strategy for multiple Paenibacillus pathogens associated with neonatal paenibacilliosis.

microbiology↗

bioRxiv2021

Evolution of Gram+ Streptococcus pyogenes has maximized efficiency of the Sortase A cleavage site

Human plasminogen (hPg)-binding M-protein (PAM), a major virulence factor of Pattern D Streptococcus pyogenes (GAS), is the primary receptor responsible for binding and activating hPg. PAM is covalently bound to the cell wall (CW) through cell membrane (CM)-resident sortase A (SrtA)-catalyzed cleavage of the PAM-proximal C-terminal LPST{downarrow}-GEAA motif present immediately upstream of its transmembrane domain (TMD), and subsequent transpeptidation to the CW. These steps expose the N-terminus of PAM to the extracellular milieu (EM) to interact with PAM ligands, e.g., hPg. Previously, we found that inactivation of SrtA showed little reduction in functional binding of PAM to hPg, indicating that PAM retained in the cell membrane (CM) by the TMD nonetheless exposed its N-terminus to the EM. In the current study, we assessed the effects of mutating the Thr4 (P1) residue of the SrtA-cleavage site in PAM (Thr355 in PAM) to delay PAM in the CM in the presence of SrtA. Using rSrtA in vitro, LPSYGEAA and LPSWGEAA peptides were shown to have low activities, while LPSTGEAA had the highest activity. Isolated CM fractions of AP53/{Delta}SrtA cells showed that LPSYGEAA and LPSWGEAA peptides were cleaved at substantially faster rates than LPSTGEAA, even in CMs with an AP53/{Delta}SrtA/PAM[T355Y] double mutation, but the transpeptidation step did not occur. These results implicate another CM-resident enzyme that cleaves LPSYGEAA and LPSWGEAA motifs, most likely LPXTGase, but cannot catalyze the transpeptidation step. We conclude that the natural P1 (Thr) of the SrtA cleavage site has evolved to dampen PAM from nonfunctional cleavage by LPXTGase. IMPORTANCEWe show in this study that functional cleavage of the sortase A (SrtA) cleavage signal for M-protein, LPST*GEAA, in the Gram+ cell membrane, which allows transpeptidation of M-protein to the cell wall, as opposed to non-functional cleavage by the highly active cell membrane nonribosomal enzyme, LPXTGase, at the downstream G-residue, is highly dependent on the presence of T at position 4. From our studies, we conclude that Streptococcus pyogenes has evolved in a manner that maximized T at this position so that SrtA preferentially cleaved the sorting signal in order that the virulence factor, M-protein, was stabilized on the cell surface through covalent attachment to the cell wall.

microbiology↗

bioRxiv2021

Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.

biochemistry↗

bioRxiv2020

Integration of prophages into CRISPR loci remodels viral immunity in Streptococcus pyogenes

CRISPR loci are composed of short DNA repeats separated by sequences that match the genomes of phages and plasmids, known as spacers. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defense, phages can produce small proteins that inhibit these nucleases. Here we demonstrate that the {Phi}AP1.1 temperate phage utilizes an alternate approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection this phage expresses a canonical anti-CRISPR, AcrIIA23 that prevents Cas9 function, allowing {Phi}AP1.1 to integrate into the direct repeats of the CRISPR locus and neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transfer of spacers, enabling a complex modulation of the type II-A CRISPR immune response.

microbiology↗

bioRxiv2019

Human blood exposure to Clostridium perfringens epsilon toxin may shed light on erythrocyte fragility during active multiple sclerosis

During active multiple sclerosis (MS), red blood cells (RBCs) harvested from patients reportedly display increased osmotic fragility and increased cellular volume (macrocytosis). The cause of these abnormalities remains unknown. We have previously proposed that Clostridium perfringens epsilon toxin (ETX) may be a blood-borne trigger for newly forming MS lesions based on its tropism for blood-brain barrier vasculature and CNS myelin. Recently, Gao et al. have reported that ETX binds to and damages human RBCs, leading to hemolysis. Moreover, the authors suggest that purinergic nucleotide (P2) receptor activation amplifies the hemolytic process. Here, we confirm that ETX indeed causes human-specific RBC lysis. However, our data suggest that the hemolytic process is mediated by metal-catalyzed oxidation of the swell-induced, nucleotide-sensitive ICln chloride channel. We use spectrophotometry, flow cytometry and Western blotting to show that ETX targets human RBCs and T lymphocytes via their shared expression of Myelin and Lymphocyte protein (MAL); a protein shown to be both necessary and sufficient for ETX binding and toxicity. ETX likely triggers T cells to release redox-active heavy metals, Cu+ and Fe3+, via the lysosomal exocytosis pathway, while RBCs likely release these heavy metals via ETX pore formation within the RBC membrane. Extracellular Cu+ and Fe3+ may then amplify hemolysis by oxidizing a previously identified heavy metal-binding site within the ICln channel pore, thus deregulating its normal conductance. Elucidating the precise mechanism of ETX-mediated hemolysis may shed light on the underlying etiology of MS, as it would explain why MS RBC abnormalities occur during active disease.\n\nIMPORTANCEDuring active MS, numerous reports suggest that circulating RBCs are larger than normal and fragment more easily. The exact trigger(s) for these RBC abnormalities and for newly forming MS lesions remains unidentified. We have proposed that ETX, secreted by the gut bacterium Clostridium perfringens, may be an environmental trigger for newly forming MS lesions. Indeed, ETX has been shown to breakdown the BBB, enter the brain and damage the myelin sheath. Because ETX is typically spread through the circulatory system, we wished to determine how the toxin affects human blood. Provocatively, there has been a recent report that ETX produces cellular abnormalities in human RBCs, reminiscent of what has been described during active MS. In our study, we sought to elucidate the precise mechanism for how ETX causes RBC damage. In addition to triggering BBB breakdown and CNS demyelination, ETX might also explain why RBCs appear abnormal during MS attacks.

microbiology↗