Biology subjects

Ferraro, N. J.

Publications and source records attributed to Ferraro, N. J..

6 recordsLinked to original sources

bioRxiv2024

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophil influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5-monophosphate-N-acetylneuraminic acid (CMP-NANA) scavenged from the host using LOS sialyltransferase (Lst), since Gc cannot make its own sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or {Delta}lst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCENeisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subvert protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampen neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

microbiology↗

bioRxiv2021

Selective Display of a Chemoattractant Agonist on Cancer Cells Activates the Formyl Peptide Receptor 1 on Immune Cells

Current immunotherapeutics often work by directing components of the immune system to recognize biomarkers on the surface of cancer cells to generate an immune response. However, variable changes in biomarker distribution and expression can result in uneven patient response. The development of a more universal tumor-homing strategy has the potential to improve selectivity and extend therapy to cancers with decreased expression or absence of specific biomarkers. Here, we designed a bifunctional agent that exploits the inherent acidic microenvironment of most solid tumors to selectively graft the surface of cancer cells with a formyl peptide receptor ligand (FPRL). Our approach is based on the pH(Low) Insertion Peptide (pHLIP), a unique peptide that selectively targets tumors in vivo by anchoring onto cancer cells in a pH-dependent manner. We establish that selectively remodeling cancer cells with a pHLIP-based FPRL activates formyl peptide receptors on recruited immune cells, potentially initiating an immune response towards tumors.

immunology↗

bioRxiv2021

Transposon Screen of Surface Accessibility in S. aureus

Bacterial cell walls represent one of the most prominent targets of antibacterial agents. These agents include natural products (e.g., vancomycin) and proteins stemming from the innate immune system (e.g., peptidoglycan-recognition proteins and lysostaphin). Among bacterial pathogens that infect humans, Staphylococcus aureus (S. aureus) continues to impose a tremendous healthcare burden across the globe. S. aureus has evolved countermeasures that can directly restrict the accessibility of innate immune proteins, effectively protecting itself from threats that target key cell well components. We recently described a novel assay that directly reports on the accessibility of molecules to the peptidoglycan layer within the bacterial cell wall of S. aureus. The assay relies on site-specific chemical remodeling of the peptidoglycan with a biorthogonal handle. Here, we disclose the application of our assay to a screen of a nonredundant transposon mutant library for susceptibility of the peptidoglycan layer with the goal of identifying genes that contribute to the control of cell surface accessibility. We discovered several genes that resulted in higher accessibility levels to the peptidoglycan layer and showed that these genes modulate sensitivity to lysostaphin. These results indicate that this assay platform can be leveraged to gain further insight into the biology of bacterial cell surfaces. Table of Contents Figure O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2021

SaccuFlow: High-throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions

Bacterial cell walls are formidable barriers that protect bacterial cells against external insults and oppose internal turgor pressure. While cell wall composition is variable across species, peptidoglycan is the principal component of all cell walls. Peptidoglycan is a mesh-like scaffold composed of crosslinked strands that can be heavily decorated with anchored proteins. The biosynthesis and remodeling of peptidoglycan must be tightly regulated by cells because disruption to this biomacromolecule is lethal. This essentiality is exploited by the human innate immune system in resisting colonization and by a number of clinically relevant antibiotics that target peptidoglycan biosynthesis. Evaluation of molecules or proteins that interact with peptidoglycan can be a complicated and, typically, qualitative effort. We have developed a novel assay platform (SaccuFlow) that preserves the native structure of bacterial peptidoglycan and is compatible with high-throughput flow cytometry analysis. We show that the assay is facile and versatile as demonstrated by its compatibility with sacculi from Gram-positive bacteria, Gram-negative bacteria, and mycobacteria. Finally, we highlight the utility of this assay to assess the activity of sortase A from Staphylococcus aureus against potential anti-virulence agents.

microbiology↗

bioRxiv2020

Packed Like Sardines - How Surface Crowdedness Impacts Accessibility to Peptidoglycan of Staphylococcus aureus

Bacterial cell walls are essential barriers that protect bacteria against the onslaught of potentially lethal molecules from the outside. Small molecule therapeutics, proteins from bacterial foes, and host immune proteins must navigate past a dense layer of bacterial biomacromolecules (e.g., capsular proteins, teichoic acids, and anchored proteins) to reach the peptidoglycan (PG) layer of Gram-positive bacteria. A subclass of molecules (e.g., antibiotics with intracellular targets) must also permeate through the PG (in a molecular sieving manner) to reach the cytoplasmic membrane. In the case of Staphylococcus aureus (S. aureus), teichoic acids are the major biopolymers that decorate bacterial cell surfaces. Despite the biological and therapeutic importance of surface accessibility, systematic analyses in live bacterial cells have been lacking. We describe a novel live cell fluorescence assay that reports on the permeability of molecules to and within the PG scaffold. The assay has robust reproducibility, is readily adoptable to any Gram-positive organism, and is compatible with high-throughput sample processing. Analysis of the factors controlling permeability to S. aureus and the methicillin resistant MRSA revealed that molecular flexibility plays a central role in molecular permeability. Moreover, teichoic acids impeded permeability of molecules of a wide range of sizes and chemical composition.

microbiology↗

bioRxiv2019

pH-Dependent Grafting of Cancer Cells with Antigenic Epitopes Promotes Selective Antibody-Mediated Cytotoxicity

A growing class of immunotherapeutic agents work by redirecting components of the immune system to recognize specific markers on the surface of cancer cells and initiate a selective immune response. However, such immunotherapeutic modalities will remain confined to a relatively small subgroup of patients until two major hurdles are overcome: (1) the specific targeting of cancer cells relative to healthy cells, and (2) the lack of common targetable tumor biomarkers among all patients. Here, we designed a unique class of agents that exploit the inherent acidic microenvironment of solid tumors to selectively graft the surface of cancer cells with immuno-engager epitopes for directed destruction by components of the immune system. Specifically, conjugates were assembled using an antigen that recruit antibodies present in human serum, and the pH(Low) Insertion Peptide (pHLIP), a unique peptide that selectively target tumors in vivo by anchoring onto cancer cell surfaces in a pH-dependent manner. We established that conjugates can recruit antibodies from human serum to the surface of cancer cells, and induce complement-dependent and antibody-dependent cellular cytotoxicity by peripheral blood mononuclear cells and also an engineered NK cell line. These results suggest that these agents have the potential to be applicable to treating a wide range of solid tumors and to circumvent the problem of narrow windows of selectivity.

immunology↗