Biology subjects

Raymond, B. B. A.

Publications and source records attributed to Raymond, B. B. A..

4 recordsLinked to original sources

bioRxiv2025

Flow cytometry-based isolation combined with ultra-sensitive proteomics of Salmonella-containing phagosomes reveals novel insights into host-pathogen interactions

Macrophages engulf pathogens into dynamic phagosomes, which many bacteria manipulate for survival. However, isolating pure pathogen-containing phagosomes remains challenging. Here, we developed a novel flow cytometry-based isolation and ultrasensitive proteomics approach to analyse phagosomal and bacterial proteomes from macrophages infected with wild-type (WT) Salmonella enterica serovar Typhimurium (STM) or a {Delta}phoP mutant at 30 min and 4 hrs post-infection. Our approach provides higher throughput, requires lower cell numbers and quantifies more proteins than previous techniques. Our data reveals key host-pathogen interactions, showing induction of PhoP-dependent virulence factors and novel putative proteins that shape STMs intracellular niche. Moreover, our data indicates that bacteria-containing phagosomes recruit mitochondrial membrane for production of reactive oxygen species. These findings provide new insights into Salmonellas manipulation of phagosomal maturation and intracellular niche formation.

microbiology↗

bioRxiv2024

The human and mouse dendritic cell receptor DCIR binds to LRP1 N-glycans containing terminal galactoses, including the α-Gal antigen

The dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor expressed by myeloid cells that plays a key immunoregulatory role in a wide range of diseases, from inflammation to cancer. However, the ligand(s) of DCIR remain(s) unknown, hampering our understanding of the exact function of this immune receptor. Here, we found that both human DCIR and mouse DCIR1 bind specifically to the low-density lipoprotein receptor-related protein 1 (LRP1), a heavily glycosylated receptor mediating the clearance of various molecules from the extracellular matrix and apoptotic cells. This interaction is mediated by galactose-terminated biantennary complex-type N-glycans, including those carrying the immunogenic -Gal epitope. Our study provides a deeper understanding of the role of DCIR in immune regulation and its potential impact on a range of immune disorders, highlighting its specificity in ligand recognition which is crucial for developing therapeutic strategies.

immunology↗

bioRxiv2023

Proteomic mapping of macrophages in response to the clearance of apoptotic cells reveals a unique reprogramming profile.

During the daily process of healthy cellular turnover, billions of cells undergo apoptosis in the human body. These cells are removed by phagocytic cells, namely macrophages through a process known as efferocytosis, which triggers a cascade of reprogramming events in the cell, with a shift towards a pro-resolving or wound healing phenotype. To date, no study has attempted to investigate these phenotypic changes from a proteomic perspective. Here, we present a novel and robust workflow for the investigation of proteome and secretome changes in bone marrow-derived macrophages (BMDMs) following efferocytosis using stable isotope labelling by amino acids in cell culture (SILAC) combined with data-independent acquisition (DIA) mass spectrometry. We show that using this workflow we can dissect the mixed proteomes of BMDMs and apoptotic cells to specifically map the reprogramming events occurring in macrophages in the later stages of efferocytosis. Specifically, we find that efferocytic macrophages adopt an alternatively activated phenotype underpinned by an increase in efferocytic and anti-inflammatory markers. We also show that the secretome contains factors that can reprogram naive BMDMs towards an efferocytosis-like, pro-resolving, phenotype. Our results provide an unprecedented view of the efferocytic landscape of macrophages and will aid in further understanding this important immunological process in the larger context of immune homeostasis and inflammatory disorders.

cell biology↗

bioRxiv2023

Bi-functional particles for real-time acidification and proteolysis multiplex assay in macrophages

Phagosome acidification and proteolysis are essential processes in the immune response to contain and eliminate pathogens. In recent years, there has been an increased desire for a rapid and accurate method of assessing these processes in real-time. Here, we outline the development of a multiplexed assay that allows simultaneous monitoring of phagosome acidification and proteolysis in the same sample using silica beads conjugated to pHrodo and DQ BSA. We describe in detail how to prepare the bi-functional particles and show proof of concept using differentially activated macrophages. This multiplexed spectrophotometric assay allows rapid and accurate assessment of phagosome acidification and proteolysis in real-time and could provide valuable information for understanding the immune response to pathogen invasion.

biochemistry↗