Biology subjects

Raab, J. E.

Publications and source records attributed to Raab, J. E..

3 recordsLinked to original sources

bioRxiv2024

A translocation-competent pore is required for Shigella flexneri to escape from the double membrane vacuole during intercellular spread

Type 3 secretion systems (T3SSs) enable bacterial virulence by translocating virulence proteins (effectors) into host cells. Shigella flexneri require T3SS to invade and to spread between cells in the colon. In order to spread, S. flexneri forms membrane protrusions that push into the adjacent host cell. These protrusions are resolved into double membrane vacuoles (DMVs) that the bacteria quickly escape. The mechanisms required for escape from the DMV are poorly understood, but the T3SS translocon pore protein IpaC is essential. Here, we show IpaC forms a pore that is competent for translocation of T3SS effectors as bacteria spread between cells. To do so, we used a genetic approach to test mutations of IpaC that disrupt its ability to translocate and to form pores. We show that during spread, IpaC is efficiently inserted into the plasma membrane, the membrane-embedded IpaC forms pore complexes, and the IpaC-dependent pores translocate effectors that are necessary for S. flexneri to escape the DMV. We further show that T3SS activation is regulated through a distinct mechanism at spread compared to at invasion; activation of T3SS secretion does not require pore formation during spread. Thus, we show that a distinct regulation of the T3SS during S. flexneri intercellular spread enables the placement of effectors both around S. flexneri and across membranes of the DMV. Altogether, this study provides new insights into how S. flexneri escapes the DMV. IMPORTANCEThe type 3 secretion system (T3SS) is required for virulence in many bacterial pathogens that infect humans. The T3SS forms a pore through which virulence proteins are delivered into host cells to enable bacterial infection. Our work investigates the Shigella translocon pore protein IpaC, which is essential not only for bacteria to invade cells, but also for bacteria to spread between cells. An ability to spread between cells is essential for pathogenesis, thus understanding the mechanisms that enable spread is important for understanding how S. flexneri infection causes illness. We show that IpaC delivers virulence factors across the host membrane for S. flexneri to efficiently spread. This study furthers our understanding of the mechanisms involved in T3SS secretion and of translocon pore function during S. flexneri intercellular spread.

microbiology↗

bioRxiv2023

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase I clinical trial investigating a ferritin nanoparticle displaying H2 hemagglutinin in H2-naive and H2-exposed adults. Therefore, we could perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited after H2 vaccination. We temporally map the epitopes targeted by serum antibodies after first and second vaccinations and show previous H2 exposure results in higher responses to the variable head domain of hemagglutinin while initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryo-EM and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the hemagglutinin head including the receptor binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses. HighlightsO_LISerum Abs after first H2-F vaccination in H2-exposed donors bound variable HA head epitopes C_LIO_LISerum Abs after first H2-F vaccination in H2-naive donors bound conserved HA head and stem epitopes C_LIO_LIRBS-targeting VH1-69 cross-reactive antibodies were induced in H2-naive individuals C_LIO_LIThe medial junction is a previously uncharacterized conserved epitope on the HA head C_LI

immunology↗

bioRxiv2023

Synaptopodin is necessary for Shigella flexneri intercellular spread

For many intracellular pathogens, their virulence depends on an ability to spread between cells of an epithelial layer. For intercellular spread to occur, these pathogens deform the plasma membrane into a protrusion structure that is engulfed by the neighboring cell. Although the polymerization of actin is essential for spread, how these pathogens manipulate the actin cytoskeleton in a manner that enables protrusion formation is still incompletely understood. Here, we show that butyrate responsive pathways promote intercellular spread by Shigella flexneri. We identify the mammalian actin binding protein synaptopodin, a butyrate responsive gene, as required for efficient intercellular spread of S. flexneri and Listeria monocytogenes. We show synaptopodin enhances the recruitment of actin to bacteria and stabilizes the actin tail. We show that, for S. flexneri, synaptopodin presence enables protrusions to form and to resolve at a greater rate, indicating that greater stability of the actin tail enables the bacteria to push against the membrane with greater force. We demonstrate that synaptopodin recruitment around bacteria requires the bacterial protein IcsA, and we show that this recruitment is further enhanced in a type 3 secretion system dependent manner. These data establish synaptopodin as required for intracellular bacteria to stabilize the actin cytoskeleton in a manner that enables efficient protrusion formation and identify for the first time that synaptopodin contributes to bacterial pathogenesis.

microbiology↗