Biology subjects

Ortega, F. E.

Publications and source records attributed to Ortega, F. E..

3 recordsLinked to original sources

bioRxiv2020

Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall

Cells across all kingdoms of life actively partition molecules between discrete cellular compartments. In Gram-positive bacteria, a thick and highly cross-linked peptidoglycan cell wall separates the bacterial membrane from the extracellular space, imposing a barrier that must be crossed by proteins whose functions require that they be exposed on the bacterial cell surface1,2. Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA3, remain associated with the bacterial membrane yet somehow thread through tens of nanometers of dense, cross-linked cell wall to expose their N-terminus on the outer surface4,5. Here, we show that entropy can drive the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We develop a physical model predicting that the entropic constraint imposed by a thin periplasm is sufficient to drive translocation of an intrinsically disordered protein like ActA across a porous barrier similar to the cell wall. Consistent with this scenario, we demonstrate experimentally that translocation depends on both the dimensions of the cell envelope and the length of the disordered protein, and that translocation is reversible. We also show that disordered regions from eukaryotic nuclear pore complex proteins are capable of entropy-driven translocation through Gram-positive cell walls. These observations suggest that entropic forces alone, rather than chaperones or chemical energy, are sufficient to drive translocation of certain Gram-positive surface proteins for exposure on the outer surface of the cell wall.

biophysics↗

bioRxiv2018

Listeria monocytogenes cell-to-cell spread in epithelia is heterogeneous and dominated by rare pioneer bacteria

L. monocytogenes hijacks host actin to promote its intracellular motility and intercellular spread. While L. monocytogenes virulence hinges on cell-to-cell spread, little is known about the dynamics of bacterial spread in epithelia at a population level. Here, we use live microscopy and statistical modeling to demonstrate that L. monocytogenes cell-to-cell spread proceeds anisotropically in an epithelial monolayer in culture. We show that boundaries of infection foci are irregular and dominated by rare pioneer bacteria that spread farther than the rest. We extend our quantitative model for bacterial spread to show that heterogeneous spreading behavior can improve the chances of creating a persistent L. monocytogenes infection in an actively extruding epithelium. Thus, our results indicate that L. monocytogenes cell-to-cell spread is heterogeneous, and that rare pioneer bacteria determine the frontier of infection foci and may promote bacterial infection persistence in dynamic epithelia.

microbiology↗

bioRxiv2018

Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection (1). Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadins involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes {Delta}inlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP DLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

microbiology↗