Biology subjects

Radhakrishnan, P.

Publications and source records attributed to Radhakrishnan, P..

5 recordsLinked to original sources

bioRxiv2025

Listeria monocytogenes cell-to-cell spread bypasses nutrient limitation for replicating intracellular bacteria

Listeria monocytogenes is an intracellular bacterial pathogen that obtains nutrients from the mammalian host cell to fuel its replication in cytosol. Sparse infection of epithelial monolayers by L. monocytogenes results in the formation of distinct infectious foci, where each focus originates from the initial infection of a single host cell followed by multiple rounds of active bacterial cell-to-cell spread into neighboring host cells in the monolayer. We used time-lapse microscopy to measure changes in bacterial growth rate in individual foci over time and found that intracellular bacteria initially replicate exponentially, but then bacterial growth rate slows later in infection, particularly in the center of the infectious focus. We found that the intracellular replication rate of L. monocytogenes is measurably decreased by limiting host cell glucose availability, by decreasing the rate of intracellular bacterial oligopeptide import, and, most interestingly, by alterations in host cell junctional proteins that limit bacterial spread into neighboring cells without directly affecting bacterial growth or metabolism. By measuring the carrying capacity of individual host cells, we found that the nutritional density of cytoplasm is comparable to rich medium. Taken together, our results indicate that the rate of intracellular L. monocytogenes replication is governed by a balance of the rate of nutrient depletion by the bacteria, the rate of nutrient replenishment by the metabolically active host cells, and the rate of bacterial cell-to-cell spread which enables the bacteria to seek out "greener pastures" before nutrient availability in a single host cell becomes limiting. SIGNIFICANCEListeria monocytogenes is a foodborne pathogen that is capable of facultative intracellular growth in a wide variety of mammalian cell types. It uses actin-based motility to spread directly from the cytoplasm of one infected host cell into another. After initially infecting cells in the small intestine, L. monocytogenes can circulate inside of infected macrophages, and subsequently spread to cells of distal organs, where it can cause spontaneous abortions in pregnant women and meningitis in immunocompromised individuals. In addition to facilitating body-wide dissemination, the actin-based cell-to-cell spread of L. monocytogenes contributes to the ability of the pathogen to evade the cell-mediated arm of the immune system. In this work, we find that cell-to-cell spread also benefits the bacteria in a third way, by enabling the bacteria to overcome the nutrient limitation they would face if they remained confined to a single host cell, such that they never enter stationary phase.

microbiology↗

bioRxiv2022

Listeria monocytogenes Co-opts Caveolin-Mediated E-cadherin Trafficking and Macropinocytosis for Epithelial Cell-to-Cell Spread

Listeria monocytogenes is an intracellular bacterial pathogen that spreads directly between adjacent host cells without exposure to the extracellular space. Recent studies have identified several host cell factors that promote L. monocytogenes cell-to-cell spread in epithelial monolayers, but details of the mechanism remain unclear. We find that the adherens junction protein, E-cadherin, promotes L. monocytogenes cell-to-cell spread at the recipient side of cell contacts. In particular, two point mutations in E-cadherins cytoplasmic domain that prevent its ubiquitination hinder bacterial cell-to-cell spread efficiency without reducing the extent of contact between neighboring cells. As ubiquitination induces E-cadherin endocytosis, we hypothesize that E-cadherin promotes protrusion engulfment, where donor cell protrusions containing L. monocytogenes are taken up by the recipient cell concurrently with E-cadherin internalization. In support of this hypothesis, we show that inhibiting caveolin-mediated membrane trafficking reduces L. monocytogenes cell-to-cell spread only under conditions where E-cadherin can be ubiquitinated. Additionally, we demonstrate that macropinocytosis also contributes to dissemination of L. monocytogenes through an epithelial monolayer.

microbiology↗

bioRxiv2020

Bromelain Inhibits SARS-CoV-2 Infection in VeroE6 Cells

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19. Highlights O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/297366v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@b7afb0org.highwire.dtl.DTLVardef@16f8185org.highwire.dtl.DTLVardef@1a07df8org.highwire.dtl.DTLVardef@1ae33fa_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIBromelain inhibits / cleaves the expression of ACE-2 and TMPRSS2 C_LIO_LIBromelain cleaves / degrades SARS-CoV-2 spike protein C_LIO_LIBromelain inhibits S-Ectodomain binding and SARS-CoV-2 infection C_LI

microbiology↗

bioRxiv2020

Mutations in MYLPF cause a novel segmental amyoplasia that manifests as distal arthrogryposis

We identified ten persons in six consanguineous families with Distal Arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T, p.(Cys157Phe) or c.469T>C, p.(Cys157Arg)) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A, p.(Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly-inherited DA, a c.98C>T, p.(Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

genetics↗

bioRxiv2020

Mechanical competition triggered by innate immune signaling drives the collective extrusion of bacterially-infected epithelial cells

Multiple distinct types of intracellular bacterial pathogens have been shown to alter the mechanics of their mammalian host cells to promote cell-to-cell spread. Conversely, host cells may respond by altering their own mechanical behavior to limit infection. We monitored epithelial cell monolayers sparsely infected with the intracellular bacterial pathogens Listeria monocytogenes or Rickettsia parkeri over the course of several days. Under conditions where these pathogens were able to trigger innate immune signaling through the NF-{kappa}B pathway and to use actin-based motility to spread non-lytically from cell to cell, domains of infected cells formed enormous three-dimensional mounds, where uninfected cells surrounding the infected cells became stiffer and actively moved toward the site of infection, collectively squeezing the softer and weaker infected cells up and ejecting them from the epithelial monolayer. Bacteria in mounds were less able to spread laterally in the monolayer, limiting the growth of the focus, while mounded cells eventually underwent cell death. Cells in infected monolayers exhibited behavioral and molecular signatures of the epithelial to mesenchymal transition (EMT), such that coordinated forceful action by uninfected bystander cells actively eliminated large domains of infected cells, consistent with the hypothesis that this collective cell response represents an innate immune response.

microbiology↗