Person: Higgins, Darren
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Higgins
First Name
Darren
Name
Higgins, Darren
6 results
Search Results
Now showing 1 - 6 of 6
Publication Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection(Landes Bioscience, 2013) Lam, Grace Y.; Cemma, Marija; Muise, Aleixo M.; Higgins, Darren; Brumell, John H.Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3+ bacteria were present within single-membrane phagosomes that are LAMP1+. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3+ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.Publication Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread(2014) Czuczman, Mark A.; Fattouh, Ramzi; van Rijn, Jorik; Canadien, Veronica; Osborne, Suzanne; Muise, Aleixo M.; Kuchroo, Vijay; Higgins, Darren; Brumell, John H.Efferocytosis, the process by which dying/dead cells are removed by phagocytosis, plays an important role in development, tissue homeostasis and innate immunity1. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes (Lm), can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells using the pore-forming toxin Listeriolysin O (LLO) and two phospholipases C2. Expression of the cell surface protein ActA allows Lm to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. We show that protrusion formation is associated with plasma membrane damage due to LLO’s pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 contributes to efficient cell-to-cell spread by Lm in macrophages in vitro and growth of these bacteria is impaired in TIM-4−/− mice. Thus, Lm promotes its dissemination in a host by exploiting efferocytosis. Our study suggests that PS-targeted therapeutics may be useful in the fight against infections by Lm and other bacteria that utilize similar strategies of cell-to-cell spread during infection.Publication Identification of Listeria monocytogenes Determinants Required for Biofilm Formation(Public Library of Science, 2014) Alonso, Almaris N.; Perry, Kyle James; Regeimbal, James M.; Regan, Patrick M.; Higgins, DarrenListeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.Publication Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells(Public Library of Science, 2015) Cemma, Marija; Lam, Grace Y.; Stöckli, Martina; Higgins, Darren; Brumell, John H.Listeria monocytogenes is an intracellular bacterial pathogen that can replicate in the cytosol of host cells. These bacteria undergo actin-based motility in the cytosol via expression of ActA, which recruits host actin-regulatory proteins to the bacterial surface. L. monocytogenes is thought to evade killing by autophagy using ActA-dependent mechanisms. ActA-independent mechanisms of autophagy evasion have also been proposed, but remain poorly understood. Here we examined autophagy of non-motile (ΔactA) mutants of L. monocytogenes strains 10403S and EGD-e, two commonly studied strains of this pathogen. The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface. However, only strain EGD-e ΔactA displayed colocalization with the autophagy marker LC3 at 8 hours post infection. A bacteriostatic agent (chloramphenicol) was required for LC3 recruitment to 10403S ΔactA, suggesting that these bacteria produce a factor for autophagy evasion. Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells. However, deletion of inlK in either the wild-type or ΔactA background of strain 10403S had no impact on autophagy evasion by bacteria, indicating it does not play an essential role in evading autophagy. Replication of ΔactA mutants of strain EGD-e and 10403S was comparable to their parent wild-type strain in macrophages. Thus, ΔactA mutants of L. monocytogenes can block killing by autophagy at a step downstream of protein ubiquitination and, in the case of strain EGD-e, downstream of LC3 recruitment to bacteria. Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.Publication Immunoprofiling of T cell responses in melanoma patients undergoing CPI therapy(BioMed Central, 2015) Talarico, Lee Ann; Grubaugh, Daniel; Yan, Zheng; Alami, Aula; Bodmer, Jean-Luc; Higgins, Darren; Hodi, F Stephen; Flechtner, JessicaPublication Invasion of the Brain by Listeria monocytogenes Is Mediated by InlF and Host Cell Vimentin(American Society for Microbiology, 2018) Ghosh, Pallab; Halvorsen, Elizabeth M.; Ammendolia, Dustin A.; Mor-Vaknin, Nirit; O’Riordan, Mary X. D.; Brumell, John H.; Markovitz, David M.; Higgins, DarrenABSTRACT Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.