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Pathogen entry mechanisms and endocytic responses to plasma membrane damage
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Richard Lundmark)ORCID iD: 0000-0003-1861-9058
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Endocytosis is a fundamental cellular process by which cells transport material from the outside to the inside of the cell through the formation of membrane invaginations that bud off from the plasma membrane. This process is important for nutrient uptake, regulating cell surface receptors and the overall plasma membrane composition. Cells have several different types of endocytic pathways where clathrin- mediated endocytosis is the most studied. Importantly, pathogens and secreted virulence factors bind to cell surface receptors and hijack the endocytic pathways in order to enter host cells. Depending on their size and molecular composition, pathogens and virulence factors are thought to make use of distinct endocytic pathways into the cell. This thesis focuses on early host cell interactions with virus, bacterial membrane vesicles and a pore-forming toxin, with a particular emphasis on endocytic mechanisms and plasma membrane repair.

During entry of pathogens, it is thought that interactions with specific cell surface molecules drive the recruitment of endocytic proteins to the plasma membrane. Viruses possess a very defined molecular composition and architecture, which facilitate specificity to these interactions. We found that Adenovirus 37, a human ocular pathogen, binds to αVβ1 and α3β1 integrins on human corneal epithelial cells and that this interaction is important for infection. In contrast to viruses, membrane vesicles shed from Helicobacter pylori are heterogeneous in size and molecular composition. These vesicles harbour various adhesins and toxins that may facilitate binding to the cell surface and recruitment of different endocytic pathways. We developed a quantitative internalization assay and showed that the H. pylori vesicles were internalized mainly via clathrin-mediated endocytosis but were also capable of exploiting other endocytic pathways.

Damage to the plasma membrane disrupts cellular homeostasis and can lead to cell death if not repaired immediately. Although endocytic mechanisms have been shown to be important for plasma membrane repair, little is known about their specific role. Listeriolysin O (LLO) is a bacterial toxin that can form pores in the plasma membrane and disrupt cellular homeostasis. We developed a reporter system for real-time imaging of the endocytic response to LLO pore formation. We found that two clathrin-independent endocytic pathways were important for plasma membrane repair. However, they were not directly involved in removing LLO pores from the plasma membrane. Our data suggests that these endocytic systems might rather influence membrane repair by their ability to regulate the plasma membrane composition, shape and tension.

In conclusion, this thesis describes how pathogens and their virulence factors make use of specific mechanisms to enter host cells as well as revealing new insights on the role of the endocytic pathways in plasma membrane repair. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2017. , 51 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1897
Keyword [en]
Endocytosis, plasma membrane, pathogens, virus, bacterial membrane vesicles, Helicobacter pylori, adenovirus 37, listeriolysin O, pore-forming toxins, membrane repair
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-134049ISBN: 978-91-7601-702-9 (print)OAI: oai:DiVA.org:umu-134049DiVA: diva2:1091058
Public defence
2017-05-19, N320, Naturvetarhuset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2017-04-28 Created: 2017-04-25 Last updated: 2017-05-05Bibliographically approved
List of papers
1. Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways
Open this publication in new window or tab >>Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways
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2014 (English)In: mBio, ISSN 2150-7511, Vol. 5, no 3, e00979-14- p.Article in journal (Refereed) Published
Abstract [en]

UNLABELLED: Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.

IMPORTANCE: Bacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-90953 (URN)10.1128/mBio.00979-14 (DOI)000338875900050 ()24846379 (PubMedID)
Available from: 2014-07-04 Created: 2014-07-04 Last updated: 2017-04-25Bibliographically approved
2. Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Open this publication in new window or tab >>Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
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2017 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, no 5, e02019-16Article in journal (Refereed) Published
Abstract [en]

Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVβ3, αVβ5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVβ5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, β1, and β4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, β3, or β5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVβ1 and α3β1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and β1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVβ5 in order to enter nonocular human cells. In this study, we found that αVβ5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVβ1 and α3β1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.

Keyword
adenoviruses, cornea, epidemic keratoconjunctivitis, integrins
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-133501 (URN)10.1128/JVI.02019-16 (DOI)000394356400016 ()27974569 (PubMedID)
Available from: 2017-04-11 Created: 2017-04-11 Last updated: 2017-04-27Bibliographically approved
3. Endocytic response to the pore-forming toxin listeriolysin O
Open this publication in new window or tab >>Endocytic response to the pore-forming toxin listeriolysin O
(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-134046 (URN)
Available from: 2017-04-25 Created: 2017-04-25 Last updated: 2017-04-27

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