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Roles of secreted bacterial factors in modulation of host cell signalling
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Sun Nyunt Wai)ORCID iD: 0000-0002-0103-0696
2023 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Roller för utsöndrade bakteriella faktorer i modulering av värdcellsignalering (Swedish)
Abstract [en]

Pathogenic bacteria employ several secretion systems to release or inject virulence factors that may alter host cell processes, generate a replicative niche, and aid bacterial survival in adverse environments. This thesis presents my investigations on how bacterial factors can modulate host cell signalling mechanisms. 

We investigated possible signalling pathways involved in targets of the Vibrio cholerae protein MakA that was found to mediate inhibition of tumour cell proliferation. Caenorhabditis elegans grazing on MakA-producing bacteria revealed that MakA may affect lipid-mediated signalling in the nematodes by affecting the level of PPK-1, a homologue of eukaryotic PIP5K1α. We studied the possible effects of MakA on eukaryotic PIP5K1α in human colon cancer cell lines and found decreased levels of PIP5K1α and pAkt in the lipid-signalling pathway. Immunoblot analyses demonstrated that MakA inhibited cyclin-dependent kinase 1 and increased p27 expression in the colon cancer cells, resulting in G2/M cell cycle arrest. MakA also caused downregulation of Ki67 and cyclin D1, limiting cancer cell proliferation. MakA is the first reported bacterial protein targeting the PIP5K1α lipid signalling pathway, thereby displaying anti-cancer capabilities. 

We discovered that phosphatidic acid (PA)-mediated MakA binding to host cell plasma membranes generated endomembrane-rich aggregates that caused host target cell autophagy and cytotoxicity. PA binding and cell toxicity by MakA required its N-terminal domain. 

The MakA genetic determinant is located within a novel pathogenicity island that also encodes the MakB, MakC, MakD, and MakE proteins. In most V. cholerae and Vibrio anguillarum genomes, mak genes form an operon, makCDBAE. The immunoblot analyses showed that wild-type V. cholerae A1552 released the MakA, MakB, and MakE proteins via the flagellum, while a flagellum-deficient mutant released very little or none. Structurally, MakA, MakB, and MakE belong to a superfamily of bacterial alpha-pore-forming toxins. Identification and structural analysis of V. cholerae Mak proteins revealed that the MakA/B/E toxin is common to several pathogenic Vibrionaceae strains, and this previously unrecognised tripartite toxin may increase their fitness and pathogenicity in various environments and host organisms. 

Bacteria release spherical lipid nanostructures, extracellular membrane vesicles, that may play many biological roles. Previously, Escherichia coli was shown to release physiologically active cytolysin A (ClyA) via outer membrane vesicles (OMVs). ClyA, the first recognised member of the bacterial alpha-pore-forming proteins, has become a model for how oligomerization and pore formation occur in membranes. The clyA gene is cryptic in commensal non-pathogenic E. coli bacteria displaying no cytolytic activity. We found that the sublytic concentration of ClyA released via OMVs by non-pathogenic E. coli profoundly affected host cells. The ClyA+ OMVs were rapidly internalised into colon cancer cells by macropinocytosis and clathrin-mediated, dynamin-dependent endocytosis. The OMV-associated ClyA caused reduced levels of cancer-activating proteins like EZH2, H3K27me3, CXCR4, STAT3, and MDM2 via the EZH2/H3K27me3/miR622/CXCR4 signalling axis. Evidently, sublytic levels of ClyA in OMVs from non-pathogenic E. coli can modulate epigenetics by targeting EZH2 protein stability and we hypothesised that E. coli in colorectal cancer microbiomes may preferentially lack this protein. Given our current understanding of ClyA interactions in cancer cell signalling, it will be intriguing to determine if and how the status of the clyA locus is involved in the aetiology of colorectal cancer. 

Abstract [sv]

Patogena bakterier använder flera utsöndringssystem för att frigöra eller injicera virulensfaktorer som kan förändra värdcellsprocesser, generera en replikativ nisch och hjälpa bakteriell överlevnad i ogynnsamma miljöer. Denna avhandling presenterar mina undersökningar om hur bakteriella faktorer kan modulera värdcellers signaleringsmekanismer. 

Vi undersökte möjliga signalvägar involverade i mål av Vibrio cholerae- proteinet MakA som visade sig förmedla hämning av tumörcellsproliferation. Caenorhabditis elegans som betar på MakA-producerande bakterier avslöjade att MakA kan påverka lipidmedierad signalering i nematoderna genom att påverka nivån av PPK-1, en homolog av eukaryot PIP5K1α. Vi studerade möjliga effekter av MakA på eukaryot PIP5K1α i humana tjocktarmscancercellinjer och fann minskade nivåer av PIP5K1α och pAkt i lipid-signaleringsvägen. Immunoblotanalyser visade att MakA hämmade cyklinberoende kinas 1 och ökade p27-uttryck i tjocktarmscancercellerna, vilket resulterade i G2/M-cellcykelstopp. MakA orsakade också nedreglerad Ki67 och cyklin D1, vilket begränsar cancercellsproliferation. MakA är det första rapporterade bakteriella proteinet som riktar sig mot PIP5K1α-lipidsignaleringsvägen och därmed visar anti-cancerförmåga.Vi upptäckte att fosfatidinsyra (PA)-medierad MakA-bindning till värdcellplasmamembran genererade endomembranrika aggregat som orsakade värdmålcellsautofagi och cytotoxicitet. För PA-bindning och celltoxicitet av MakA behövs dess N-terminala domän. 

MakA genetiska determinanten är belägen inom en ny patogenicitetsö som också kodar för MakB-, MakC-, MakD- och MakE-proteinerna. I de flesta genomen hos V. cholerae och Vibrio anguillarum bildar mak generna ett operon, makCDBAE. Immunoblotanalyserna visade att vildtyp V. cholerae A1552 utsöndrar MakA-, MakB- och MakE- proteinerna via flagellen, medan en flagell-defekt mutant utsöndrade mycket lite eller inget. Strukturellt sett tillhör MakA, MakB och MakE en superfamilj av bakteriella alfa-porbildande toxiner. Identifiering och strukturell analys av V. cholerae Mak-proteiner avslöjade att MakA/B/E- toxinet är gemensamt för flera patogena Vibrionaceae-stammar, och detta, tidigare okända trekomponent toxin kan bidra till bakteriernas överlevnadsförmåga och patogenicitet i olika miljöer och värdorganismer. 

De flesta bakterier frisätter sfäriska lipidnanostrukturer, extracellulära membranvesiklar, som kan spela många biologiska roller. Tidigare visades Escherichia coli frisätta fysiologiskt aktivt cytolysin A (ClyA) via yttre membranvesiklar (OMV). ClyA, den första medlemmen i en familj av bakteriella alfa-porbildande proteiner, har blivit en modell för hur oligomerisering och porbildning sker i membran. clyA-genen är kryptisk i kommensala icke-patogena E. coli bakterier som inte uppvisar någon cytolytisk aktivitet. Vi fann att den sublytiska koncentrationen av ClyA frisatt via OMVs av icke-patogena E. coli påverkade värdceller på ett påtagligt sätt. ClyA+ OMVs internaliserades snabbt i tjocktarmscancerceller genom makropinocytos och clathrin-medierad, dynaminberoende endocytos. Detta OMV-associerade ClyA orsakade minskade nivåer av canceraktiverande proteiner som EZH2, H3K27me3, CXCR4, STAT3 och MDM2 via EZH2/H3K27me3/miR622/CXCR4- signalaxeln. Uppenbarligen kan sublytiska nivåer av ClyA i OMV från icke-patogena E. coli modulera epigenetik genom effekter som påverkar EZH2-proteinstabilitet och vi antog att E. coli i mikrobiom hos individer med kolorektalcancer företrädesvis kan sakna detta protein. Med tanke på vår nuvarande förståelse av ClyA-interaktioner i cancercellssignalering, kommer det att bli intressant att avgöra om och hur statusen för clyA-lokuset är involverat i etiologin för kolorektal cancer. 

Place, publisher, year, edition, pages
Umeå: Umeå University , 2023. , p. 138
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2250
Keywords [en]
bacteria-host interaction, motility-associated killing factor A, outer membrane vesicles, cytolysin A, phosphatidylinositol 4-phosphate 5-kinase alpha, enhancer of zeste homologue 2, epigenetic modulation
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology; Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-208208ISBN: 978-91-8070-091-7 (print)ISBN: 978-91-8070-092-4 (electronic)OAI: oai:DiVA.org:umu-208208DiVA, id: diva2:1756365
Public defence
2023-06-09, Major Groove (Byggnad 6L), 6K och 6L, Sjukhusområdet, Umeå universitet, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 2018-02914The Kempe Foundations, JCK-1728Swedish Cancer Society, 2017-419Available from: 2023-05-17 Created: 2023-05-11 Last updated: 2023-05-11Bibliographically approved
List of papers
1. Bacterial protein MakA causes suppression of tumour cell proliferation via inhibition of PIP5K1α/Akt signalling
Open this publication in new window or tab >>Bacterial protein MakA causes suppression of tumour cell proliferation via inhibition of PIP5K1α/Akt signalling
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2022 (English)In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 13, no 12, article id 1024Article in journal (Refereed) Published
Abstract [en]

Recently, we demonstrated that a novel bacterial cytotoxin, the protein MakA which is released by Vibrio cholerae, is a virulence factor, causing killing of Caenorhabditis elegans when the worms are grazing on the bacteria. Studies with mammalian cell cultures in vitro indicated that MakA could affect eukaryotic cell signalling pathways involved in lipid biosynthesis. MakA treatment of colon cancer cells in vitro caused inhibition of growth and loss of cell viability. These findings prompted us to investigate possible signalling pathways that could be targets of the MakA-mediated inhibition of tumour cell proliferation. Initial in vivo studies with MakA producing V. cholerae and C. elegans suggested that the MakA protein might target the PIP5K1α phospholipid-signalling pathway in the worms. Intriguingly, MakA was then found to inhibit the PIP5K1α lipid-signalling pathway in cancer cells, resulting in a decrease in PIP5K1α and pAkt expression. Further analyses revealed that MakA inhibited cyclin-dependent kinase 1 (CDK1) and induced p27 expression, resulting in G2/M cell cycle arrest. Moreover, MakA induced downregulation of Ki67 and cyclin D1, which led to inhibition of cell proliferation. This is the first report about a bacterial protein that may target signalling involving the cancer cell lipid modulator PIP5K1α in colon cancer cells, implying an anti-cancer effect.

Place, publisher, year, edition, pages
Springer Nature, 2022
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-201753 (URN)10.1038/s41419-022-05480-7 (DOI)000895373300001 ()36473840 (PubMedID)2-s2.0-85143300255 (Scopus ID)
Funder
Swedish Research Council, 2018-02914Swedish Research Council, 2019-01720Swedish Research Council, 2019-01318Swedish Research Council, 2016-05009Swedish Cancer Society, CAN-2017-419Swedish Cancer Society, 2020-711Swedish Cancer Society, CAN-2017-381The Kempe Foundations, JCK-1728The Kempe Foundations, SMK-1553The Kempe Foundations, JCK2931.1U9Malmö University
Available from: 2022-12-21 Created: 2022-12-21 Last updated: 2023-09-05Bibliographically approved
2. Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA
Open this publication in new window or tab >>Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA
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2021 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 17, no 3, article id 1009414Article in journal (Refereed) Published
Abstract [en]

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.

Place, publisher, year, edition, pages
Public Library of Science, 2021
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-181991 (URN)10.1371/journal.ppat.1009414 (DOI)000631027700007 ()33735319 (PubMedID)2-s2.0-85103129339 (Scopus ID)
Available from: 2021-04-12 Created: 2021-04-12 Last updated: 2023-05-11Bibliographically approved
3. A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion
Open this publication in new window or tab >>A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion
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2021 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 47, article id e2111418118Article in journal (Refereed) Published
Abstract [en]

Vibrio cholerae, responsible for outbreaks of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe how the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto-unrecognized genomic island. The proteins MakA/B/E can form a tripartite toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the cytolytic activity of the Mak proteins was obtained by X-ray crystallography. Flagellum-facilitated secretion ensuring spatially coordinated delivery of Mak proteins revealed a role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-191257 (URN)10.1073/pnas.2111418118 (DOI)000727697700014 ()34799450 (PubMedID)2-s2.0-85121209218 (Scopus ID)
Funder
Swedish Research Council, 2016-05009Swedish Research Council, 2018-02914Swedish Research Council, 2019-01720Swedish Research Council, 2007-08673The Kempe Foundations, SMK-1756.2The Kempe Foundations, SMK-1553The Kempe Foundations, JCK-1728Swedish Cancer Society, 2017-419The Kempe Foundations, SMK-1961Swedish Research Council
Available from: 2022-01-12 Created: 2022-01-12 Last updated: 2023-05-11Bibliographically approved
4. Sublytic activity of a pore-forming protein from commensal bacteria causes epigenetic modulation of tumor-affiliated protein expression
Open this publication in new window or tab >>Sublytic activity of a pore-forming protein from commensal bacteria causes epigenetic modulation of tumor-affiliated protein expression
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cytolysin A (ClyA) is a pore-forming protein expressed at sublytic levels by a strongly silenced gene in non-pathogenic Escherichia coli, including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction, the ClyA-expressing bacteria display a cytolytic phenotype. However, it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli-host interactions in vivo. Here, we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and can affect host cells in a profound and remarkable manner. OMVs isolated from ClyA+ E. coli were rapidly internalised into cultured colon cancer cells. The OMV-associated ClyA inhibited the expression of cancer-activating proteins such as H3K27me3, CXCR4, STAT3, and MDM2 via the EZH2/H3K27me3/miR622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non-pathogenic E. coli can target the stability of the EZH2 protein to modulate epigenetics of colon cancer cells 

Keywords
outer membrane vesicles, pore-forming toxin ClyA, cancer cell epigenetics, non-pathogenic E. coli
National Category
Microbiology in the medical area
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:umu:diva-208205 (URN)
Available from: 2023-05-11 Created: 2023-05-11 Last updated: 2023-05-11

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