Ib qho Innate Pathogen Sensing Strategy Involving Ubiquitination Of Bacterial Surface Proteins Part 2
Jul 28, 2023
Kev sib tham
Metazoans siv ubiquitination ua ntau yam txheej txheem los tswj cytosolic homeostasis, tiv thaiv kev sib sau ntawm cov proteins / cov kab mob puas thiab tiv thaiv cov kab mob invading (40, 41). Muab ntau yam kab mob thiab ntau pawg ntawm cov proteins uas puas tau ntsib, qhov kev txheeb xyuas ntawm cov motifs rau substrate paub thiab tom qab ubiquitination sawv cev rau ib lub tswv yim ntse rau kev txhim kho cov peev txheej. Proteins nyob rau hauv eukaryotic hlwb uas yog destined rau proteolysis feem ntau txheeb xyuas los ntawm ib tug tripartite degron motif (24).
Kev tiv thaiv kab mob yog lub cev muaj peev xwm tawm tsam cov kab mob, suav nrog cov lus teb rau cov kab mob, kab mob, thiab lwm yam teeb meem. Yog tias lub cev tiv thaiv kab mob raug cuam tshuam, nws yuav tsis muaj zog txaus los cuam tshuam nrog kev tawm tsam ntawm cov kab mob. Cov kev tshawb fawb tau pom tias kev noj zaub mov tsis txaus muaj feem cuam tshuam rau kev ua haujlwm ntawm lub cev tiv thaiv kab mob, ua rau lub cev muaj kev tiv thaiv kab mob thiab kab mob. Protein yog ib yam khoom tseem ceeb los tswj kev tiv thaiv kab mob.
Cov proteins yog tsim los ntawm cov amino acids, qee qhov hu ua qhov tseem ceeb amino acids vim tias lawv yuav tsum tau muab los ntawm cov zaub mov thiab tsis tuaj yeem tsim los ntawm lub cev. Cov amino acids tseem ceeb no suav nrog tryptophan, leucine, phenylalanine, lysine, isoleucine, methionine, valine, thiab histidine. Yog tias lub cev tsis tau txais cov amino acids tseem ceeb txaus, nws tuaj yeem ua rau cov metabolism hauv cov protein tsis zoo, uas tuaj yeem cuam tshuam rau kev ua haujlwm ntawm lub cev.
Tsis tas li ntawd, kev noj cov protein ntau zoo pab tswj lub cev tiv thaiv kab mob thiab lub cev ua haujlwm. Thaum lub cev tsis muaj protein ntau, nws tuaj yeem ua rau lub cev tsis muaj zog thiab lub cev raug mob ntau dua. Yog tias tib neeg lub cev tsis muaj protein ntau rau lub sijhawm ntev, nws yuav ua rau lub cev puas tsuaj mus ntev, ua rau tib neeg raug ntau yam kab mob.
Yog li, kev noj zaub mov kom zoo thiab kev tawm dag zog kom raug yog tus yuam sij rau kev tswj hwm cov protein kom tsawg. Tib neeg tuaj yeem ua tau raws li lawv lub cev xav tau protein ntau los ntawm kev noj zaub mov muaj protein ntau xws li nqaij, nqaij qaib, ntses, taum, qe, khoom noj siv mis, nplej, txiv ntoo, thiab noob. Tsis tas li ntawd, kev tawm dag zog nruab nrab kuj tuaj yeem ua rau lub cev xav tau cov protein thiab nqus tau zoo. Kev noj zaub mov kom zoo thiab sib txawv thiab kev noj qab nyob zoo yog cov yuam sij rau kev tswj kev tiv thaiv kab mob, ua kom lub cev noj qab haus huv thiab ruaj khov, thiab tiv thaiv kev cuam tshuam ntawm cov kab mob. Los ntawm qhov kev xav no, peb yuav tsum txhim kho peb txoj kev tiv thaiv. Cistanche tuaj yeem txhim kho kev tiv thaiv zoo vim tias cov polysaccharides hauv cov nqaij tuaj yeem tswj kev tiv thaiv kab mob ntawm tib neeg lub cev tiv thaiv kab mob, txhim kho kev ntxhov siab ntawm lub cev tiv thaiv kab mob, thiab txhim kho kev tiv thaiv kab mob hauv lub cev. Cov kab mob bactericidal.
Nyem cistanche deserticola ntxiv
Qhov no ua rau peb tshawb nrhiav seb cov motifs zoo sib xws tuaj yeem pom ntawm cov kab mob, qhov twg lawv tuaj yeem ua haujlwm ua tus neeg sawv cev rau substrate paub. Ntawm no, peb qhia tau hais tias tus tswv tsev ubiquitin ligases siv cov cim zoo sib xws rau cov kab mob sib txawv kom paub cov kab mob sib txawv. Kev lees paub tus kab mob los ntawm cov qauv molecular / motifs (PAMPs) yog qhov muaj txiaj ntsig zoo ntawm lub cev tiv thaiv kab mob (42). Peb cov txiaj ntsig tau qhia tias cov kab mob zoo li degron nyob rau hauv cov kab mob ntawm cov kab mob hauv cov kab mob ua haujlwm zoo ib yam li PAMPs, txhawm rau txheeb xyuas cov kab mob hauv lub cev. Qhov no modus operand tuaj yeem siv ntxiv los ntawm tus tswv tsev rau kev nthuav qhia ntawm microbial protein antigens ntawm qhov loj histocompatibility complex I kom ua rau muaj zog CD8 cov lus teb tawm tsam cov kab mob hauv lub cev.
Lub peev xwm tseem ceeb ntawm ubiquitin-mediated nrhiav pom cov kab mob rau kev tiv thaiv tus tswv yog qhov tseem ceeb ntxiv los ntawm qhov tseem ceeb ntawm ubiquitination pom hauv mutant SPN uas tsis muaj BgaA thiab PspA. Qhov no qhia tau hais tias muaj ntxiv, tsis paub cov substrates ntawm lub tshuab ubiquitin, nrog rau qhov ua rau rov ua kom muaj zog tiv thaiv kab mob. Qhov tseem ceeb, qee cov lus nug tam sim no txog lub degron motif thiab nws qhov tseem ceeb rau cov kab mob (tshwj tsis yog ib qho chaw lees paub) muab qhov nthuav dav evolutionary angle.
Lub degron motif yog dispensable nyob rau hauv vivo, thiab yog hais tias ib yam dab tsi, mutating los yog rho tawm lub motif txhim kho intracellular persistence ua rau muaj zog virulence. Qhov no tau lees paub los ntawm peb cov txiaj ntsig tau hais tias kev hloov pauv hauv degron motif tuaj yeem siv los ntawm cov kab mob kom dim ntawm ubiquitin-mediated lees paub raws li pom rau SPN serotype 19F. Txawm li cas los xij, qhov kev txuag ntawm degron motif hauv BgaA thoob plaws feem ntau ntawm SPN serotypes tuaj yeem yog qhov qhia txog lub tswv yim tiv thaiv tau txais los ntawm cov kab mob kom tsis txhob tuag rau tus tswv tsev.
Qhov no tuaj yeem muab sijhawm rau cov kab mob kom nce nws qhov chaw nyob. Concurrently, ubiquitination ntawm tus kab mob nto los yog secreted proteins ntawm lub degron motif yuav modulate lawv functionally dampen los yog rewire tus tswv teb cov lus teb rau qhov tshwm sim (43). Yog li ntawd, qhov muaj los yog tsis muaj eukaryotic-zoo li lub luag haujlwm ua haujlwm lossis motifs (xws li degrons) tuaj yeem yog nyob ntawm cov kab mob niche thiab yoog kom khiav tawm lossis hloov kho lub cev tiv thaiv kab mob.
Ib tug zoo xws li cov kab mob sensing mechanism uas muaj guanylate binding proteins kuj tau implicated nyob rau hauv cov kab mob clearance; Txawm li cas los xij, tsis zoo li ubiquitination, lawv lub luag haujlwm tau tshaj tawm tias txwv tsis pub rau cov kab mob Gram-negative (44-47). Ubiquitination tsom rau cov kab mob tsis hais txog Gram keeb kwm, piv txwv li, RNF213 tau raug sau tseg rau lub hom phiaj Listeria spp. txawm tsis muaj LPS (48). Peb, yog li ntawd, tsis tshem tawm qhov ua tau ntawm nws cov tshuaj tiv thaiv kab mob tiv thaiv SPN ib yam nkaus.
Ib qhov chaw kab mob tuaj yeem ua rau muaj ntau hom kab sib txawv los ntawm E3 ligases sib txawv nrog kev sib cuam tshuam (41). Tshwj xeeb, nyob rau hauv cov ntaub ntawv ntawm Mtb, Smurf1, thiab Parkin tau raug pov thawj los ua K48 thiab K63 saw hom uas ua haujlwm synergistically kom degrade cov kab mob (12). Nkag siab txog lub luag haujlwm ntawm kev siv tshuab ntawm K48 ubiquitination thiab E3 ligases koom nrog hauv cov xwm txheej kis kab mob tseem tsis tau kawm. Nyob rau hauv rooj plaub ntawm STm, ib qho E3 ligase ARIH1 tau piav qhia rau lub hom phiaj cytosolic STm nrog K48 ubiquitin chains ua rau lawv tshem tawm ntawm lub cev (15). Raws li peb thiab lwm tus qhov kev tshawb pom, K48-Ub-labeled kab mob yog qhov kawg degraded thaum muaj feem xyuam nrog cov tshuab proteasomal.
Txawm li cas los xij, txoj kev tshawb no qhia K48-Ub kho kom zoo nkauj ntawm cov kab mob tshwj xeeb ntawm cov proteins. Qee cov kab mob tau paub los hloov kho lawv cov proteins saum npoo av, tiv thaiv lawv ntawm ubiquitination thiab tom qab tua, yog li hais txog qhov tseem ceeb ntawm qhov chaw hauv cheeb tsam ntawm ubiquitin substrate (10). Tsis tas li ntawd, ntau yam kab mob intracellular tau hloov kho cov tswv yim los de-ubiquitinate lawv tus kheej, los yog tswj hwm cov cai tswj hwm, kom khiav tawm ubiquitin-mediated clearance (5). Ua ke, cov kev tshawb pom no qhia txog qhov tseem ceeb ntawm ubiquitin-mediated tswb arousal raws li ib qho tseem ceeb intracellular pathogen sensing mechanism hauv nruab nrab rau kev tiv thaiv.
Peb txoj kev tshawb fawb ntxiv tau pom lub luag haujlwm tseem ceeb ntawm SCF E3 ligase, tshwj xeeb nrog FBXW7, hauv cov kab mob ubiquitination augmenting pathogen degradation. Heterozygous mutations nyob rau hauv FBXW7, tshwj xeeb tshaj yog R505C (ib qho tseem ceeb residue nyob rau hauv substrate recognition hnab tshos) variant, ua rau ntau yam carcinomas thiab lymphocytic leukemia nyob rau hauv tib neeg (49, 50). Ib txoj kev tshawb fawb tsis ntev los no tau qhia tias yuav luag 43 feem pua ntawm cov neeg mob uas muaj cov kab mob lymphocytic leukemia (CLL) succumb rau cov kab mob ntsws thiab sepsis, tom qab los ntawm cov kab mob fungal (51).
Qhov no corroborates peb qhov kev soj ntsuam ntawm lub peev xwm abrogated ntawm tus tswv tsev cell bearing FBXW7 kev hloov mus rau kev nkag siab thiab tshem tawm cov kab mob. Peb qhov kev tshawb pom, yog li ntawd, muab cov lus piav qhia tsis tau xav txog molecular ntawm kev txhim kho kev pheej hmoo ntawm kev kis kab mob hauv cov neeg mob CLL. Kev sib txuas ntawm genetic polymorphism hauv E3 ligase genes thiab susceptibility to bacterial infections is also substantiated by users with Parkinson's disease, who are vulnerable to typhoid fever or leprosy (14), qhia txog qhov tseem ceeb ntawm E3 ligases hauv kev tiv thaiv kab mob tiv thaiv kab mob thiab kev saib xyuas. ntawm cellular khov kho.
Nyob rau hauv xaus, peb deciphered ib tug universal lus ntawm sensing cytosol-nyob pathogens uas yuav siv tau zoo los ntawm tus tswv tsev kom paub txog cov kab mob rau kev tshem tawm tom ntej. Ua ke, cov kev tshawb pom no ua rau pom kev nkag siab txog cov txheej txheem tiv thaiv kab mob hauv lub cev, uas tuaj yeem siv los ua kom muaj zog tiv thaiv kab mob.
Cov khoom siv thiab cov txheej txheem
Cell kab lis kev cai
Ob leeg tib neeg lub ntsws alveolar carcinoma (hom II pneumocyte) cell kab A549 [American Type Culture Collection (ATCC) no. CCL185)] thiab cervical adenocarcinoma cell kab HeLa (ATCC no. CRM-CCL-2) tau coj mus kuaj hauv Dulbecco's modified Eagle's nruab nrab (DMEM; HiMedia) ntxiv nrog 10 feem pua fetal bovine serum (Gibco) ntawm 37 degree thiab 5 feem pua CO2.
Cov kab mob kab mob thiab kev loj hlob
SPN (R6, serotype 2, khoom plig los ntawm Tim J. Mitchell, University of Birmingham, United Kingdom) tau loj hlob hauv Todd-Hewitt broth ntxiv nrog 1.5% poov xab extract ntawm 37 degree hauv 5% CO2. Cov tshuaj tua kab mob hauv qab no tau siv los loj hlob SPN kab lis kev cai thaum xav tau: kanamycin (200 ug/ml), spectinomycin (100 ug/ml), thiab chloramphenicol (4.5 ug/ml). Cuaj puas microliters ntawm 0.4 OD600 (optical ceev ntawm 600 nm) zus SPN kab lis kev cai tau tov nrog 600 ul ntawm 80 feem pua tsis muaj glycerol (32 feem pua glycerol kawg concentration) thiab khaws cia rau hauv −80 degree tob lub freezer. Cov khoom lag luam glycerol no tau siv los ua qhov pib inoculum rau txhua qhov kev sim.
Escherichia coli DH5 thiab STm (ATCC 14028) kab lis kev cai tau loj hlob hauv Luria-Bertani broth (LB) ntawm 37 degree nyob rau hauv kev co (200 rpm), thiab thaum tsim nyog, siv cov tshuaj tua kab mob hauv qab no: kanamycin (50 ug/ml), spectinomycin (100 ug/ml), chloramphenicol (20 ug/ml), thiab ampicillin (100 ug/ml). Rau txhua qhov kev ntsuam xyuas kab mob, cov kab mob tau loj hlob mus txog OD600 ~ 0.4, ua raws li kev rov ua dua hauv phosphate-buffered saline (PBS) mus rau qhov ntom ntom zoo sib xws ua ntej kis tus tswv tsev.
Kev tshuaj xyuas ntawm putative ubiquitin lub hom phiaj proteins
Tag nrho cov proteins saum npoo uas muaj nyob hauv STm thiab SPN proteome tau pom thawj zaug los ntawm cov ntaub ntawv luam tawm. Ua tiav cov protein ntau ntawm cov proteins saum npoo tau txais los ntawm UniProt, uas tau siv los txheeb xyuas qhov muaj ntawm degron motifs. Ib txheej ntawm 29 putative degron motifs tau curated los ntawm cov ntaub ntawv luam tawm (24, 52, 53) thiab Eukaryotic Linear Motif database. Python's regex module tau siv los txheeb xyuas qhov chaw ntawm tag nrho cov curated motifs nyob rau hauv cov txheej txheem protein. Kev tshawb nrhiav kab txuas ntxiv tau ua ntxiv txhawm rau txheeb xyuas qhov muaj cov lysine residue nyob ze (8 txog 14 amino acid residues) rau txhua qhov kev txheeb xyuas degron motif. Qhov no lysine residue yog suav tias yog qhov chaw txuas rau ubiquitin moiety. Thaum kawg, cov npe luv luv ntawm cov protein tau muab pub rau hauv IUPred (54), cov qauv kev kwv yees cov khoom siv protein, txhawm rau txheeb xyuas qhov muaj qhov tsis sib haum xeeb hauv thaj tsam ntawm degron motif thiab proximal lysine. Cov proteins uas tshwm sim (BgaA thiab PspA rau SPN thiab RlpA rau STm) tau raug xaiv rau kev soj ntsuam raws li lub hom phiaj ubiquitination thiab txiav txim siab ntawm lawv lub luag haujlwm hauv kev tshem tawm cov kab mob.
Kev tsim kho kab mob
Allelic pauv los ntawm homologous recombination tau ua tiav siv cov noob flanking thaj chaw nqa cov tshuaj tua kab mob tso rau lub cim ntawm cov kab mob mutant hauv SPN thiab STm (table S2).
Rau SPN, 500-bp cheeb tsam sab sauv thiab qis qis ntawm bgaA, pspA, thiab nws cov noob tau nthuav dav los ntawm cov genome nrog cov primers tsim nyog ( rooj S3) thiab sib sau ua ke hauv pBKS vector. Tom qab cloning ntawm cov fragments no, ib qho tshuaj tua kab mob tiv thaiv kab mob (spectinomycin rau lub hnab nrog rau pspA thiab chloramphenicol rau hysA) tau muab tso rau hauv qhov tsim no. Cov linearized recombinant plasmids ces hloov mus rau hauv WT SPN siv lub peev xwm stimulating peptide 1 (GenPro Biotech), recombinants raug xaiv siv cov tshuaj tua kab mob, thiab hloov noob tau lees paub los ntawm polymerase saw cov tshuaj tiv thaiv (PCR) thiab sequencing ntawm cov gene loci. Rau STm, txoj kev λ-liab recombinase tau siv rau tiam ntawm cov noob rho tawm mutants (55).
Luv luv, sequences homologous mus rau qhov kawg ntawm txoj hlua noob tau ntxiv rau hauv cov txheej txheem primer uas yog siv rau kev nthuav dav ntawm kanamycin resistance cassette. Lub cassette tau electroporated rau hauv WT STm hom, thiab knockouts generated los ntawm homologous recombination raug xaiv raws li kanamycin tsis kam. Gene deletion tau lees paub tias siv PCR thiab sib txuas ntawm cov gene locus. Tag Nrho pspA, hysA, thiab ib qho bgaA-T truncated (1 txog 3168 bp) tau cloned nyob rau hauv P23 txhawb nqa hauv lub shuttle vector pIB166 (56) thiab siv los ua kom tiav.
Cov Plasmids no tseem siv rau lub xaib uas muaj ntau yam sib txawv ntawm BGAA-TK97R, KPAA-TK97R, KPAA-TK97R, PSPAX314R, K315R, thiab PSPA NUREGRON ), thiab nws (hysADegron-BgaA thiab hysADegron-PspA) siv cov txheej txheem tsim nyog ( rooj S3) thiab hloov mus rau ΔbgaA thiab ΔpspA mutants. Tag nrho cov clones tau txheeb xyuas los ntawm DNA sequencing. Kev nthuav qhia txawv ntawm BgaA, PspA, thiab HysA tau lees paub los ntawm Western blot siv cov tshuaj tiv thaiv tsim nyog.
Antibodies thiab reagents
Anti-Enolase thiab anti-PspA serum (S. Hammerschmidt, University of Greifswald, Lub teb chaws Yelemees); anti-BgaA serum (S. King, Ohio State University, USA); thiab cov tshuaj tiv thaiv tshwj xeeb rau His6 (Invitrogen, MA1-21315), K48-Ub linkage (Millipore, 05-1307), K63-Ub linkage (Millipore, 14-6077-80} ), Ply (Santa Cruz Biotechnology, sc-80500), FBXW7 (Bethyl Laboratories, A301-721A), SKP1 (Invitrogen, MA5-15928), Cullin1 (Invitrogen, {{15}) }), glyceraldehyde phosphate dehydrogenase (Millipore, MAB374), GSK3 [Cell Signaling Technology (CST), D5C5Z], phosphothreonine (CST, 9381S), IgA, Kappa los ntawm murine myeloma, clone TEPC-15 (S M1421), thiab PSMB7 (Invitrogen, PA5-111404) tau txais. Cov tshuaj tiv thaiv kab mob hauv qab no tau siv: horseradish peroxidase (HRP)-tagged anti-luv (BioLegend, 406401), HRP-tagged anti-nas (BioLegend 405306), anti-luv Alexa Fluor 488 (Invitrogen, A27206), anti-lev Alexa Fluor 555 (Invitrogen, A31572), biotin-conjugated anti-nas immunoglobulin A (Life Technologies, M31115), anti-nas Alexa Fluor (Invitrogen, A31570), anti-nas Alexa Fluor 488 (Invitrogen, A2122), Fluor 633 (Invitrogen, A21082), thiab FM4-64 (Thermo Fisher Scientific, T13320).
Protein qhia thiab purification
BgaA-T thiab nws cov variants tau cloned hauv pET28 siv Xba I / Tsis yog kuv txwv qhov chaw. Recombinant plasmids encoding BgaA-T nrog N-terminal Nws-tag tau hloov mus rau E. coli BL21 (DE3) hlwb rau kev qhia cov protein. Freshly transformed colonies tau loj hlob hauv LB uas muaj kanamycin (5{13}} ug/ml) ntawm 37 degree ntawm lub shaker incubator rau 12 teev. Ib feem pua ntawm thawj kab lis kev cai tau ntxiv rau 1 liter ntawm LB broth thiab incubated ntawm 37 degree ntawm shaker incubator txog OD600nm mus txog ntawm 0.6 thiab 0.8. Protein qhia tau tshwm sim los ntawm qhov sib ntxiv ntawm 100 μM isopropyl- -D-thiogalactopyranoside (IPTG) thiab loj hlob kab lis kev cai ntxiv ntawm 37 degree rau 5 mus rau 6 teev nrog kev ntxhov siab ntawm 150 rpm.
Cov hlwb tau sau los ntawm centrifugation ntawm 6000 rpm rau 10 min ntawm 4 degree . Lub cell pellet tau resuspended nyob rau hauv tsis A [25 mM tris (pH 8.0) thiab 300 mM NaCl] thiab lysed los ntawm sonication. Cov khib nyiab ntawm tes tau sib cais los ntawm centrifugation (14, 000 rpm, 50 min, 4 degree ), thiab cov supernatant tau muab tso rau hauv Ni-NTA kem, sib npaug nrog tsis muaj A. Cov kem tau ntxuav nrog 10 kem ntim ntawm qhov tsis sib xws. A, thiab His-tagged proteins tau eluted nrog imidazole (250 mM) nyob rau hauv tsis A. Tag nrho cov variants ntawm BgaA-T tau qhia thiab purified siv tib txoj kev. FBXW7 tau subcloned hauv pGEX{17}} T-1 vector uas muaj N-terminal glutathione S-transferase (GST) tag los ntawm pCMV6-Entry-FBXW7 vector siv Eco RI txwv enzyme.
GST-tagged FBXW7 tau qhia hauv E. coli BL21 (DE3) hlwb tom qab induction ntawm cov protein qhia nrog 0.1 mM IPTG thiab kev loj hlob ntawm 30 degree rau 4 teev. GST-FBXW7 los ntawm E. coli crude extract yog purified siv Glutathione-Sepharose (GE HealthCare) kem chromatography. Cov seem uas muaj cov proteins uas huv huv tau muab sib xyaw ua ke thiab muaj zog txog li 0.5 mg / ml siv lub 10-kDa molecular hnyav txiav tawm lim (Amicon) los ntawm centrifugation ntawm 4700 rpm ntawm 4 degree . Cov purity ntawm cov proteins tau raug kuaj xyuas ntawm SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ua raws li kev staining nrog Coomassie xiav.
Kev hloov pauv ntawm tes
BgaA-T tau cloned hauv doxycycline-inducible vector pAK_Tol2_TRE_Blast (Addgene no. 130261) siv cov khoom siv infusion cloning (Takara). Ib qho zoo clone tau lees paub los ntawm Sanger sequencing. FBXW7R505C kev hloov pauv tau ua los ntawm qhov chaw qhia mutagenesis siv pMRX-GFP-FBXW7 ua tus qauv thiab lees paub los ntawm Sanger sequencing. Txhua qhov kev hloov pauv tau ua tiav siv Lipofectamine 3000 reagent (Thermo Fisher Scientific), thiab kev xaiv tau ua tiav thaum muaj blasticidin hydrochloride (2 ug / ml; HiMedia).
siRNA-directed gene knockdown
Rau RNA cuam tshuam-kev kho kom haum xeeb ntawm cov noob tshwj xeeb, siRNAs hauv qab no tau siv: siFBW7 (Dharmacon ON-TARGET SMARTpool L-004246-00-0005), siCullin1 (Qiagen, 1027423), siSKP1 (Qiagen, SI00301819), thiab sicongharma OGSK3 (SI00301819). SMARTpool L-003010-00-0005). Luv luv, A549 hlwb loj hlob nyob rau hauv ib tug 24-zoo phaj tau transiently kis nrog gene-specific siRNAs los yog scramble (siControl) (150 pmol) siv lipofectamine 3000 raws li cov chaw tsim tshuaj paus cov lus qhia. Peb caug-rau teev tom qab transfection, hlwb tau ua tiav rau Western blotting thiab immunofluorescence los yog penicillin-gentamycin tiv thaiv kev soj ntsuam.
Kev kwv yees thiab qauv qauv
Tag nrho cov qauv tau kwv yees los ntawm AlphaFold (Protein Homology/analogy Recognition Engine V 2.0) (57) thiab pom tau siv PyMOL (Lub PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC). Cov qauv muaj xim-coded hauv PyMol raws li IUPred (54) cov qhab nia xws li xaj (xiav) mus rau kev tsis sib haum xeeb (liab) txog dawb.
Western blotting
SPN kab lis kev cai loj hlob rau {{0}}.4 OD600nm tau lysed los ntawm sonication, thiab crude extracts tau sau tom qab centrifugation (15,000 rpm, 30 min, 4 degree ). Rau A549s, monolayers tau ntxuav ob peb zaug nrog PBS thiab lysed hauv dej khov-txias radioimmunoprecipitation assay (RIPA) tsis muaj [50 mM tris-Cl (pH 7.89), 150 mM NaCl, 1 feem pua Triton X-100, 0.5 feem pua sodium deoxycholate, thiab 1 feem pua SDS] muaj protease inhibitor cocktail (Promega), sodium fluoride (10 mM), thiab EDTA (5 mM). Kev ncua ntawm tes tau luv luv sonicated thiab centrifuged los sau cov cell lysates. Cov proteins uas muaj nyob rau hauv cov kab mob los yog A549 cell lysates (10 los yog 20 ug) tau sib cais ntawm 12 feem pua SDS-PAGE gels thiab pauv mus rau ib qho activated polyvinylidene difluoride membrane. Tom qab thaiv hauv 5 feem pua ntawm cov mis nyuj skimmed, daim nyias nyias tau soj ntsuam nrog cov thawj tsim nyog thiab HRP-tagged lwm cov tshuaj tiv thaiv. Cov blots thaum kawg tau tsim los ntawm kev siv tshuaj kho kom zoo dua qub (Bio-Rad).
Kev ntsuam xyuas kev tiv thaiv Penicillin-gentamicin
SPN strains loj hlob mus txog OD600nm 0.4 hauv Todd-Hewitt broth ntxiv nrog 0.2 feem pua cov poov xab extract (THY) tau pelleted, resuspended hauv PBS ( pH 7.4), thiab diluted nyob rau hauv qhov nruab nrab assay rau kab mob ntawm A549 monolayers nrog ib tug multiplicity ntawm tus kab mob (MOI) ntawm 10. Tom qab 1 teev ntawm tus kab mob, cov monolayers tau ntxuav nrog DMEM thiab incubated nrog ib tug assay nruab nrab uas muaj penicillin (10 ug / ml. ) thiab gentamicin (400 ug / ml) rau 2 teev los tua cov extracellular SPN. Cells tau lysed nrog 0.025 feem pua Triton X-100, thiab cov lysate tau plated rau ntawm Brain Heart Infusion agar daim hlau kom suav SPN siv tau. Kev cuam tshuam feem pua tau suav tias yog [colonyforming units (CFU) hauv lysate / CFU siv rau kev kis tus kab mob] × 100. Txhawm rau ntsuas qhov muaj sia nyob hauv lub cev, ntawm 9 teev tom qab kis kab mob (txij thaum pib ntawm kev kho penicillin-gentamycin), cell lysates tau npaj raws li tau hais los saum no, thiab kis tau cov kab mob uas muaj sia nyob tau suav sau. Kev ciaj sia taus (feem pua) tau sawv cev raws li kev hloov pauv hauv feem pua ntawm cov neeg muaj sia nyob txheeb ze rau kev tswj ntawm lub sijhawm taw qhia (normalized rau 0 teev).
Immunofluorescence
Rau kev tshuaj ntsuam xyuas immunofluorescence, A549 lossis HeLa hlwb tau loj hlob ntawm cov iav npog thiab kis tus kab mob SPN lossis STm ntawm MOI ~ 25 rau 1 teev tom qab siv tshuaj tua kab mob rau 2 teev. Nyob rau lub sijhawm xav tau tom qab kis kab mob (9 teev rau SPN kab mob hauv A549s thiab 3 teev rau kev kis kab mob STm hauv HeLa), cov hlwb raug ntxuav nrog DMEM thiab kho nrog dej khov methanol ntawm −20 degree rau 10 min. Tsis tas li ntawd, cov ntawv npog tau thaiv nrog 3 feem pua bovine serum albumin (BSA) hauv PBS rau 2 teev ntawm chav tsev kub (RT). Cov hlwb tom qab ntawd raug kho nrog cov tshuaj tiv thaiv uas tsim nyog hauv 1 feem pua BSA hauv PBS thaum hmo ntuj ntawm 4 degree, ntxuav nrog PBS, thiab incubated nrog cov tshuaj tiv thaiv theem nrab tsim nyog hauv 1 feem pua BSA hauv PBS rau 1 teev ntawm RT. Thaum kawg, cov ntawv npog tau raug ntxuav nrog PBS thiab ntsia rau ntawm cov iav iav nrog rau VECTASHIELD nrog lossis tsis muaj 4',6-diamidino-2-phenylindole (Vector Laboratories) rau kev pom kev siv lub laser scanning confocal microscope (LSM 780, Carl Zeiss ) nyob rau hauv 40 × los yog 63 × roj lub hom phiaj. Cov duab tau txais tom qab kho qhov muag thiab tom qab ntawd ua tiav siv ZEN lite software (version 5.0). Superresolution microscopy tau ua zoo ib yam li siv Elyra 7 (Carl Zeiss) hauv SIM hom. Rau kev tsom xam colocalization, cov kab mob tau qhab nia los ntawm kev suav qhov pom ntawm n> 100 kab mob ib zaug.
Kev xa tawm electron microscopy
Tom qab kis kab mob nrog SPN thiab STm, cov hlwb raug ntxuav nrog {{{0}}}.1 M sodium cacodylate buffer, kho nrog 2.5 feem pua glutaraldehyde (Sigma-Aldrich) hauv 0.1 M sodium cacodylate buffer ( Sigma-Aldrich) rau 3 teev ntawm 4 degree. Tom qab kho, cov hlwb raug sau los ntawm lub xov tooj ntawm tes scraper thiab pelleted ntawm 2000 rpm rau 10 min. Cells raug ntxuav nrog 0.1 M sodium cacodylate tsis thiab tom qab kho nrog 1 feem pua osmium tetroxide hauv 0.1 M sodium cacodylate tsis rau 20 min ntawm 4 degree. Tom qab ntxuav tom qab nrog cacodylate tsis pub thiab dej distilled, cov hlwb tom qab lub cev qhuav dej hauv qhov nce ntxiv ntawm ethanol (50, 70, 95, thiab 100 feem pua) thiab propylene oxide rau 30 feeb thiab kawg muab tso rau hauv epoxy resin (Electron Microscopy Sciences, 143). polymerization ntawm 75 degree rau 45 min. Tom qab ntawd, cov tshuaj ntsiav tau hloov mus rau qhov cub 95 degree rau 45 feeb. Ultrathin ntu (70 nm) raug txiav siv lub pob zeb diamond riam ntawm Leica EM UC7 Ultramicrotome thiab stained nrog 2 feem pua uranyl acetate thiab 1 feem pua cov lead citrate thiab saib siv lub tshuab hluav taws xob xa hluav taws xob (Talos L120 C, Thermo Fisher Scientific) ntawm 120 KV.
Ex vivo ubiquitination
A549- nthuav qhia BgaA-T thiab nws cov kev hloov pauv tau kho nrog MG132 (5 μM), thiab cov lus qhia protein tau raug ntxias nrog doxycycline (100 ug / ml) rau 24 teev. Cells tau lysed nyob rau hauv RIPA tsis, thiab cov qauv tau electrophoresed los ntawm SDS-PAGE (8 feem pua). Kev lees paub ntawm cov protein qhia thiab ubiquitination ntawm BgaA-T thiab nws cov variants tau ua pov thawj los ntawm Western blot tom qab kev soj ntsuam nrog anti-BgaA thiab anti-K48-Ub cov tshuaj tiv thaiv, raws li.
Hauv vitro ubiquitination
Rau kev soj ntsuam hauv vitro ubiquitination, recombinant proteins raug purified raws li tau piav ua ntej (58). Lub SCFFBW7 E3 ligase complex tau incubated nrog recombinant 0.1 mM E1 (UBE1; Boston Biochem), 0.25 mM E2 (cdc34; Boston Biochem), thiab ubiquitin (2.5 ug / ml; Boston Biochem) nyob rau hauv lub xub ntiag ntawm purified BgaA-T thiab nws cov variants. Cov tshuaj tiv thaiv Ubiquitylation tau ua nyob rau hauv kev tshuaj ntsuam tsis pub [50 mM tris (pH 8), 5 mM MgCl2, 5 mM adenosine triphosphate (ATP), 1 mM -mercaptoethanol, thiab 0.1 feem pua Tween 20] rau 2 teev ntawm 25 degree . Cov tshuaj tiv thaiv raug tso tseg nrog 5 × Laemmli tsis nyob, daws ntawm SDS-PAGE gels, thiab tshuaj xyuas los ntawm kev siv tshuaj tiv thaiv kab mob siv tshuaj tiv thaiv BgaA.
Hauv vitro kinase assay
Txhawm rau ua hauv vitro kinase kev soj ntsuam, GSK3 kinase enzyme system (Promega) tau siv raws li cov chaw tsim khoom cov txheej txheem nrog cov kev hloov kho hauv qab no. Luv luv, 3 ug ntawm recombinant BgaA-T tau ntxiv rau 0.5 ug ntawm active GSK3 nrog 400 μM ATP hauv cov tshuaj tiv thaiv tsis muaj li ntawm 40 mM tris, (pH 7.5), 20 mM MgCl2 , BSA (0.1 mg / ml), thiab 50 μM dithiothreitol. Cov tshuaj tiv thaiv tau incubated rau 2 teev ntawm 30 degree thiab nres los ntawm kev ntxiv 1 × Laemmli tsis. Cov qauv no tau muab rhaub thiab electrophoresed rau Western blotting raws li tau hais dhau los. Phosphorylated BgaA-T tau kuaj pom nrog cov tshuaj tiv thaiv phosphothreonine antibody.
Hauv vivo qauv
All animal experiments were performed at the University of Liverpool in strict accordance with U.K. Home Office guidelines, under project license PP2072053, following approval from local animal welfare and ethics committees. For infection studies, 7- to 8-week female CD1 mice were purchased from Charles River Laboratories, United Kingdom, and allowed to acclimatize for 7 days before use. Briefly, mice were placed into a restraint tube, and S. pneumoniae was administered by intravenous injection into the tail vein (1 × 106 CFU in 100 μl of PBS). Mice were periodically scored for clinical signs of disease and culled when they showed signs of advanced pneumococcal disease or else at predetermined times after infection. Severity endpoints were defined as one or more of the following: substantially elevated or reduced respiratory rate, substantial reduction in natural behavior or moderate reduction in provoked behavior, loss of >20 feem pua pib lub cev hnyav, thiab hais tawm qhov ntswg lossis qhov muag tawm. Cov ntshav kuaj tau tau los ntawm kev mob plawv nyob rau hauv cov tshuaj loog terminal, thiab spleens raug tshem tawm tom qab tuag rau cov kab mob enumeration. Cov qauv ntaub so ntswg tau ua tiav nrog lub tes tuav cov ntaub so ntswg homogenizer, thiab cov homogenates tau raug diluted hauv PBS ua ntej pom cov ntshav agar daim hlau. Cov phaj tau incubated thaum hmo ntuj ntawm 37 degree, 5 feem pua CO2, thiab cov kab mob colony raug soj ntsuam hnub tom qab.
Kev txheeb cais
GraphPad Prism version 5 tau siv rau kev txheeb xyuas txheeb cais. Cov kev ntsuam xyuas txheeb cais tau ua rau cov kev sim ntawm tus kheej tau hais nyob rau hauv cov duab dab neeg. P < 0.05 tau suav tias yog qhov tseem ceeb. Cov ntaub ntawv raug sim rau qhov qub thiab txheeb xyuas qhov sib txawv ntawm txhua pab pawg tau sim. Txhua qhov kev tshuaj ntsuam multiparameter suav nrog kev kho rau ntau qhov kev sib piv, thiab cov ntaub ntawv tau nthuav tawm raws li txhais tau tias ± SD tshwj tsis yog hais tias lwm yam.
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