脆弱拟杆菌-课件

脆弱拟杆菌

Bacteroides

fragilisBacteroides

fragilis

概述

生物学特性与疾病相关性

健康相关性

文献报告2Taxonomy•

真细菌Eubacteria•

放线菌门Actinobacteria

放线菌纲放线菌目•

棒状杆菌亚目•

分枝杆菌科•

诺卡菌科•

棒状杆菌科长链分枝酸，抗酸阳性中长分枝酸，抗酸弱阳性短链分枝酸，抗酸阴性G+•

放线菌亚目放线菌科•

链霉菌亚目链霉菌科•

厚壁菌门Firmicutes无分枝酸，抗酸阴性•

杆菌纲Bacilli：葡萄球菌，链球菌，乳杆菌科，肠球菌科，芽孢杆菌，李斯特菌•

荚膜梭菌纲Clostridia•

柔膜菌纲Mollicutes：支原体，脲原体

无细胞壁，质膜含胆固醇•

变形菌门Proteobacteria•

-变形菌纲：立克次体目，巴通体，布鲁菌•

-变形菌纲：奈瑟菌，伯克菌，鲍特菌•

-变形菌纲：肠杆菌科，嗜血杆菌，军团菌，不动杆菌，假单胞菌，弧菌•

-变形菌纲：螺杆菌，弯曲菌G-•

梭杆菌门Fusobacteria•

螺旋体门Spirochaetes•

衣原体门Chlamydiae•

拟杆菌门Bacteroidetes•

拟杆菌纲拟杆菌目：卟啉单胞菌科，普雷沃菌科，拟杆菌科拟杆菌属3Bacteroides

genus•

=Bacteroidesfragilis

group(BFG)•

~20species•

Type

species:B.fragilis•

G-,obligateanaerobic

(?),motile

ornon-motilerods•

One

ofthemostabundantbacteriaingutfloraandfeces•

The

mostimportantandmostcommonly

foundanaerobes

inhuman

infections,associatedwithmortality4Bacteroides

fragilis

脆弱拟杆菌•拟杆菌属代表种，研究最多•人群肠道定植率高，但存在地区、种族等差异•肠道菌群中不占明显优势，即使在拟杆菌属中通常也非优势种，且所占比例受食物组成等因素影响•但在肠外厌氧菌感染中最常被分离到•可能是肠外“毒力”最强的肠道菌群成员5肠道正常菌群肠外机会致病

肠道壁物理损伤（肠道炎症、肿瘤，腹腔手术、结肠镜检查…）

肠道菌逸出（混合感染），异位定植于腹腔等处

炎症反应导致脓肿

(abscess)形成

脓肿增大可致肠梗阻，侵蚀血管可产瘘

脓肿破裂可导致菌血症，感染扩散腹腔脓肿中B.

fragilis分离率可高达80%6Bacteroides

fragilis

概述

生物学特性与疾病相关性

健康相关性

文献报告7B.fragilis

特殊生物学性状•

Aerotolerance

耐氧•Spingolipid

synthesis

鞘脂合成•LowendotoxityLPS

低内毒素活性脂多糖•Zwitterionicpolysacchridecapsule

两性离子多糖荚膜•BFT

外毒素•

Antibioticresistance

抗生素抗性拟杆菌属其它种亦可能具备某些上述性状8细菌与氧•

Aerobesandanaerobes•Oxygenrequirement:

terminaloxidase•High-affinity

oxidase:

microxic/nanoxic

respiration•Low-affinity

oxidase:

oxic

respiration•Oxygentolerance:reactiveoxygenspecies(ROS)•ROS:O

+e

O

…-22•SOD:2O

+2H

H

O

+O

,

Catalase:

2H

O-+

22

222

22H2O+

O2•ROS

DNA

andproteindamagerepair9细菌与氧Morris,R.L.and

T.

M.

Schmidt

(2013).

NatRev

Microbiol

11(3):

205-21210Oxygen

in

animalintestineE.coli

expressing

luciferase

reporter

grown

anaerobically

were

injectedwith

luciferase

substrateintoligated

rabbit

ileal

loops.

Luciferase

requiresoxygen

and

ATPtoproduce

detectableluminescence

from

substrate.11Marteyn,

B.,et

al.(2010).

Nature465(7296):

355-358.Oxygen

gradient

in

animalintestineShigella

flexneri

expressing

GFP

reporter

grown

anaerobically

were

injected

intorabbit

ligatedileal

loops.

GFP

covalently

binds

oxygen

to

fold

intoamature,fluorescent

conformation.

Immunofluorescent

imagingofclamped

loops

shows

littleornoGFP

signals

(notshown).12Marteyn,

B.,

et

al.(2010).

Nature465(7296):

355-358.B.fragilis

-

an

aerotolerant

obligate

anaerobe•

SOD+,catalase+•

Keymetabolism

enzymesonlyreversiblyinhibitedby

oxygen•

Some

aerotolerance-related

proteinsareencodedonasingleoperonandinduced

byoxygen13B.fragilis

-

ananaerobe?B.f.

encodes

ahigh-affinity

oxidase

enabling

itto

growin

thepresenceofnanomolar

oxygen.Baughn,

A.D.and

M.

H.Malamy

(2004).Nature

427(6973):

441-444..14B.fragilis

-

ananaerobe?Inactivationofasingle

gene

in

B.f.

enables

growthathigheroxygen

levels.Meehan,

B.M.,etal.

(2012).ProcNatl

Acad

SciUS

A

109(30):

12153-12158.15B.fragilis

-ananaerobe

Fairlylong-termsurvivalinaerated

environment

Quickrecoveryofgrowth

when

oxygenis

nearlyexhausted

B.

f.outgrowscommon

facultative

anaerobes

likeE.

coli

under

anaerobicconditions

How

mightthesepropertiesaffect

colonizationandpathogenesis?16B.fragilis

特殊生物学性状•

Aerotolerance

耐氧•Spingolipid

synthesis

鞘脂合成•LowendotoxityLPS

低内毒素活性脂多糖•Zwitterionicpolysacchridecapsule

两性离子多糖荚膜•BFT

外毒素•

Antibioticresistance

抗生素抗性17Sphingolipid

鞘脂•

真核生物生物膜重要组分及信号分子•

细菌中仅有的鞘脂合成菌：

拟杆菌门

变形菌门-变形菌纲Sphingomonadales目(其中Sphingomonas属无LPS，以糖鞘脂代之)18(GSL)Bacterial

glycolipids

require

APC

processing19/courses/bio141/lecguide/unit6/intro/iNKTcell/microbial_lipids_DC_iNKTcell.htmlGlycolipid

antigens

arepresented

toiNKT20Van

Kaer,L.,

et

al.(2011).

Cell

TissueRes

343(1):

43-55.NKT

modulates

innate

&adaptive

responsesNKT

recognition

of

glycolipid

leads

to

a

cascade

of

events

that

results

in

theactivation

of

APCs

and

many

other

bystander

NK,

T

and

B

cells.

The

effects

rangefrom

Th1-type

responses

to

Th2-type

responses.

In

most

experimental

cases,

theresponse

is

advantageous

to

the

host

(indicated

in

green),

however,

in

some

cases,theresponse

canbedeleterious

(indicated

inred).Godfrey,D.I.

and

S.P.

Berzins(2007).

NatRev

Immunol

7(7):505-518.21B.fragilis

GSL

and

iNKT

activationAn,

D.,etal.(2014).

Cell

156(1-2):

123-133.B.fragilis

-

aGSL-producer

B.

fragilis

GSL

apparentlydoesnotactivateiNKT(Spingomonas

GSL,however,

activatesiNKT)

B.

fragilis

GSL

apparentlyevenantagonizesiNKTactivationbyotherglycolipids

How

mightthesepropertiesaffect

colonizationandpathogenesis?23B.fragilis

特殊生物学性状•

Aerotolerance

耐氧•Spingolipid

synthesis

鞘脂合成•LowendotoxityLPS

低内毒素活性脂多糖•Zwitterionicpolysacchridecapsule

两性离子多糖荚膜•BFT

外毒素•

Antibioticresistance

抗生素抗性24G-bacterial

cellwall25/~caflab/lpxc2.htmLipid

A

structuresLipid

A

isthe‘endotoxic’

partofendotoxin

(LPS).Ogawa

et

al.Frontiers

inBioscience

12,

3795-3812,

May1,

2007.26Toll-likereceptors

(TLRs)27Yang,

L.and

E.Seki

(2012).Frontiers

inPhysiology

3Lipid

A

is

sensedby

TLR428Maeshima,

N.andR.Fernandez(2013).Frontiersin

Cellular

andInfectionMicrobiology

3.Lipid

A

structure

affects

TLR4

agonist

activity29/review-lps-tlr4.Yersiniapestis

lipid

AMatsuura,

M.(2013).

FrontImmunol

4:109.30B.fragilis:

low-endotoxic

lipid

ANon-classicallipid

A

structure•Low

acylation

butlonger

acyl

chains•Lowphosphorylation

Pooractivity

as

TLR4

agonist(mayevenfunctionas

TLR4

antagonist)

Muchlower

endotoxicitycompared

to

e.g.E.

coli

How

mightthesepropertiesaffect

colonizationandpathogenesis?31B.fragilis

特殊生物学性状•

Aerotolerance

耐氧•Spingolipid

synthesis

鞘脂合成•LowendotoxityLPS

低内毒素活性脂多糖•Zwitterionicpolysacchridecapsule

两性离子多糖荚膜•BFT

外毒素•

Antibioticresistance

抗生素抗性32B.fragilis

polysaccharide

capsuleWhite

arrows:

outer

membrane.

Black

arrows:

capsule.Patrick,S.,etal.

(2010).Microbiology

156(Pt

11):

3255-3269.33B.fragilis

polysaccharide

A

(PSA)两性离子多糖

Zwitterionicpolysaccharide(ZPS)重复单元Mazmanian,

S.K.and

D.

L.

Kasper

(2006).NatRevImmunol

6(11):

849-858.34B.fragilis

and

itscapsule•荚膜多糖合成基因突变后出现生长缺陷，持续传代后自发突变回复•B.f.可合成至少8个不同类型荚膜多糖，至少PSA及PSB为ZPS型，其中PSA含量最丰富•合成单一类型荚膜多糖的突变株定植能力差，无法与合成多种类型荚膜多糖的野生株竞争

B.

fragilis

荚膜多糖及其复杂性为生长、定植必须Liu,C.H.,et

al.(2008).

ProcNatl

Acad

Sci

US

A

105(10):

3951-3956.35B.fragilis

capsule

and

abscess

in

mice•腹腔内单独注射纯化荚膜或ZPS可诱发脓肿•静脉注射荚膜多糖或ZPS可诱生免疫保护•过继免疫实验表明过继血清（抗体）不足以提供免疫保护，必须过继脾细胞（细胞免疫），特别是抑制性Treg•无胸腺小鼠、/

TCR基因敲除小鼠腹腔注射ZPS不出现脓肿

B.

fragilis

ZPS通过激活T细胞诱生腹腔脓肿，特异性Treg可抑制脓肿形成36Most

polysaccharides

are

poor

immunogensPatrick,S.,etal.

(2010).Microbiology

156(Pt

11):

3255-3269.37ZPS

ispotent

B-

and

T-cell

immunogenPatrick,S.,etal.

(2010).Microbiology

156(Pt

11):

3255-3269.38Bacteorides

ZPS

is

not

uniqueZPS

hasalso

been

identified

inotherbacteria:•Capsule

ofS.

pneumoniae

type1

(Sp1)•Capsules

andtechnoic

acids

ofcertaintypes

of

S.

aureus•O-antigen

of

Morganella

moganiiOnlyB.

f.

ZPS

hasbeen

linked

toabscess-formation.Schumann,

B.,

etal.(2014).

Chemical

Science

5(5):1992-2002.39ZPS

as

vaccineadjuvantPPV:

polysaccharide

pneumococcal

vaccinePCV:

pneumococcal

conjugated

vaccineFitzgerald

Tet

al.Western

Pacific

Surveillance

and

Response

Journal,

2012,

3(2):33-38.40B.fragilis

特殊生物学性状•

Aerotolerance

耐氧•Spingolipid

synthesis

鞘脂合成•LowendotoxityLPS

低内毒素活性脂多糖•Zwitterionicpolysacchridecapsule

两性离子多糖荚膜•BFT

外毒素•

Antibioticresistance

抗生素抗性41Enterotoxigenic

B.fragilis

(ETBF)•流行病学证据表明家畜腹泻与特定株型B.

f.相关•家畜及实验动物模型中证明培养物及无菌上清可引起腹泻等症状•病理表现为远端回肠及结肠上皮损伤，基底层中性粒细胞浸润•BFT为分泌型锌金属蛋白酶，基因位于~6kb小型毒力岛，后者位于大型接合性转座子中•BFT已发现3种同源异构体，但任一ETBF株仅携带其中一种•BFT可能与真核锌金属蛋白酶具有共同起源42ETBF-induced

murinecolitisSears,

C.L.

(2009).

ClinMicrobiol

Rev

22(2):349-36943BFT-induced

CPEEffectsofBFT

oncultured

HT29/C1

cells

(ahuman

colonic

carcinoma

continuouscell

line).

The

effectsarenot

permanent

and

reverseduponcontinuedculture.Sears,

C.L.

(2009).

ClinMicrobiol

Rev

22(2):349-36944Mechanisms

of

BFT

toxicity•BFT

treatmentresultsincleavageof

plasmamembrane-bound

E-cadherin-catenin

released

fromplasma

membrane

→Wntpathwayactivation→proliferation

(hyperplasia)•MAPK/NF-B

pathway

activation→cytokine

release•Disruptionof

zonulaadherensand

zonulaoccludens(intermediate

andtightjunctions)•IntracellularredistributionofF-actin

Intestinalepithelialcellmorphology

changes

Intestinalepithelial

permeability

increases

Diarrhea,

inflammation,

hyperplasia45ETBF

and

diseases•小鼠中ETBF定植力与NTBF无明显差别•ETBF致C57/B6小鼠急性肠炎转慢性亚临床肠炎•ETBF全球分布，成人无症状携带多见•与成人腹泻相关性不显著，主要见于1-5岁小儿•与炎性肠病IBD、结肠直肠癌CRC关系日益明显•可能参与其它目前病因不明的肠道疾病：肠易激综合征IBS，抗生素相关腹泻AAD，麸质过敏症/乳糜泻CD，新生儿坏死性小肠结肠炎NEC

…46ETBF

enhances

tumor

formationin

ApcMin

miceAPCmin

isamouse

model

for

CRCinwhich

intestinal

neoplasiaspontaneously

occur

as

a

result

of

the

single

remainingApcallelemutating.The

effect

ofETBFis

Th17-dependent.Wu,

S.,etal.

(2009).

NatMed

15(9):101467-1022.B.fragilis

特殊生物学性状•

Aerotolerance

耐氧•Spingolipid

synthesis

鞘脂合成•LowendotoxityLPS

低内毒素活性脂多糖•Zwitterionicpolysacchridecapsule

两性离子多糖荚膜•BFT

外毒素•

Antibioticresistance

抗生素抗性48Antibiotic-resistance

of

B.fragilis

group•

耐药机制丰富，对多类抗生素不敏感•

-内酰胺酶阳性•

氨基糖苷类几乎不吸收•

四环素类拮抗蛋白TetQ及外排泵•

临床用药：-lactam/-lactamaseinhibitor,carbapenem,

clindamycin,metronidazole•

碳青霉烯类、林可酰胺类、硝基咪唑类、喹诺酮类抗性上升中，多重耐药株临床有发现49Antibiotic-resistance

of

B.fragilis

group•

BFG抗药机制繁多•

肠道内BFG之间DNA水平传递频繁•

序列分析显示BFG抗药基因与其它肠道菌、可能偶尔经过肠道的人体其它部位感染/定植菌之间高度同源，提示发生过基因水平传递•

通过外膜囊泡分泌抗性酶，为无抗性共栖菌提供表型抗性

BFG可能是肠道中抗药基因周转中心50Other

features

of

B.

fragilis•

粘附因子：菌毛，凝集素•

酶类神经氨酸酶NA，透明质酸酶，硫酸软骨素酶胆盐水解酶（耐高浓度胆盐）•

溶血素•

外膜囊泡（文献报告）51Culture

of

B.fragilisBacteroidesBileEsculin(BBE)•

TrypticSoy

Agar45.0gm•

Oxbile

(Oxgall)

20.0gm•

Reducing

Agents/PeroxideInhibitors

1.5gm•

Esculin

1.0gm•

Ferric

AmmoniumCitrate0.5gm•

Hemin

12.0mg•

Gentamicin

100.0mg(Perliter)52Bacteroides

fragilis

概述

生物学特性与疾病相关性

健康相关性

文献报告53Benefits

of

B.fragilis

group

to

host•

肠道生境占位硬膜菌门和拟杆菌门为最主要肠道菌群成员Colonization

resistance•

物质能量代谢营养体重54Benefits

of

B.fragilis

to

host•

宿主免疫系统发育及功能PSA

promotes

CD4+

T

celldevelopmentand

Th1/Th2

balancePSA

promotes

Treg

developmentGSL

antagonizesiNKT

activation•

宿主神经系统发育及功能B.

f.影响肠道菌群代谢产物55GF

miceareimmunocompromised•

Low

splenicCD4+

T

cellcounts•

Underdeveloped

gutandGALTGutis

animmune

organaswell

asadigestiveorgan•

Th2-skewed

immune

responses•

Higher

susceptibilitytopathogen

infection,sometimes

withworse

outcomes•

Colonization

withnormal

gutflorareversesatleastsome

oftheabovephenotypes

‘Hygiene’

hypothesis56Intestinal

deficiencies

in

GF

mice57PSA

restores

splenic

CD4+

Tlevel

inGF

miceIntraperitonealtreatmentMazmanian,

S.K.,et

al.(2005).

Cell

122(1):

107-118.58PSA

rectifies

Th1/Th2

imbalance

in

GF

mice←

Th2cytokineCytokine

production

bysplenicCD4+

T

cells

followinganti-CD3/anti-CD28

stimulation←

Th1cytokineMazmanian,

S.K.,et

al.(2005).

Cell

122(1):

107-118.59PSA

rectifies

Th1/Th2

imbalance

in

GF

mice60Patrick,

S.,etal.

(2010).

Microbiology

156(Pt

11):

3255-3269.Benefits

of

B.fragilis

to

host•

宿主免疫系统发育及功能PSA

promotes

CD4+

T

celldevelopment

and

Th1/Th2

balancePSA

promotes

Treg

developmentGSL

antagonizesiNKT

activation•

宿主神经系统发育及功能B.

f.影响肠道菌群代谢产物61B.f.

and

inflammatory

bowl

disease

(IBD)•IBDUlcerativecolitisCrohn’s

disease•Mousemodels

ofIBD:•DSS(dextransodiumsulphate)•

TNBS

(Trinitrobenzene

sulphonicacid):

Th17-dependent•Oxazolone:NKT-dependent•Helicobacterhepaticus

inRag-/-immunodeficientmice62PSA

protects

against

TNBS-induced

colitisRectal

TNBS

plusoralPSA.Mazmanian,

S.K.,et

al.(2008).

Nature

453(7195):

620-625.63PSA

induces

IL-10

and

suppresses

IL-17PSA

→Treg

(IL-10)

→Th17

(IL-17)

?Mazmanian,

S.K.,et

al.(2008).

Nature

453(7195):

620-625.64PSA

and

multiple

sclerosis

(MS)•

MSisaninflammatory

demyelinating

diseaseaffectingCNS

functions•

Experimentalautoimmuneencephalomyelitis(EAE)isconsidered

amousemodel

of

MS•

Colonization

withwildtype,butnotPSA-deficient,B.

tectsantibiotics-treatedmice

againstEAE•

OralPSA

protects

mice

frominducedEAE•

PSA-induced

protectionis

correlatedwith

higherDC,

Treg,

IL-10andlower

Th1/Th17

cytokinesOchoa-Reparaz,

J.,

et

al.(2010).

Mucosal

Immunol

3(5):487-495.Ochoa-Reparaz,

J.,

et

al.(2010).

JI