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Appl Microbiol Biotechnol (2015) 99:88838890

DOI 10.1007/s00253-015-6946-x

MINI-REVIEW

More than a locomotive organelle: flagella in Escherichia coli


Mingxu Zhou 1,2 & Yang Yang 1,2 & Panlin Chen 1,2 & Huijie Hu 1,2 & Philip R. Hardwidge 3 &
Guoqiang Zhu 1,2

Received: 14 April 2015 / Revised: 17 August 2015 / Accepted: 20 August 2015 / Published online: 8 September 2015
# Springer-Verlag Berlin Heidelberg 2015

Abstract The flagellum is a locomotive organelle that allows


bacteria to respond to chemical gradients. This review summarizes the current knowledge regarding Escherichia coli flagellin variants and the role of flagella in bacterial functions
other than motility, including the relationship between flagella
and bacterial virulence.

Keywords Escherichia coli . Flagella . Pathogenicity .


Virulence

* Guoqiang Zhu
yzgqzhu@yzu.edu.cn
Mingxu Zhou
zhoumingxu@outlook.com
Yang Yang
yy@yzu.edu.cn
Panlin Chen
chen_panlin@outlook.com
Huijie Hu
hhj0417@163.com
Philip R. Hardwidge
hardwidg@gmail.com
1

College of Veterinary Medicine, Yangzhou University, 12 East


Wenhui Road, Yangzhou 225009, China

Jiangsu Co-innovation Center for Prevention and Control of


Important Animal Infectious Diseases and Zoonoses,
Yangzhou 225009, China

College of Veterinary Medicine, Kansas State University,


Manhattan, KS 66506, USA

Introduction
Escherichia coli (E. coli) is a Gram-negative, rod-shaped, facultatively anaerobic bacterium that resides in the gastrointestinal tract of warm-blooded animals (Donnenberg 2013). Most
E. coli are motile, typically with peritrichous flagella. The
length of the typical flagellum of E. coli is about 10 m and
the diameter is 20 nm. Flagellin, also known as H-antigen
(FliC protein, encoded by fliC gene in E. coli), is a major
surface antigen for E. coli serotyping (Nataro and Kaper
1998). Fifty-three H-antigens are recognized while a designation of NM (H) indicates the absence of flagellin (Orskov
and Orskov 1984). About 20,000 flagellin monomers polymerize to form a filament (OBrien and Bennett 1972). A
bacterial flagellum also consists of a basal body that acts as
a rotary motor and a hook that connects the motor and propeller (Terashima et al. 2008). Over 50 genes participate in flagellar biosynthesis (Macnab 2003). The genetic regulation of
flagellar assembly has been reviewed recently (Deane et al.
2010; Kazmierczak and Hendrixson 2013) and will not be
discussed in detail here. The flagellum is primarily a locomotive organelle, allowing bacteria to move from nutrient-poor
environments toward nutrient-rich ones (Wadhams and
Armitage 2004). In the past few years, studies have revealed
that flagella also affect bacterial virulence by delivering virulence factors and mediating biofilm formation, bacterial adhesion, and invasion. This review describes the different flagellin variants in E. coli and highlights the role of flagella in
bacterial functions other than motility.
E. coli flagellin variants
Based on considerable variability of E. coli flagellin in ultrastructure and 53 different H-antigens recognized, Lawn et al.
grouped 50 different flagella H serotypes into six

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morphotypes (designated as type A to type F) (Lawn 1977;


Lawn et al. 1977). Sequence analysis revealed that the flagellin proteins of E. coli and several other species (e.g.,
Salmonella) are conserved in the N- and C-terminal regions,
while the central region is highly variable (Kuwajima et al.
1986; Reid et al. 1999). Crystal structures of monomeric FliC
showed that the protein is composed of four domains
(Samatey et al. 2001). The D0 and D1 domains comprise the
conserved N- and C-termini of FliC, respectively, and form
packed -helical structures in the filament core (Yonekura
et al. 2001). The D2 and D3 hypervariable domains are
inserted into the D1 domain and are located at the outer surface of the filament (Yonekura et al. 2003).
H serogrouping is commonly determined using an agglutination test, but cross-reactivity is frequently observed among
different E. coli H-antigens (He et al. 1996; Seah and Kwang
2000). The central variable region of flagellin carries H serotype specific epitopes and PCR- or microarray-based methods
are also used for serotyping. Wang et al. sequenced 43 of the
53 H flagellin serotypes (fliC encoded) and performed phylogenetic analysis of three regions of the E. coli flagellin genes,
which could be used for further serotype determination (Wang
et al. 2003). Beutin et al. analyzed the fliC genes in representative H8 and H40 types and found that both serotypes have
two genotypes, which can be differentiated further using restriction fragment length polymorphisms (RFLPs) (Beutin
and Strauch 2007). Although most E. coli H-antigens are
encoded by fliC, the H3, H35, H36, H47, and H53-antigens
are encoded by flkA, and both the H44 and H55-antigens are
encoded by fllA (Feng et al. 2008; Ratiner 1982, 1987, 1998).
The flnA and flmA genes encode H17 and H54-antigens, respectively (Ratiner 1998; Ratiner et al. 2010). These new
dominant flagellin are located at a distance from the chromosomal fli cluster, but the two terminal regions of these genes
are just as conserved as the fliC gene (Ratiner 1983). The 53 H
flagellin serotypes are summarized in Table 1.
Some E. coli strains contain two alternatively expressed flagellin genes, giving rise to two different H phases (Ratiner 1983,
1987). Bilateral flagellar phase variation, first described in
Salmonella enterica serovar Typhimurium, is linked to bacterial
virulence (Ikeda et al. 2001; Zieg et al. 1977), whereas the flagellar phase variation in flk- and fln-positive E. coli strains is
unilateral (Feng et al. 2008; Liu et al. 2012). The flk region is
on a genomic island (GI) in flk-positive strains and the principle
for phase variation is that when the flk GI is present in the chromosome, both the FlkA flagellin and the repressor protein FlkB
are expressed, thus repressing fliC expression. When the flk GI is
excised from the chromosome, flkAB is irreversibly deleted, causing fliC repression to be released (Feng et al. 2008). The mechanism of flagellar phase variation in fln-positive strains is similar
to that of flk-positive strains, but the fln region is not on a GI and
its excision is mediated by integrase Int1157 (Liu et al. 2012).
Although the flk/fln region excised in some strains, the expressed

Appl Microbiol Biotechnol (2015) 99:88838890

FliC flagellin is still non-functional, as the fliC genes in these


strains are disrupted or defective (Feng et al. 2008). The flagellar
phase variation mechanisms in other E. coli strains carrying the
fll and flm genes remain to be defined.
Flagellar type III secretion system
Gram-negative bacteria utilize type III secretion systems (T3SS)
to deliver effector proteins into the cytoplasm of infected host
cells (Galan et al. 2014; Tosi et al. 2013). AT3SS is encoded by
enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli
(EHEC), while a less studied E. coli type III secretion system 2
(ETT2) exists in the majority of E. coli strains (Jarvis and Kaper
1996; Zhou et al. 2014a). The E. coli flagellar T3SS (fT3SS)
apparatus shares high homology with the T3SS and consists of
six integral membrane proteins (FlhA, FlhB, FliO, FliP, FliQ,
FliR) and three soluble components that form the ATPase complex for substrate delivery (FliH, FliI, FliJ) (Table 2) (Evans
et al. 2014). The former six proteins interact to form a multisubunit complex (with FlhA as the major subunit) in the central
pore of the supramembrane (MS) ring, working as an export
gate for the ATPase FliI and its regulator FliH, while FlhB plays
a role in determining export substrate specificity (Minamino
2014). FliO maintains FliP stability through a transmembrane
domain interaction in S. enterica (Barker et al. 2010). The functions of FliP, FliQ, and FliR are less well characterized but are
known to be required for fT3SS activity. FlgN, FliS, and FliT act
as substrate-specific chaperones not only to protect their cognate
substrates from degradation and aggregation in the cytoplasm
but also to transfer the substrates to the export apparatus (Fraser
et al. 1999; Yokoseki et al. 1995).
The fT3SS secretes flagellar components and can also secrete other virulence factors in some bacteria. The phospholipase YplA, which enhances bacterial virulence, was observed
as a non-flagellar protein secreted through the fT3SS by
Yersinia enterocolitica (Young et al. 1999). The flagellar export apparatus secretes the virulence-associated proteins HBL
and PC-PLC from Bacillus thuringiensis (Ghelardi et al.
2002). The cell-invasion-associated virulence factors FlaC,
FspA, and CiaC are also secreted by Campylobacter jejuni
through the fT3SS (Neal-McKinney and Konkel 2012; Poly
et al. 2007; Song et al. 2004). Modified E. coli and S. enterica
fT3SSs have been used to secrete heterologous polypeptides
in high concentration and purity (Majander et al. 2005; Singer
et al. 2012), and may be useful in producing antigens and in
generating live vaccine strains.
Flagella affecting bacterial adhesion
Bacteria utilize fimbrial surface structures to attach to host
cells (Croxen et al. 2013; Fleckenstein et al. 2010). Recent
reports also suggest that the flagella of some pathogenic
E. coli also bind directly to host cells. Flagella-deficient strains

Appl Microbiol Biotechnol (2015) 99:88838890


Table 1

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Genetic and morphologic information of E. coli H serotypes

H serotype

GenBank accession number

ORF length (bp)

Morphotypea

H1

CP007799

1788

H2
H3

AY249138
AB128916

1494
1590

C
U

H4
H5

AY249989
AY249990

1050
1311

A
B

H6

AY249991

1647

H7
H8

AY249992
AJ865465
AJ884569

1758
1479

E
D

AY249994
AY249995

2013
1263

U
C

-a
-b

H9
H10
H11

FN649414

1464

H12

AY249997

1788

H14
H15

AY249998
AY249999

1653
1689

F
F

H16
H17

AB128919
NC017663

1575
1524

U
A

H18
H19
H20

AY250001
AY250002
AY250003

1665
1842
1731

F
F
F

H21
H23
H24
H25
H26
H27

DQ862122
AY250005
AY250006
LM996317
AY250008
AF345848

1476
1767
1479c
1332
1674
1464

D
E
C
B
F
D

H28
H29
H30
H31
H32

AY250010
AY250012
AY250011
AY250013
AY250014

1740
1332
1713
1668
1713

F
C
F
F
F

H33
H34
H35
H36
H37
H38
H39
H40

1287
1638
1509
1671
1686
1344
1299
1479

B
E
U
U
F
B
B
D

H41
H42
H43
H44
H45
H46
H47

AY250015
AY250016
EF392692
EF392693
AY250017
AY250018
AY250019
AJ884568
AJ865464
AY250020
AY250021
AY250022
AB269770
AY250023
AY250024
EF392694

1680
1281
1506
1725
1707
1719
1107

F
B
C
F
E
F
B

H48
H49

AY250025
AY250026

1497
1695

C
E

-a
-b

Descriptionb

flkA

fliC; 2 genotypes

flnA

flkA
flkA

fliC; 2 genotypes

fllA

flkA

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Appl Microbiol Biotechnol (2015) 99:88838890

Table 1 (continued)
H serotype

GenBank accession number

ORF length (bp)

Morphotypea

Descriptionb

H51

AY250027

1818

H52
H53

AY250028
AB128917

1344
1272

B
C

flkA

H54

AB128918

1551

ND

flmA

H55
H56

AB269771
AY250029

1869
1344

ND
ND

fllA

Morphotypes: A thin, long pitch helix; B thin, subunit pattern; C rough subunit pattern; D polar subunit pattern; E short pitch loop pattern; F long pitch
loop pattern. Serotypes labeled BU^ have a unique surface structure not readily grouped with those of any other serotype. The morphotype of three
serotypes (H54, H55, and H56) are not determined (ND)

Except H3, H17, H35, H36, H44, H47, H53, H54, and H55 serotypes, the other 44 H-antigens are all encoded by fliC genes

As lacking of N- and C-terminal regions sequence for the H24-antigen, the ORF length here is based on the conserved N- and C-terminal length of other
serotypes

of neonatal meningitis E. coli (NMEC) associate with human


brain microvascular endothelial cells (HBMECs) less avidly
than isogenic wild-type strains, and pre-treating HBMECs
with purified recombinant flagellin prevents NMEC adhesion
(Parthasarathy et al. 2007). Flagella also mediate porcine
ETEC adhesion to piglet intestinal epithelial cell lines and
purified FliCH1 and FliCH19 bind directly to host cells (Duan
et al. 2012, 2013a). In EPEC/EHEC, H6 and H7 flagella have
adhesive properties to HeLa cells and to the bovine intestinal
epithelium, respectively (Giron et al. 2002; Mahajan et al.
2009), probably through their interactions with host mucins.
The hypervariable domain D2 of FliCH1 from the probiotic
E. coli strain Nissle 1917 interacts with gluconate in mucus
(Troge et al. 2012). EtpA, a two-partner secretion (TPS) protein identified in ETEC H10407 (H11), functions as a molecular bridge that binds to both flagella and to host cell receptors
(Fleckenstein et al. 2006; Roy et al. 2009). Also, the EtpA is
not encoded in all pathogenic types of E. coli (unpublished
data).
Table 2

EHEC and ETEC outbreaks are linked to contaminated


fresh produce, and recent studies of the mechanisms by which
different E. coli serotypes bind fresh salad leaves have implicated flagella as major adherence factors. Mutation of fliC in
EHEC EDL933 reduced adherence to leaves, similar to observations with ETEC H10407 (Shaw et al. 2011; XicohtencatlCortes et al. 2009). Berger et al. suggested that multiple adherence factors, including flagella, also play a role in
enteroaggregative E. coli (EAEC) attachment to leaves
(Berger et al. 2009). Recent data obtained from using plant
glycan arrays has also supported these observations (Rossez
et al. 2014).
Flagella may also contribute to bacterial invasion into
mammalian cells. Aflagellated mutants of avian pathogenic
E. coli (APEC) O78:K80 exhibited reduced colonization and
invasion in vivo (La Ragione et al. 2000). fliC deletion mutants of F18+ ETEC O139:H1 had a reduced ability to invade
IPEC-1 and IPEC-J2 cells (Duan et al. 2013b). Flagellin of
EHEC O113:H21 was also reported to be involved in bacterial

Homologous proteins of the E. coli fT3SS and T3SSs from different bacteria species

Flagellar T3SS (E. coli)

S. enterica SPI-1 encoded T3SS

Shigella ssp.

Description

FliH
FliI
FliJ
FlhA
FlhB
FliO
FliP
FliQ
FliR
FlgN
FliS
FliT

OrgB
InvC
InvI
InvA
SpaS
N/A
SpaP
SpaQ
SpaR
N/A
SicA
PipC

MxiN
Spa47
Spa13
MxiA
Spa40
N/A
Spa24
Spa9
Spa29
N/A
IpgC
IpgF

ATPase regulator
ATPase
Chaperone
Major export apparatus protein, interacts with soluble components
Export switch protein, interacts with soluble components
Minor export apparatus protein, maintains stability of FliP
Minor export apparatus protein, cleaved signal sequence
Minor export apparatus protein
Minor export apparatus protein
Chaperone
Chaperone
Chaperone

Appl Microbiol Biotechnol (2015) 99:88838890

invasion into HCT-8 cells (Luck et al. 2006; Rogers et al.


2012). However, the mechanism by which flagella may mediate invasion is unclear.

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coordinate bacterial functions that promote survival in infected (Kansal et al. 2013). The myriad relationships between
flagella and bacterial virulence remain an active area of
investigation.

Flagella-mediated biofilm formation


Bacteria that form biofilms can be highly resistant to antibiotics and are capable of evading the host immune system
(Yang et al. 2012). Biofilm formation is a complex process
involving multiple cell surface proteins, such as fimbriae,
autotransporters, and flagella (Allsopp et al. 2012; Hossain
and Tsuyumu 2006; Ulett et al. 2007). We found a 50 %
reduction in biofilm formation in fliC mutants of K88+
and F18+ ETEC strains, with a reduced ability of these mutants to form microcolonies (Duan et al. 2012; Zhou et al.
2014b). Hung et al. determined that uropathogenic E. coli
(UPEC) flagella mutants are only able to form sparse collections of bacteria, which do not mature to biofilms (Hung et al.
2013). A motA mutant (non-motile but with flagella) and a
fliA mutant (lacking flagella) show decreased biofilm formation as compared with a wild-type E. coli K12 strain (Wood
et al. 2006). Motility may be related to biofilm transitions
through c-di-GMP (Guttenplan and Kearns 2013). Quorum
sensing pathways are also believed to affect flagellamediated biofilm formation (Quinones et al. 2005; Yang
et al. 2013).
Flagellin induces host pro-inflammatory responses
and apoptosis
Innate immunity serves as an essential first-line defense
against microbial pathogens. Toll-like receptor 5 (TLR5), a
pattern-recognition receptor (PRR) expressed on the surface
of mammalian cells, recognizes bacterial flagellin and triggers
a pro-inflammatory response (Hayashi et al. 2001; VijayKumar and Gewirtz 2009). TLR5 interacts with the conserved
domain of monomeric but not polymeric flagellin (Smith et al.
2003), causing downstream activation of the MyD88dependent signaling pathway (Hayashi et al. 2001). Flagellin
can also induce host responses via two intracellular PRRs,
Ipaf and Naip5 driving caspase-1 activation and IL-1 and IL18 processing, which can also affect apoptosis pathways
(Miao et al. 2006; Ren et al. 2006).

Conclusions
The flagellum controls many important functions other than
motility, including protein secretion, adhesion, biofilm formation, and pro-inflammatory host responses as well as apoptosis. Flagellar master genes also coregulate other virulence factors in pathogenic E. coli (Lane et al. 2007; Lehti et al. 2012;
Simms and Mobley 2008; Zhou et al. 2013), which may help

Acknowledgments This study was funded by the 948 programme


grant no. 2011-G24 from the Ministry of Agriculture of the Peoples
Republic of China, the Genetically Modified Organisms Technology Major Project of China (2014ZX08006-001B), grants from the Chinese National Science Foundation Grant (no. 31270171, no. 31072136, and no.
30771603), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, a project funded by
China Scholarship Council (201500090062), and a fund of excellent
doctorial dissertations from Yangzhou University.
Conflict of interest The authors declare that they have no competing
interests.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.

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