Microbiology (2015), 161, 1592–1599
DOI 10.1099/mic.0.000123
Roles of the TonB1 and TonB2 proteins in
haemin iron acquisition and virulence in
Riemerella anatipestifer
Shuang Miao, Linlin Xing, Jingjing Qi, Hui Yu, Pan Jiang, Bingqing Sun,
Junsheng Cui, Changcan Ou and Qinghai Hu
Correspondence
Qinghai Hu
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences,
518 Ziyue Road, Shanghai 200241, PR China
[email protected]
Received 3 March 2015
Accepted 15 June 2015
Two TonB systems in Riemerella anatipestifer were found and characterized as ExbB1–ExbD1–
TonB1 and ExbB2–ExbD2–ExbD29–TonB2, but the significance of two sets of TonB complexes
in R. anatipestifer is not clear. In this study, by deleting the tonB1 or tonB2 gene of
R. anatipestifer strain CH3, we investigated the roles of the TonB1 and TonB2 proteins in iron
acquisition and virulence. The results showed that strain CH3 could utilize haemin as the sole
iron source in the presence of L -cysteine, but haemin iron acquisition was defective in the
CH3DtonB1 mutant, and the deletion of either tonB1 or tonB2 significantly reduced adhesion
to and invasion of Vero cells. Animal experiments indicated that the LD50 of the CH3DtonB1
and CH3DtonB2 mutants in ducklings was ,224- and ,87-fold, respectively, higher than that
of the WT CH3 strain. Additional analysis indicated that blood bacterial loading of ducklings
infected with CH3DtonB1 or CH3DtonB2 decreased significantly compared with that found for
WT CH3-infected ducklings. Thus, our results indicated that the TonB1, but not TonB2 protein,
is involved in haemin iron acquisition and that both TonB proteins are necessary for optimal
bacterial virulence.
INTRODUCTION
Iron is an essential cofactor for several enzymes and metabolic
pathways in microbes and their eukaryotic hosts (Huynh &
Andrews, 2008), and a number of high-affinity iron transport
systems have been identified in Gram-negative bacterial pathogens (Andrews et al., 2003; Braun & Hantke,
2011). The TonB–ExbB–ExbD complex is required for
the energy-dependent, active transport of iron-bound substrates across the outer membrane of Gram-negative bacteria. The ExbB and ExbD proteins anchor TonB in the
cytoplasmic membrane, whilst TonB, which serves as the
energy-transducing protein, spans the periplasmic space
as a dimer to interact with high-affinity outer membrane
receptors (Krewulak & Vogel, 2011; Noinaj et al., 2010).
Many Gram-negative bacteria harbour one or more sets
of functional TonB systems, which specifically or preferentially drive the acquisition of various iron sources.
Abbreviation: DIP, 2,29-dipyridyl.
The GenBank/EMBL/DDBJ accession numbers for the sequences of
the TonB1 and TonB2 systems in Riemerella anatipestifer strain CH3
are KM393215 and KM393216, respectively.
One supplementary table and two supplementary figures are available
with the online Supplementary Material.
1592
In addition, TonB systems in Gram-negative bacteria are
also involved in the uptake of vitamin B12, nickel, carbohydrates, phages and colicins (Blanvillain et al., 2007;
Schauer et al., 2007). Furthermore, reflecting the importance of iron to the survival of bacterial pathogens within
the host environment and as one of the components
involved in the transduction of environmental signals,
TonB protein has been shown to be essential for virulence
in many bacterial pathogens (Beddek et al., 2004; Stork
et al., 2004; Wang et al., 2008).
Riemerella anatipestifer, a member of the family Flavobacteriaceae, causes epizootic infectious disease in poultry and
serious economic losses, especially to the duck industry,
worldwide (Sandhu, 2008), and its iron acquisition systems
are not well defined. In our previous reports, we found
that 23 of 24 (95.83 %) virulent isolates and one of three
avirulent R. anatipestifer isolates were positive for the sip
gene, which encodes a siderophore-interacting protein that
is involved in iron acquisition and is required for the virulence of the R. anatipestifer CH3 strain (Tu et al., 2014).
In addition, the TonB-dependent receptor TbdR1 is
involved in haemin iron acquisition and is necessary for optimal bacterial virulence (Lu et al., 2013). These previous
results suggested that TonB-dependent pathways may play
important roles in R. anatipestifer iron acquisition.
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R. anatipestifer TonB1 and TonB2
According to the full genome sequences of four
R. anatipestifer strains in GenBank, this bacterium is
predicted to contain two TonB–ExbB–ExbD systems
(Mavromatis et al., 2011; Wang et al., 2014; Yuan et al.,
2011). In this study, by deleting the tonB1 or tonB2 gene
from R. anatipestifer strain CH3, the roles of the two TonB
proteins in haemin iron acquisition and pathogenesis were
investigated.
2,29-dipyridyl (DIP; Sigma-Aldrich) at 0, 50, 100, 150, 200 and
250 mM. All bacterial strains were grown at 37 uC with shaking and
growth curves were constructed as described previously (Hu et al.,
2002). Iron utilization assays were performed in liquid culture as
described elsewhere (Hagan & Mobley, 2009), with slight
modifications.
Prior to inoculation, strains were washed with PBS containing 250 mM
DIP and then *105 c.f.u. inoculated into TSB containing 200 mM DIP
supplemented with FeCl3 (200 mM) or haemin (1–100 mM; SigmaAldrich) as the sole iron source and incubated at 37 uC with shaking.
The OD600 was determined as indicated.
METHODS
Bacterial cultures. R. anatipestifer strain CH3 and its derivatives were
cultured at 37 uC in tryptic soy broth (TSB) or agar (TSA) (BD) in a
5 % CO2 atmosphere. Escherichia coli strains were grown at 37 uC in
Luria–Bertani broth or agar. For selective growth of bacterial strains,
antibiotics were added at the following concentrations, unless otherwise
stated: ampicillin, 100 mg ml21; kanamycin, 50 mg ml21; spectinomycin,
80 mg ml21; chloramphenicol, 34 mg ml21; erythromycin, 1 mg ml21.
DNA manipulation and analysis. Genomic DNA was extracted
using a QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions. Plasmid DNA was extracted using a TIANprep
Mini Plasmid kit (Tiangen Biotech). PCRs were performed using a
high-fidelity PCR system (Roche Diagnostics). All the sequences of
the primers used are listed in Table S1 (available in the online Supplementary Material). PCR products were cloned into a pGEM-T Easy
vector (Promega). Automated DNA sequencing was performed at
Invitrogen. DNA analysis was completed using DNASTAR 7.01 software
(DNASTAR ) and BLAST network services.
Construction of deletion mutants CH3DtonB1 and CH3DtonB2.
The tonB1 or tonB2 genes of R. anatipestifer strain CH3 were deleted by
allelic exchange in which a recombinant suicide vector was used to
replace a 577 bp fragment of the 861 bp tonB1 ORF or a 529 bp fragment
of the 846 bp tonB2 ORF fragment with a 1119 bp spectinomycin
resistance cassette, as described previously (Lu et al., 2013; Tu et al.,
2014) (Fig. S1). Upstream and downstream regions (700–1200 bp)
flanking each gene were amplified with specific primers (Table S1). The
1119 bp spectinomycin resistance cassette was PCR-amplified from
plasmid pFW5 using the primers Spc-F1 and Spc-F2 (Hu et al., 2011).
The PCR-derived fragments were ligated into the suicide vector
pDS132 (Philippe et al., 2004). E. coli S17-1lpir was transformed with
the pDS132 derivatives and conjugated with the R. anatipestifer strain
CH3, and the transconjugants were selected on TSA agar containing
kanamycin and spectinomycin. Mutants were confirmed by PCR
amplification of the 16S rRNA, spec, tonB1 or tonB2 genes using specific
pri-mers (Table S1). The mutant strains with the phenotype
16S rRNA+Spec+tonB12 and 16S rRNA+Spec+tonB22 were
designated CH3D tonB1 and CH3DtonB2 respectively.
Construction of the complemented strains. For complementation
analysis, recombinant plasmids were generated by cloning the intact
tonB1 or tonB2 ORF into the E. coli–Riemerella anatipestifer shuttle
vector pRES (Hu et al., 2013). The tonB1or tonB2 ORF was ligated
into plasmid pRES to generate pRES-TonB1 and pRES-TonB2, respectively. The expression of tonB1or tonB2 in the complementing
plasmids was under the control of the putative promoter pSR. Then,
plasmid pRES-TonB1 or pRES-TonB2 was introduced into the corresponding mutant by conjugation, as described previously (Hu et al.,
2011), to generate the complemented strains CH3DtonB1(pRESTonB1) for the mutant CH3DtonB1 and CH3DtonB2(pRES-TonB2)
for the mutant CH3DtonB2.
Iron utilization assays. The CH3 and derivative strains were
tested for iron-limited growth in TSB containing the iron chelator
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Bacterial adherence and invasion assays. To determine the in-
fluence of tonB1 or tonB2 deletion on the adherence and invasion
capacity of the R. anatipestifer strain CH3 to African green monkey
kidney epithelial cells (Vero cells; ATCC CCL-81), a quantitative
adherence assay was performed using the CH3DtonB1 and
CH3DtonB2 mutants, the complemented strains, and the WT CH3
strain, as described previously (Hu et al., 2011). All assays were performed in duplicate and repeated on at least 3 separate days.
Virulence assays. Cherry Valley ducklings (1 day old) were obtained
from Zhuanghang Duck Farm (Shanghai, PR China) and housed in
cages under controlled temperatures ranging from 28 to 30 uC and a
12 h light/dark cycle, with free access to food and water. Animal experiments in this study were carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory
Animals of the Institutional Animal Care and Use Committee set
by Shanghai Veterinary Research Institute, Chinese Academy of
Agricultural Sciences. The protocol was approved by the Committee
on the Ethics of Animal Experiments of Shanghai Veterinary Research
Institute (Permit 13-10).
To determine whether inactivation of the tonB1 or tonB2 gene influenced R. anatipestifer virulence, the LD50 values of the CH3DtonB1,
CH3DtonB2, CH3DtonB1(pRES-TonB1), CH3DtonB2(pRES-TonB2)
and WT CH3 strains were measured as described previously (Hu
et al., 2011). Briefly, for each strain, Cherry Valley ducklings (8 days old)
were divided randomly into five groups (10 ducklings per group)
and injected intramuscularly with 107, 108, 109 or 1010 c.f.u. each
bacterial strain. Ducks that became moribund were killed humanely
and counted as dead. Dead ducks were subjected to R. anatipestifer
identification. Ducks were monitored daily for clinical symptoms and
death rate until 10 days post-infection and LD50 values were calculated
using the Reed–Muench method (Reed & Muench, 1938).
Bacterial loads in the blood from CH3DtonB1-, CH3DtonB2-, CH3D
tonB1(pRES-TonB1)-, CH3DtonB2(pRES-TonB2)- and WT CH3infected ducklings were compared. Cherry Valley ducklings (8 days old)
were injected intramuscularly with 109 c.f.u. each bacterial strain, and
blood samples were collected at 12 and 24 h post-inoculation (six
ducklings per group per time point), and plated on TSA agar plates for
counting.
Statistical analysis. The statistical significance of the data were
determined by one-way ANOVA using Prism 5 (GraphPad). Pv0.05
was considered statistically significant.
RESULTS
Genetic analysis of the TonB1 and TonB2 systems
of R. anatipestifer strain CH3
In this study, we cloned the genes encoding two putative
TonB systems in R. anatipestifer strain CH3 according to
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S. Miao and others
the whole-genome sequence of strain DSM15868 (GenBank
accession number CP002346). The TonB1 system in
R. anatipestifer CH3 consisted of three ORFs (ExbB1–
ExbD1–TonB1) and the TonB2 system included four
closely linked ORFs (ExbB2–ExbD2–ExbD29–TonB2; Fig.
S2). The two TonB systems and their flanking genes in
strain CH3 were the same as those found in strains
DSM15868, RA-GD, CH-1 and CH-2. Moreover, both the
TonB1 and TonB2 genes of R. anatipestifer were located
downstream of the exbB and exbD genes. There are two alleles
that were found in both tonB1 and tonB2 ORFs, due to eight
and two amino acid replacements, respectively. In both
tonB1 and tonB2 genes, allele 1 was shared by strains CH3
and CH-1, and allele 2 was found in strains DSM15868,
CH-2 and RA-GD. TonB1 and TonB2 proteins from
R. anatipestifer strain CH3 shared 15.8 % amino acid
sequence identity. In addition, although R. anatipestifer
strain HXb2 does not carry the sip gene (Tu et al., 2014), it
does carry the TonB1 and TonB2 systems.
The putative conserved domains of the TonB1 and TonB2
proteins from R. anatipestifer strain CH3 were analysed
with the National Center for Biotechnology Information
Conserved Domain Database tools. The TonB1 and TonB2
proteins were shown to contain a TonB domain (COG0810)
at aa 14–163 (TonB1) and 47–275 (TonB2), respectively,
whilst TonB2 contained a TonB_C superfamily (pfam03544)
domain at aa 209–279. In E. coli, the TonB_C domain
could interact with the N-terminal TonB box of the outer
membrane transporter that binds the Fe3+–siderophore
complex (Pawelek et al., 2006). This indicated that the
TonB2 protein may be involved in siderophore utilization
in R. anatipestifer.
Phenotypic characteristics of the tonB deletion
mutants
The mutants CH3DtonB1 and CH3DtonB2 were phenotypically characterized and compared with the WT CH3 strain.
The colony morphologies of the TonB mutants on TSA agar
were similar to that of the WT CH3 strain (data not shown).
The growth curves of the WT CH3 strain, the CH3DtonB1
mutant and its complement strain CH3DtonB1(pRESTonB1), and the CH3DtonB2 mutant and its complement
strain CH3DtonB2(pRES-TonB2) were determined in TSB
medium at 37 uC with shaking. The results showed that
the growth of the mutant CH3DtonB2 was significantly
slower than that of CH3 parent strain (Pv0.05; Fig. 1a)
and the complemented plasmid pRES-TonB2 could restore
the growth ability of the mutant CH3DtonB2 (Pw0.05).
Both TonB proteins play a critical role in iron
acquisition
To determine whether the TonB1 and TonB2 systems play
a role in iron acquisition in R. anatipestifer strain CH3,
the growth of the mutants CH3DtonB1 and CH3DtonB2,
and the complemented strains CH3DtonB1(pRES-TonB1)
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and CH3DtonB2(pRES-TonB2) in TSB supplemented with
different concentrations of DIP was measured. As shown
in Fig. 1(b), the growth of both CH3DtonB1 and CH3D
tonB2 was significantly slower compared with that of the
WT CH3 strain when grown in the presence of 50 mM DIP
(Pv0.05) and the growth defect of the mutant CH3DtonB2
was more severe than that of CH3DtonB1. In addition, the
growth of CH3DtonB2 was almost non-existent in TSB supplemented with 100 mM DIP and so was that of CH3DtonB1
in TSB with 200 mM DIP. Furthermore, although the WT
CH3 strain, the CH3DtonB1 and CH3DtonB2 mutants, and
the complemented strains CH3DtonB1(pRES-TonB1) and
CH3DtonB2(pRES-TonB2) did not grow in TSB containing
200 mM DIP, the addition of 200 mM FeCl3 to the cultures
could restore the growth of the WT CH3 strain and the complemented strains (Fig. 1c). These results suggested that both
TonB1 and TonB2 were involved in iron acquisition by
R. anatipestifer strain CH3, and that the tonB2 mutant was
more impaired in its ability to utilize iron. Moreover, the
growth defect of the CH3DtonB1 and CH3DtonB2 mutants
may have been due to their decreased capacity for iron
acquisition.
TonB1, but not TonB2, plays an important role
in haemin utilization
As shown in Fig. 2, the growth of strain CH3 was unaffected
in TSB supplemented with 0–10 mM haemin, but decreased
in TSB containing 20 mM haemin and was non-existent in
TSB containing 40 mM haemin. Additionally, in TSB containing 250 mM DIP, the growth of strain CH3 was almost
non-existent in TSB supplemented with 0–40 mM haemin
(Fig. 2a). It was suggested that R. anatipestifer cannot utilize
haem as the sole iron source under these conditions. To test
whether L -cysteine plays a role in haemin utilization in
R. anatipestifer, the growth of CH3 in TSB supplemented
with 250 mM DIP, 10 mM haemin and 0–6 mM cysteine
was measured. The results showed that the growth defect
of CH3 in iron-depleted TSB could be rescued by haemin
in the presence of 1–6 mM L -cysteine (Fig. 2b). Growth of
CH3 in TSB containing 4 mM cysteine, 250 mM DIP and
0.5–20 mM haemin was almost the same as that in TSB
medium, whilst it was defective in the presence of 50 mM
haemin (Fig. 2c). This suggested that R. anatipestifer CH3
could utilize haemin as the sole available iron source in the
presence of L -cysteine.
To further explore the roles of the TonB1 and TonB2 proteins in haemin transport in R. anatipestifer, the WT CH3
strain, the CH3DtonB1 and CH3DtonB2 mutants, and
the complemented strains CH3DtonB1(pRES-TonB1) and
CH3DtonB2(pRES-TonB2) were grown in iron-depleted
TSB supplemented with 10 mM haemin as the sole available
iron source in the presence of 4 mM L -cysteine. The results
showed that the WT CH3 strain, CH3DtonB2 and the
complemented strain CH3DtonB2(pRES-TonB2) grew normally under these conditions, whilst CH3DtonB1 and the
complemented strain CH3DtonB1(pRES-TonB1) displayed
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Microbiology 161
R. anatipestifer TonB1 and TonB2
(b) 3.5
Growth (OD600)
3.0
Growth (OD600)
(a) 3.6
3.3
3.0
2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0
2.5
2.0
1.5
1.0
0.5
0
0
1
2
3
4
5
6
7
8
9 10 11 12
0
1
2
3
4
Time (h)
6
7
8
9
10 11 12
Time (h)
Growth (OD600)
(c) 3.6
3.3
3.0
2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0
5
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (h)
Fig. 1. Growth of the tonB mutants, the complemented strains and the WT CH3 strain. (a) Growth in TSB medium. (b)
Growth in TSB medium supplemented with 50 mM DIP. (c) Growth in TSB medium supplemented with 200 mM DIP and
200 mM FeCl3. X, CH3; %, CH3DtonB1; &, CH3DtonB1(pRES-TonB1); g, CH3DtonB2; m, CH3DtonB2(pRES-TonB2).
The OD600 was determined at the indicated time points. Data represent mean¡SD of three determinations.
a severe growth defect (Fig. 2d); however, the recombinant
shuttle plasmid pRES-TonB1 could not restore the growth
defect of mutant CH3DtonB1. The results indicated that
TonB1, but not TonB2, of R. anatipestifer strain CH3 was
involved in haemin iron transport.
TonB1 or TonB2 inactivation leads to decreased
adherence to and invasion of Vero cells
To investigate whether the deletion of tonB1 or tonB2
played a role in adhesion to a biotic surface, we assessed
the capacity of the WT CH3 strain, the CH3DtonB1 and
CH3DtonB2 mutants, and the complemented strains
CH3DtonB1(pRES-TonB1) and CH3DtonB2(pRES-TonB2)
to adhere to Vero cells. The adhesion and invasion capacities
of the CH3DtonB1 and CH3DtonB2 mutants to Vero cells
were significantly decreased compared with those of the
WT CH3 strain (Pv0.01; Fig. 3), and complementation of
the mutants with plasmids pRES-TonB1 or pRES-TonB2
significantly restored their adhesion and invasion capacities
(Pv0.01). These results suggested that the deletion of tonB1
http://mic.sgmjournals.org
or tonB2 influenced the interaction between R. anatipestifer
and host cells.
TonB1 and TonB2 systems are required for the
virulence of R. anatipestifer
The ability to acquire iron under low-iron conditions
is required for the virulence of a variety of bacterial
pathogens (Tai et al., 1993). To determine the effect of the
tonB1 or tonB2 deletions on the virulence of the R. anatipestifer strain CH3, groups of Cherry Valley ducklings (8 days
old) were inoculated with the WT CH3 strain, the tonB
mutants or the complemented strains. All of the strains
were able to invade and infect deeper tissues, and to cause
systemic infection and death. At 10 days post-infection,
the calculated LD50 values of the WT, CH3DtonB1,
CH3DtonB1(pRES-TonB1), CH3DtonB2 and CH3DtonB2(pRES-TonB2) strains were 2.29|107, 5.13|109,
4.9|107, 2.0|109 and 2.14|108 c.f.u., respectively. For
the CH3DtonB1 and CH3DtonB2 mutants, the *224and *87-fold increases, respectively, in the LD50 values
compared with that of the WT CH3 strain indicated that
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S. Miao and others
(a) 3.6
3.3
3.0
2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0
(b) 2.7
Growth (OD600)
Growth (OD600)
2.4
1.8
1.5
1.2
0.9
0.6
0.3
TS
B
C
ys
ys
m
M
6
4
m
M
C
C
ys
ys
C
m
M
2
1
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M
C
ys
ys
C
0.
5
m
M
m
M
0.
25
0
m
M
C
ys
0
0. μM
25
h
0. μM ae
5 h m
μM a in
1 h em
2. μM ae in
5 h mi
μ a n
5 M h em
10 μM ae in
h m
20 μM aem in
h
μ
40 M aemin
μM hae in
ha min
0
0. μM emi
25
n
h
0. μM ae
5 h m
in
μ
1 M haem
μ
2. M ae in
5 h mi
μ a n
5 M h em
μ
10 M ae in
h m
20 μM aem in
h
40 μM aemin
μM hae in
ha min
em
in
0
TSB
TSB+250 μM DIP
(c)
(d) 2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Growth (OD600)
3.0
2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0
0
0. μM
25 h
a
μ em
0. M h in
5
a
μM em
i
1 ha n
μM em
i
2.
5 hae n
μM m
in
5 hae
μM m
10 ha in
μM em
20 ha in
μM em
50 h in
μ ae
10 M min
0 hae
μM m
ha in
em
in
TS
B
Growth (OD600)
2.1
2
1
B2)
B1)
onB
onB
-Ton
-Ton
3Δt
S
3Δt
S
H
E
H
E
C
C
pR
1(pR
B2(
tonB
Δton
Δ
3
3
H
C
CH
3
CH
Fig. 2. The TonB1 protein is involved in haemin utilization in the presence of L -cysteine. (a) Effect of 0–40 mM haemin on the
growth of the WT CH3 strain in TSB medium or TSB supplemented with 250 mM DIP. (b) Effect of 0–6 mM L -cysteine on the
growth of the WT CH3 strain in TSB medium supplemented with 250 mM DIP and 10 mM haemin. (c) Effect of 0–100 mM
haemin on the growth of the WT CH3 strain in TSB medium supplemented with 250 mM DIP and 4 mM L -cysteine. (d) The
mutant CH3DtonB1 and the complemented strain CH3DtonB1(pRES-TonB1) displayed a severe growth defect in TSB medium
supplemented with 250 mM DIP, 4 mM L -cysteine and 10 mM haemin. The bacteria were cultured for 10 h at 37 8C with shaking
and growth was monitored by measuring OD600. The error bars represent means ¡standard deviations from three determinations.
Asterisks indicate statistically significant differences compared with that of wild-type CH3 (**P,0.01).
disruption of either tonB1 or tonB2 resulted in
the attenuation of R. anatipestifer virulence. Meanwhile, the LD50 values of the complemented strains CH3D
tonB1(pRES-TonB1) and CH3DtonB2(pRES-TonB2) were
similar to that of the CH3 strain.
In addition, to investigate the effect of the deletion of
tonB1 or tonB2 during systemic R. anatipestifer infections
in vivo, bacteria were recovered from the blood of infected
ducklings at 12 and 24 h post-inoculation and bacterial
colonies were determined by plate counting. As shown in
Fig. 4, the bacterial loads in the blood of ducklings infected
with the CH3DtonB1 or CH3DtonB2 mutants decreased
significantly in comparison with those from ducklings infected with the WT CH3 strain (Pv0.01). Taken
1596
together, these results provided evidence that both TonB1
and TonB2 were involved in R. anatipestifer pathogenesis.
DISCUSSION
In Gram-negative bacteria, TonB, ExbB and ExbD form a
cytoplasmic membrane complex required for high-affinity
iron transport. In E. coli and most other Gram-negative bacteria, one single set of the TonB–ExbB–ExbD complex mediates the assimilation of multiple types of iron-loaded
siderophores or ligands (Faraldo-Gómez & Sansom, 2003).
However, R. anatipestifer, similar to some other bacteria,
such as Vibrio cholerae, Vibrio anguillarum, Actinobacillus
pleuropneumoniae and Pseudomonas aeruginosa, has been
shown to harbour two or more sets of functional TonB
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Microbiology 161
No. of bacteria (c.f.u.) (×103)
R. anatipestifer TonB1 and TonB2
18
16
14
12
10
5
4
3
2
1
0
**
**
**
**
3
1
2 2)
3
1
2 2)
1)
1)
CH tonB onB tonB onB
CH tonB onB tonB onB
3Δ S-T 3Δ S-T
3Δ S-T 3Δ S-T
CH pRE CH pRE
CH pRE CH pRE
(
(
(
(
B2
B1
B1
B2
on
on
on
on
t
t
t
t
3Δ
3Δ
3Δ
3Δ
CH
CH
CH
CH
Adherence
Invasion
Fig. 3. Adherence and invasion assays for the CH3DtonB1 and
CH3DtonB2 mutants, the complemented strains, and the WT CH3
strain. The data are presented as the number of bacteria bound
to the surface or located in the cytosol of Vero cells in each well
of 24-well plates. Data represent mean¡SD from three independent
experiments. Asterisks indicate statistically significant differences
compared with that of wild-type CH3 (**P,0.01).
Bacterial load [c.f.u.
(ml blood)–1] (×104)
systems. The significance of two sets of TonB complexes in
R. anatipestifer is not clear. Generally, two distinct TonB
systems may play different roles in iron acquisition. The
two TonB systems in V. cholerae were shown to have
unique, as well as common, functions. Both TonB systems
functioned well in the transport of the siderophore vibriobactin, whilst the TonB2 system was required for utilization
of enterobactin and for haem transport via HasR (Seliger
400
350
300
250
200
35
30
25
20
15
10
5
0
**
**
**
**
3
2
1
3
2
)
1
1)
2)
2)
CH tonB onB tonB onB
CH tonB onB1 tonB onB
T
T
T
Δ
Δ
Δ
T
Δ
3
3
3
3
S
S
S
S
H
CH (pRE CH (pRE
CH (pRE C (pRE
B1
B2
B2
B1
n
n
n
n
to
to
to
Δt o
3Δ
3Δ
3Δ
H3
CH
CH
CH
C
12 h post-inoculation
24 h post-inoculation
Fig. 4. Blood bacterial loadings of ducklings infected with the
CH3DtonB1 and CH3DtonB2 mutants, the complemented strains,
and the WT CH3 strain. Data represent mean¡SD from six ducklings. Asterisks indicate statistically significant differences between
two groups (**P,0.01).
http://mic.sgmjournals.org
et al., 2001; Wyckoff et al., 2007). In addition, although theTonB1 system specifically contributed to haemin and haemoglobin utilization in Vibrio alginolyticus MVP01, both
TonB systems showed the ability to support the utilization
of iron sources from ferrichrome and vibrioferrin, the
endogenous siderophore of V. alginolyticus (Wang et al.,
2008). In this study, our results suggested that although the
two TonB systems are involved in iron utilization by
R. anatipestifer CH3, the TonB1, but not TonB2, system
may play an important role in haemin iron utilization.
It was noteworthy that R. anatipestifer CH3 utilized
haemin as an iron source in the presence of L -cysteine.
This may be due to the specific culture requirements of
R. anatipestifer. Most R. anatipestifer strains grew better on
TSA in a 5 % CO2 incubator or in a candle jar than that on
TSA without additional CO2, but also grew well with shaking
in TSB without additional CO2. In addition, the fact that the
tonB1 mutant was more attenuated in virulence than the
tonB2 mutant may be related to a haemin iron acquisition
defect in the mutant CH3DtonB1.
Whether the TonB1 or TonB2 systems are involved in the
uptake of other substances, such as haemoglobin, holotransferrin, vitamin B12, nickel or carbohydrates, remains
to be clarified. Some R. anatipestifer strains can secret
siderophores (our unpublished data), but we have not purified siderophores from R. anatipestifer to test which TonB
system contributes to the import of iron–siderophore complexes into the cytosol. In addition, we also did not determine why the tonB2 deletion in R. anatipestifer strain CH3
exhibited a greater growth defect than that of the tonB1
deletion. These questions remain to be clarified in future
studies.
The control of iron homeostasis is central in the host–
pathogen interplay and influences the course of an infectious
disease in favour of either the host or the pathogenic invader
(Nairz et al., 2010). In our previous studies, we showed that
the deletion of the tbdR1 or sip genes, which were also
involved in iron acquisition, reduced the pathogenicity to
ducklings, biofilm formation, and adherence to and invasion
of Vero cells (Lu et al., 2013; Tu et al., 2014). In this study,
consistent with their roles in iron acquisition, both TonB1
and TonB2 were found to be involved in host infections.
In fact, this is a common phenomenon. Many previous
studies of Gram-negative bacteria suggested that iron
may serve as the ‘critical determinant’ that decides the outcome of host–bacteria interactions (Banin et al., 2005;
Crosa, 1989; Weinberg, 1978) and that iron is an important environmental signal that controls the differential
expression of a large number of genes, some of which
encode important bacterial virulence factors (Rhodes et al.,
2007). In addition, the expression of adherence factors by
several bacterial species is influenced by the environmental
iron supply (Alves et al., 2010). In a previous study, iron
was shown to have a regulatory role in the adhesion of
diphtheria bacilli to cells of the human respiratory tract
(HEp-2 cells) and blood (erythrocytes), and low iron availability modulated the expression of Corynebacterium
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1597
S. Miao and others
diphtheriae surface carbohydrate moieties (Moreira et al.,
2003). In this study, we investigated the adherence or invasion capacity of the tonB mutants to Vero cells. The results
of our study demonstrated that the adherence or invasion
capacity of the tonB1 or tonB2 mutants to Vero cells was
significantly reduced compared with that of the WT CH3
strain. Therefore, it is tempting to speculate that low iron
acquisition by the tonB1 and tonB2 mutants may adversely
affect one or more adhesion/invasion factor(s).
In conclusion, our results showed that L -cysteine plays an
important role in haemin utilization in R. anatipestifer and
that TonB1, but not TonB2, was involved in haemin iron
acquisition in the presence of L -cysteine. Moreover, both
the tonB1 and tonB2 mutants showed an impaired adherence
capacity to Vero cells, as well as reduced virulence.
Hu, Q., Miao, S., Ni, X., Lu, F., Yu, H., Xing, L. & Jiang, P. (2013).
Construction of a shuttle vector for use in Riemerella anatipestifer.
J Microbiol Methods 95, 262–267.
Huynh, C. & Andrews, N. W. (2008). Iron acquisition within host cells
and the pathogenicity of Leishmania. Cell Microbiol 10, 293–300.
Krewulak, K. D. & Vogel, H. J. (2011). TonB or not TonB: is that the
question? Biochem Cell Biol 89, 87–97.
Lu, F., Miao, S., Tu, J., Ni, X., Xing, L., Yu, H., Pan, L. & Hu, Q. (2013).
The role of TonB-dependent receptor TbdR1 in Riemerella anatipestifer in iron acquisition and virulence. Vet Microbiol 167, 713–718.
Mavromatis, K., Lu, M., Misra, M., Lapidus, A., Nolan, M., Lucas, S.,
Hammon, N., Deshpande, S., Cheng, J. F. & other authors (2011).
Complete genome sequence of Riemerella anatipestifer type strain
(ATCC 11845). Stand Genomic Sci 4, 144–153.
Moreira, L. O., Andrade, A. F., Vale, M. D., Souza, S. M., Hirata, R., Jr,
Asad, L. M., Asad, N. R., Monteiro-Leal, L. H., Previato, J. O. & MattosGuaraldi, A. L. (2003). Effects of iron limitation on adherence and cell
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ACKNOWLEDGEMENTS
Nairz, M., Schroll, A., Sonnweber, T. & Weiss, G. (2010). The struggle
This work was supported by the National Natural Science Foundation
of China (31272590 and 31472224).
for iron – a metal at the host–pathogen interface. Cell Microbiol 12,
1691–1702.
Noinaj, N., Guillier, M., Barnard, T. J. & Buchanan, S. K. (2010).
TonB-dependent transporters: regulation, structure, and function.
Annu Rev Microbiol 64, 43–60.
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