Helicobacter pylori CagL Y58/E59 Mutation Turns-Off Type IV Secretion-Dependent Delivery of CagA into Host Cells
Backert S (2014) Helicobacter pylori CagL
Y58/E59 Mutation Turns-Off Type IV Secretion-Dependent Delivery of CagA into
Host Cells. PLoS ONE 9(6): e97782. doi:10.1371/journal.pone.0097782
Helicobacter pylori CagL Y58/E59 Mutation Turns-Off Type IV Secretion-Dependent Delivery of CagA into Host Cells
Nicole Tegtmeyer 0
Judith Lind 0
Benedikt Schmid 0
Steffen Backert 0
Jun Sun, Rush University Medical Center, United States of America
0 1 Friedrich Alexander University Erlangen, Department of Biology, Division of Microbiology, Erlangen, Germany, 2 Friedrich Alexander University Erlangen, Department of Biology, Division of Biotechnique , Erlangen , Germany
The type IV secretion system (T4SS) is a major virulence determinant of the gastric pathogen Helicobacter pylori. The CagL protein is a specialized adhesin of the corresponding T4SS pilus, which establishes initial contact with the integrin b1 receptor on host target cells. Recent studies proposed that Y58 and E59 amino acid polymorphisms in CagL increase the virulence of H. pylori strains by enhanced translocation and phosphorylation of the CagA effector protein. These polymorphisms were therefore correlated with an increased risk of gastric cancer development. Here we show that the Y58/E59 motif, which is located in a loop connecting two a-helices, and corresponding polymorphisms could influence the function of CagL. However, expression of isogenic CagL Y58/ E59 variants in H. pylori strain 26695 significantly blocked the translocation and phosphorylation of CagA as compared to complemented wild-type CagL. These results suggest that the function of the T4SS for delivery of CagA is turned-off by the Y58/E59 mutation in CagL. This activity appears to be similar to the one recently described for another T4SS pilus protein, CagY, which is also sufficient to cause gain or loss of T4SS function. These data support the hypothesis that certain mutations in CagL or recombination events in CagY may serve as a sort of molecular switch or perhaps rheostat in the T4SS, which could alter the function of the pilus and "tunes" injection of CagA and host pro-inflammatory responses, respectively.
In this Formal Comment we refer to the recent publication in
PLOS ONE by Yeh and co-workers . This report claims that
the Helicobacter pylori CagL amino acid polymorphisms Y58 and
E59 increase the virulence of corresponding clinical strains. CagL
is a specialized adhesin encoded by the cag pathogenicity island
(cagPAI). During infection, CagL is recruited to the surface of a
type IV secretion system (T4SS) pilus structure, mediating contact
with the integrin a5b1 receptor and translocation of the virulence
factor CagA . Yeh and co-workers produced H. pylori CagL
mutants in one strain (Hp1033) followed by infection of AGS
gastric epithelial cells and investigated the expression of integrin
a5b1, CagA translocation/phosphorylation and other parameters
. It was claimed that H. pylori CagL Y58/E59 point mutants
retain active integrin b1 with stronger binding affinity and
significantly enhance CagA translocation and phosphorylation as
Formal Comments are critiques of specific published articles.
compared to wild-type CagL. The above study was based on an
earlier publication by the same group, reporting that CagL
sequence polymorphisms in H. pylori correlated with
clinicohistological outcomes and gastric a5b1 integrin expression . In
this study, 145 patients with H. pylori infection and different gastric
diseases were investigated. The isolates from the gastric cancer
(GC) group of patients revealed a higher rate of CagL Y58/E59
amino acid sequence polymorphisms than those in non-GC
patients (P , 0.05). The authors therefore correlated the Y58/
E59 polymorphisms in CagL with an increased risk of GC
development , which was now interpreted to arise by higher
binding capacities of CagL to integrin a5b1 and enhanced
injection of CagA into gastric epithelial cells .
We have previously reported that CagL contains an
RGDmotif, like the human extracellular matrix protein fibronectin, and
is able to trigger RGD-dependent binding to integrin a5b1 during
infection . It was also shown that purified CagL alone, in an
RGD-dependent fashion, can directly induce intracellular
signaling pathways upon contact with mammalian cells including kinase
activation , gastric acid suppression [5,6], b-defensin-1
suppression  and production of IL-8 . During interaction
with various human and mouse cell lines, CagL mimics fibronectin
in triggering cell spreading, focal adhesion formation, and
activation of several tyrosine kinases in an RGD-dependent
manner. Among the activated factors are the kinases FAK and
Src, but also the actin-binding protein cortactin and EGF receptor
members EGFR and Her3/ErbB3 [4,5,9]. It was also
demonstrated that CagL can interact with the integrin member avb3
with yet unknown consequences for the host cell . In addition,
CagL can bind to the integrin member avb5 to induce signaling
leading to gastrin production in an RGD-independent manner
. Very recently, we presented the crystal structure of CagL
revealing an elongated four-helix bundle [12,13]. The RGD-motif
is surface-exposed but located within a long a helix (Figure 1A,B),
which is unprecedented as previously characterized
integrinbinding RGD-motifs are located within extended or flexible loops
. Comparison of seven crystallographically-independent CagL
molecules revealed substantial structural flexibility, leading to the
hypothesis that CagL may partly unfold during receptor binding
. Here we aimed to investigate the role of Y58/E59 amino
After the first publication by Yeh and co-workers in 2011 ,
we got interested in the potential role of CagL Y58/E59
polymorphisms in host cell interactions by the H. pylori T4SS.
The CagL crystal structure reveals that the amino acids at position
58 and 59 are located at the end of a loop between two
neighboring helices, a1 and a2 (Figure 1A/B). Considering the
structural flexibility in this region , we assumed that mutation
to the Y58/E59 residues may cause a change in CagL structure,
leading to altered integrin interaction which could influence CagA
translocation. To investigate this hypothesis, we first deleted the
entire cagL gene in the cagPAI of H. pylori and complemented
wildtype cagL gene of strain 26695 in the urease gene locus using a
socalled double cross-over construct as described [9,14]. While
wild-type H. pylori was able to produce phosphorylated CagA in
infected AGS cells, the cagL deletion mutant did not as expected
(Figure 1C/D and Figure S1). Complementation of the cagL
mutant strain with wild-type cagL carrying a hemagglutinin
(HA)tag restored CagA translocation to wild-type levels, indicating that
our complementation approach works (Figure 1C/D and Figure
S1). We have then noted that the CagL Y58/E59 region of the
used GC strains contains some additional amino acid exchanges at
position 6062  (Figure 1B, yellow box). We therefore
introduced the amino acids 5862 of GC strains in CagL of
strain 26695 in order to generate a CagL Y58/E59 expressing
mutant, CagLYE (Figure 1B, bottom). We have collected four
different clones, which were confirmed by PCR and standard
sequencing, indicating that the mutation was introduced correctly
into H. pylori. As a further control, we monitored the expression of
the 26 kDa CagL protein using a-HA antibodies. The results show
that CagL is produced in similar amounts between the
complemented wild-type and Y58/E59 expressing mutants (Figure 1C).
However, to our great surprise, infection experiments have shown
that the CagLYE variant completely suppressed the production of
phosphorylated CagA as compared to complemented wild-type
CagL as monitored in a time course of 18 hours (Figure 1C and
data not shown). Three different experiments with four individual
CagLYE clones (14) gave the same results (Figure 1C/D). Thus,
our experiments revealed entirely different data to those reported
by Yeh and co-workers .
CagL is one of the most studied factors of the H. pylori T4SS
encoded by the cagPAI . We show here that expression of
an isogenic CagL Y58/E59 variant in H. pylori strain 26695
significantly blocked the translocation and phosphorylation of
CagA as compared to complemented wild-type CagL. These data
are in contrast to a recently published paper . As a possible
explanation for the conflicting data, we cannot exclude the
possibility of specific genetic differences between strains 26695 and
Hp1033. However, we have noted that Yeh and co-workers
performed a very unusual procedure to introduce point mutants in
H. pylori . The cagL gene was first disrupted by introduction of a
chloramphenicol resistance (ChlR) cassette as selective marker.
Thus, the cagL gene was not deleted from the chromosome, which
offers the possibility of unwanted single cross-over events during
transformation. It is well known that recombination in H. pylori by
single cross-over leads to integration of the entire plasmid into the
chromosome , with the result that two copies of cagL genes will
appear in the transformants. This would mean that both the cagL
wild-type and cagL mutant are possibly present in the
chromosome, and both genes can easily recombine with each other during
cultivation. However, to generate Y58/E59 mutants, this clone
was then transformed with the cagL gene carrying the various
mutations, and the authors screened for one clone that lost the
ChlR cassette . Since chromosomal transformation rates in H.
pylori are commonly between 1025 to 1026 per microgram DNA
, this would indicate that Yeh and co-workers must have
screened at least 100,000 colonies per experiment in order to get
one ChlRnegative clone. This experimental design is technically
extremely difficult and was not described. However, it also
remained unclear how correct introduction of the Y58/E59
mutations and exclusion of wild-type cagL gene presence in the
Hp1033 chromosome has been confirmed . In addition, the
sequence of wild-type cagL from strain Hp1033 is not provided by
the authors nor deposited in a gene database, and cagL expression
was not tested by Western blotting or RT-PCR . It is therefore
very likely that the generated mutant clone can still express
functional CagL wild-type protein, which could explain their
results. By comparison, we excluded single cross-over events by
deleting entire cagL from the chromosome before introducing the
cagL mutant allele [9,14]. In addition, we have confirmed our cagL
mutagenesis and correct expression by several independent
approaches including PCR, sequencing and Western blotting.
We also tested various individual cagL YE mutant clones, which
always gave the same results as described above. We therefore
suggest that the function of the T4SS for delivery of CagA is
turned-down by the Y58/E59 mutation in CagL of 26695 and
does not enhance T4SS functions as claimed by Yeh and
We think that the above example of contradictory data
underlines the important necessity that extreme care should be
taken when performing mutagenesis in H. pylori, which is the
proper basis for subsequent functional studies. Based on the above
results, our Formal Comment offers putative reasons for the
conflicting data and should help to diminish uncertainty in the
scientific community. Interestingly, another recent report
demonstrated in murine and non-human primate models that
immunedriven host selection for recombination in another H. pylori cagPAI
protein, CagY, is also sufficient to cause gain or loss of T4SS
function with regard to IL-8 induction . These data together
support the hypothesis that certain variations in both proteins,
CagY and CagL, may function as sort of molecular switches or
perhaps rheostats in the T4SS which can alter the function of the
pilus and "tunes" injection of CagA and host pro-inflammatory
responses, respectively. In future experiments the function of such
polymorphisms for the infection process should be studied in more
detail using a large number of H. pylori strains from patients with
different disease outcome.
Materials and Methods
AGS cell culture
The human gastric adenocarcinoma cell line AGS (ATCC
CRL-1739TM) was cultivated in RPMI 1640 medium, which was
supplemented with 10% fetal calf serum (Gibco, Paisley, UK).
Cells were grown at 37uC and 5.0% (v/v) CO2 and subcultivated
in a ratio of 1:31:5 every 23days at a confluence of 70% to 80%.
H. pylori strains and infection studies
We are using cagL of the fully sequenced strain 26695 as a model
(accession number NC_000915). The H. pylori wild-type strain
and isogenic HpDcagL deletion mutant were generated and grown
as described . To complement the HpDcagL mutant strain,
the wild-type cagL gene was introduced into the chromosomal
ureA locus, using a pAD1-derived plasmid . CagL proteins
expressed from the ureA promoter contain a hemagglutinin (HA)
tag introduced following the signal sequence at amino acid
position 22 . For infection experiments, H. pylori were grown
for 2days in thin layers and added at a multiplicity of infection
(MOI) of 100 . All experiments were done in triplicate.
Site-directed mutagenesis of CagL
Site-directed mutagenesis of CagL was performed using the
pAD1 vector as DNA template . As shown in Figure 1B, CagL
of GC strains contain the amino acids Tyr-Glu-Ile-Gly-Lys
(YEIGK) at position 5862 of the protein, while strain 26695
has the amino acids Asn-Glu-Met-Gly-Glu (NEMGE) at this
position. To generate a CagL 26695YE mutant (Figure 1B,
bottom), we performed PCR reactions with the following primers:
628F 59-GGTAAAGAAGATGCTCTAAACATC and 628R
59GATTTCATAATTAGCACTAGGGCTAG to open and
amplify the entire construct. For amplification, PhusionH High-Fidelity
DNA Polymerase (NEB, Ipswich, USA) was used, followed by
PCR purification (MinElute PCR Purification Kit, Qiagen,
Hilden, Germany), digestion with DpnI (Promega, Madison,
USA), and ligation using T4 DNA Ligase (Promega).
Resequencing and Western blotting of E. coli or H. pylori lysates,
respectively, verified the appropriate expression of CagL mutant
variants from the resulting plasmids.
Antibodies and Western blotting
Infected cells were harvested in ice-cold PBS containing 1 mM
Na3VO4 (Sigma-Aldrich). Western blotting was done as previously
described . Rabbit a-CagL antiserum was raised against the
C-terminal peptide (C-RSLEQSKRQYLQER) of the protein and
was prepared by Biogenes (Berlin, Germany). The a-HA-tag
antibody (NEB Cell Signaling, Frankfurt/M., Germany) was also
used to detect tagged CagL. The pan-a-phosphotyrosine antibody
PY-99 (Santa Cruz) and a-CagA (Austral Biologicals, San Ramon,
CA, USA) were used to investigate the phosphorylation of CagA
. The a-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) antibody (Santa Cruz) served as loading control in
each Western blot (data not shown). As secondary antibodies,
horseradish peroxidase conjugated a-mouse, a-rabbit, or a-goat
polyvalent sheep immunoglobulin were used and antibody
detection was performed with the ECL Plus chemiluminescence
kit (Amersham Pharmacia Biotech) . Band intensities were
quantitated with the Lumi-Imager F1 (Roche Diagnostics,
Mannheim, Germany) . The data are representative from
three independent experiments.
All data were evaluated using Student t-test with SigmaStat
statistical software (version 2.0). Statistical significance was defined
by P#0.05 (*) and P#0.005 (**). All error bars shown in Fig. 1 and
those quoted following the 6 signs represent standard deviation.
Figure S1 Role of Helicobacter pylori CagL Y58/E59
mutation in type IV secretion-dependent delivery of
CagA in host cellAs.GS gastric epithelial cells were infected
with the indicatedH. pylori strains and cagL mutants for 8
hours using a multiplicity of infection of 100. Resulting protein
lysates were probed with the indicated antibodies as described.
This figure shows the original uncropped Western blots underlying
Figure 1C. The cut sections are marked with boxes. The red asterisk
in thea -PY-99 blot marks the phosphorylated 125 kDa host
cell protein vinculin, which always runs below the phospho-CagA
band at about 140 kDa .
We kindly thank Dr. Wolfgang Fischer (Pettenkofer Institute Munich,
Germany) for discussion of the data and Dr. Hartmut Niemann (University
Bielefeld, Germany) for his support concerning the CagL structure.
Conceived and designed the experiments: NT JL BS SB. Performed the
experiments: NT JL BS. Analyzed the data: NT JL BS SB. Wrote the
paper: NT SB. Made figures: NT BS SB.
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