Electron Microscopic, Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger
Genetic and Protein Expression Analyses of
Helicobacter acinonychis Strains from a Bengal Tiger. PLoS ONE 8(8): e71220. doi:10.1371/journal.pone.0071220
Electron Microscopic, Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger
Nicole Tegtmeyer 0 1
Francisco Rivas Traverso 0 1
Manfred Rohde 0 1
Omar A. Oyarzabal 0 1
Norbert Lehn 0 1
Wulf Schneider-Brachert 0 1
Richard L. Ferrero 0 1
James G. Fox 0 1
Douglas E. Berg 0 1
Steffen Backert 0 1
Michael Hensel, University of Osnabrueck, Germany
0 a Current address: Department of Biology, Chair of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany b Current address: Department of Medicine, University of California San Diego , San Diego, California , United States of America
1 1 Institute of Medical Microbiology, Otto von Guericke University Magdeburg , Magdeburg, Germany , 2 Helmholtz Centre for Infection Research , Braunschweig, Germany , 3 Institute for Environmental Health, Inc., Seattle, Washington, United States of America, 4 Institute for Medical Microbiology and Hygiene, University of Regensburg , Regensburg, Germany , 5 Centre for Innate Immunity & Infectious Diseases, Monash Institute of Medical Research , Clayton , Australia , 6 Division of Comparative Medicine, Massachusetts Institute of Technology , Cambridge, Massachusetts , United States of America, 7 Department of Molecular Microbiology, Washington University School of Medicine , St. Louis, Missouri , United States of America
Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 mm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, c-glutamyltranspeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.
Funding: The work of SB is supported through a DFG grant (project B10 of CRC-796). FRT was supported by a stipend of SENACYT (Secretaria Nacional de Ciencia
y Tecnologia, Panama). RLF is a Senior Research Fellow of the NHMRC. Research at MIMR is supported by the Victorian Governments Operational Infrastructure
Support Program. The research in DBs group has been supported by US National Institutes of Health Grants (R21 AI078237 and R21 AI088337). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: OAO is employed by Institute for Environmental Health Inc and declares no conflict of interest. This does not alter the authors adherence
to all the PLOS ONE polices on sharing data and materials.
The genus Helicobacter comprises a heterogeneous group of
Gram-negative bacteria that colonise different mammalian hosts,
including domestic and wild animals, non-human primates and
humans [1,2]. Currently there are 33 validated Helicobacter species
and several other described isolated candidates . Best known is
the human gastric pathogen, Helicobacter pylori , which is highly
motile using a unipolar bundle of two to six sheathed flagella .
During co-evolution with humans, a multitude of
pathogenicityassociated factors were developed to adapt and survive in the
challenging gastric milieu. Helicobacter pyloris hallmark enzyme is a
potent multi-subunit urease complex, which is fundamental for
neutralizing the acidic pH in the stomach . Other bacterial
factors such as the blood-group antigen binding protein BabA ,
sialic acid-binding adhesin SabA , outer inflammatory protein
OipA , neutrophil activating protein NapA , lytic
transglycosylase Slt , duodenal ulcer promoter protein A
(DupA)  and c-glutamyl transpeptidase (GGT) 
contribute significantly to successful H. pylori pathogenesis. In
addition, the secreted protease HtrA (high temperature
requirement A) may disrupt epithelial cell barrier functions as it can
cleave the host tumor suppressor and cell adhesion protein
Ecadherin [17,18]; and two potential relaxases, the VirD2 homologs
Rlx1 and Rlx2, are involved in DNA transfer [19,20]. The best
studied virulence factors in H. pylori, however, are the vacuolating
cytotoxin VacA and the effector protein CagA [21,22]. Mature
VacA is a multifunctional toxin implicated in perturbing
endosomal trafficking, mitochondrial apoptosis and inhibition of
T-cell proliferation [23,24]. The cagA gene, located in the cag
pathogenicity island (cagPAI), is a marker of a type IV secretion
system, a molecular syringe-like structure (composed of VirB1 to
VirB11, VirD4 and several other Cag proteins) through which
CagA can be delivered into host target cells .
Established genetic polymorphisms in cagA and vacA genes affect
H. pylori infection outcomes and also exhibit clear
phylogeographical structural differences that reflect both ancient and recent
human migrations and contacts [22,26]. More generally, H. pylori
is genetically highly diverse; independent isolates (from unrelated
persons) are usually distinguishable from one another by DNA
fingerprinting ; and strains typically differ from one another by
some 25% in sequences of essential housekeeping genes and 5%
or more in overall gene content [28,29]. This stems from frequent
point mutation, differences in restriction-modification systems, and
recombination between divergent strains and species. H. pylori is
transmitted preferentially within families and communities ,
and phylogenetically distinct sets of DNA sequences are found in
strains from different parts of the world .
Genetic studies indicate that H. pylori co-migrated with humans
from east Africa around 58,000 years ago, and its present
worldwide genetic diversity reflects the isolation by distance that
has shaped this bacterial species over time . However, it is still
not fully clear when exactly H. pylori became adapted to the human
gastric niche. The most favoured idea is that Helicobacter species
have been universally part of humans and our non-human primate
ancestors microbiota since long before modern Homo sapiens
appeared on Earth [31,32]. Alternatively, a host jump theory 
suggested acquisition of H. pylori infections in humans more
recently, ca. 10,000 years ago, when the first agricultural societies
started, as the result of frequent contacts with infected
domesticated animals . Besides scattered reports of natural H. pylori
infection associated gastritis in domestic cats [2,3537], the main
natural hosts for H. pylori seem to be humans and some
nonhuman primates [38,39]. The potential for species jumps between
hosts is illustrated, however, by lab reports of human H. pylori
strains that were adapted to mice, dogs, cats, piglets and
mongolian gerbils . In addition, a number of other gastric
non-pylori Helicobacter spp. have been identified in various
mammalian hosts in recent years, including H. felis, H. acinonychis,
H. salomonis, H. bizzozeronii, H. mustelae, H. suis and H. aurati as well
as some extragastric Helicobacter spp. such as H. canis, H. pullorum,
H. cinaedi, H. fennelliae, H. typhlonius, H. bilis, H. hepaticus and H.
Based on genome sequencing and other reports, the closest
relative of H. pylori is H. acinonychis, which has been found in
stomachs of big predator cats such as lions or cheetahs
[31,33,44,4852]. Complete sequencing of H. acinonychis strain
Sheeba and comparisons to European and African H. pylori strains
exhibited similar core genes and also distinctively different features
including an unusually high number of fragmented genes for VacA
and outer membrane proteins (OMPs). A host jump from early
humans to large felines, probably about 200,000 years ago, was
proposed . However, our knowledge of non-pylori Helicobacter
spp. such as H. acinonychis is still very incomplete, as illustrated by a
total of only two deposited 16S rRNA sequences (accession
numbers AM260522.1 and AF057163.1) for this species. We are
interested in identifying novel gastric and extra-gastric Helicobacter
spp., and characterising their genetics, bacterial pathogenicity
factors and gastric disease-associated processes to better
understand mechanisms of bacterial adaptation to host environments
and associated health and disease . Here we report on the
isolation and detailed molecular characterisation of novel H.
acinonychis strains from a Bengal tiger (Panthera tigris tigris).
Isolation and Visualisation of Helicobacter Strains from a
Five single Helicobacter-like colonies (called SB-1 to SB-5) from
diarrheic fecal samples of a captive Bengal tiger from Thailand
were grown under microaerobic growth conditions using either
Columbia agar plates with 5% sheep blood or GC agar plates with
10% horse serum. Gram-staining indicated that these bacteria
were Gram-negative. Scanning electron microscopic investigation
revealed spiral-shaped Helicobacter-like organisms (Fig. 1A), about
0.250.45 mm in diameter and 2.56 mm in length. The majority
of these bacteria contained 14 monopolar flagella with lengths of
about 1.54.5 mm, although some were non-flagellated (Fig. S1).
This suggests that some bacteria may have lost their flagella during
sample processing. Negative stained samples revealed similar
results (Fig. 1B). The visualized flagella were commonly sheathed
and about 4348 nm in diameter, although non-sheathed flagella
(1517 nm in diameter) were also observed. In addition, we often
found that the flagella ends were thickened and had a bulb-like
shape (see enlargement panels in Fig. 1B, arrows).
16S rRNA, 23S rRNA and RAPD Fingerprinting Analysis of
Helicobacter Genomic DNA
Next, we investigated the 16S rRNA of these isolates in direct
comparison with various Helicobacter species including H. pylori,
H. acinonychis, H. felis, H. fennelliae, H. hepaticus, H. mustelae, H.
salomonis, H. bilis, H. cinaedi, H. typhlonius, H. magdeburgensis, H.
bizzozeroni, H. canis and H. aurati. For this purpose, we amplified a
1.2 kb DNA fragment of the 16S rRNA region which is highly
conserved within the genus Helicobacter . All Helicobacter
species revealed the expected PCR product, while Campylobacter
jejuni or other controls did not (Fig. 2A and data not shown). To
confirm the specificity of these fragments, all PCR products were
then digested with the restriction endonuclease HhaI or AluI,
which yield specific band patterns for known Helicobacter species
. The RFLP pattern was similar between various Helicobacter
isolates (Fig. 2B and Fig. S2AB), and SB-1 was most similar to
those of H. pylori and H. acinonychis, which suggests that our Bengal
tiger isolates are closely related to these species. This conclusion is
in good agreement with the RAPD fingerprinting profiles using
various primers (Fig. 2C and Fig. S2CD). The 16S rRNA genes
from our individual isolates were then sequenced (GenBank
accession number JN251811.1). They belong phylogenetically to a
specific 16S rRNA gene cluster, which includes isolates of the
species H. acinonychis and H. pylori (Fig. 3A). Sequencing of a
23S rRNA gene fragment yielded a dendrogram which was also in
full agreement with that generated by the analysis of the 16S
rRNA gene (Fig. S3).
Bengal Tiger Helicobacter Isolates Express a Functional
An active urease is considered essential for all Helicobacters that
colonize the acidic mammalian stomach, whereas extra-gastric
Helicobacters are generally without urease [1,2]. To test the idea that
our new Helicobacters are likely to be stomach colonizers despite
having been isolated from feces, we next tested if our strains have a
functional urease. For this purpose, bacteria were grown on
selective acidified agar plates supplemented with urea, the
substrate of H. pylori urease . These experiments yielded
functional urease enzymes allowing urea hydrolyzation to a high
extent in all H. acinonychis strains including SB-1 to SB-5, similar to
that of H. pylori control strain 26695, while retarded growth and no
urea hydrolysis was seen in the 26695DureA mutant as expected
(Fig. 4A and data not shown). This confirms that the Helicobacter
isolates exhibit strong urease activity, much like that of H. pylori
and other H. acinonychis isolates, and thus, are likely of gastric
origin although they also survived passage through the intestine.
Helicobacter Isolates Contain Conserved Urease and
Flagellin but not vacA or cagA Genes
Next we amplified and sequenced urease and flagellin genes
(Table S1). Their sequences (deposited in the NCBI GenBank;
accession numbers listed in Materials and Methods) are highly
similar to those from H. acinonychis strain Sheeba (100% and 98%
DNA identity, respectively), and less similar to corresponding H.
pylori genes (94% and 90% identity, respectively, to those of strain
26695) (data not shown). As reported for various H. acinonychis
strains from other big cats such as lions and cheetahs , analyses
of PCR products using vacA gene specific primers indicated that
vacA is fragmented, which implies that a functional vacuolating
cytotoxin is not synthesized, an inference confirmed by
immunoblotting (data not shown). Collectively, these data indicate that
these tiger isolates belong to the novel H. pylori-derived species, H.
acinonychis. The VacA results seem particularly remarkable since
VacA is expressed in virtually all H. pylori strains and seems to
contribute importantly to bacterial fitness during colonization
. Furthermore, we also failed to PCR amplify conserved
fragments of cagA and other cagPAI genes such as virB10 and
virB11 (Table S1), in accord with the lack of a cagPAI in the
genome-sequenced H. acinonychis strain .
Prophage Genes are Potential Genetic Markers for
Helicobacter acinonychis Isolates
To further test our inference that these novel strains belong to
the H. acinonychis group, we investigated the presence of several
other genetic markers. Previous genome sequencing of the Sheeba
isolate and subsequent microarray analyses had identified
prophage genes , now known to be related to recently
discovered plaque forming temperate phage from East Asia [56
58]. In general, certain prophages have been implicated in
bacterial virulence  and it has been suggested that prophage
genes of H. acinonychis might potentially affect host adaptation and
specificity . Forty one of the imported coding sequences
(CDSs) in the Sheeba strain are actually present within two
prophages, called prophage I and prophage II, but such prophages
or remnants are present in only a very few H. pylori genomes .
A PCR assay developed for one of these prophage genes
[prophage I ORF3, a helicase (Hac1336), Table S1],
demonstrated this genes presence in SB-1 to SB-5, Sheeba and other H.
acinonychis isolates, while its absence from several H. pylori in our
collection (Fig. 4B). These results provide further evidence that
SB1 to SB-5 represent H. acinonychis strains.
Genetic Comparison of Helicobacter acinonychis Isolates
from Europe, US and Asia
To investigate genetic relatedness among different H. acinonychis
isolates, we compared 16S rRNA genes and RAPD fingerprint
typing patterns of our five isolates (SB-1 and SB-5) with those of
two other H. acinonychis strains from a Sumatran tiger maintained
in captivity in a German zoo . RFLP of the 16S rRNA PCR
products yielded identical fragment patterns (Fig. S4AD). The
nucleotide sequence of the 16S rRNA gene was also highly similar
between isolates from Bengal and Sumatran tigers (accession
number AF057163) (Fig. 3, bottom). Thus, we next scored RAPD
fingerprint patterns, which are more effective than the focused
analysis of individual genes at discriminating between related
strains. The RAPD patterns were also almost identical between
our five strains and those from the Bengal and Sumatran tigers
(Fig. S5AC), indicating that very closely related H. acinonychis
strains have colonized different tiger subspecies from very different
Next, we compared the RAPD fingerprint patterns of our H.
acinonychis strain SB-1 with those of strains from other big cats:
namely three cheetahs from a US zoo, two lions, one lion-tiger
hybrid and another tiger housed at a European circus (Table 1).
The results show that the fingerprinting patterns of our isolates
from the Bengal tiger are also very similar to those of the cheetah
and lion isolates, which were categorised in group I , while are
distinct from those of the lion-tiger hybrid and other tiger isolates,
classified as group II H. acinonychis (Fig. 4C and Fig. S6AC) .
Total Protein Profiling and Expression of Homologous
Pathogenicity Factors from H. pylori
The close relatedness of our H. acinonychis isolates with H. pylori
allowed us to screen for the presence or absence of well-known
colonization and pathogenicity factors by immunoblotting. First,
we compared the total protein profiles from our Bengal tiger
isolate SB-1 with that of the fully-sequenced H. pylori strains 26695
and J99. Coomassie-blue staining of separated total proteins
revealed the presence of typical bands with sizes matching those of
urease subunits A and B (Fig. 5A), whereas a band in the size range
of CagA (ca. 130150 kDa) was only observed in the H. pylori
strains, but not in SB-1 (Fig. 5A), in agreement with the PCR
results described above. Second, Western blot analysis also
confirmed the presence of urease A (ca. 30 kDa) and urease B
(ca. 60 kDa) subunits and the absence of a CagA band in SB-1
extracts (Fig. 5B). In agreement with the finding of flagella by
electron microscopy, we also found a ,60 kDa flagellin
component recognised by an H. pylori-specific anti-flagellin antibody in
extracts of our strains (Fig. 5C, top).
The availability of antibodies against a series of other H. pylori
proteins led us to screen more systematically for several adhesins
(BabA, SabA or OipA), other virulence factors (NapA, HtrA, Slt,
DupA), cagPAIencoded proteins (VirB10/CagY, Cag3/Cagd,
CagM or CagN) and DNA transfer proteins (the VirD2 orthologs,
Rlx1 and Rlx2) [19,20,62]. All antibodies were proven to
specifically recognise the corresponding proteins in the H. pylori
strains 26695 and J99, respectively (Fig. 5C and Fig. S7AB), and
our recently genome-sequenced H. pylori strains Shi470 and Cuz20
(accession numbers NC010698.2 and CP002076.1), which, unlike
genetic relatedness. Panel C: RAPD fingerprinting of the Helicobacter isolates using primer D9355 was performed as described previously . A
typical RAPD fingerprinting profile is shown and revealed the relatedness between H. pylori, H. acinonychis and Bengal tiger isolate. Asterisks indicate
three major bands which were identical among the latter three samples. M, DNA size marker.
26695 and J99, do encode full-length DupA (,80 kDa) and Rlx
(,70 kDa) proteins (Fig. S7B). We found that while no SabA,
OipA, DupA and none of the cagPAI proteins are expressed in H.
acinonychis, bands corresponding in size to BabA, GGT, HtrA,
NapA and Slt were produced, indicating that SB-1 contains genes
for these proteins (Fig. 5C and Fig. S7AB).
Numerous Helicobacter species have been identified in a wide
range of mammals, possibly reflecting long evolutionary
coexistence [1,2,32,38]. The main hosts of H. pylori are humans and
non-human primates, although this species has also been isolated
from domestic cats [2,3537], and laboratory experiments have
shown that it can also infect rodents (mice and gerbils), dogs, cats
Figure 4. Urease expression and genetic relatedness of various Helicobacter acinonychis isolates from different big cats from the US,
Europe and Asia. Panel A: Selection of bacteria producing functional urease on acidified agar supplemented with urea . Left samples
correspond to H. acinonychis strain Sheeba (top) and the Bengal tiger isolate SB-1 (bottom). The observed color change from orange to red indicated
that bacterial colonies were producing functional urease and growing. Right samples are the H. pylori wild-type (wt) 26695 (top) and isogenic DureA
mutant (bottom). Color change did not occur in the DureA mutant, indicating that functional urease was not being produced. Panel B: PCR of the
prophage gene helicase (Hac1336, Supplemental Table S1) shows a specific 1.2 kb product for the Bengal tiger isolate SB-1 and other H. acinonychis
strains, but not H. pylori. Panel C: RAPD fingerprinting of the indicated H. acinonychis isolates from tigers, cheetahs, lions and lion-tiger reveals the
close relatedness between strains in two specific groups, called I and II, as indicated. The RAPD primer D1254  has been used in this experiment.
M, DNA size marker.
and pigs [1,2]. The stomachs of mammalian carnivores (e.g. cats,
dogs, lions and cheetahs) are often naturally infected by non-pylori
Helicobacter species, including H. felis, H. bizzozeronii and H.
salomonis, which are very different from H. pylori [1,2,38], and
interestingly, the stomachs of large felines, can also be infected
with H. acinonychis, which is closely related to H. pylori
[31,33,44,4852,61,63]. However, compared to H. pylori we know
very little about H. acinonychis, with most of our knowledge about
Zoo, Columbus, Ohio, USA
Zoo, Columbus, Ohio, USA
Zoo, Columbus, Ohio, USA
aall animals were kept in captivity.
its genetics deriving from the genome sequence of only one strain,
Sheeba . The many fragmented genes in this strain Sheeba,
which are functional in H. pylori, led to the proposal that H.
acinonychis was separated from H. pylori lineages some 200,000
years ago, perhaps after a large feline ate an H. pylori-infected
human, thereby allowing a jump between mammalian hosts .
H. acinonychis has been isolated from captive American and
European lions and cheetahs suffering from chronic gastritis and
vomiting [33,44,63], as well as tigers and one lion-tiger hybrid
[33,61]. In the present report, we have isolated for the first time
live H. acinonychis from diarrheic feces of a Bengal tiger (Panthera
tigris tigris) from Thailand. Although usually Helicobacters have not
been culturable from normal feces, our data are in accord with the
success in culturing H. pylori from feces from humans with diarrhea
[64,65] or H. mustelae in feces from ferrets suffering from
We characterised five individual strains at the molecular level.
Electron microscopy revealed a typical spiral-shaped
Helicobacterlike organism with 14 monopolar sheathed or non-sheated
flagella. PCR amplification, sequencing and phylogenetic analyses
based on similarity values of the 16S rRNA and 23S rRNA genes
identified our Bengal tiger strains as H. acinonychis.
Prophagespecific PCR and RAPD fingerprinting confirmed that these new
strains are very closely related to other H. acinonychis isolates. The
very low diversity of H. acinonychis strains from different animals
(tigers, cheetahs, lions and lion-tiger hybrid) and geographic
origins (US, Europe and Asia), which have been placed in only two
subgroups (I and II) with highly similar RAPD fingerprints, is
remarkable, given that H. pylori isolates are extremely diverse
[26,27,68]. The reason for this difference between the two species
in strain diversity is unknown, but may reflect the relatively
younger age of H. acinonychis as a species, or evolutionary
constraints imposed in its special big cat hosts.
The availability of H. pylori specific antibodies enabled us for the
first time to screen H. acinonychis for the expression of certain
pathogenicity associated proteins by Western blotting. Our data
indicate that Bengal tiger isolates express a flagellin (,60 kDa),
but the flagella structures seen by electron microscopy were
morphologically distinct from that of H. pylori. Another factor
important for colonisation in the stomach is the urease complex,
which consists of two major subunits (UreA and UreB) and some
accessory proteins. Urease activity neutralising gastric pH is
required to survive in an acid milieu, and also may play a role in
H. pylori metabolism , disruption of transepithelial resistance
 and pro-inflammatory responses . Both proteins are
highly conserved, bands corresponding to both UreA and UreB
proteins were expressed, and functional assays demonstrated a
highly active urease complex in our H. acinonychis isolates.
Adherence of H. pylori to specific glycan receptors in the human
gastric mucosa by outer membrane protein family members BabA,
SabA and OipA adhesins is widely assumed to be adaptive, to
contribute importantly to initial colonization and long-term
persistence in human hosts [68,71,72]. In contrast, the potential
adhesins of H. acinonychis are completely unknown. In agreement
with the host jump theory, the frequency of fragmented genes is
particularly high in the sequenced H. acinonychis Sheeba strain as
compared to H. pylori genomes, and this includes 12 OMPs, VacA
and others . Thus, it is probably not surprising that we could
not detect a Western blot band specific for SabA in H. acinonychis.
Interestingly, we observed a ,72 kDa band reacting with BabA
antiserum in H. acinonychis. A full-length ortholog of BabA has not
been noted in the Sheeba genome , but some of the OMPs in
the Sheeba strain exhibit homologous stretches to BabA, which
may explain our Western blot results. Furthermore, no protein
band corresponding to OipA was found in H. acinonychis, which is
in line with the observation that one gene in the Sheeba genome
(OMP-7, fragment 2), showing extensive homology to OipA, is
fragmented and therefore unlikely to be expressed in H. acinonychis.
In agreement with the absence of cagPAI and cagA genes in
previously analysed H. acinonychis isolates [31,33], we were also
unable to detect any protein expression for CagA and well-known
other cagPAI components such as Cagd, CagM, CagN or VirB10.
Furthermore, we also failed to PCR amplify conserved fragments
of vacA, cagA and other cagPAI genes. However, we were able to
detect full-length proteins in H. acinonychis for a series of other
wellknown H. pylori pathogenicity factors including NapA, GGT, HtrA
and Slt. The detection of GGT, NapA and Slt may explain the
chronic gastritis as characterized by the occurrence of
inflammatory cells in the gastric mucosa observed of some infected felines
[44,49,61]. The finding of an HtrA ortholog in H. acinonychis also
raises the possibility that this protein may disturb epithelial barrier
functions by cleaving E-cadherin [17,18,73], which also could be
involved in the gastric pathology observed in big cats.
In H. pylori there recently has been considerable interest in
strain-specific genes in the so-called plasticity regions, which are
large, possibly conjugative, transposons or transposon remnants
. Recent work has shown that they encode putative
pathogenicity factors, such as the duodenal ulcer-promoting gene
A (dupA), which has been associated with duodenal ulceration .
It has been noted that extensive size variation exists in the dupA
genes among clinical H. pylori isolates, which may interfere with
their putative activity [14,15]. Other genes include those encoding
putative DNA transfer enzymes, such as the relaxases Rlx1 and
Rlx2 [19,20,62]. Interestingly, in H. acinonychis we found proteins
of similar size to those of H. pylori that cross-reacted with antisera
to both Rlx1 and Rlx2, but no cross-reactivity with an anti-DupA
antibody. The role of Rlx1 and Rlx2 is not fully clear, but they
may be involved in the exchange of genetic material between
bacteria, which warrant further investigations [19,20,62,74].
Taken together, we have morphologically and genetically
characterised new Helicobacter spp. strains isolated from a Bengal
tiger in Thailand, which were classified as H. acinonychis and which
show similar genetic background to those of previously isolated H.
acinonychis strains from captive tigers, lions and cheetahs located in
Europe or the US. Currently there is only one H. acinonychis
genome sequence available, strain Sheeba, isolated from a lion
housed in a Russian circus , and there is a lack of other genetic
information in databases referring to different H. acinonychis strains
isolated from big cats located in various geographic locations.
Thus, this is the first report of an H. acinonychis isolate from an
Asian tiger. In addition, we have shown remarkably similar RAPD
fingerprinting patterns between worldwide H. acinonychis isolates
and screened for known pathogenicity factors from H. pylori such
as flagellin, ureaseA/B, NapA, HtrA, GGT, Slt, two relaxases and
probably a BabA-like protein. Most of these genes are expressed in
H. acinonychis strains isolated from the Bengal tiger. However,
CagA and cagPAI factors, as well as VacA, OipA, SabA and
DupA, were not detected. An important challenge for the future
will be to identify the function of proteins involved in colonization
and disease development. The use of mouse-adapted H. acinonychis
strains  should be a valuable approach for analyzing the
interplay between this human-derived animal pathogen and its
host. These studies could reveal the specificity of infections and
help us understand the evolutionary routes used by these gastric
Materials and Methods
Colonies of Helicobacter strains were isolated from diarrheic feces
of a captive Bengal tiger (Panthera tigris tigris) suffering from gastritis
in a zoo in Bangkok/Thailand. The samples were collected in
sterile tubes, incubated with brain heart infusion (BHI) medium
(5 mL per gram material), shaken for 20 min at 37uC in 50 mL
Falcon tubes at 1,0006g. The mixture was then centrifuged for
10 min at 2,0006g to remove larger particles and non-digested
material. The supernatant was removed and passed through sterile
filter paper (Whatman, GE Healthcare, UK limited Amersham
Place, UK) to further remove debris. Bacteria were then cultured
in different amounts (100, 50, 25 or 5 mL) on different agar plates
(H. pylori selective agar plates, GC agar plates with 10% horse
serum, Campylobacter selective plates, M uller-Hinton agar plates,
and Columbia agar plates containing 5% sheep blood). These
plates were incubated for 2, 3, 4, and 7 days, respectively. The gas
generating systems Campygen, Anaerogen (both from Oxoid/
Fisher Scientific, Germany), Anaerocult (Merck, Darmstadt,
Germany), and an anaerobic chamber with a mix of nitrogen,
carbon dioxide and hydrogen (90%, 5% and 5%, respectively)
were used for incubation at 37uC. Single bacterial colonies (called
SB-1, SB-2, SB-3, SB-4 and SB-5) were isolated and grown on
Columbia agar plates containing 5% sheep blood and Campygen
for further analyses.
Grown bacterial colonies were screened by standard
Gramstaining (Crystal violet, Grams iodine solution, acetone/ethanol
(50:50 vol/vol), 0.1% basic fuchsin solution) . This method
was applied as an initial step to investigate the morphology,
homogeneity and culture purity of the isolated bacterial
Bacterial Strains and Culture Conditions
We included in our studies some other described Helicobacter
species as controls: H. acinonychis (ATCC51101), H. felis
(ATCC49179), H. fennelliae (ATCC35684), H. hepaticus strain
1549/00 , H. mustelae (strain NL03-2004, unpublished), H.
salomonis (strain NL07-2005, unpublished), H. bilis (ATCC43879),
H. cinaedi (DSM5359, DSMZ Braunschweig, Germany), H.
typhlonius (MIT 97-6810), H. magdeburgensis (strain HM-007) ,
H. bizzozeronii (strain NL07-2005, unpublished), H. canis
(ATCC51401) and H. aurati (MIT 97-5075). In addition, we
included the four fully sequenced H. pylori strains 26695, J99 ,
Cuz20 and Shi470 , and a series of H. acinonychis strains
(Table 1) including two other widely uncharacterised isolates from
a Sumatran tiger, Panthera tigris sumatrae . All strains were
grown under standard conditions as described. To test for
functional urease activity, bacteria were grown on selective
acidified agar plates supplemented with urea, the substrate of H.
pylori urease, and phenol red as indicator .
The fecal samples were collected with permission and help by a
zookeeper. An Ethics statement was not necessary as the sample
was not collected by an invasive method disturbing the tiger in any
aspect. The tiger was actually not affected in any way nor harmed.
DNA Isolation, PCR Analyses and Sequencing
Plate-grown Helicobacter sp. were harvested with a sterile cotton
swab and suspended in 200 mL of lysis buffer (50 mM Tris-HCl
(pH 7.6), 100 mM EDTA, 0.5% Tween-20, 20 mg of proteinase
K per mL) and incubated at 58uC for 2 hours . The proteinase
K was inactivated by conventional phenol/chloroform extraction
method. Purified DNA was then precipitated with 2.5 volume of
96% ethanol and washed with 70% ethanol. To investigate the
presence of certain genes, we performed PCR assays using the
primers summarised in Table S1. DNA sequences were
determined by standard sequencing procedures [47,76]. The following
gene sequences from strain SB-1 were deposited in GenBank
databases: 16S rRNA (accession number JN251811.1), 23S rRNA
(KC470072.1 and KC470071.1), flagellin (KC470069.1), urease
(KC470068.1) and helicase (KC470070.1). Sequence comparison
was performed using NCBI database tools (http://blast.ncbi.nlm.
16S and 23S rRNA Sequence Analyses
16S and 23S rRNA sequence data from the tiger strain and
from closely related species, collected from GenBank, were aligned
using BioEdit . Aligned sequences were then imported as
FASTA to MEGA5  to determine DNA relatedness using the
Neighbor-Joining method  and to construct the optimal tree
using the Maximum Composite Likelihood method .
Restriction Fragment Length Polymorphism (RFLP) of the
16S rRNA Gene
For restriction fragment analysis of the 16S rRNA gene, we
amplified by PCR a specific and conserved 1.2 kb subfragment as
described  (Table S1). RFLP patterns of amplified PCR
products were obtained with each of the following restriction
enzymes, AluI and HhaI [54,81]. Digests were performed in the
appropriate 16buffers as recommended by the manufacturer
(New England Biolabs, Acton, MA, USA).
Randomly Amplified Polymorphic DNA (RAPD)
The RAPD fingerprinting method established to study H. pylori
strains , was used to compare the diversity of the DNA
sequences among the Helicobacter strains tested. This method uses
arbitrary oligonucleotide sequences to prime DNA fragments from
the whole genome. We used 20 ng genomic DNA from each strain
as template, 20 pmol of each primer (Table S1), 1U Taq
DNApolymerase (Qiagen, Hilden, Germany) and 250 mM from each
dNTP, 16buffer, and sterilized double distilled water for a total
volume of 50 mL. A Perkin-Elmer thermal cycler model 9700 was
used for amplification reactions. The cycling program was four
cycles of 94uC, 5 min; 40uC, 5 min; 72uC, 5 min; low stringency
amplification, and a final incubation at 72uC for 10 min.
Field Emission Scanning Electron Microscopy (FESEM)
Bacterial cells were harvested and fixed in a sterile solution
containing 5% formaldehyde, 2% glutaraldehyde in cacodylate
buffer (0.1 mM cacodylate, 0.01 mM CaCl2, 0.01 mM MgCl2,
0.09 mM sucrose, pH 6.9) for 1 hour on ice [82,83]. The solution
was centrifuged and passed through a sterile filter. After several
washes with cacodylate buffer and TE buffer (20 mM Tris, 1 mM
EDTA, pH 6.9), samples were dehydrated in serial dilutions of
acetone (10%, 30%, 50%, 70%, 90%, and 100%) on ice for
15 min each step. Samples were then allowed to reach room
temperature before another change of 100% acetone, after which
they were subjected to critical-point drying with liquid CO2
(CPD030; Bal-Tec, now Leica, Wetzlar, Germany). Samples were
finally covered with a ca. 10.0 nm thick gold film by sputter
coating (SCD500; Bal-Tec) and examined in a field emission
scanning electron microscope (Zeiss DSM 982 Gemini) using an
Everhart Thornley SE detector and in-lens detector in a 50:50
ratio at an acceleration voltage of 5.0 kV.
Electron Microscopic Analysis by Negative Staining
For negative staining, thin carbon support films were prepared
by indirect sublimation of carbon on freshly cleaved mica. Samples
were then absorbed to the carbon film and negatively stained with
1% (wt/vol) aqueous uranyl acetate (pH 4.5). After air drying,
samples were examined by transmission electron microscopy
(TEM) in a Zeiss TEM 910 at an acceleration voltage of 80 kV
and at calibrated magnifications using a line grid replica. Images
were recorded digitally with a Slow-Scan CCD-Camera (ProScan,
102461024, Scheuring, Germany) with ITEM-Software
(Olympus Soft Imaging Solutions, M unster, Germany).
A previously published method was adapted . Briefly, a
washed pellet of the strains Hp 26695, J99, Shi470 or Cuz20 and
Bengal tiger isolate was suspended in 0.5 mL of sodium dodecyl
sulfate (SDS) buffer (50 mM Tris hydrochloride (pH 6.8), 5%
bmercaptoethanol (vol/vol), 1% sodium dodecyl sulphate (wt/vol),
15% glycerol (vol/vol), and 0.01% bromophenol blue). The
homogenate was heated for 5 min at 95uC. Insoluble debris was
removed by centrifugation at 10,0006g for 5 min. Supernatants
were subjected to 6% and 10% SDS polyacrylamide gel
electrophoresis (SDS-PAGE) gels and blotted by Semi dry blotting.
Antibodies and Immunoblotting Analyses
The following primary antibodies were used: Rabbit polyclonal
anti-CagA antibody was purchased from Austral Biological (San
Ramon, CA, USA). The mouse polyclonal anti-urease antibodies
and anti-CagN antibodies were described elsewhere [85,86].
Polyclonal rabbit antibodies recognizing a series of other H. pylori
proteins, were raised against peptides corresponding to the
following conserved amino acid (aa) residues derived from strain
26695: BabA (aa 126140: CGGNANGQESTSSTT), SabA (aa
172186: CAMDQTTYDKMKKLA), OipA (aa 275282:
NYYSDDYGDKLDYK), NapA (aa 105118:
EFKELSNTAEKEGD), Slt (aa 492505: LRRWLESSKRFKEK), HtrA (aa 90
103:DKIKVTIPGSNKEY), FlaA (aa 93106:
KVKATQAAQDGQTT), GGT (aa 175188: RQAETLKEARERFL),
DupA (aa 551564: MLNIDSDNQQDNKA), VirB10/CagY
(repeat region: VSRARNEKEKKE), Cagd (aa 3245:
IKATKETKETKKEA), and Rlx2 (aa 131144:
HLVFSIDENSNEKN). Rabbit anti-Rlx1 and anti-CagM antibodies were raised
against the entire recombinant Rlx1 or CagM proteins,
respectively. All antibodies were affinity-purified and prepared according
to standard protocols by Biogenes GmbH (Berlin, Germany).
Horseradish peroxidase-conjugated anti-mouse or anti-rabbit
polyvalent sheep immunglobulin was used as secondary antibody
(DAKO Denmark A/S, DK-2600 Glostrup, Denmark) and blots
were developed with ECL Plus Western blot reagents (GE
Healthcare, UK limited Amersham Place, UK) .
Figure S1 Morphological analyses of novel
Helicobacters from a Bengal tiger by scanning electron
microscopy. The majority of bacteria contained either no or 14
monopolar sheated flagella as shown. Representative pictures are
shown from two preparations. Each bar corresponds to 1 mm.
Figure S2 Analysis of 16SrRNA by RFLP and RAPD
fingerprinting of different Helicobacter species. Panel A:
Schematic representation of the 1.2 kb 16S rRNA gene PCR
product with indicated restriction sites for endonuclease HhaI.
Panel B: DNA isolated from various Helicobacter species, including
the Bengal tiger isolate SB-1, was amplified followed by RFLP
using HhaI. Similar bands were obtained in the RFLP pattern of
H. pylori, H. acinonychis, H. mustelae, H. bilis, H. magdeburgensis and H.
canis (lanes marked with asterisks), indicating their close genetic
relatedness. Panels C/D: RAPD fingerprinting of the Helicobacter
isolates using primer D-9355 (panel C) and D-8635 (panel D) was
performed as described . Typical RAPD fingerprinting profiles
are shown and revealed the relatedness between H. pylori, H.
acinonychis and SB-1. M, DNA size marker.
Figure S3 Phylogenetic tree of the 23S ribosomal RNA
gene from the tiger strain SB-1 and the most closely
related sequences from different Helicobacter species.
The alignment was performed with BioEdit using gap penalties of
10 for gap opening, 5 for gap extension and a bootstrap value of
1,000. MEGA5 was used to infer DNA relatedness using the
Neighbor-Joining method. The evolutionary distances were
computed using the Maximum Composite Likelihood method
and are in the units of the number of base substitutions per site.
The optimal tree with the sum of branch length was equal to
0.3061 for 23S rRNA. Helicobacter sp. and Wolinella succinogenes were
used as outgroups. The phylogenetic tree shows that the 23SrRNA
gene of our Bengal tiger strain (accession number KC470072.1)
branched together with Helicobacter acinonychis from a Sumatran
tiger, thus demonstrating close relatedness among them.
Figure S4 Analysis of individual Helicobacter colonies
from Sumatran and a Bengal tiger by 16SrRNA PCR and
RFLP. Panel A: Schematic representation of the 1.2 kb 16S
rRNA gene PCR product with indicated restriction sites for AluI
and HhaI, respectively. Panel B: A conserved 1.2 kb DNA
fragment of the 16S rRNA gene in the genus Helicobacter 
was amplified from two H. acinonychis colonies from a Sumatran
tiger  and five colonies from a Bengal tiger investigated in this
study. Panels C/D: To confirm the specificity of these fragments,
Figure S5 Analysis of individual Helicobacter colonies
from Sumatran and a Bengal tiger by RAPD
fingerprinting. Panel AC: To investigate the genetic relatedness among
individual colonies isolated from tigers, total DNA isolated from
two H. acinonychis colonies from a Sumatran tiger  and five
colonies from our Bengal tiger was subjected to RAPD
fingerprinting analysis as described elsewhere . This method
uses a set of single primers (D-14307, D-9355 or D-8635 as shown
in panels AC) which arbitrarily anneal and amplify genomic
DNA resulting in strain-specific fingerprinting patterns . The
RAPD patterns were highly similar but not fully identical among
all investigated clones indicating their close genetic relatedness.
Figure S6 Genetic relatedness of various Helicobacter
acinonychis isolates from different big cats from the US,
Europe and Asia as analysed by RAPD fingerprinting.
Panel AC: RAPD fingerprinting PCR of the indicated H.
acinonychis isolates from tigers, cheetahs, lions and lion-tiger
(compare Fig. 4C and Table 1) reveals the close relatedness
between strains in two specific groups, called I and II, as indicated.
The RAPD primers D-1281, D-1283 and D-1290  have been
used in this experiment and are shown in panels A, B and C,
respectively. M, DNA size marker.
Figure S7 Western blotting analysis of the Bengal tiger
isolate SB-1 for well-known pathogenicity-associated
factors reported in H. pylori. Panel A: Total proteins were
isolate from SB-1 and H. pylori strains 26695 and J99, separated by
SDS-PAGE and stained with the indicated antibodies against
typical H. pylori proteins of the cag type IV secretion system,
showing their presence in both H. pylori strains but absence in
1. Panel B: Total proteins were isolated from SB-1 and H. pylori
strains Cuz20 and Shi470, separated by SDS-PAGE and stained
with the indicated antibodies against two potential DNA transfer
proteins (relaxases), Rlx1 and Rlx2 [19,20], and the duodenal
ulcer promoting gene A (DupA). Both H. pylori strains express all
three proteins, while SB-1 only exhibits a band for Rlx1 and Rlx2,
but not DupA.
We thank Ina Schleicher (HZI Braunschweig/Germany) for excellent
technical assistance in electron microscopy and Karen Guillemin
(University of Oregon, Eugene/USA) for providing the anti-CagN
Conceived and designed the experiments: NT FRT OAO MR SB.
Performed the experiments: NT FRT OAO MR. Analyzed the data: NT
FRT OAO MR DEB SB. Contributed reagents/materials/analysis tools:
NL RLF JGF DEB WSB. Wrote the paper: NT SB. Made figures: NT
FRT MR OAO SB.
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