The high-pathogenicity island is absent in human pathogens of Salmonella enterica subspecies I but present in isolates of subspecies III and VI.
ABSTRACT In this study we tested 74 Salmonella strains of all eight Salmonella groups and were able to demonstrate the presence of two high-pathogenicity island types in strains of Salmonella groups IIIa, IIIb, and VI. Most high-pathogenicity island-positive isolates produced yersiniabactin under iron-limited conditions and were positive for the high-molecular-weight proteins HMWP1 and HMWP2.
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ABSTRACT: ZET Salmonella türleri fakültatif hücre içi yerleşim gösteren patojen bakterilerdir. Makrofajlara, dendritik ve epitelyal hücrelere invazyon yapabilmekte ve bütün Salmonella türleri patojen olarak kabul edilmekte-dir. İnvazyondan, hücre içinde canlılığını sürdürebilmesinden ve ekstraintestinal yayılımdan sorumlu olan virülans faktörlerini kodlayan genler Salmonella patojenite adaları (SPA) içinde yer alır. SPA'nın horizontal gen transferi ile kazanılmış olduğu düşünülmektedir. Bazı patojenite adaları Salmonella cinsi içinde korun-muş olarak bulunurken, bazıları belirli serotipler için özgüldür. SPA'nın varlığı ve özelliklerine göre Salmo-nella serotipleri arasında konak hücreye adaptasyon, virülans faktörleri ve oluşturdukları enfeksiyonların şiddeti açısından farklılıklar bulunmaktadır. Salmonella virülans gen kümeleri 12 patojenite adası içinde yerleşim göstermektedir. Enfeksiyonun intestinal fazını kapsayan virülans genleri SPA-1 ve SPA-2'de yer almakta; hücre içinde sağkalım, fimbriyal ekpresyon, çoklu antibiyotik direnci, magnezyum ve demir alı-mı ve sistemik enfeksiyon oluşumu için gerekli olan virülans faktörlerini kodlayan genler diğer SPA içinde bulunmaktadır. SPA dışında, alternatif sigma faktör σ s (RpoS) regülatörü ile adaptif aside tolerans yanıtı (ATY) da Salmonella serotiplerinin diğer önemli virülans faktörleridir. Salmonella türlerinin virülan suşların-da bulunan RpoS ile ATY, enfeksiyonun intestinal fazı sırasında, mide asiditesi, safra tuzu, yetersiz oksijen, besin yetersizliği, antimikrobiyal peptidler, mukus ve doğal mikrobiyotanın varlığı gibi olumsuz çevresel koşullar altında canlılığını sürdürebilmesini ve aynı zamanda etkenin fagozom veya fagolizom gibi olum-suz koşullarda varlıklarını sürdürmesini sağlamaktadır. Bu derleme yazıda, Salmonella serotiplerinin virü-lans faktörlerini belirleyen önemli patojenite adaları ve virülans genlerinin regülasyonunda etkili bazı fak-törler ile ilgili bilgiler özetlenmiştir. Anahtar sözcükler: Salmonella; patojenite adası; sigma faktörü; adaptif aside tolerans yanıtı. Geliş Tarihi (Received): 23.08.2012 • Kabul Ediliş Tarihi (Accepted): 06.09.2012 ABSTRACT Salmonella species are facultative intracellular pathogenic bacteria. They can invade macrophages, dendritic and epithelial cells. The responsible virulence genes for invasion, survival, and extraintestinal spread are located in Salmonella pathogenicity islands (SPIs). SPIs are thought to be acquired by hori-zontal gene transfer. Some of the SPIs are conserved throughout the Salmonella genus, and some of them are specific for certain serovars. There are differences between Salmonella serotypes in terms of adaptation to host cell, virulence factors and the resulting infection according to SPA presence and cha-racteristics. The most important Salmonella virulence gene clusters are located in 12 pathogenicity is-lands. Virulence genes that are involved in the intestinal phase of infection are located in SPI-1 and SPI-2 and the remaining SPIs are required for intracellular survival, fimbrial expression, magnesium and iron uptake, multiple antibiotic resistance and the development of systemic infections. In addition SPIs, Sig-ma σ s (RpoS) factors and adaptive acid tolerance response (ATR) are the other two important virulence factors. RpoS and ATR found in virulent Salmonella strains help the bacteria to survive under inapprop-riate conditions such as gastric acidity, bile salts, inadequate oxygen concentration, lack of nutrients, an-timicrobial peptides, mucus and natural microbiota and also to live in phagosomes or phagolysosomes. This review article summarizes the data related to pathogenicity islands in Salmonella serotypes and so-me factors which play role in the regulation of virulence genes.Mikrobiyoloji bülteni 01/2013; 47(1):181-188. · 0.61 Impact Factor
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ABSTRACT: In the pregenomic era, the acquisition of pathogenicity islands via horizontal transfer was proposed as a major mechanism in pathogen evolution. Much effort has been expended to look for the contiguous blocks of virulence genes that are present in pathogenic bacteria, but absent in closely related species that are nonpathogenic. However, some of these virulence factors were found in nonpathogenic bacteria. Moreover, and contrary to expectation, pathogenic bacteria were found to lack genes (antivirulence genes) that are characteristic of nonpathogenic bacteria. The availability of complete genome sequences has led to a new era of pathogen research. Comparisons of genomes have shown that the most pathogenic bacteria have reduced genomes, with less ribosomal RNA and unorganized operons; they lack transcriptional regulators but have more genes that encode protein toxins, toxin-antitoxin (TA) modules, and proteins for DNA replication and repair, when compared with less pathogenic close relatives. These findings questioned the paradigm of virulence by gene acquisition and put forward the notion of genomic repertoire of virulence.Briefings in functional genomics 06/2013; · 4.21 Impact Factor
- Microbial siderophores, Edited by Ajit Varma, Sudhir Chincholkar, 07/2007: pages 67-89; Springer., ISBN: 978-3-540-71159-9
JOURNAL OF BACTERIOLOGY, Feb. 2003, p. 1107–1111
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 185, No. 3
The High-Pathogenicity Island Is Absent in Human Pathogens of
Salmonella enterica Subspecies I but Present in Isolates of
Subspecies III and VI
T. A. Oelschlaeger,1D. Zhang,1S. Schubert,2E. Carniel,3W. Rabsch,4
H. Karch,5and J. Hacker1*
Institut fu ¨r Molekulare Infektionsbiologie, University of Wu ¨rzburg, Wu ¨rzburg,1Max-von Pettenkofer-Institut fu ¨r Hygiene
und Mikrobiologie, Munich,2Robert-Koch-Institut, Wernigerode,4and Institut fu ¨r Hygiene, Universita ¨t Mu ¨nster,
Mu ¨nster,5Germany, and Institut Pasteur, Paris, France3
Received 10 July 2002/Accepted 13 November 2002
In this study we tested 74 Salmonella strains of all eight Salmonella groups and were able to demonstrate the
presence of two high-pathogenicity island types in strains of Salmonella groups IIIa, IIIb, and VI. Most
high-pathogenicity island-positive isolates produced yersiniabactin under iron-limited conditions and were
positive for the high-molecular-weight proteins HMWP1 and HMWP2.
The presence of pathogenicity islands in the genomes of
bacterial pathogens is one of the main features that differen-
tiate them from closely related nonpathogenic strains or spe-
cies. Furthermore, particular pathogenicity islands are specific
for certain pathotypes. The high-pathogenicity island (HPI),
however, has been found in many species of the family Enter-
obacteriaceae (27). The term high-pathogenicity island was
coined because this island was shown to be present in all strains
of Yersinia that are highly virulent for mice or humans but
absent from strains with lower virulence (10, 11). This island
consists of 12 genes and encodes a highly efficient iron acqui-
sition system driven by the siderophore yersiniabactin. A P4-
like integrase (int) gene is located at the 5? end of HPI. The
genes ybtS (irp9), irp1, irp2, ybtT (irp4), and ybtE (irp5) form the
yersiniabactin synthesis gene cluster. Two genes, ybtQ (irp7)
and ybtP (irp6), encode proteins of the ABC transporter family.
The receptor protein for yersiniabactin and pesticin in the
outer membrane is encoded by fyuA, located at the 3? end of
the HPI core. The expression of HPI-specific genes is induced
by iron-limiting conditions and is regulated by the ybtA product
(13, 22). The gene termed ybtX (irp8) is of unknown function.
Besides the three pathogenic Yersinia species, the HPI was
identified in various pathotypes of Escherichia coli and in E.
coli isolates from stool samples of healthy individuals. In ad-
dition, HPI was detected in some strains of Citrobacter spp.,
Enterobacter cloacae, and Klebsiella spp. (3, 28). However, not
all HPI-positive strains of the different species of the family
Enterobacteriaceae produce the iron chelator yersiniabactin or
the yersiniabactin receptor (27, 28). Despite several previous
attempts, the HPI could not be detected in any Salmonella
strains belonging to Salmonella enterica serovars Typhimurium,
Enteritidis, or Typhi, which are pathogenic for humans (3, 27).
In the present work, we investigated various Salmonella
strains for the presence of the HPI not only of S. enterica group
I, comprising the majority of salmonellae pathogenic for hu-
mans and other warm-blooded animals, but also of the other
groups of S. enterica as well as strains of the species Salmonella
HPI is present in particular subspecies of S. enterica. The
genus Salmonella consists of two species, S. enterica, with seven
subspecies, also termed taxonomic groups (I to IV, VI, and
VII), and S. bongori (group V). The presence of HPI was
investigated for 74 Salmonella strains, including all strains in
Salmonella reference collection C (SARC) (5). This was per-
formed by PCRs with primers specific for HPI genes (Fig. 1).
The arrangement of the HPI genes was determined by addi-
tional PCRs with primer pairs of which each primer was spe-
cific for one of two adjacent genes (Fig. 1). All primers were
designed according to the HPI sequence of Yersinia pestis strain
KIM6? (14). The sequences of the primers employed and the
conditions used are listed in a table which can be found on our
home page (http://www.uni-wuerzburg.de/infektionsbiologie
Of the 74 strains tested, one strain (no. 1649) of nine of
group IIIa, all 11 strains of group IIIb, and two of eight strains
of group VI were positive, i.e., resulted in PCR products of the
expected sizes in the initial screening (Table 1). For all these
strains, the genes covering the HPI from ybtS to fyuA mapped
at the same positions as the corresponding genes of Y. pestis
strain KIM6? (14). The only exception was strain 1380 of
group IIIb. Its HPI lacked 80% of irp2 and all of the genes irp1,
irp3 (ybtU), irp4 (ybtT), irp5 (ybtE), and fyuA. The presence of
only one HPI per genome of the HPI-positive strains was
demonstrated by Southern blot analysis (29) of BssHII-di-
gested chromosomal DNA probed either with a PCR fragment
representing part of the fyuA sequence (PCR 30, Fig. 1A; Fig.
2) or with a PCR fragment obtained with the ybtS-specific
primers ybtS4 and ybtSlp (PCR 31, Fig. 1A) (blot not shown).
Characterization of Salmonella HPI core region. Typically,
the HPI identified in Yersinia spp. and E. coli contains at its 5?
end an int gene highly homologous to the P4-like integrase
gene of E. coli (4, 6, 7, 17). This integrase gene located down-
stream of ybtS was only present in the HPI-positive strains
* Corresponding author. Mailing address: Institut fu ¨r Molekulare
Infektionsbiologie, Universita ¨t Wu ¨rzburg, Roentgenring 11, D-97070
Wu ¨rzburg, Germany. Phone: 931 31 2575. Fax: 931 31 2578. E-mail:
SARC13 and 1443 of group VI (Fig. 1A). The nucleotide
sequence of int was identical to that of Y. pestis strain KIM6?.
Nucleotide sequences were compared to those in the data
bases with the programs BlastN and BlastX (1).
The HPI of strains SARC13 and 1443 was termed type 1. In
contrast, the int gene was missing in all HPI-positive strains of
groups IIIa and IIIb. Other differences between the HPIs of
groups IIIa and IIIb compared to those of group VI and Yer-
sinia strains were determined by complete (int and ERIC ele-
ment) or partial sequencing (ybtSXQPA, irp1 to irp5, and fyuA)
for all HPI-positive strains of groups IIIa and IIIb. These
differences were (i) a ybtS gene with 12 additional nucleotides
at its 3? end, (ii) an additional 206 bp (i.e., an ERIC element)
between ybtP (irp6) and ybtA, and (iii) a mutation in irp1
replacing the adjacent amino acids Asp-Ala with Gly-Tyr (Fig.
1). The HPI of S. enterica strains of groups IIIa and IIIb was
termed type 2 HPI in order to differentiate this type of HPI
from that identified in Yersinia spp. and other enterobacteria,
including S. enterica group VI.
Expression of HPI genes. The functionality of yersiniabactin
biosynthesis was evaluated by detecting the presence of yersi-
niabactin in the cell-free supernatant of iron-starved bacteria
by using a translational reporter gene fusion system with lucif-
erase (20, 28). Synthesis and release of yersiniabactin were
observed in all HPI-positive strains tested except 1380, which
carries a truncated island, and 50/90 and HPI-negative control
strains (Table 1). In addition, expression of the high-molecu-
lar-weight proteins HMWP1 and HMWP2, representing mul-
tidomain enzymes involved in synthesis of the iron chelator
yersiniabactin (9), was detected by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) analysis of
total proteins extracted from five iron-starved but not iron-
replete HPI-positive Salmonella strains (IIIa, 1649; IIIb, 845/
96, SARC 7, and SARC 8; and VI, 1443) and from Yersinia en-
terocolitica strain Ye8081 (positive control) (data not shown).
Determination of integration site of Salmonella HPIs by
analysis of flanking region on 5? side. The HPI element in
most enterobacteria is inserted into an asparagine-specific
FIG. 1. Physical maps of the two types of HPI with locations of primer pairs and corresponding PCRs (indicated by arabic numbers close to
thin arrows) for analysis of the presence and order of HPI genes and (19, 20, 21, and 22 are inverse PCRs) for analysis of sequences flanking the
HPI element in the Salmonella strains. Flanking regions of HPI in Salmonella strains are not drawn to scale. Different patterns indicate different
sequences of the flanking regions. (A) The type 1 HPI (S. enterica group VI, strains 1443 and SARC13) is identical to HPI of Yersinia spp. and
E. coli (cross-hatched box, flanking sequence at the 5? side; vertically striped box, flanking sequence at the 3? side). (B) The type 2 HPI (S. enterica
group IIIa and IIIb strains) lacks an int gene, harbors a 3?-extended ybtS gene, and contains an ERIC element and three nucleotide exchanges in
irp1 (checkered box, flanking sequence at the 5? side; horizontally striped box ending as dashed-line open box, flanking sequence at the 3? side).
For further details, see the text.
1108 NOTESJ. BACTERIOL.
tRNA gene, preferentially asnT (6, 21, 27). In order to analyze
the HPI site of insertion in the Salmonella strains, PCRs spe-
cific for the asn tRNA, int, or ybtS gene were performed. PCR
products of the expected sizes were observed for the two HPI-
positive strains of group VI (1443 and SARC13) only (Fig.
1A). In addition, analysis of the DNA sequence upstream of
the HPI-containing asn tRNA gene was performed after in-
verse PCR with primer pair int7 and int8 (PCR 21, Fig. 1A)
and revealed 86 to 97% identity with the genomic DNA se-
quence of S. enterica serovar Typhimurium strain LT2 (bases
2081500 to 2083248). Within this region, a sequence 973 bp
upstream of the asn tRNA gene was identified that was 100%
identical with the serU tRNA gene of E. coli (Fig. 1).
In order to determine the site of insertion of the type 2 HPI
in the strains not belonging to S. enterica group VI, inverse
PCR starting inside ybtS (PCR 19, Fig. 1B) was performed.
Cloning and sequencing of the resulting amplification products
confirmed the extension of the ybtS gene and the absence of an
int gene at the 3? end of the HPI in these strains (Fig. 1B).
Furthermore, 113 nucleotides after the stop codon of ybtS were
identical between all HPI-positive strains of Salmonella group
IIIa and IIIb and showed no homology to any sequences in the
databases. Starting 114 nucleotides downstream of the ybtS
gene, the sequence was ?90% identical to the genome se-
quence of S. enterica serovar Typhimurium strain LT2 in all
these strains. Identity began with the sequence 5?-TTATG
AGA-3?. The first nucleotide of this sequence corresponds to
nucleotide 1882950 of the complete genome of S. enterica and
is 41 nucleotides downstream of the stop codon of gene ychF.
Obviously, the HPI of the HPI-positive strains of Salmonella
groups IIIa and IIIb was inserted at a different location of the
chromosome and not into any of the asn tRNA genes.
Determination of region flanking 3? end of HPI in Salmo-
nella spp. A primer pair (Fig. 1A, PCR 22) for inverse PCR in
fyuA yielded an amplification product of the same size as in
Yersinia pestis for strains SARC13 and 1443 of group VI only.
Sequence analysis of the amplified fragment (about 1 kb)
showed 96% identity with the corresponding region down-
stream of fyuA in Yersinia pseudotuberculosis strain PB1 (24)
(Fig. 1A). The 3?-flanking region of the HPI-positive strain
845/96 of group IIIb was determined from a cosmid clone
carrying the right half (3? part) of the HPI and about 30 kb of
DNA downstream of fyuA. Sequence analysis of 5,237 bp start-
FIG. 2. Southern blot with chromosomal DNA from HPI-positive
Salmonella strains 1649 (lane 1) (group IIIa), group IIIb strains 50/90
(lane 2), 461/95 (lane 3), 658/95 (lane 4), 845/96 (lane 5), 1376/95 (lane
6), 1380 (lane 7), SARC8 (lane 8), 1474 (lane 9), 1757 (lane 10),
SARC7 (lane 11), and 1494 (lane 12), and group VI strains 1443 (lane
13) and SARC13 (lane 14). Strain SARC14 (lane 15) was the negative
control, and Y. pestis strain KIM6? (lane 16) served as the positive
control. Chromosomal DNA was digested with BssHII and probed
with the amplification product of PCR 30 (see table at http://www.uni
-wuerzburg.de/infektionsbiologie/PCR-HPI.htm), representing part of
the fyuA sequence.
TABLE 1. Determination of the presence of HPI in salmonellaea
I S. enterica serovar
S. enterica serovar
S. enterica serovar
S. enterica serovar
S. enterica subsp.
S. enterica subsp.
S. enterica subsp.
IIIaS. enterica subsp.
IIIbS. enterica subsp.
IVS. enterica subsp.
V S. bongori
VIS. enterica subsp.
aThe strain number of each strain that was positive in the HPI gene detection
assays is presented. Salmonella strains were tested for the presence of HPI by
PCRs specific for the genes ybtS-ybtX, irp1-irp2, and irp4-fyuA. Yersiniabactin
production was determined with an indicator strain expressing luciferase under
the control of the fyuA promoter (20). Strain 1380 of group IIIb was only positive
in PCRs specific for ybtS-ybtX and for part of irp2. ND, not determined. With the
exception of the SARC strains, all but one Salmonella strain (S. enterica serovar
Typhimurium strain C17) were from the strain collection of the Robert Koch
Institute, Wernigerode Branch, National Reference Center for Salmonellae and
VOL. 185, 2003NOTES1109
ing with the first nucleotide after the stop codon of fyuA re-
vealed no homology to any nucleotide sequence in the genome
databases. However, the deduced amino acid sequence of the
last 557 nucleotides of the 5,237-bp region downstream from
fyuA exhibited 72% identity with a putative sulfatase of Y.
pestis encoded by gene YPO3047 (Fig. 1B).
The sequence obtained from the cosmid containing the re-
gion downstream from fyuA was used to design primers for
PCR amplification of DNA of other HPI-positive Salmonella
strains. The 5,237-bp fragments that were obtained for all
HPI-positive group IIIb strains (50/90, 461/95, 658/95, 845/96,
1376/95, 1494, 1474, 1757, SARC7, and SARC8) and for the
HPI-positive group IIIa strain 1649 had a sequence identical to
that of the 3?-flanking region of strain 845/96 (Fig. 1B). The
only exception was strain 1380, which contains a 3?-truncated
HPI and for which no amplified fragment was detected. These
results show that the 3?-flanking regions of all HPI-positive
strains of groups IIIa and IIIb are identical (Fig. 1B).
The yersiniabactin gene cluster is viewed as an entity which
can be transferred horizontally between bacteria of the family
Enterobacteriaceae. Its presence in Yersinia spp. is correlated
with high virulence for mice and humans (9, 18, 19, 25). There-
fore, it was termed the high-pathogenicity island. With differ-
ent methods, it was shown that S. enterica group I strains did
not contain the HPI. Furthermore, in other pathogenic enter-
obacteria such as Shigella boydii, Shigella dysenteriae, Shigella
flexneri, and Shigella sonnei, HPI has not been detected (27).
However, in other enterobacteria such as E. coli, HPI was also
found in substantial numbers in isolates from healthy individ-
uals and in strains from ECOR groups A and B1, which are
considered nonpathogenic (12, 28). It can be concluded that
HPI might not be a virulence factor in every strain background
but might have other functions in commensal and environmen-
tal bacteria (16, 21).
Consequently, this study was designed to probe for the pres-
ence of HPI in Salmonella group I strains as well as in Salmo-
nella strains of other groups. First, we confirmed the previously
published data that the HPI is not present in S. enterica group
I. Second, of 53 strains of Salmonella groups II to VII, 14
strains (26.4%) were identified as containing the HPI. All but
one strain harbored all the HPI genes from ybtS to fyuA. In
contrast to the HPI in other members of the Enterobacteriaceae
family, the int gene encoding a P4-like integrase was only
present in two HPI-positive Salmonella strains of group VI.
This was surprising because this gene is responsible for the
integration of HPI into the host chromosome (23).
Further analysis of the Salmonella HPIs revealed that two
types of HPI can be distinguished in salmonellae. Type 1,
found in strains of Salmonella group VI, is identical to the HPI
of Yersinia pestis strain KIM6? and other members of the
enterobacteria. The other HPI type (type 2) of strains of Sal-
monella groups IIIa and IIIb lacks the int gene but encodes a
3?-extended ybtS gene containing an insertion of a 206-bp
ERIC-like element and a mutation in irp1. How was the second
Salmonella HPI type inserted into the chromosome? Either it
was inserted by the product of the int gene, which was lost later
on, or its insertion relied on another gene product. This might
be an integrase encoded somewhere else in the chromosome or
an integrase provided by a phage during the integration pro-
cess. The former hypothesis is unlikely, because the type 2 HPI
of Salmonella spp. differs not just in the lack of the int gene
from type 1 HPI but also in the other alterations mentioned.
Furthermore, the integration site typical of HPI in Yersinia
species as well as in E. coli is an Asn-specific tRNA gene,
preferentially asnT. However, only the type 1 Salmonella HPI
is integrated into an asn tRNA gene, most likely asnT (26). The
Salmonella HPI of type 2 was inserted not adjacent to any
tRNA but at a position 41 nucleotides downstream of the ychF
gene in the genome of S. enterica serovar Typhimurium, be-
cause there the sequence becomes almost identical to the Sal-
Nevertheless, HPI might have been acquired only once by
Salmonella spp. This hypothesis is based on the fact that HPI
was found to be present in the three closely related Salmonella
subgroups IIIa, IIIb, and VI. In turn, this might be the result of
HPI acquisition by a Salmonella ancestor which was the pre-
decessor of these three subgroups. The iron-sequestering func-
tion encoded by HPI is not controversial (8), and most HPI-
positive Salmonella strains produced yersiniabactin. However,
this function might be of importance not only in warm-blooded
animals but also in other animals or the environment as well.
The technique used to identify yersiniabactin relied on the fact
that yersiniabactin acts as a regulator in a complex with YbtA
for the expression of its receptor, FyuA (13, 20). This regula-
tory function might work not only on other genes of the HPI
(e.g., irp genes) but on non-HPI genes in the same bacterial cell
and/or on gene expression of other bacteria expressing the
In conclusion, HPI was found in Salmonella strains of groups
IIIa, IIIb, and VI. In these strains, it might not only function as
an iron-sequestering system in host animals and the environ-
ment but might also influence the activity of other genes as
This work was supported by the Deutsche Forschungsgemeinschaft,
Sonderforschungsbereich 479, and the Fond der Chemischen Indus-
We thank W.-D. Hardt (Munich) for the kind gift of the SARC
strains and C. Albert (Wu ¨rzburg) for excellent technical assistance.
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