The Yersinia high-pathogenicity island.

A pathogenicity island present only in highly pathogenic strains of Yersinia (Y. enterocolitica 1B, Y. pseudotuberculosis I and Y. pestis) has been identified on the chromosome of Yersinia spp. and has been designated High-Pathogenicity Island (HPI). The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin. The major function of this island is thus to acquire iron molecules essential for in vivo bacterial growth and dissemination. The presence of an integrase gene and att sites homologous to those of phage P4, together with a G + C content much higher than the chromosomal background, suggests that the HPI is of foreign origin and has been acquired by chromosomal integration of a phage. The HPI can excise from the chromosome of Y. pseudotuberculosis and is found inserted into any of the three copies of the asn tRNA loci present in this species. A unique characteristic of the HPI is its wide distribution in various enterobacteria. Although first identified in Yersinia spp., it has subsequently been detected in other genera such as E. coli, Klebsiella and Citrobacter.     

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis . In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis , has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis . A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica , the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica , is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS 100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.     

Characterization of the integration site of Yersinia high-pathogenicity island in Escherichia coli

The high-pathogenicity island (HPI) of virulent Yersiniae consists of (i) a functional core encoding for biosynthesis and uptake of the siderophore yersiniabactin and (ii) a 5- to 13-kb AT-rich region of unknown function. This Yersinia HPI has been shown to be widely distributed among different pathotypes of Escherichia coli. In this study, the insertion site of the HPI was defined in three different E. coli strains: The enteroaggregative E. coli (EAggEC) strain 17-2, the uropathogenic (UPEC) E. coli strain 536, and the probiotic E. coli DSM6601. We demonstrated that in all three E. coli isolates the HPI is associated with the asnT tRNA (5'-extremity) and truncated in the AT-rich region (3'-extremity) since the 17-bp direct repeat (DR) of the asn tRNA that flanks the HPI in Yersinia is missing in E. coli. Moreover, in comparison to the HPI-negative E. coli K-12 strain, a uniform deletion must have taken place in the E. coli chromosome adjacent to the 3'-border of the HPI.     

Horizontal transfer of the high-pathogenicity island of Yersinia pseudotuberculosis

The horizontal transfer of genetic elements plays a major role in bacterial evolution. The high-pathogenicity island (HPI), which codes for an iron uptake system, is present and highly conserved in various Enterobacteriaceae, suggesting its recent acquisition by lateral gene transfer. The aim of this work was to determine whether the HPI has kept its ability to be transmitted horizontally. We demonstrate here that the HPI is indeed transferable from a donor to a recipient Yersinia pseudotuberculosis strain. This transfer was observable only when the donor and recipient bacteria were cocultured at low temperatures in a liquid medium. When optimized conditions were used (bacteria actively growing in an iron-deprived medium at 4 degrees C), the frequency of HPI transfer reached approximately 10(-8). The island was transferable to various serotype I strains of Y. pseudotuberculosis and to Yersinia pestis, but not to Y. pseudotuberculosis strains of serotypes II and IV or to Yersinia enterocolitica. Upon transfer, the HPI was inserted almost systematically into the asn3 tRNA locus. Acquisition of the HPI resulted in the loss of the resident island, suggesting an incompatibility between two copies of the HPI within the same strain. Transfer of the island did not require a functional HPI-borne insertion-excision machinery and was RecA dependent in the recipient but not the donor strain, suggesting that integration of the island into the recipient chromosome occurs via a mechanism of homologous recombination. This lateral transfer also involved the HPI-adjacent sequences, leading to the mobilization of a chromosomal region at least 46 kb in size.     

A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high-pathogenicity island

Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.     

Excision of the high-pathogenicity island of Yersinia pseudotuberculosis requires the combined actions of its cognate integrase and Hef, a new recombination directionality factor

The Yersinia high-pathogenicity island (HPI) encodes the siderophore yersiniabactin-mediated iron uptake system. The HPI of Yersinia pseudotuberculosis I has previously been shown to be able to excise precisely from the bacterial chromosome by recombination between the attB-R and attB-L sites flanking the island. However, the nature of the Y. pseudotuberculosis HPI excision machinery remained unknown. We show here that, upon excision, the HPI forms an episomal circular molecule. The island thus has the ability to excise from the chromosome, circularize and reintegrate itself, either in the same location or in another asn tRNA copy. We also demonstrate that the HPI-encoded bacteriophage P4-like integrase (Int) plays a critical role in HPI excision and that, like phage integrases, it acts as a site-specific recombinase that catalyses both excision and integration reactions. However, Int alone cannot efficiently promote recombination between the attB-R and attB-L sites, and we demonstrate that a newly identified HPI-borne factor, designated Hef (for HPI excision factor) is also required for this activity. Hef belongs to a family of recombination directionality factors. Like the other members of this family, Hef probably plays an architectural rather than a catalytic role and promotes HPI excision from the chromosome by driving the function of Int towards an excisionase activity. The fact that the HPI, and probably several other pathogenicity islands, carry a machinery of integration/excision highly similar to those of bacteriophages argues for a phage-mediated acquisition and transfer of these elements.     

The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae

A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. Recently, the HPI was also detected in various strains of Escherichia coli. In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated. Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island. These isolates were three E. coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca). As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2. In the K. oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed. In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp. This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.     

Yersiniabactin and other siderophores produced by clinical isolates of Enterobacter spp. and Citrobacter spp

We analyzed the ability of extraintestinal strains of Enterobacter spp. and Citrobacter spp. to employ different siderophore-mediated strategies of iron acquisition. All strains produced iron-chelating compounds. Cross-feeding assays indicated that most isolates of both Enterobacter spp. and Citrobacter spp. excreted catecholate siderophore enterobactin, less produced aerobactin, and single strains excreted hydroxamates different from aerobactin. Besides, we analyzed if the strains had the ability to produce the siderophore yersiniabactin coded by the Yersinia high-pathogenicity island (HPI). The presence of HPI genes was observed in single isolates of three species: E. cloaceae, E. aerogenes and C. koseri. A detailed polymerase chain reaction analysis revealed differences in the genetic organization of the HPIs; however, in a cross-feeding test we proved that yersiniabactin was produced and the island was functional.     

The Yersinia HPI is present in Serratia liquefaciens isolated from meat

AIMS: The aim of the study was to screen the Enterobacteriaceae flora of meat for the presence of bacteria harbouring the Yersinia high-pathogenicity island (HPI). METHODS AND RESULTS: Bacteria from 29 meat and 29 liver samples were isolated on violet-red bile glucose agar. A total of 197 isolates were screened for the presence of the irp2 gene, encoded within the HPI, by PCR. One isolate that was positive for irp2 gene was also positive for the fyuA, irp1, ybtP/ybtQ, ybtX/ybtS and int/asn tRNA genes by PCR. The presence of fyuA, irp1 and irp2 genes was confirmed by Southern hybridization. CONCLUSIONS: The isolate was identified as Serratia liquefaciens by sequencing of the 16S rRNA gene and by ribotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a Serratia harbouring the Yersinia HPI. Serratia is a frequently occurring Enterobacteriaceae genus in chill-stored meat.     

The Yersinia high-pathogenicity island is highly predominant in virulence-associated phylogenetic groups of Escherichia coli

The high-pathogenicity island (HPI) present in pathogenic Yersinia and encoding the siderophore yersiniabactin, has recently been identified in the asnT tRNA region of various Escherichia coli pathotypes, especially those responsible for bacteremia and urosepsis. Most E. coli strains causing such extra-intestinal infections belong to phylogenetic groups B2 and D. In this study we investigated (i) the distribution and localization of HPI among the different E. coli phylogenetic groups, using the ECOR reference collection; and (ii) the prevalence of HPI among a set of 124 phylogenetically characterized E. coli strains responsible for neonatal meningitis. Ninety-three percent of the ECOR strains belonging to groups B2 and D harbored HPI. In contrast, the island was present in 32% and 25% of strains belonging to groups A and B1, respectively, which are considered to be non-pathogenic. HPI was found in 100% of the neonatal meningitis strains, 13 of which belonged to groups A and B1, suggesting that HPI might contain virulent factors required for the development of neonatal meningitis. Moreover, we observed for the first time that HPI can be inserted in a site different from the asnT tRNA region.     

The Yersinia high-pathogenicity island: an iron-uptake island.

Highly pathogenic Yersinia carry a pathogenicity island termed high-pathogenicity island (HPI). The Yersinia HPI comprises genes involved in the synthesis of the siderophore yersiniabactin and can thus be regarded as an iron-uptake island. A unique characteristic of the HPI is its wide distribution among different enterobacteria such as Escherichia coli, Klebsiella, Citrobacter and Salmonella. Other types of iron-uptake systems are also carried by different pathogenicity islands in enterobacteria.     

The Yersinia high-pathogenicity island in Escherichia coli and Klebsiella pneumoniae isolated from polymicrobial infections

We examined 12 pairs of strains of Escherichia coli and Klebsiella pneumoniae isolated from mixed infections in human for the presence of the Yersinia high-pathogenicity island (HPI). In one case both isolates carried the HPI, whereas in 11 cases one strain of the pair was HPI-positive. Although there were differences in the organization of the Yersinia HPI, all HPI-positive isolates were able to produce yersiniabactin. The presence of the Yersinia HPI may enhance the capability of strains involved in mixed infections to replicate in iron-deprived conditions in the host.     

Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniae strain isolated from a primary liver abscess

Genomic heterogeneity has been shown to be associated with Klebsiella pneumoniae strains causing pyogenic liver abscesses (PLA) and metastatic infections. In order to explore the mechanism responsible for genomic heterogeneity in K. pneumoniae, we compared the complete genomic sequences of strains NTUH-K2044 and MGH78578. An ~76-kbp DNA fragment located adjacent to an asparagine (asn) tRNA gene was present in NTUH-K2044 but not in MGH78578. This fragment could be divided into three regions with different functions, and structurally it resembled a functional integrative and conjugative element (ICE), ICEEc1, in Escherichia coli. The 5′ region of this fragment contained genes similar to a high-pathogenicity island (HPI) of Yersinia pestis and Yersinia pseudotuberculosis. The middle region was similar to part of a large plasmid in K. pneumoniae, and the 3′ region contained genes responsible for DNA conjugative transfer. Therefore, this DNA fragment was designated ICEKp1. Precise excision and extrachromosomal circularization of ICEKp1 were detected in K. pneumoniae wild-type strain NTUH-K2044. ICEKp1 could integrate into the asn tRNA loci of the chromosome of another K. pneumoniae isolate. The prevalence of ICEKp1 was higher in PLA strains (38 of 42 strains) than in non-tissue-invasive strains (5 of 32 strains). Therefore, ICEKp1 may contribute to the transmission of the HPI and result in K. pneumoniae PLA infection-associated genomic heterogeneity.     

禽致病性大肠埃希菌强毒力岛（HPI）全岛缺失株的构建

目的构建大肠埃希菌强毒力岛（HPI）全岛缺失突变株,为进一步评价大肠埃希菌HPI的功能打下基础。方法根据已知大肠埃希菌HPI基因序列设计PCR敲除引物,引物5′端有50 bp的拟敲除基因的同源臂,3′端为扩增引物,以pKD3为模板,扩增两侧含FRT位点的氯霉素抗性基因,利用pKD46的λ重组系统替换E.coli ZL基因组上的毒力岛全岛基因,再利用表达Flp重组酶的质粒pCP20,可将FRT位点之间的氯霉素抗性基因删除,用鉴定引物进行鉴定并测序。结果构建的全岛缺失株与预期一致。结论成功构建了禽致病性大肠埃希菌强毒力岛（HPI）全岛缺失突变株。     

Proteobactin and a yersiniabactin-related siderophore mediate iron acquisition in Proteus mirabilis

Proteus mirabilis causes complicated urinary tract infections (UTIs). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly upregulated genes (P ≤ 0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596-2605, encode putative siderophore systems. PMI0229-0239 encodes a non-ribosomal peptide synthetase-independent siderophore system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis, and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore have reduced fitness in vivo. The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis.     

Integron cassette insertion: a recombination process involving a folded single strand substrate

Integrons play a major role in the dissemination of antibiotic resistance genes among Gram-negative pathogens. Integron gene cassettes form circular intermediates carrying a recombination site, attC, and insert into an integron platform at a second site, attI, in a reaction catalyzed by an integron-specific integrase IntI. The IntI1 integron integrase preferentially binds to the 'bottom strand' of single-stranded attC. We have addressed the insertion mechanism in vivo using a recombination assay exploiting plasmid conjugation to exclusively deliver either the top or bottom strand of different integrase recombination substrates. Recombination of a single-stranded attC site with an attI site was 1000-fold higher for one strand than for the other. Conversely, following conjugative transfer of either attI strand, recombination with attC is highly unfavorable. These results and those obtained using mutations within a putative attC stem-and-loop strongly support a novel integron cassette insertion model in which the single bottom attC strand adopts a folded structure generating a double strand recombination site. Thus, recombination would insert a single strand cassette, which must be subsequently processed.     

Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8

The high-pathogenicity island (Yen HPI) of Yersinia enterocolitica biogroup (BG) 1B strains is associated with mouse virulence. Three repeated sequences are clustered on the A+T-rich part of the Yen HPI downstream of the fyuA yersiniabactin receptor gene in Y. enterocolitica O:8 strains WA-314 and 8081. In addition to IS1328 and IS1400, the RS3 repeated sequence consists of a novel insertion sequence, IS1329, inserted into the remnants of IS1222. This partial IS retains both 44-bp inverted terminal repeats (ITRs) of IS1222 but has suffered deletions of different sizes in strains WA-314 and 8081. IS1329 is 1243-bp long, carries 25-bp imperfect ITRs and two consecutive orfs capable to encode 110-amino acid (aa) and 249-aa proteins, respectively. IS1329 is present only in BG 1B Y. enterocolitica strains. Similarly to IS1400, IS1329 and IS1222 belong to the IS3 group of mobile elements and seem to have preference for the 'local hopping' into the A+T-rich part of the Yen HPI. These insertion sequences may be responsible for the imprecise deletions of the Yen HPI in strain WA-314.     

Independent acquisition and insertion into different chromosomal locations of the same pathogenicity island in Yersinia pestis and Yersinia pseudotuberculosis

We show that Yersinia pestis and pesticin-sensitive isolates of Y. pseudotuberculosis possess a common 34 kbp DNA region that has all the hallmarks of a pathogenicity island and is inserted into different asparaginyl tRNA genes at different chromosomal locations in each species. This pathogenicity island (YP-HPI) is marked by IS 100 , has a G + C content different from its host, is flanked by 24 bp direct repeats, encodes a putative, P4-like integrase and contains the iron uptake virulence genes from the pgm locus of Y. pestis . These findings indicate independent horizontal acquisition of this island by Y. pestis and Y. pseudotuberculosis . The two YP-HPI locations and their possession of an integrase gene support a model of site-specific integration of the YP-HPI into these bacteria.     

Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4′-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.