Различные сидерофоры обусловливают фенотип высокой патогенности иерсиний Текст научной статьи по специальности «Биологические науки»

УДК 616.9

A.Rakin, D.Garzetti

DIFFERENT SIDEROPHORES CONTRIBUTE TO THE HIGH-PATHOGENICITY PHENOTYPE IN YERSINIA

Max von Pettenkofer-Institute, LMU, Munich; Germany

For successful proliferation in the hostile mammalian environments, the highly pathogenic Yersiniae (Y pestis, Y. pseudotuberculosis and Y. enterocolitica subsp. enterocolitica) have to sequester ferric iron from the host iron storage molecules. Siderophores, low molecular weight molecules, that demonstrate high affinity for the iron, are responsible for Fe3+ capture and transport in bacteria. At least one endogenous siderophore system, yersiniabactin, is known to be involved in iron acquisition in highly virulent Yersiniae. Its inactivation in Y. pestis and Y. enterocolitica subsp. enterocolitica results in significant attenuation of virulence. However, the yersiniabactin is not present in all highly virulent Yersiniae. Indeed, the large group of Y. pseudotuberculosis serotypes O2, O3, O4, O5 as well as serotype O1 Far East Scarlet like fever (FESLF) strains carry an alternative, iron acquisition system, pseudochelin, encoded by the Yersinia non-ribosomal peptide ynp locus. Thus, the yersiniabactin activity is not the only one associated with the high-pathogenicity phenotype of the human pathogenic Yersiniae.

Key words: pathogenic Yersiniae, high-pathogenicity phenotype, siderophores

А.Ракин, Д.Газетти

Различные сидерофоры обусловливают фенотип высокой патогенности иерсиний

Институт Макса фон Петтенкофера, Мюнхен, Германия

Высоковирулентным Yersiniae (Y. pestis, Y. pseudotuberculosis и Y. enterocolitica subsp. enterocolitica) для успешного размножения в неблагоприятных условиях хозяйского организма необходимо трехвалентное железо, которое связано железосодержащими молекулами хозяина. Низкомолекулярные молекулы, сидерофоры, обладающие высоким сродством к железу, отвечают за эффективный захват и транспорт Fe3+ в бактериальную клетку. По крайней мере одна сидерофорная система, а именно, система синтеза и транспорта йерсиниобактина, отвечает за активный транспорт железа у йерсиний. Инактивация этой системы приводит к значительному снижению вирулентности у Y. pestis и Y. enterocolitica subsp. enterocolitica. Однако, йерсиниобактин присутствует далеко не у всех представителей рода йерсиний. Так, Y. pseudotuberculosis серотипов O2, O3,

O4, OS, а также штаммы O1, вызывающие Дальневосточную скарлитиноподобную лихорадку, содержат альтернативную систему транспорта железа, псевдохелин, которая кодируется локусомynp (нерибосомный синтез пептидов). Следовательно, как минимум две системы снабжения железом ассоциированы с фенотипом высокой патогенности у йерсиний, а именно, системы йерсинибактина и псевдохелина.

Ключевые слова: патогенные иерсинии, фенотип высокой патогенности, сидерофоры.

The highly-pathogenic Yersiniae are defined as bacteria, which are able to kill mice in low infectious doses. Yersinia pestis, Y pseudotuberculosis and Y enterocolitica subsp. enterocolitica belong to this highly virulent group. Presence of the so-called High-Pathogenicity Island (HPI), encoding a siderophore yersiniabactin iron acquisition system (Ybt), is a main prerequisite for the high-pathogenicity phenotype in Yersinia. Loss or inactivation of this system results in significant virulence attenuation of Y pestis and Y enterocolitica subsp. enterocolitica [3, 5, 7, 11, 14, 15]. Thus, the yersiniabactin-mediated ferric iron uptake system is accepted as one of the main virulence-associated determinants in human pathogenic Yersiniae. However, although all highly pathogenic Y. pseudotuberculosis serotypes were initially suspected to possess the ybt cluster this is not the case. It has been shown that only strains of serotype O1 possess the complete yersiniabactin gene cluster [15] while other highly virulent strains of serotypes O2, O4, O5 lack ybt. The situation becomes even more intriguing with strains of serotype O3, which partly contain the ybt genes and partly are devoid of ybt. Moreover, the ybt cluster is not

functional in the O3ybt group, due to a partial truncation of the ybt genes including the fyuA gene encoding the yersiniabactin receptor [9, 15]. Accordingly, the O3ybt strains are unable to produce the yersiniabactin, while the other O3 strains demonstrate siderophore activity on the chrome azurol S siderophore detection CAS agar [15, 19]. The O3ybt strains show also reduced virulence in animals compared to their siderophore positive counterpart O3 group [9]. Moreover, the strains of other highly virulent, human pathogenic Y. pseudotuberculosis serotypes, O2, O4, and O5, also produce siderophores in the absence of the yersiniabactin genes. Previously we have applied suppression subtractive hybridization to two serotype O3 strains of both groups (O3ybt and O3), the siderophore-negative Yp346 with a truncated ybt and the prototype highly virulent siderophore-positive, but ybt-negative YPIII strain, widely used in Yersinia pathogenicity research [17]. Several gene sequences with high similarities to iron siderophore biosynthetic genes were uncovered in the YPIII genome (e.g. YPO0776, according to Y. pestis CO92 annotation). These data strongly support the presence of an alternative iron siderophore system

in O3, a system that is able to substitute the Ybt and to support the high-virulent phenotype of Y. pseudotuberculosis. Indeed, sequence of the YPIII strain (http:// www.ncbi.nlm.nih.gov/genome/510?project_id=59151) demonstrated the presence of siderophore iron acquisition systems in the O3 group and the complete absence of the ybt cluster. The functionality of at least some of these systems is supported by the ability of O3 strains to synthesize and produce a siderophore. In contrast, one of the potential alternative siderophore clusters, initially designated as HPI-2 (High-Pathogenicity Island-2, due to its similarity with the ybt cluster located on a mobile HPI structure) is evidently non-functional in Y pestis, due to insertion of the IS100 element, a frameshift disrupting the biosynthetic ORF and / or its disruption into two separated parts [8]. However, the HPI-2 cluster, designated ynp (Yersinia non-ribosomal peptide locus) is evidently intact in Y pseudotuberculosis but entirely absent in Y pestis Pestoides F strain. In Y pseudotuberculosis serotype O1 IP32953 strain, the ynp locus (YPTB3290-3298) contains putative siderophore assembly genes (YPTB3296-3297) coding for the mixed nonribosomal peptide synthetase (NRPS) / polyketide synthase (PKS) pathway and transport genes (YPTB3290-3291) and TonB-dependent outer membrane receptor (YPTB3298) (Fig. 1). The ynp locus does not contain a phosphopan-tetheinyl (P-pant) transferase, which is necessary for the assembly of the NRPS-PKS complex. Therefore, one of the two P-pants encoded within the Yersinia genomes is necessary for the siderophore biosynthesis (Bobrov et al, 2002).

Here we aimed to look for distribution of the Yersinia pseudochelin Ynp system in Y. pseudotuberculosis with special impact on serotype O3 strains and address its possible implication in siderophore synthesis and iron uptake.

Materials and methods

Bacterial strains were obtained from the collection of the Max von Pettenkofer-Institute, LMU, Munich, Germany.

Siderophore production was demonstrated in a colorimetric chrome azurol S assay using a solidified liquid CAS agar developed by Schwyn and Neilands (1987).

Conditions of iron deprivation were achieved in NBD medium (Nutrient Broth, 5 g of NaCl with addition of 0,1 mM 2-2’-dipyridyl).

Primer walking was used to close the deletion gap in the ybt cluster sequence in siderophore-negative but irp2-positive Y. enterocolitica O3 genomes (O3ybt group).

A genome wide hybridization microarray [12] was applied to compare gene contents of the selected Yersinia genomes.

High-quality Y pseudotuberculosis genome sequences were obtained from the publicly accessible databases http://www.ncbi.nlm.nih.gov and draft genome sequences of Y pseudotuberculosis serotype O4 and O5 strains were obtained in cooperation with BGI-Hongkong Co. (Hong Kong) and annotated with the RAST server [2]. Circular representation of the genome comparisons was performed with BRIG [1].

Results

We have compared the strains of serotype O3 available in our collection (Table 1) for the presence of the yersiniabactin biosynthetic irp2 gene and the ability to produce a siderophore on the CAS agar, to grow in iron-deficient NBD medium and to support growth of E. coli H1884 entD,F (siderophore enterochelin -negative mutant unable to grow in iron depleted NBD medium). Only strains of the siderophore-positive O3 group, but not of the O3ybt group, were able to grow in iron depleted

Fig. 1. The pseudochelin iron uptake operon (Yersinia non-ribosomal peptide locus ynp, YPTB3296-3297) in Y. pseudotuberculosis IP32953 strain (NCBI Reference Sequence: NC_006155.1). The picture is generated using Geneious program 6.1.5

Table l

Comparison of Y. pseudotuberculosis O3 strains according to their CAS activity, presence of the ybt genes and ability to grow and support growth in iron-depleted media

Strain, isolation place CAS activity Presence of the irp2 gene Growth in NBD, support of H1884 entD,F growth

1216/93, Norway No No

14240, Turmenistan No No

200/90, Germany No No No

298/89, Australia No No

1134/90, Japan No No

2887, Argentina No No

Turku, Finland No No

Act-2, Russia No No

307, New Zealand No No No

346, Denmark No No No

201/90, Germany Yes No

201, Denmark Yes No

1401, Sweden Yes No

YPIII, U.S.A. Yes No

146, Germany Yes No Yes

445/73, Russia Yes No Yes

483/71, Russia Yes No Yes

714, Japan Yes No

medium as well as to support the growth of the H1884 entD,F mutant.

Using the whole genome hybridization microarray [12] we tested the presence of the siderophore-associ-ated sequences (ybt and ynp) in the O3 strains. It turns out that, while the strains of the O3ybt group contain the int, ybtS-ybtP, ybtA and irp2-irpl genes (YPO1911 -YPO1916, according to Y pestis CO92 annotation [13], they completely lack the ynp genes (clustered in two groups, YP00770 - YP00778 and YP01011 - YP01012 in Y pestis CO92, or YPTB3290-YPTB3298, according to IP32953 annotation). Alternatively, the O3 group siderophore-positive strains possess the complete ynp gene clusters but none of the ybt associated genes.

Using primer walking we have defined the deletion end point in the ybt cluster in O3ybt strains (Fig. 2). The deletion includesybtU-fyuA sequences and terminates 30 bp downstream the irpl stop codon.

Several completed and draft Yersinia genomes are now available in the public databases (http://www.ncbi.

int* irp6-9 ybtA irp2 irpl lrp3-5 fyuA to се Co

YesHPI » >»i=h^

int ybtS,X,Q,PybtA irp2 irpl ybtU,T,E fyuAxis \S100

YP03 HPI ) »

int lrp6-9 ybtA irp2 irpl

Fig.2. Structure of the yersiniabactin mobile gene cluster (HPI) in different Yersiniae. Note different designations of the ybt genes in Y. pestis /Y. pseudotuberculosis. Yps HPI stands for Y. pestis HPI and YpO3 HPI - for Y. pseudotuberculosis O3ybt HPI and Yen HPI - for Y. enterocolitica subsp. enterocolitica HPI

nlm.nih.gov/genome). For genome comparison we have included two draft genome sequences obtained from Y. pseudotuberculosis serotype O4 and O5 strains in cooperation with BGI, China. This allows analysis and comparison of the corresponding human-pathogenic Y. pseudotuberculosis for the presence of the sidero-phore iron uptake systems on the whole genome scale (Figure 3).

The partial deletion of the yersiniabactin ybt gene locus is evident in serotype O3ybt strains B6796_O3, B6862_O3, B6863O3 and B6864_O3 while the complete ybt cluster is missing in strain YPIII serotype O3; strains 66, O4 and 68, O5 and, to our surprise, also in the serotype O1 Far East Scarlet like fever (FESLF) strain IP31758 [6, 10]. Further sequencing of additional eight FESLF strains supports the absence of the ybt genes in this group of serotype O1 strains (data not shown). In contrast, the ynp cluster was absent in all O3ybt isolates but present in the strains of the other serotypes under comparison. Moreover, another potentially active siderophore iron acquisition system, aerobactin (iuc), is absent in the O3ybt strains. Thus, none of the three siderophores possibly supporting iron acquisition, the yersiniabactin, the pseudochelin (ynp) and the aerobactin (iuc) is present or functional in the O3ybt group. Interestingly, both iron uptake systems, ynp and iuc, are located in the integration hot spot that might represent another Plasticity Zone (PZ2) in Y pseudotuberculosis genome. In contrast, sporadic Y. pseudotuberculosis O1 strains carry all three siderophore ferric iron acquisition systems. However, the functionality of these systems has to be demonstrated [8].

P-pant transferases are necessary for assembly of the NRP/PKS complex and synthesis of the pseudo-chelin. Indeed, all analyzed Y. pseudotuberculosis strains analyzed have both unaltered genes encoding P-pant transferases, ybtD, which initiates the YBT system and acpP, required for the fatty acid synthesis. Thus, the two O3 groups do not differ by their ability to support the synthesis of the Ynp siderophore due to the absence of the supporting P-pants.

Discussion

The so-called High-Pathogenicity Island supplies bacteria with the ability to synthesize the yersiniabac-tin (Ybt), a siderophore sequestering bound ferric iron from the host iron sources. The HPI represents an archetypical genetic mobile structure, a genomic island that transposes to any available free asn-tRNA genes in the host genome using a site-specific recombination. The HPI originally described in Yersinia is widely disseminated in Enterobacteriaceae (20) due to its mobile nature. However, the Ybt system is not present in all highly pathogenic Yersiniae (as it was initially suspected) and it is restricted to Y pestis and Y enterocolitica subsp. enterocolitica, both of which demonstrate their absolute dependence on Ybt for in vivo survival and virulence [3, 5, 7, 11, 14].

Fig. 3. Whole genome alignment of Y. pseudotuberculosis strains. Significant differentiating gene regions are represented. The white color indicates regions present in strain IP32953 but absent from the other strains

In contrast, most representatives of another group of highly virulent Yersinia, Y pseudotuberculosis, do not possess the Ybt iron supply system. Only sporadic Y pseudotuberculosis O1 strains demonstrate the presence of the ybt gene cluster while strains of the other highly virulent serotypes, O2, O4, O5, as well as epidemic FESLF O1 strains, lack the ybt genes. They also carry the pseudochelin Fe3+ acquisition system (Ynp) as an alternative to the yersiniabactin [18]. Moreover, serotype O3 strains likely exhibit competition between the Ybt and the Ynp ferric iron systems; they possess either of the systems, but not both of them. The similarity of the Ybt and Ynp systems and their potential biochemical and genetic cross talk might explain such competition of the two iron scavenging systems. Simultaneous presence and expression of these two related non-ribosomal peptide synthesized siderophore systems in sporadic O1 strains awaits its explanation.

Taken together, the alternative non-ribosomal peptide synthesized siderophore iron supply system Ynp (designated as “pseudochelin” due to its preferential distribution in Y pseudotuberculosis [18]), is also responsible for the ferric iron siderophore mediated acquisition and, thus, for the highly-virulent phenotype of the vast majority of Y pseudotuberculosis strains and serotypes. Indeed, inactivation of the siderophore biosynthetic

gene YPBT3297 by homologous recombination with a chloramphenicol resistance cassette containing DNA fragment in YPIII, serotype O3 resulted in its inability to produce the siderophore (data not shown).

The ferric iron supply is not directly bound to pathogenicity of bacteria but it is a prerequisite for their successful multiplication in host and virulence potential. The Fur-dependent regulation of both iron supply and virulence supports a cross link between these traits. Bacteria. including pathogenic ones, possess different siderophore iron systems. Some of them have been acquired through a horizontal gene transfer and might be exposed in the available global gene pool and, thus, become available for import by other microorganisms to exploit alternative pathways.

Summary

1. The yersiniabactin ferric iron acquisition system, traditionally supposed to be responsible for the high-pathogenicity phenotype of Yersinia, is restricted

to Y pestis and Y. enterocolitica subsp. enterocolitica strains.

2. Human highly pathogenic Y pseudotuberculosis

O2, O4, O5, O3ynp and epidemic FeSLF O1 demonstrate alternative siderophore activity in the absence of

the yersiniabactin system.

3. The alternative iron acquisition system designated as pseudochelin (originally annotated as Yersinia non-ribosomal peptide locus, ynp) is responsible for the production of the siderophore in Y. pseudotuberculosis

02, O4, 05, O3ynp and FESLF O1 non-Ybt group.

4. Strains of two groups of Y. pseudotuberculosis

03, namely O3ybt and O3, demonstrate differences in their siderophore activity. Strains of the O3ybt group do not exhibit any siderophore activity and are of reduced virulence due to the absence of functional iron acquisition systems (the pseudochelin, ynp; the aerobactin, iuc and truncation of the yersiniabactin locus, ybt). In turn, the O3ynp strains are siderophore pseudochelin positive but lack the yersiniabactin locus, ybt.

5. The present designation of the ybt genomic island as the High-pathogenicity Island [4] as the only one associated with the high-pathogenicity phenotype in Yersinia is misleading and must be withdrawn.

References

1. Alikhan N.F., Petty N.K., Ben Zakour N.L., Beatson S.A. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011; 12:402.

2. Aziz R.K., Bartels D., Best A.A., DeJongh M., Disz T., Edwards R.A., Formsma K., Gerdes S., Glass E.M., KubalM., Meyer F., Olsen G.J., Olson R., Osterman A.L., OverbeekR.A., McNeil L.K., Paarmann D., Paczian T., Parrello B., Pusch G.D., Reich C., Stevens R., Vassieva O., Vonstein V., Wilke A., Zagnitko O. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics.

3. Bearden S.W., Fetherston J.D., Perry R.D. Genetic organization of the yersiniabactin biosynthetic region and construction of avir-ulent mutants in Yersinia pestis. Infect Immun. 1997; 65:1659-68.

4. Carniel E. The Yersinia high-pathogenicity island: an iron-uptake island. Microbes Infect. 2001; 3:561-9.

5. deAlmeida A.M., Guiyoule A., Guilvout I., Iteman I., Baranton G., Carniel E. Chromosomal irp2 gene in Yersinia: distribution, expression, deletion and impact on virulence. Microb Pathog. 1993; 14:9-21.

6. Eppinger M., Rosovitz M.J., Fricke W.F., Rasko D.A., Kokorina G., Fayolle C., Lindler L.E., Carniel E., Ravel J. The complete genome sequence of Yersinia pseudotuberculosis IP31758, the causative agent of Far East scarlet-like fever. PLoS Genet. 2007; 3(8):e142.

7. Fetherston J.D., Kirillina O., Bobrov A.G., Paulley J.T., Perry R.D. The yersiniabactin transport system is critical for the pathogenesis of bubonic and pneumonic plague. Infect Immun. 2010; 78:2045-52.

8. Forman S., Paulley J.T., Fetherston J.D., Cheng Y.Q., Perry R.D. Yersinia ironomics: comparison of iron transporters among Yersinia pestis biotypes and its nearest neighbor, Yersinia pseudotu-

berculosis. Biometals. 2010; 2:275-94.

9. Fukushima H. Molecular epidemiology of Yersinia pseudotuberculosis. Adv. Exp. Med. Biol. 2003; 529:357-8.

10. Gurleva G.G., Domaradskii I.V, Smolikova L.M., Khaliapina E.E., Grigor ’ian E.G. [Biological properties of the causative agent of pseudotuberculosis isolated from scarlatina-like fever patients]. Zh. Mikrobiol. Epidemiol. Immunobiol. 1973; 50:125-9.

11. Heesemann J., Hantke K,. Vocke T., Saken E., Rakin A., Stojiljkovic I., Berner R. Virulence of Yersinia enterocolitica is closely associated with siderophore production, expression of an iron-repressible outer membrane polypeptide of 65,000 Da and pesticin sensitivity. Mol. Microbiol. 1993; 8:397-408.

12. Hinchliffe S.J., IsherwoodK.E., StablerR.A., PrenticeM.B., Rakin A., Nichols R.A., Oyston P.C., Hinds J., Titball R.W., Wren B.W. Application of DNA Microarrays to Study the Evolutionary Genomics of Yersinia pestis and Yersinia pseudotuberculosis. Genome Res. 2003; 13(9):2018-29.

13. Parkhill J., Wren B.W., Thomson N.R., Titball R.W., Holden M.T., Prentice M.B., SebaihiaM., James K.D., Churcher C., Mungall K.L., Baker S., Basham D., Bentley S.D., Brooks K., Cerdeno-Tarraga A.M., Chillingworth T., Cronin A., Davies R.M., Davis P., Dougan G., Feltwell T., Hamlin N., Holroyd S., Jagels K., KarlyshevA.V, Leather S., Moule S., Oyston P.C., Quail M., RutherfordK., Simmonds M., Skelton J., Stevens K., Whitehead S., Barrell B.G. Genome sequence of Yersinia pestis, the causative agent of plague. Nature. 2001; 413:523-7.

14. Perry R.D., Fetherston J.D. Yersiniabactin iron uptake: mechanisms and role in Yersinia pestis pathogenesis. Microbes Infect. 2011; 13:808-17.

15. Rakin A., Urbitsch P., Heesemann J. Evidence for two evolutionary lineages of highly pathogenic Yersinia species. J. Bacteriol. 1995; 177:2292-8.

16. Rakin A., Schubert S., Pelludat C., Brem D., Heesemann J. The High-Pathogenicity Island of Yersiniae. In: Pathogenicity islands and other mobile virulence elements. Kaper J. B., Hacker, editors. Washington D.C.: ASM-Press; 1999. P. 77-90.

17. Rakin A., Golubov A., Iwobi A., Heesemann J. Tracing acquisitions and losses in Yersinia genomes. In: The Genus Yersinia. Entering the Functional Genomic Era. Skurnik M., Bengoechea J.A., Granfors K., editors. NY: Kluwer Academic/Plenum Publishes; 2003. P. 19-23.

18. Rakin A., Schneider L., Podladchikova O. Hunger for iron: the alternative siderophore iron scavenging systems in highly virulent Yersinia. Front Cell Infect. Microbiol. 2012; 2:151.

19. Schwyn B., Neiland J.B. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 1987; 160:47-56.

20. Schubert S., Rakin A., Heesemann J. The Yersinia high-pathogenicity island (HPI): evolutionary and functional aspects. Int. J. Med. Microbiol. 2004; 294:83-94.

Authors:

Rakin A., Garzetti D. Max von Pettenkofer-Institute, LMU, Pettenkofer str. 9a, 80336 Munich, Germany. E-mail: rakin@mvp.uni-muenchen.de

Об авторах:

РакинА., Газетти Д. Институт Макса фон Петтенкофера. Мюнхен, Германия. E-mail: rakin@mvp.uni-muenchen.de

Поступила 05.08.13.