Genomic islands in Photorhabdus

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Genomic islands in Photorhabdus Nicholas R. Waterfield, Phillip J. Daborn and Richard H. ffrench-Constant Genomic islands are responsible for unique aspects of bacterial behavior such as symbiosis and pathogenicity. Photorhabdus luminescens is a pathogen of insects that spends part of its lifecycle in symbiosis with a nematode. Here, we describe novel genomic islands from Photorhabdus that are involved in symbiosis and pathogenicity, and discuss the inter-relationship between virulence factors used against invertebrates and vertebrates.

however, not only killing the insect but also providing food for the growing nematodes [8]. Thus, both bacteria and nematodes undergo rounds of replication within the cadaver, forming an effective ‘symbiosis of pathogens’. Here, we describe genomic islands from Photorhabdus luminescens subsp. akhurstii W14, as a basis for a functional dissection of their role in pathogenicity and symbiosis. Genomic islands of Photorhabdus

Genomic islands encode specific phenotypic traits such as pathogenicity and symbiosis, and ‘pathogenicity islands’ (PAIs) are unstable regions present in pathogens but absent from non-pathogens [1]. Described from pathogens of humans [2], PAIs often show shifts in GC content and tRNA linkage. Invertebrate hosts, however, constitute a larger group of extant organisms than vertebrates and have also been exposed to the ‘pathosphere’ [3] of bacterial virulence factors for a longer evolutionary period. Moreover, invertebrates are often secondary hosts of pathogens of vertebrates, for example, the flea and Yersinia pestis, the causative agent of bubonic plague [4]. With the current genomic focus on pathogens of vertebrates [5], we are left uncertain as to the nature of genomic islands in pathogens of invertebrates and their role in the evolution of vertebrate pathogenesis. We are interested in identifying genomic islands in Photorhabdus, a pathogen of insects but a nematode symbiont [6–8]. Lifecycle of Photorhabdus

Photorhabdus lives within the guts of nematodes that infect insects [8]. Following invasion of the insect, the bacteria are released into the bloodstream where they grow unrestricted by the insect immune system, inhibiting their own phagocytosis by phagocytes (hemocytes) and releasing toxins and exoenzymes that kill and bio-convert the host [9–11]. The bacteria are still mutualistic with the nematodes, http://tim.trends.com

We estimated that ~53% of the ~5.5 Mb Photorhabdus genome was distinct from Escherichia coli [12]. To isolate islands unique to Photorhabdus, we end-sequenced an arrayed cosmid library and compared the end-sequences with those in public databases using BLAST algorithms. Unique cosmids were sequenced and checked for pathogenic phenotypes, such as the ability to persist within, or kill, insects [11]. As all Photorhabdus strains (even clinical isolates) are pathogenic to insects, we identified genomic islands by gene homology (BLASTX), relative location in the genome (tRNA linkage or within E. coli-like core sequence) or altered GC content (estimated as 41.5% for the W14 core). The first unique island carries multiple copies of toxin complex (tc) genes [13,14] inserted at an AspV tRNA (Fig. 1). The tc genes encode high molecular weight insecticidal toxin complexes or Tcs, which destroy the insect midgut [13,15]. The island carries multiple tcdA-like (tcdA1, tcdA2 and tcdA3) and tcdB-like (tcdB1 and tcdB2 ) genes. The region also carries multiple tccC-like genes (tccC2, tccC3, tccC4 and tccC5), enteric repetitive intergenic consensus (ERIC; [16])-like sequences, duplicated ORFs (luxR-like regulators), and a truncated tcdA-like gene, all suggesting recent mobility and rearrangement; this hypothesis is also supported by the deletion of two tc genes from this region in Photorhabdus strain HB [14]. The tcd island is adjacent to nrgA, a gene encoding

4′-phosphoantetheinyl (Ppant) transferase (an enzyme necessary for enterobactin production), deletion of which causes P. luminescens to fail to support nematode growth [17]. Further, nrgA is also adjacent to a fimbrial operon encoding strain-specific adhesins potentially involved in nematode association (N.R. Waterfield et al., unpublished). This linkage of pathogenicity and putative symbiosis genes suggests a ‘hot spot’ for the recombination of horizontally acquired genes. Additionally, given the close association of tccC elements with other tc loci and their relationship to recombinational hot spot (rhs) elements [18], we suggest they might be directly involved in such rearrangements. Given its demonstrated instability and linkage to a tRNA, the tcd island is a candidate PAI, although its GC content is not strikingly different from the rest of the genome, suggesting its acquisition could be ancient. Also consistent with a PAI, the region contains genes often found in phage, including holin and ner-like (phage Mu regulator-like) sequences. Holins are associated with tc-like genes in other Gram-negative bacteria [14] and ner has been implicated in variation in the phenotypes of Photorhabdus colonies [19]. Furthermore, AspV tRNA is the site of insertion of PAIs in several other bacteria, including PAI-6 of Salmonella enterica and O-island 7 of E. coli O157, which also contain rhs/tccC-like genes. A second island, inserted at a Phe tRNA, encodes the novel toxin ‘makes caterpillars floppy’ or Mcf, a large toxin with little similarity to known proteins [11]. The mcf gene is striking in that E. coli expressing this gene can both persist within and kill insects [11]. The Mcf toxin causes apoptosis in insect phagocytes, helping the bacteria avoid phagocytosis, and also destroys the midgut, causing the caterpillar to lose body turgor and become ‘floppy’ [11]. This region carries genes encoding proteins from the ShlA/HecA/FhaA hemagglutinin-like family (Fig. 2a) and a fragment of a CP4-like integrase. The same Phe tRNA

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54.3% 1 2

4 5

3

6

Insect pathogenesis

Core

Nematode symbiosis

44.8%

7

8 9 10 11 12 13 1415 16

23 25 27 17 1819 20 21 22 24 26 28 29

30

31 32 33

34

tccC4 Phf fimbriae

nrgA

ERIC′

tc-toxin island

tcdA fragment

42.7% 35

36

tcdA3

37

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tcdA2

tcdB2

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Holin tccC3

tcdA4 Lipase pdl2

ERIC′

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tccC5

51

tcdA1

52 53 54 55

56

tcdB1 Lipase pdl1

57 58 59 60 61 6263 64

tccC2 ERIC′

Phage

AspV tRNA 38.5% 65 66 67 68 697071 72 73

74 75 76

luxR-like

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78 79 80 81

82 83 84 85 86

luxR-like

luxR-like

87 88

Tc genes Putative symbiosis Bacteriophage derived Genomic core seq orthologs (e<-70) Unknown or homologs (e>-50) 2

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Fig. 1. The toxin complex d (tcd) island from Photorhabdus luminescens W14 (accession no. AY144119). The tcd genes form tandem arrays of tcdA-like (ORFs 36, 37, 45 and 50) and tcdB-like (ORFs 38 and 51) genes, bordered by a tccC-like sequence (ORFs 30, 40, 46 and 56). The island is inserted at an AspV tRNA and is separated from nrgA (ORF 10), involved in nematode symbiosis, by a section of core sequence showing homology to Escherichia coli (ORFs 11–21) and Yersinia pestis (ORFs 22–28). The nrgA gene lies next to a fimbrial operon encoding Phf fimbriae (ORFs 4–8). The junctions of the islands with the core sequence show directly duplicated ORFs (arrows), including multiple copies of luxR-like regulators (ORFs 64–66, 77–79 and 81–83) and two copies of a ner phage Mu-like regulator gene (ORFs 67 and 84). The presence of other genes often found in phage, including holins (ORF 39), suggests that the tc genes themselves could be phage associated. Boxes represent ORFs transcribed in either direction (above and below line) and are color-coded based on their putative functions. The percentage GC content (between arrows) is shown above the line.

is also associated with the She PAI from Shigella flexneri 2a and O-island 122 from E. coli O157, which also carry CP4-like integrases, suggesting a common mechanism of acquisition. A third island contains a gene encoding a cytotoxic necrotising factor (CNF)-like toxin, designated Pnf. The CNF1 protein from uropathogenic E. coli is a Rho GTPase-activating toxin whose carboxyl terminus is an enzyme that deamidates Rho GTP-binding proteins causing rearrangement of the actin cytoskeleton [20,21]. Although the specific http://tim.trends.com

role of Pnf in Photorhabdus is unknown, the carboxy-terminal region is conserved and contains the active site [22] (Fig. 2b), but the cell-targeting and translocation domains are absent. Although the region surrounding Pnf is not tRNA linked, it does show altered GC content and contains genes with similarity to the cyanobacterium Nostoc. A fourth island with a skewed GC content carries two copies of a macrophage-toxin-like encoding gene and has similarities in genomic organization to E. coli O157 EDL933 0-island 7,

with the presence of an rhs element and a CP4-like integrase gene. This region is linked to the previously reported phlAB hemolysin Photorhabdus locus [23], and a second integrase gene, suggesting that it might form part of a larger region involved in pathogenicity (Fig. 2a). Finally, a fifth unique region encodes a type III secretion system (TTSS). TTSSs are ‘molecular syringes’ used to deliver effector proteins from bacteria across host cell membranes and have, again, largely been described from pathogens of vertebrates. The order of genes in the TTSS island is similar to that in Y. pestis but differs from that in Pseudomonas aeruginosa as the pscU-exsB region is inverted (Fig. 3). Predicted proteins include homologs of Y. pestis YopBD, YopR, YopT and LcrV. Antibodies raised against Y. pestis LcrV play a protective role in mice against infection by plague [24], suggesting LcrV-like proteins might also be important in the interaction

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palB (activator)

mcf

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palA (hemolysin/hemagglutinin)

Phet RNA 1314 16

(a) Mcf 40.5%

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pnf 9 10 11 12

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(b) Pnf 39.72%

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Integrase

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phlAB

(c) Mt 58.4% Putative virulence Mobile element/bacteriophage like Unknown or homologs (e>-50)

36.1%

62%

20–30 kb

Rhs

Repeat Integrase Genomic core seq orthologs (e<-70) 2kb

(d) 610

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- - - - - - - - - M L K Y F T A - - - D N - - - - - K L N K G - - - - - - - - - H I S P L K - - - - - - R K G L L V G S - - - - - - - - - D N - - - - - - - - - - - - A P I D I P V I A H R - - - - - -

599

G A T E E E A W N I A S Y H T A G G S T E E L H E I L L G Q G P Q S S L G F T E Y T S N V N S A D A A S R R H F L V V I K V H V K Y I T N N N V S Y V N H W A I P D E A P V E V L A V V D R R F N F P E

Ec Cnf1

599

G T T E E E A W N I A R Y H T A G G S T E E L H E I L L G Q G P Q S S L G F T E Y T S N I N S A D A A S R R H F L V V I K V Q V K Y I N N N N V S H V N H W A I P D E A P V E V L A V V D R R F N F P E

Ec Cnf2

600

G T T E E E A W N I A N Y K T A G G S N K D L E E N F I E A G P Q F N L S F S E Y T S S I N S A D T A S R K H F L V I I K V Q V K Y I S N D N V L Y A N H W A I P D E A P V E V L A V V D R R F I F P E

Yp Cnf

710 42

720

730

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750

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770

780

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Pl pnf

800

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y D S N N Q L E Q A S - - - - - - S L R N S D S G Q E I P F H D V V T G F R G D Q

Pl pnf

699

P S T P P D I S T I R K L L S L R Y F K E S I E S T S K S N F Q K L S R G N I D V L K G R G S I S S T R Q R A I Y P Y F E A A N A D E Q Q P L F F Y I K K D R F D N H G Y D Q Y F Y D N T V G L N G I P

Ec Cnf1

699

P S T P P N I S I I H K L L S L R Y F K E N I E S T S R L N L Q K L N R G N I D I F K G R G S I S S T R Q R A I Y P Y F E S A N A D E Q Q P V F F Y I K K N R F D D F G Y D Q Y F Y N S T V G L N G I P

Ec Cnf2

700

P P V K P K L S F I Q K I A N - R F L T E N V A E I S S I N F R R L N S G N I N V L K G R G V F S S R R L R E I Y L R F D A A N A D E L R P G D V Y V K K T K F D S M G Y D S H F Y N E G I G I N G A P

Yp Cnf

810 77

820

830

840

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860

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V T S S E S G - - - - - S G A I G - K H W G K N K L D H N I T G I N V V N G A S G T V G I K I A L R D I R P G Y P I I V T S G A L S G C T M V Y A V K D N Y F F A Y H T G Q K P G D D E W R T G Q D G V

Pl pnf

799

T L N T Y T G E I P S D S S S L G S T Y W K K Y N L T N E T S I I R V S N S A R G A N G I K I A L E E V Q E G K P V I I T S G N L S G C T T I V A R K E G Y I Y K V H T G T T K S L A G F - T S T T G V

Ec Cnf1

799

T L N T Y T G E I L S D A S S L G S T Y W K K Y N L T N E T S I I R V S N S A R G A N G I K I A L E E V Q E G K P V I I T S G N L S G C T T I V A R K G G Y L Y K V H T G T T I P L A G F - T S T T G V

Ec Cnf2

799

T L N T Y T G E Y V A D S S S Q G A T Y W L K Y N L T N E T S I I K V S N S A R G A N G I K I A L E E I E E N K P V V I T S G T L T G C T V V F A R K G E Y F Y A V H T G N S E S L I G F - T S T S G V

Yp Cnf

910

920

930

940

950

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980

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V T T A Q S H K A L L S D S K P I A V N K Q N N D - L V N I F A E - Y D Q S V I T Y M G - - - K Q A V V I D N T A E N V S V F N Y D E I K P G K P A I R A G Y S Y A L L A N D N G Q V S V K V L S E D A

Pl pnf

898

K K A V E V L E L L T K E P I P R V E G I M S N D F L V D Y L S E N F E D S L I T Y S S S E K K P D S Q I T I I R D N V S V F P Y - - F L D N I P E H G F G T S A T V L V R V D G N V V V R S L S E S Y

Ec Cnf1

898

K K A V E V F E L L T N N P M P R V E G V M N N D F L V N Y L A E S F D E S L I T Y S S S E Q K I G S K I T I S R D N V S T F P Y - - F L D N I P E K G F G T S V T I L V R V D G N V I V K S L S E S Y

Ec Cnf2

898

A K A I E V L S S L S E L E V P A L P D V I N N N T L V E Y L S D N F D S A L I S Y S S S S L K P N S M I N I S R E N V S T F S Y - - Y T D D I Q L P S F G T S V T I L V R T N D N T V V R S L S E S Y

Yp Cnf

171

1010

1020

266

I V S P G K N G N S I K V I N S L K K R L L .

996 996

S L N A - - D A S E I S V L K V F S K K F S L N V - - E N S N I S V L H V F S K D F

996

T M N S - - N S S K M V V F N V L Q K D F

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Fig. 2. (a–c) Comparison of three genomic islands in Photorhabdus W14 (AF503504, AY144120, AY144118 and AY144117). (a) Island encoding a novel insecticidal toxin termed ‘makes caterpillars floppy’ or Mcf (ORF 9). The mcf gene lies within a region of altered GC content (encompassing ORFs 9–11), also containing hemagglutinin/hemolysin-like genes (palBA). The region is linked to a Phe tRNA and an integrase gene fragment (ORF 15). (b) Island containing a cytotoxic necrotising factor-like gene (pnf) and genes (ORFs 1–26) similar to Nostoc sp. PCC 7120 (accession no. NC_003272), bacteriophage (14–16 and 20) and transposases (ORF 12). The region is flanked by two large repeats (ORFs 1–4 and 23–26; dashed arrows). (c) Island containing two copies of a macrophage like-toxin similar to that found in pathogenic strains of Escherichia coli. Note the presence of an rhs element, an integrase, and linkage to the phlAB hemolysin locus. (d) The predicted amino acid sequence of Pnf from Photorhabdus aligned with the carboxy-terminal domains of other Cnf homologs showing conservation of the Rho-deamidase active site (boxed). Sequences are E. coli CNF1 and CNF2 and Yersinia pseudotuberculosis CNF (accession nos S37405, A55260 and AAG45433).

of Photorhabdus with its invertebrate hosts. P. luminescens W14 produces a factor capable of suppressing its own phagocytosis [10] and recent studies show that the YopT-like effector can be delivered into insect hemocytes on contact [25], suggesting that the TTSS might play an important role in modulating the interaction of Photorhabdus with phagocytes. Implications for the evolution of insect and vertebrate virulence

The concept of a ‘pathosphere’ [3], describes a pool of potential virulence http://tim.trends.com

factors readily transferred between different bacterial pathogens. However, to embrace the appearance and evolution of vertebrate hosts we must include the period when bacteria could only infect non-animals or invertebrates, that is, before vertebrates themselves evolved. In this ‘pre-vertebrate’ pathosphere, virulence factors would have been selected for virulence within invertebrate and non-animal (plants and other microorganisms) hosts. Given extensive transfer of virulence factors between pathogens of invertebrates and vertebrates, and the bias of existing

databases to pathogens of vertebrates, this history is hard to interpret. However, the genomic islands presented here begin to describe the diversity of anti-invertebrate virulence factors and their similarity to those used against vertebrates. Moreover, as both the vertebrate and invertebrate immune systems rely heavily on phagocytes, it would not be surprising if similar virulence factors, such as Photorhabdus Mcf, Pnf, RtxA-like, macrophage-toxinlike, hemolysin-like and type III effector-like proteins [11,12], could act on both immune systems. In fact, strains of Photorhabdus asymbiotica that are still pathogenic to insects have been isolated from human wounds, suggesting that the same virulence factors can indeed be used to survive both immune systems [26,27]. In conclusion, we are presently unable to assess the true inter-relationship between anti-invertebrate and anti-vertebrate virulence factors. However, some bacterial pathogens of

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Phage and O-antigen genes

Phage and capsule genes

W14 TTSS 46.76%

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Fig. 3. Operons encoding a Photorhabdus type III secretion system (TTSS; AY144116). Predicted proteins similar to YopR (ORF 4), YopBD (ORF 30 and 31) and YopT are labeled. The TTSS is linked to a region containing phage and capsule genes (ORFs 34–43), which might have been involved in its acquisition. The gene order is compared with that found in Pseudomonas aeruginosa (accession nos AF010149 and AF010150). Note that a similar inversion (dashed lines from ORFs 12–33) is also found in equivalent Yersiniae loci.

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humans, such as Y. pestis, use invertebrates as secondary hosts or vectors. Moreover, the majority of homologs and orthologs of Photorhabdus genes are most similar to those of the Yersiniae, suggesting a recent common ancestor. We will continue to address this question by comparing genomic islands present in Photorhabdus strains found in association with their nematode partners with those found in clinical isolates. By understanding which subset of genes can be used against either the invertebrate and/or vertebrate immune system, we hope to begin to unravel this complex question. References 1 Hacker, J. and Kaper, J.B. (2000) Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54, 641–679 2 Hacker, J. et al. (1990) Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vitro and in vivo in various extraintestinal Escherichia coli isolates. Microb. Pathog. 8, 213–225 3 Burland, V. et al. (1998) The complete DNA sequence and analysis of the large virulence http://tim.trends.com

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plasmid of Escherichia coli O157:H7. Nucleic Acids Res. 26, 4196–4204 Perry, R.D. and Fetherston, J.D. (1997) Yersinia pestis – etiologic agent of plague. Clin. Microbiol. Rev. 10, 35–66 Fitzgerald, J.R. and Musser, J.M. (2001) Evolutionary genomics of pathogenic bacteria. Trends Microbiol. 9, 547–553 Forst, S. and Nealson, K. (1996) Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol. Rev. 60, 21–43 Forst, S. et al. (1997) Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51, 47–72 Forst, S. and Clarke, D. (2001) Bacteria–nematode symbiosis. In Entomopathogenic Nematology (Gaugler, R., ed.), pp. 57–77, CAB International Dowds, B.C.A. and Peters, A. (2002) Virulence mechanisms. In Entomopathogenic Nematology (Gaugler, R., ed.), pp. 79–98, CABI Publishing Silva, C.P. et al. (2002) Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cell. Microbiol. 4, 329–339 Daborn, P.J. et al. (2002) A single Photorhabdus gene makes caterpillars floppy (mcf) allows Escherichia coli to persist within and kill insects. Proc. Natl. Acad. Sci. U. S. A. 99, 10742–10747 ffrench-Constant, R.H. et al. (2000) A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14:

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potential implications for virulence. Appl. Environ. Microbiol. 66, 3310–3329 Bowen, D. et al. (1998) Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129–2132 Waterfield, N.R. et al. (2001) The toxin complex genes of Photorhabdus: a growing gene family. Trends Microbiol. 9, 185–191 Waterfield, N. et al. (2001) Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli. Appl. Environ. Microbiol. 67, 5017–5024 Versalovic, J. et al. (1991) Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19, 6823–6831 Ciche, T.A. et al. (2001) A phosphopantetheinyl transferase homolog is essential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic nematode Heterorhabditis bacteriophora. J. Bacteriol. 183, 3117–3126 Wang, Y.D. et al. (1998) Rhs elements comprise three subfamilies which diverged prior to acquisition by Escherichia coli. J. Bacteriol. 180, 4102–4110 O’Neill, K.H. et al. (2002) Phenotypic switching in the entomopathogenic bacterium Photorhabdus sp. strain K122 is induced by the ner gene. J. Bacteriol. 184, 3096–3105 Flatau, G. et al. (1997) Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 387, 729–733 Sugai, M. et al. (1999) Cytotoxic necrotizing factor type 2 produced by pathogenic Escherichia coli deamidates a Gln residue in the conserved G-3 domain of the rho family and preferentially

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inhibits the GTPase activity of RhoA and rac1. Infect. Immun. 67, 6550–6557 22 Buetow, L. et al. (2001) Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1. Nat. Struct. Biol. 8, 584–588 23 Brillard, J. et al. (2002) The PhlA hemolysin from the entomopathogenic bacterium Photorhabdus luminescens belongs to the two-partner secretion family of hemolysins. J. Bacteriol. 184, 3871–3878 24 Fields, K.A. et al. (1999) Virulence role of V antigen of Yersinia pestis at the bacterial surface. Infect. Immun. 67, 5395–5408

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25 Zumbihl, R. et al. (2002) Xenorhabdus and Photorhabdus virulence factors and their impacts on insect cellular immunity. In Proceedings of The XXXV Annual Meeting of the Society for Invertebrate Pathology, 18–23 August, Iguassu Falls, Brazil, pp. 177–182, Society for Invertebrate Pathology 26 Colepicolo, P. et al. (1989) Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl. Environ. Microbiol. 55, 2601–2606 27 Farmer, J.J. et al. (1989) Xenorhabdus luminescens (DNA hybridization group 5)

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Nicholas R. Waterfield Phillip J. Daborn Richard H. ffrench-Constant* Dept of Biology and Biochemistry, University of Bath, South Building, Bath, UK BA2 7AY. *e-mail: [email protected]

Meeting Report

Pathogenesis and host response of Helicobacter pylori Anthony P. Moran, Ann-Mari Svennerholm and Charles W. Penn The 5th International Workshop on Pathogenesis and Host Response in Helicobacter Infections was held in Elsinore, Denmark, 4–7 July, 2002. Published online: 31 October 2002

Helicobacter pylori is a human gastroduodenal pathogen that can persist for a lifetime in the stomach of 50% of the world’s population. Many infected individuals are asymptomatic; however, for a significant number of individuals, infection with H. pylori causes the development of gastritis, gastric and duodenal ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma, loss of gastric glands (atrophy) and finally adenocarcinoma, a disease with very high morbidity and mortality. The pathogenic mechanisms of this Gram-negative bacterium and the interactions between this pathogen and its host have been the focus of intensive investigations for the past 15 years [1,2]. Evidence has emerged that the outcome of infection depends not only on host factors but also on characteristics of the infecting strain. Moreover, in the past decade, it has emerged that several helicobacters other than H. pylori are important in the pathogenesis of gastric and enterohepatic diseases of animals and humans [3]. New concepts in H. pylori pathogenesis

Adhesion to the gastric mucosa plays an important role in its initial colonization and in the subsequent persistence of infection. The group of Thomas Borén (Umeå University, Sweden) reported the purification of the Lewisb blood group http://tim.trends.com

antigen-binding adhesin, BabA, and its characterization with maintained binding activity. Importantly, expression of the protein was found to be growth-phase dependent and regulated at the transcription level. Furthermore, this group identified another adhesin, SabA, a sialic acid-binding adhesin, which, like BabA, is a member of the Hop family of outer membrane proteins. The SabA adhesin binds to inflamed, but not to healthy mucosa or Lewisb. The receptor, sialyl-Lewisx glycosphingolipid, appears to be induced with increasing inflammation and, therefore, could support infection chronicity. In other adhesion-related studies, Silvia Barone (University of Padova, Siena, Italy), using confocal microscopy, has shown that bacterial adhesion to target cells is necessary for efficient delivery of the active vacuolating toxin, VacA, to the host cell and has explored the mechanisms involved. The cag pathogenicity island [a 40-kb DNA insertion in the chromosomal glutamate racemase gene (glr)], encodes a type IV secretion system and continues to be a focus of attention. H. pylori uses the type IV secretion system to inject the CagA protein and probably other factor(s) into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates host cytoskeletal rearrangements associated with a scattering or hummingbird cell phenotype. Steffen Backert and colleagues (Max Planck Institute for Infection Biology, Berlin, Germany) produced a panel of recombinant CagA constructs that induce variations in bacterium–host

cell cross-talk as well as in colonization, attachment and internalization. Furthermore, they explored the mechanism of CagA phosphorylation and found it is mediated by tyrosine kinases of the Src family. In an elegant overview of the current knowledge of the pathogenesis of H. pylori, Manfred Kist (University of Freiburg, Germany) identified important perspectives for future research and important pathogenic concepts that must be addressed (Box 1). H. pylori genomics and proteomics

Functional genomics studies presented at the meeting included several papers making genomic-level comparisons between H. pylori isolates using microarrays or genomic-based typing methods, whereby the breadth and diversity of the species at the level of gene content is becoming clearer. Noteworthy was the description by Sebastian Suerbaum (University of Würzburg, Germany) of the genome sequence of Helicobacter hepaticus, which will provide a fascinating outgroup comparison by which to define the genes that are unique at the Helicobacter species level. Although DNA microarrays are now available from several sources, few studies involved transcriptomics. Nevertheless, a significant new advance is the use of signature-tagged mutagenesis (STM) to detect and monitor in vivo-related gene expression, a hugely powerful approach for the dissection of H. pylori pathogenesis (Rainer Haas et al., Ludwig-MaximiliansUniversity, Munich, Germany). Proteomics continues to be a powerful tool,

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