Bacteriophage–bacteriophage interactions in the evolution of pathogenic bacteria

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Bacteriophage–bacteriophage interactions in the evolution of pathogenic bacteria E. Fidelma Boyd, Brigid M. Davis and Bianca Hochhut Many bacteriophages carry virulence genes encoding proteins that play a major role in bacterial pathogenesis. Recently, investigators have identified bacteriophage–bacteriophage interactions in the bacterial host cell that also contribute significantly to the virulence of bacterial pathogens. The relationships between the bacteriophages pertain to one bacteriophage providing a helper function for another, unrelated bacteriophage in the host cell. Accordingly, these interactions can involve the mobilization of bacteriophage DNA by another bacteriophage, for example in Escherichia coli, Vibrio cholerae and Staphylococcus aureus; the host receptor for one bacteriophage being encoded by another, as found in V. cholerae; and the presence of one bacteriophage potentiating the virulence properties of another bacteriophage, as found in V. cholerae and Salmonella enterica.

E. Fidelma Boyd* Dept of Microbiology, National University of Ireland, University College Cork, Cork, Ireland. *e-mail: [email protected] Brigid M. Davis Bianca Hochhut Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center and Tufts University School of Medicine, 750 Washington Street, Boston, MA 02111, USA.

Bacteriophages are viral cellular parasites that depend on bacterial host processes to produce viral proteins and viral particles. The redirection of bacterial resources towards virion production is generally of no benefit to the bacterium; however, a subset of bacteriophage genomes encode virulence factors as well as essential viral proteins, and production of these virulence factors can enhance bacterial survival. Since it was first demonstrated that Corynebacterium diphtheriae contains a bacteriophage that encodes diphtheria toxin1, numerous bacteriophage-encoded virulence genes have been identified (Table 1), including genes encoding proteins that can enable a bacterium to colonize a new host [e.g. lipopolysaccharide (LPS) O-antigen] or that inactivate host defense mechanisms (e.g. superoxide dismutase), as well as genes encoding a range of toxins (e.g. cholera toxin, erythrogenic toxin and shiga toxin). As these virulence genes could provide benefits to their bacterial host, bacteriophages containing them are likely to be selectively maintained within the bacterial gene pool. It is becoming increasingly evident, however, that the perpetuation, mobilization and functioning of bacteriophage virulence genes can depend not only on the host bacteria but also on additional, often unrelated bacteriophages. In this review, we will discuss several recently described bacteriophage–bacteriophage interactions and their role in the evolution and manifestation of bacterial virulence. The relationships between the bacteriophages pertain to bacteriophage DNA uptake and transfer as well as their contributions to virulence. We have divided the bacteriophage–bacteriophage

interactions into three categories, based on the role of a helper bacteriophage in the relationship (Table 2). In the first category, the helper bacteriophage is involved in the transfer of unrelated bacteriophage DNA. In the second category, for which there is only one example, the helper bacteriophage supplies the host bacterium’s receptor for another bacteriophage. Finally, in the last category, the helper bacteriophage potentiates another bacteriophage’s contribution to virulence. Mobilization of a bacteriophage by a helper bacteriophage Lambdoid P2 and P4 transfer

The interaction between LAMBDOID BACTERIOPHAGES (see Glossary) P2 and P4 in Escherichia coli is the classical example of the mobilization of a defective bacteriophage (P4) by a helper bacteriophage (P2). P2 and P4 are temperate coliphages with icosahedral capsids and contractile tails with side fibers. The P4 genome (11.6 kb) is considerably smaller than the P2 genome (33.8 kb) and they share little sequence similarity, with the exception of similar cos sites essential for phage DNA packaging2. P4 is dependent on P2 or a related bacteriophage to supply the capsid, tail and lysis genes for the assembly of its own capsid, packaging of its DNA and lysis of the bacterial host cell. These requirements are essential for P4 to complete a lytic cycle and produce virions2,3. Therefore, P4 is a defective bacteriophage, which, once released as a virion, can infect, replicate and integrate its DNA within a host bacterium in the absence of P2. By contrast, P2 is not dependent upon P4 to complete a lytic cycle and produce functional viral particles. However, under certain cellular conditions, P4 can activate P2 gene expression by activating the promoters of P2 late genes2. The broad host range of P2 and P4, which includes Shigella spp., Salmonella enterica, Klebsiella pneumoniae, Pseudomonas aeruginosa and Yersinia spp., gives these phages enormous potential as vectors for the horizontal transfer of virulence-linked genes. So it is not surprising to find that several bacteriophages related to P2 and P4 have been implicated in the introduction of virulence genes into a range of enteric species. The temperate phage SopEΦ (Ref. 4), a new member of the P2 family, encodes a type III-dependent effector protein (SopE), which is involved in bacterial entry into cultured epithelial

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Table 1. Bacteriophage-encoded virulence factorsa Bacterial species

Bacteriophage

Gene

Protein or phenotype

Refs

Escherichia coli O157:H7

933, H-19B ΦFC3208 λ λ

stx hly2 lom bor

Shiga toxins Enterohemolysin Serum resistance Host cell envelope protein

40–44 45 46 46

Shigella flexneri

Sf6 sfII, sfV, sfX

oac gtrII

O-antigen acetylase Glucosyl transferase

5,6 47–49

Salmonella enterica

SopEΦ Gifsy-2 Gifsy-2 Gifsy-1 ε34

sopE sodC-1 nanH gipA rfb

Type III effector Superoxide dismutase Neuraminidase Insertion element Glucosylation

4,50 36 – 39 51

Vibrio cholerae

CTXΦ K139 VPIΦ

ctxAB glo tcp

Cholera toxin G-protein like TCP pilin

8 52 23

Pseudomonas aeruginosa

ΦCTX

ctx

Cytotoxin

53

Clostridium botulinum

Phage C1

C1

Neurotoxin

54,55

Staphylococcus aureus

NA Φ13 TSST-1

see, sel entA, sak tst

Enterotoxin Enterotoxin A, staphylokinase Toxic shock syndrome toxin-1

56 57 17,18

Streptococcus pyogenes

T12

speA

Erythrogenic toxin

58,59

tox

Diphtheria toxin

1

Corynebacterium diphtheriae β-phage aAbbreviations:

TCP, toxin co-regulated pilus; TSST-1, toxic shock syndrome type 1.

cells in S. enterica sv. Typhimurium. Similarly, the genes encoding the LPS O-antigen of Shigella flexneri are encoded on bacteriophage Sf6 (Refs 5,6), which shows homology to many lambdoid phages. Moreover, P4-like integrases are encoded within a number of PATHOGENICITY ISLANDS (PAIs) from Gram-negative and Gram-positive bacteria (Table 3), which has led to the hypothesis that P4-related bacteriophages might have contributed to the acquisition of these PAIs in these bacteria (Box 1). To date, P2–P4 interactions have not been shown to contribute directly to bacterial virulence but an intriguing speculation is the possible mobilization of PAI DNA associated with a P4-like integrase by P2 bacteriophage. Support for this hypothesis would be provided by demonstrating the transfer of PAI DNA by P2 bacteriophages. Regardless of the true significance of P4 in the acquisition of PAIs, the long history of studies of P2–P4 interactions provides a reference point with which other bacteriophage–bacteriophage interactions can be compared. Table 2. Bacteriophage–bacteriophage interactions Bacterial host

Helper phage

Associated phage

Ref.

P4 CTXΦ tst element

3 15 17

CTXΦ

23

CTXΦ Gifsy-1

31 36

Mobilization of bacteriophage DNA

Escherichia coli Vibrio cholerae Staphylococcus aureus

P2 CP-T1 80α

Host receptor for bacteriophage

V. cholerae

VPIΦ

Potentiate bacteriophage-encoded virulence genes

V. cholerae Salmonella enterica

VPIΦ Gifsy-2

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CTXΦ and CP-T1 in Vibrio cholerae

Vibrio cholerae is a Gram-negative bacterium that causes cholera, a severe profuse secretory diarrhea with a predilection for pandemic spread. Until recently, of the nearly 150 recognized serogroups of V. cholerae, only the O1 serogroup was associated with epidemic cholera. The V. cholerae O1 serogroup can be divided into classical and El Tor biotypes. In 1992, for the first time in the history of cholera, a newly recognized serogroup – O139 – emerged and temporarily replaced the predominant El Tor O1 serogroup as the main cause of epidemic cholera7. Humans become infected with V. cholerae after ingestion of food and water contaminated with the pathogen. V. cholerae colonizes and multiplies within the small intestine where it produces CHOLERA TOXIN (CT), a potent A-B-type enterotoxin that ADPribosylates proteins eliciting a secretory response. The genes encoding CT, ctxAB, are contained within the genome of the filamentous temperate bacteriophage CTXΦ (Ref. 8). Following infection of classical and El Tor V. cholerae strains lacking a CTXΦ integration site, CTXΦ can replicate as a plasmid designated the phage replicative form (RF). V. cholerae strains responsible for contemporary cholera epidemics, El Tor O1 and O139 strains, contain fully functional prophages that can produce the infectious RF of CTXΦ. These strains can transmit ctxAB to any V. cholerae strains that produce the CTXΦ receptor, the TOXIN CO-REGULATED PILIN (TCP), which is also an essential intestinal colonization factor8. As TCP is required for CTXΦ infection of V. cholerae, it was proposed that there were two crucial sequential steps in the evolution of pathogenic

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Glossary Cholera toxin (CT): A potent two-component A-B type exotoxin that is secreted by pathogenic Vibrio cholerae. CT catalyzes the transfer of an ADP-ribose from NAD to a GTP-binding regulatory component of adenylate cyclase, causing constitutive expression in enterocytes. Φ: A 6.9-kb single-stranded covalently closed DNA filamentous bacteriophage that carries CTXΦ the genes encoding cholera toxin (ctxAB). CTXΦ shows homology to the filamentous phages from Escherichia coli but, unlike M13 and f1, CTXΦ integrates into a specific site in the V. cholerae genome. Lambdoid bacteriophages: Double-stranded DNA bacteriophages that typically infect E. coli or closely related species such as Salmonella enterica. They share a common genetic map and can recombine with one another to produce viable mosaic genomes containing conserved and variable regions. Lambdoid bacteriophages lysogenize by site-specific recombination with the bacterial chromosome mediated by the integrase gene. All have life cycles similar to the coliphage λ. Pathogenicity island (PAI): A large region of chromosomal DNA (35–200 kb) that encodes several virulence factors and that is present in all pathogenic isolates and usually absent from non-pathogenic isolates of a species. Toxin co-regulated pilus (TCP): A bundle-forming pilus that belongs to the type IV class pili expressed by a number of Gram-negative bacteria, which includes the bundle-forming pilus (BFP) from enteropathogenic E. coli. TCP and CT production are co-regulated by the ToxR regulon. Vibrio pathogenicity island (VPI): A 39.5-kb region on the V. cholerae chromosome that encodes the TCP and other virulence genes. The VPI is present in all epidemic V. cholerae isolates but rarely found in non-pathogenic V. cholerae isolates. Φ: A 39.5-kb novel filamentous bacteriophage that encodes the VPI in V. cholerae isolates. VPIΦ

V. cholerae8–11. First, V. cholerae strains acquired the tcp operon (which is not present in all V. cholerae strains; see additional discussion) and second, these TCP+ strains were infected with and lysogenized by CTXΦ. However, this model of the evolution of pathogenic V. cholerae has been called into question by several recently described isolates of V. cholerae (both O1 and non-O1 strains) that lack TCP genes but contain CTXΦ sequences12,13. Some authors have suggested that such isolates arose by TCP-mediated CTXΦ infection with subsequent loss of the TCP region10,11. Alternatively, such strains might possess an as-yet-unidentified receptor and/or mode of CTXΦ acquisition. For example, a recent report by Heilpern and Waldor14 demonstrated that CTXΦ uses both

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TCP and the gene products of tolQRA to enter the host cell. They detected CTXΦ infection of TCP− V. cholerae, albeit at greatly reduced frequency, and found that TolQRA are absolutely required for phage entry into TCP− cells14. In addition, it has recently been shown that the V. cholerae generalized transducing bacteriophage CP-T1 can transfer the entire CTXΦ genome to V. cholerae strains, including TCP− strains15. Furthermore, it was found that CP-T1 transduces the CTX prophage preferentially compared with other chromosomal markers15. Several possibilities can be envisioned that might explain this preferential transfer. For example, analysis of the DNA sequence of CTXΦ and its flanking regions for CP-T1 pac sites identified many candidate sites. Alternatively, and not mutually exclusive of the presence of potential pac sites, the preferential transduction of CTXΦ DNA might in part be accounted for by the higher copy number of CTXΦ sequences in SM115, the donor strain used in the transduction assays. Both the presence of the tandemly duplicated CTX prophage and the CTXΦ RF in SM115 increase the copy number of CTXΦ DNA in this strain. Finally, it is also possible that there is preferential packaging of the CTXΦ RF by CP-T1, compared with chromosomal sequences. Regardless of the reasons for preferential transduction of CTXΦ, both classical and El Tor strains of V. cholerae could serve as CTXΦ donors, even though classical biotype strains do not produce infectious CTXΦ (Ref. 16), and both TCP+ and TCP− strains could serve as recipients of CTXΦ genes15. These data confirm that expression of a specialized CTXΦ receptor is not always essential for conversion of non-toxigenic strains to toxigenicity. However, the fact that most toxigenic isolates of V. cholerae encode

Table 3. Bacteriophage-related integrases associated with pathogenicity (PAI) and genetic islandsa Species

PAI

Gene

Phage-related integrase

tRNA

Refs

Escherichia coli

PAI III (LEE) PAI I PAI II PAI IV PAI V HPI-2

eaeB, esp hly hly, prf hly, pap hly, prs fyuA-irp

SF6, P4, ΦR73 ΦR73 P4 P4 P4 P4

selC selC leuX pheV pheR asnT

63 64 64 65 65 66

Shigella flexneri

SHI-2 NA

Aerobactin O-antigen

ΦR73 P22

selC NA

60,61 62

Yersinia enterocolitica

HPI-1

fyuA-irp

P4

asnT

70

Yersinia pseudotuberculosis

HPI-2

fyuA-irp

P4

asnT

71

Yersinia pestis

HPI-2

pgm, hms, fyuA-irp CP4

asp

72

Haemophilus influenzae

HiGI1

NA

CP4-57

leu

67

Neisseria meningitidis

Region 3

Phage genes

Mu

NA

68

Mesorhizobium loti

Symbiosis

Symbiotic genes

P4

phe

69

Staphylococcus aureus

SaPI1 SaPIbov

tst tst, sec, sel

L54a T12, T270

– –

17 18

Dichelobacter nodosus

PI

vap

ΦR73, P4

serV

73

aAbbreviations:

HPI, high pathogenicity island; LEE, locus of enterocyte effacement; NA, not applicable; PAI, pathogenicity island; SaPI, Staphylococcus aureus pathogenicity island.

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Box 1. The relationship between pathogenicity islands and bacteriophages. Pathogenicity islands (PAIs) are large chromosomal regions (35–200 kb) encoding several virulence gene clusters that are present in all pathogenic isolates and usually absent from non-pathogenic isolates. The percentage G+C content of PAIs differs from that of the host genome, they encode an integrase, insert into the chromosome adjacent to tRNA genes and are flanked by direct repeatsa. Interestingly, PAIs share several of their defining characteristics with some bacteriophages; for example, bacteriophages are known to encode a variety of virulence factors (Table 1), their phylogenetic distribution is scattered, they encode integrases, integrate at tRNA genes and are flanked by repeat sequences. In addition, bacteriophages, like PAIs, are not ancestral to their host genome but instead have been acquired by horizontal DNA transfer. These similarities between PAIs and bacteriophages have led to the hypothesis that PAIs might have a phage origin and were therefore acquired by new hosts via transduction. Data to support this hypothesis are limited and depend to a large extent on the association of bacteriophage-like integrases with PAIs. However, there are two studies that have demonstrated the mobilization of PAIs between bacterial strains: the transfer of the Vibrio pathogenicity island (VPI) and the Staphylococcus aureus pathogenicity island (SaPI1) between V. cholerae and S. aureus isolates, respectivelyb,c. Karaolis and colleagues have more recently proposed that the VPI is in fact the genome of a filamentous bacteriophage named VPIΦ (Ref. d) and it could be argued that the SaPI1 has characteristics that are more in common with a defective bacteriophage than a PAI. Hence, these studies add more credence to the hypothesis of a phage origin for PAIs. However, the presence of a bacteriophage-like

TCP as well as CTXΦ suggests that most did not arise as a result of generalized transduction, and favors the model of sequential evolution of virulence. α Staphylococcus aureus tst and bacteriophage 80α

A generalized transducing bacteriophage also plays a role in the transfer of bacteriophage-encoded virulence genes in the Gram-positive bacterium Staphylococcus aureus. S. aureus can induce a wide range of diseases, including toxic shock syndrome, which results from production of toxic shock syndrome toxin-1 (TSST-1), a potent superantigen. TSST-1 is encoded by the tst gene, which inserts into two different sites on the genome of S. aureus isolates and was therefore initially believed to be part of a transposon. However, rather than the same element at different locations, it is suggested that the tst gene is encoded on variant genetic elements with different genomic locations. Recently, Lindsay et al.17 have shown that, in S. aureus strain RN4282, tst is found http://tim.trends.com

integrase and integration site is not sufficient evidence to conclude that transduction underlies PAI mobility and acquisition by new hosts, as other mobile elements, such as conjugative transposons, are also known to integrate at tRNA genes and to utilize similar integrasesa,e,f. Moreover, the presence of an integrase and insertion site might only reflect the mechanism by which PAI DNA was integrated, and not the process by which PAI DNA was acquired. For example, complete sequence analysis of the 35.5-kb locus of enterocyte effacement (LEE) from two E. coli strains EDL933 and E2348/69 shows that LEE is inserted into selC and is highly conserved between these two strains, with the exception of the presence of a 7.5-kb cryptic P4-like prophage 933L in strain EDL933 (Ref. f) but mobilization of LEE by this prophage appears unlikely as both putative att sites occur on one side of LEE (Ref. g). References a Hacker, J. et al. (1997) Pathogenicity islands of virulent bacteria: structure, function, and impact on microbial evolution. Mol. Microbiol. 23, 1089–1097 b Karaolis, D.K. et al. (1998) A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains. Proc. Natl. Acad. Sci. U. S. A. 95, 3134–3139 c Lindsay, J.A. et al. (1998) The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Mol. Microbiol. 29, 527–543 d Karaolis, D.K. et al. (1999) A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria. Nature 399, 375–379 e Campbell, A. et al. (1992) Lambdoid phages as elements of bacterial genomes (integrase/phage21/Escherichia coli K-12/icd gene). Genetica 86, 259–267 f Hochhut, B. et al. (1997) CTnscr94, a conjugative transposon found in enterobacteria. J. Bacteriol. 179, 2097–2102 g Perna, N.T. et al. (1998) Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 66, 3810–3817

within a 15-kb genetic element. They propose that this tst element is part of a PAI, which they have termed the S. aureus pathogenicity island (SaPI1) as it possesses several PAI characteristics. For example, SaPI1 is present in pathogenic isolates and absent from non-pathogenic isolates of S. aureus, encodes the virulence factors TSST-1 and a second possible superantigen toxin as well as a putative integrase, and is flanked by 17-bp direct repeats17. Interestingly, these characteristics are also common features in many bacteriophages. Furthermore, SaPI1 has additional bacteriophage characteristics such as the presence of an integrase that is homologous to the staphylococcal bacteriophage ΦPVL integrase, including a conserved regulatory sequence, a sitespecific attachment site and a bacteriophage-like insertion–excision mechanism. Moreover, unlike PAIs from some Gram-negative bacteria, which integrate at tRNA genes and have a G+C content that differs from that of the host genome, the SaPI1 inserts

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into the tyrB gene and has a G+C content (31%) that is similar to the rest of the S. aureus chromosome. Consequently, we suggest that the SaPI1 can also be considered a defective bacteriophage. In S. aureus strain RN4282, the SaPI1 is not capable of self-mobilization; however, Lindsay et al.17 have shown that it is mobilized by propagation of the staphylococcal generalized transducing bacteriophages 13 and 80α. They demonstrate that bacteriophages 13 and 80α encapsidate and efficiently transduce the SaPI1 to mutant recA recipient strains, in which it integrates at the 17-bp site-specific att site. In strain RN4282, the SaPI1 is induced to excise and replicate specifically by 80α. The precise mechanism underlying SaPI1 transfer has not been characterized; however, it is proposed that SaPI1 recombines reversibly with the 80α genome, resulting in the high transduction frequency and bacteriophage-induced replication of the element17. The SaPI1 can integrate into the S. aureus genome but cannot excise without the presence of a functional phage; it could be argued that this is advantageous to the bacterial cell as, once SaPI1 integrates, the tst gene can be maintained on the chromosome, thus ensuring vertical transmission to progeny. To understand better the origin and evolution of the SaPI1, analysis of the distribution, integration site and structure of this region in a range of S. aureus isolates is required. Interestingly, Lindsay and colleagues suggest that the highfrequency, 80α-dependent mechanism outlined for the SaPI1 transfer in strain RN4282 is likely to be responsible for the horizontal spread of the tst gene among clinical isolates of S. aureus17. A recent study of bovine S. aureus isolates by Fitzgerald et al.18 has identified the tst gene encoded within a 15.9-kb genetic element, which they named a putative S. aureus pathogenicity island (SaPIbov). The SaPIbov region is found in most bovine S. aureus isolates, encodes multiple superantigens, contains a putative integrase gene, is bordered by 74-bp repeats and integrates adjacent to the 3′ end of the GMP synthase gene in the S. aureus chromosome18. The SaPIbov integrase-like gene has 40% identity with integrases from Streptococcus pyogenes bacteriophages T12 and T270, which carry the gene encoding streptococcus pyrogenic exotoxin. SaPIbov contains some sequence similarity to the SaPI1 described by Lindsay et al.17, particularly in the central region [open-reading frame (ORF)4–ORF11], which is identical between the two SaPIs. Fitzgerald et al.18 examined a group of closely related S. aureus strains for the presence of SaPIbov and found that some strains did not contain this region, suggesting that the element is capable of deletion and/or horizontal transfer. However, they were unable to induce transfer of SaPIbov using phages 80α, 85 and 11, suggesting that transfer of the region could be phage specific or involve a different mechanism of transfer from that associated with SaPI1. The http://tim.trends.com

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conservation of the central region of the SaPIs, which encodes the tst gene, and the divergence of sequences flanking this region not only indicates a common origin for the tst gene but more importantly also suggests that these genes are highly mobile and can recombine with diverse genetic elements. A host receptor for one bacteriophage is encoded by another TCP, the V. cholerae host receptor for CTXΦ

As mentioned above, TCP, a type IV bundle-forming pilus that functions as an essential intestinal colonization factor, is also the receptor on the V. cholerae cell for CTXΦ (Refs 8,19). Initially, the region surrounding the TCP gene cluster was shown to conform to the definition of a PAI and was termed the TCP-ACF element20,21 or VIBRIO PATHOGENICITY ISLAND (VPI)22. Hence, the 39.5-kb VPI region is present in epidemic and pandemic strains but absent from non-pathogenic strains; encodes several virulence determinants including TCP, ToxT and accessory colonization factor; has a low G+C content (35%) compared with the V. cholerae genome (47%); contains a putative integrase; is flanked by att sites; and inserts into ssrA, a tRNA-like gene21,22. Remarkably, a recent study by Karaolis and colleagues23 proposes that this island is apparently the genome of a novel lysogenic filamentous phage, VPIΦ, and the major pilin subunit of TCP, TcpA, is apparently also the phage coat protein19. Thus, it appears that the receptor for one bacteriophage (TCP, the CTXΦ receptor) is encoded within the genome of a second bacteriophage, VPIΦ. It is not yet clear whether TCP and VPIΦ are a single entity or whether VPI enables the production of two distinct structures. Accordingly, the precise nature of the CTXΦ receptor – a pilus versus a phage – is still unknown. If the pili do in fact contain DNA and are equivalent to VPIΦ, this raises the possibility that other type IV pili are also bacteriophages. It will be interesting to determine whether the protein product of tcpG, which is not encoded within VPI but is required for TCP biogenesis24, is also essential for VPIΦ biogenesis. Thus far, the finding that VPIΦ encodes the receptor (and perhaps is the receptor) for CTXΦ is the only example of this type of bacteriophage–bacteriophage interaction, and this example could prove to have been a unique development (for a more comprehensive review of these findings see Ref. 25). Interestingly, the VPIΦ genome is atypical compared with previously described filamentous bacteriophages from Gram-negative bacteria including E. coli, V. cholerae, Vibrio parahaemolyticus, Pseudomonas aeruginosa and Xanthomonas spp. VPIΦ shows neither conservation of overall gene order nor the small genome size associated with filamentous phages from these bacteria. In fact, VPIΦ does not show homology to any bacteriophage genes in the database, with the

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Questions for future research •

•

•

With the rapid sequencing of bacterial genomes and the identification of novel bacteriophages, will other additional mechanisms of bacteriophage–bacteriophage interactions be uncovered? What is the evolutionary significance or consequence of interactions between bacteriophages for the bacteriophages themselves and their hosts? Do the interactions we have described between bacteriophages represent the coevolution of bacteriophages?

exception of the integrase-like gene (int). Experimental transfer of VPIΦ between strains was demonstrated for one transductant, a serogroup O10 non-toxigenic strain, whereas transfer into the predominantly CTXΦ-positive O1 serogroup could not be shown, suggesting that uptake of VPIΦ by V. cholerae in the natural environment might be a limiting factor in the emergence of novel toxigenic strains. Regardless of these differences, the discovery of VPIΦ has broadened our understanding of the contributions of bacteriophages to the sequential acquisition of virulence genes in the emergence of pathogenic isolates of V. cholerae, and highlighted the diverse range of genetic elements that encode bacterial virulence genes. The interdependence of bacteriophages for expression of bacterial virulence tcpP, tcpH and toxT

Gene expression from both VPI and CTX prophages is regulated by a complex interaction of VPI genes and V. cholerae chromosomal genes controlled by environmental signals and growth conditions. Three proteins, ToxR, TcpP and ToxT, coordinately regulate transcription of the structural genes for CT and TCP, and other virulence genes. ToxR, a transcription activator, works synergistically with another membrane protein, ToxS, to activate expression of the VPIΦ loci tcpP, tcpH and toxT and the CTXΦ loci ctxAB (Refs 26–28). ToxT amplifies its own expression and, along with TcpP and TcpH, directly activates production of the main virulence factors of V. cholerae: CT (encoded within CTXΦ) and TCP (encoded within VPIΦ)20,29,30. Most of the experimental evidence for this V. cholerae virulence regulatory cascade is based on in vitro data. Recently, Lee and colleagues31 completed in vivo studies that show that the requirement for ToxR and TcpP in regulating CT and TCP expression in vivo differs significantly from that in vitro. They also demonstrate an even more intimate interaction between VPIΦ and CTXΦ gene expression. Using recombinase-based in vivo expression technology (RIVET), they monitored ctxA (the catalytic subunit http://tim.trends.com

of CT) and tcpA (the major TCP subunit) transcription activation during infection in the infant mouse model of cholera31. Their data suggest that production of TCP precedes production of CT, and that CT expression is induced only after increased levels of ToxR and ToxT proteins have been synthesized31. This temporal progression might enable V. cholerae to delay release of CT until the bacterium is in close proximity to host cells. In addition to the fact that TCP is the CTX receptor, such finely tuned regulation of ctxAB encoded in CTXΦ by genes encoded within VPIΦ is evidence of the coevolution of these bacteriophages. Although not mutually exclusive with this possibility, these regulatory processes might reflect conservation of regulation mechanisms among bacteriophages. Salmonella enterica Gifsy-1 and Gifsy-2

S. enterica has been implicated in a wide variety of infections ranging from life-threatening typhoid to gastroenteritis and bacteremia. S. enterica is a facultative intracellular pathogen, typically colonizing reptiles, birds and mammals, with some serovars showing remarkable host species specificity; for example, serovars Typhi and Gallinarum exclusively infect humans and birds, respectively. Therefore, it is not surprising, in view of the diversity of animal species infected and the range of diseases caused, that many virulence genes, encoded on both the chromosome and on mobile genetic elements, are required for S. enterica pathogenesis. S. enterica has been shown to harbor many temperate bacteriophages32–34, the role of which in Salmonella survival is unknown. Recently, Figueroa-Bossi and Bossi35,36 described two lambdoid bacteriophages, Gifsy-1 and Gifsy-2, that account for 2% of the S. enterica serovar Typhimurium genome and are important in Salmonella virulence. Gifsy-1 and Gifsy-2 are located at 57 and 23.5 centisomes on the chromosome, respectively, show some similarity to each other, are both fully functional and are capable of lysogenizing susceptible strains36. The role of Gifsy-1 and Gifsy-2 in Salmonella pathogenesis is complex and probably involves several as-yet-unidentified genes. Gifsy-2 is known to encode the virulence gene sodC1, the product of which, a periplasmic copper and zinc co-factored superoxide dismutase (SodCI), protects Gram-negative bacteria from exogenous oxidative damage caused by the macrophage oxidative burst37. Interestingly, the virulent S. enterica serovar Typhimurium strain ATCC14028s contains two divergent Cu, Zn-SodC enzymes, a chromosomally encoded SodCII protein, which displays only 57% amino acid sequence similarity to SodCI encoded on Gifsy-2 (Ref. 38). The chromosomally encoded sodCII gene is present in all Salmonella isolates, but the Gifsy-2 encoded sodCI is only found in certain strains belonging to the most pathogenic serotypes38, suggesting that sodCI is transferred via transduction

Review

Acknowledgements We thank Matthew Waldor for sharing his infectious enthusiasm for the subject . We are grateful to Katie Moyer and all in the Waldor laboratory for their insightful comments and suggestions. This work was supported by an Enterprise Ireland Basic Research grant to E.F.B., a NIH grant #GM20483-01 to B.M.D. and a Deutsche Forschungsgemeinschaft grant to B.H.

TRENDS in Microbiology Vol.9 No.3 March 2001

among isolates. Curing bacteria of the Gifsy-2 prophage results in substantial attenuation in mouse virulence37,38. By contrast, under most circumstances, Figueroa-Bossi and Bossi36 found that the Gifsy-1 prophage did not appear to contribute to Salmonella virulence. However, Gifsy-1 lysogens that contain sodCI but lack the remainder of the Gifsy-2 genes are more virulent than isogenic strains lacking a Gifsy-1 prophage35. Figueroa-Bossi and Bossi36 propose that Gifsy-1 might encode virulence factors whose effects are dependent upon sodCI; it could be that Gifsy-1 contributes to virulence simply by enhancing expression of SodCI. A potential candidate gene encoded on Gifsy-1 that could benefit SodCI expression is the gipA gene, recently identified by Stanley and colleagues39. The gipA locus is encoded on Gifsy-1 and is important for the survival of S. enterica serovar Typhimurium in the Peyer’s patches of BALB/c mice39. The GipA ORF shows homology at the amino acid level to a group of IS891like insertion elements that is found in a diverse group of organisms. The authors hypothesized that GifA could be a regulatory protein that is involved in the control of one or more virulence factors39; whether this includes sodCI remains to be determined. Another potential virulence factor that maps to within Gifsy-2 on the S. enterica serovar Typhimurium genome is neuraminidase (encoded by nanH), a protein that plays a role in virulence in several pathogenic microorganisms. To date, the

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The acquisition of foreign DNA conferring novel phenotypes to a range of diverse bacteria was a major step in the evolution of pathogenic bacteria from their non-pathogenic ancestors. Among the major players in the horizontal transfer of virulence genes are a range of mobile genetic elements, which include plasmids, transposons, conjugative transposons and bacteriophages. The recent studies described in this review indicate that, although bacteriophages are important vectors for the transmission of virulence genes within bacterial populations, they also serve as important precursors for the expression of bacterial virulence. Furthermore, the interactions between bacteriophages can promote the sequential acquisition of virulence genes and lead to the emergence of new pathogenic strains, as seen in V. cholerae, in which the VPIΦ encoding TCP is first acquired before CTXΦ can lysogenize V. cholerae. Relationships between bacteriophages and their role in bacterial virulence seems likely to become an emerging theme in the study of bacterial pathogenesis.

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