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An analysis of type-III secretion gene clusters in Chromobacterium violaceum
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Genome Analysis
An analysis of type-III secretion gene clusters in Chromobacterium violaceum Helen J. Betts, Roy R. Chaudhuri and Mark J. Pallen Bacterial Pathogenesis and Genomics Unit, Division of Immunity and Infection, Institute for Biomedical Research, Medical School, University of Birmingham, Birmingham B15 2TT UK
Chromobacterium violaceum is an environmental Gramnegative bacterium that is common in soil and water in tropical and sub-tropical regions. It is also a model organism for studying quorum-sensing and is a rare but deadly human pathogen. Recent completion of the genome sequence of C. violaceum strain ATCC 12472 revealed the presence of genes associated with type-III secretion systems (TTSSs). One of these systems resembles the Spi-1 system found in Salmonella enterica, whereas another is similar to the Spi-2 system from the same organism. Here, we present a detailed analysis and a fresh annotation of the two gene clusters. Moreover, we highlight the presence of several genes encoding putative type-III effector proteins that lead us to predict that this organism can manipulate vesicular trafficking, the actin cytoskeleton and apoptotic pathways within mammalian cells to its own advantage. Type-III secretion is one of at least five different types of secretion systems that are used by Gram-negative bacteria to export proteins across the cell envelope [1]. During type-III secretion, a multi-protein complex spans the inner and outer membranes and delivers proteins to the exterior in an ATP-dependent manner. An additional key feature of virulence-associated type-III secretion systems (TTSSs) is their ability to deliver effector proteins into the cytoplasm of a target eukaryotic cell through a translocation apparatus that connects the bacterial and eukaryotic cells and facilitates passage of proteins through the target cell plasma membrane. Among animal and human pathogens, research has focused on a small group of intensively studied TTSSs, which include the Ysc-Yop system from Yersinia, the Mxi-Spa system from Shigella, the system encoded by the locus for enterocyte effacement (LEE) in Escherichia coli and Citrobacter rodentium (Box 1), and two systems in Salmonella enterica encoded by the two Salmonella pathogenicity islands Spi-1 and Spi-2. Studies on these systems and the gene clusters encoding them have clarified many of the mechanistic details of type-III secretion and identified numerous secreted effector proteins [1]. Genome sequencing efforts have recently delivered a dramatic increase in the number of known TTSSs [2]. Although many of these newly discovered systems have not been linked to virulence – or been shown to be functional – their Corresponding author: Mark J. Pallen ([email protected]). Available online 22 September 2004 www.sciencedirect.com
discovery prompts the question of how representative the better-characterised systems are of TTSSs in general. Chromobacterium violaceum: a rare but deadly pathogen Chromobacterium violaceum is a motile environmental b-proteobacterium that is common in soil and water in tropical and sub-tropical regions. In response to autoinducers, it produces a purple pigment, known as violacein, which has led to its adoption in the research laboratory as a tool for studying quorum-sensing. C. violaceum is also a cause of sporadic infection in humans and other mammals, typically presenting as a fulminant septicaemia that resembles melioidosis [3]. Despite its rarity, C. violaceum infection is associated with a high mortality rate [4]. However, very little is known about the molecular basis of virulence in this organism. The Brazilian National Genome Project Consortium recently reported the genome sequence of the C. violaceum type strain ATCC 12472. One unexpected finding was the presence of genes capable of encoding two TTSSs [5,6]. One of these systems resembles the Spi-1 system from S. enterica, whereas the other resembles systems encoded by the Spi-2 gene cluster in S. enterica and by the LEE in E. coli. Rather confusingly, in the C. violaceum genome sequence the second system has been annotated with designations taken haphazardly from both the Spi-2 and the LEE systems. Here, we present a more detailed analysis and a fresh annotation of the two gene clusters, which we have termed Chromobacterium pathogenicity islands 1 and 2 (or Cpi-1 and Cpi-2), and highlight some of the insights and questions they evoke. Chromobacterium pathogenicity island 1 Cpi-1 lies directly downstream of Cpi-2 and its homology with Spi-1 spans 26 genes from CV2615 to CV2642, although the island might extend upstream to encompass CV2613 and CV2614 (Figure 1a). The island extends 183 base pairs downstream of CV2642 to end at a tRNA-Leu gene. As type-III secretion gene clusters are commonly inserted into tRNA genes, this might suggest that Cpi-1 was acquired by horizontal gene transfer and inserted into the tRNA-Leu gene. However, the GCC content of Cpi-1 resembles the chromosomal average, suggesting that it has been resident in the C. violaceum genome for a long time and might even have originated in this species or in a close relative. We have renamed the genes in Cpi-1 using the approach that was adopted during the assignment of
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Box 1. The type-III secretion system (TTSS) encoded by the locus for enterocyte effacement (LEE) from Escherichia coli and Citrobacter rodentium similarity to EspB and EspD. However, to date, an equivalent to the EspA pilus has not been visualized on the surface of S. enterica cells; thus, despite compelling support from sequence homology, it remains an open question whether SseB polymerizes into a filamentous structure. An SseB homologue has been described in Edwardsiella tarda, but has not been characterised. EspA and its associated pilus show structural and sequence similarities to flagellins and to the flagellar filament; this probably indicates weak homology. Effector molecules subvert host cellular functions: † Tir acts as receptor for intimin. † Tir, EspH and Map modulate actin cytoskeleton. † EspF triggers apoptosis. † Map targets mitochondria. † Cif blocks cell cycle G2/M transition.
The basal secretion apparatus of highly conserved proteins spanning the inner and outer membranes and periplasmic space is shown in Figure I. Components of the needle and of the translocation apparatus are secreted through a molecular syringe into the host cell cytoplasm. Effectors and translocators require molecular chaperones for stabilization before secretion. The LEE-encoded system is unusual because it possesses a pilus (made from EspA) that connects the needle to the translocation pore – in most TTSSs, the needles and pore are in direct contact. Translocators EspB and EspD are required to form the pore in the host cell plasma membrane and to contribute to EspA pilus formation. Proteins from the Spi-2 system of Salmonella enterica show sequence homology to translocation-associated proteins encoded by the LEE: SseB resembles EspA, whereas SseD and SseC show weak
Translocation of effectors through a molecular syringe into host cell cytoplasm
Eukaryotic host cell plasma membrane
Translocation pore in plasma membrane Made from EspB and EspD EspA filament Made from EspA
Needle OM
Basal secretion apparatus spanning bacterial inner and outer membranes and periplasmic space
IM
Bacterial cytoplasm
Chaperones bind to translocators and effectors in bacterial cytoplasm TRENDS in Microbiology
Figure I. A schematic representation of the type-III secretion system encoded by the LEE (locus for enterocyte effacement) region.
gene names in the recently discovered cryptic Spi-1-like gene cluster in E. coli (known as ETT2 for E. coli type-III secretion system 2) [7]. Thus, where the Spi-1 genes begin with an initial to represent their origin within the genus Salmonella, we have in Cpi-1 substituted the corresponding initial from Chromobacterium (e.g. sipA becomes cipA, spaT becomes cpaT); elsewhere we have abbreviated the www.sciencedirect.com
three-letter gene name to two letters prefaced with a ‘c’ (e.g. invF becomes civF, prgK becomes cprK, orgABC becomes corABC; Figure 1a). Cpi-1 contains homologues of the inv-spa genes encoding the secretion apparatus in an arrangement identical to that in Spi-1. However, comparison of the Cpi-1 system with Spi-1, and with ETT2, raises several points. First,
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(a)
TRENDS in Microbiology
Cpi-1A
Vol.12 No.11 November 2004
Cpi-1 cicA
iacP cor
cpr
cip A
C B A KJ I H
D
iag B cpa
C
B
civ
S RQ PO N M C B
A
E
arm dsb S R D G
G F
cilA
C. violaceum
Spi-1 avrA
hilC
hilD or g
C B A KJ I H
sprB
sip hilA
A
sptP
invH
sicA
iag B sicP iacP
pr g D
spa
C
B
inv
S RQ PO N M C B
A
E
G F
S. typhimurium LT2
ETT2
eip island
iag B eilA
D
eip X
B eicA
yge
or g
epr
G H
B A
K JI H
epa
eiv
S RQ PO J I C B
A
E
G
F
pkg A
E. coli EAEC 042
(b)
Cpi-2 slt
cse cscB cseB cseB csa D E 1 2 C 1 2 3 4 H J csaL GI
csa CV2593
csaK D C
csr B A
csa N OPQ RST U
M V
umuC
C. violaceum
Spi-2 B
ssr A
ssa HJ K L M V GI
ssa B C DE
ssa N OPQ RST U
S. typhimurium LT2
sepD escJ
LEE ler
esc RST U
cesD escC
sepZ esc sepQ V N
sepL cesD2 esp eae escD A D B espF
cesT tir
E. coli O157 Sakai
Type III apparatus protein
Chaperone
Other TTSS protein
Secreted effector/translocator protein
Regulator
Non-TTSS protein TRENDS in Microbiology
Figure 1. Chromobacterium pathogenicity islands. (a) Chromobacterium pathogenicity island 1 (Cpi-1) compared with homologous type-III secretion gene clusters from Salmonella enterica serovar Typhimurium strain LT2 and enteroaggregative Escherichia coli strain 042. (b) Chromobacterium pathogenicity island 2 (Cpi-2) compared with homologous type-III secretion gene clusters from S. enterica serovar Typhimurium strain LT2 and enterohaemorrhagic E. coli O157:H7 Sakai strain. Genes are coloured according to functional category. Abbreviations: LEE, locus for enterocyte effacement; TTSS, type-III secretion system.
Cpi-1 lacks an invH homologue (whereas, by comparison, ETT2 contains only an invH pseudogene fragment [8]). Notably, there are no homologues of InvH in any other known TTSS. Thus, in evolutionary terms, InvH appears to be a optional feature of type-III secretion confined to Spi-1; however, within the Spi-1 TTSS it appears to play a key role in secretion through interactions with InvG and peptidoglycan [9,10]. This prompts the question: how and why was invH recruited to this role in this one system? A second interesting observation is the absence of needle-complex genes within the Cpi-1 cluster. Instead, www.sciencedirect.com
homologues of the needle-complex genes prgHIJK and of the orgABC genes are found in a separate gene cluster (CV2417–2423), located more than 200 kilobases upstream of Cpi-1, which we have named Cpi-1a (Figure 1a). A third noteworthy feature of the Cpi-1 system is that it includes novel members of otherwise sparsely populated type-III secretion protein families. These include CpaN, a novel fourth member of the SpaN-EivJ-Spa32 family of proteins (implicated in the control of needle length in Salmonella and Shigella TTSSs [11,12]) and CorC, the only known homologue of the
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Table 1. Putative Chromobacterium violaceum effectora CV number CV0296 CV2292 CV0974
Homologue SopE SopD SptP
E value 2e-18 2e-09 7e-26
% ID 34% 31% 35%
CV2266
SseJ
6e-49
38%
CV2274
OspG
3e-04
28%
CipA
SipA
4e-39
26%
CipB CipC CV2638
SipB SipC Vpa1373
0.0 3e-73 4e-18
62% 44% 22%
Comments on function SopE is a guanine nucleotide exchange factor for host-cell Rho-GTPases SopD is required for fluid accumulation in host cells SptP contains GTPase-activating domain at the N-terminus and tyrosinephosphatase domain at the C-terminus. CV0974 possesses C-terminal tyrosine phosphatase domain, with N-terminal domain of unknown function SseJ induces large membranous conglomerations in host cells; assists in regulating dynamics of the vacuolar membrane in infected cells OspG is a putative Shigella effector containing an unusual protein kinase domain SipA induces host cytoskeletal reorganization by locally inhibiting ADF/cofilinand gelsolin-directed actin disassembly, and by stimulating pathogeninduced actin polymerization; works in concert with SipC SipB disrupts mitochondria, inducing autophagy and cell death SipC works with SipA in polymerising actin Hypothetical Vibrio parahaemolyticus protein, clusters with type-III secretion genes, putative effector?
a
Putative effectors from C. violaceum identified by homology with proven or suspected type-III secretion effectors from other organisms. E value is the expect value and %ID is the percentage identity reported by BLASTP searches.
recently characterised Salmonella protein OrgC (hypothesized to be a secreted repressor of the SPI1 virulence genes [13]). Similar to Spi-1, the Cpi-1 cluster contains genes for the translocation apparatus (cipABCD) and for a SicA-like molecular chaperone (cicA), which is likely to bind to the translocators. Some of the Cpi-1 translocators are, by analogy with Spi-1, likely to have additional functions as virulence effectors (Table 1). However, Cpi-1 contains no other obvious effector genes; more specifically, it lacks homologues of avrA and sptP, although interestingly, in contrast to ETT2, Cpi-1 does encode a homologue of InvB, which chaperones several effectors in S. enterica [14]. Cpi-1 also encodes a tetratricopeptide-repeat TTSS regulator [2], although the cilA gene in Cpi-1 is at the opposite end of the island to hilA in Spi-1 (Figure 1). CilA represents a novel member of the HilA family of TTSS regulators, joining HilA from Spi-1, YgeH from ETT2 and EilA from the ETT2-associated eip cluster [8,15]. Cpi-1 also encodes an InvF-EivF-like regulator, CivF, and a twocomponent regulator system (CV2634–5), which lacks orthologues in Spi-1. Additional Spi-1-like genes in Cpi-1 include homologues of iagB and iacP. There are six other Cpi-1 genes that lack equivalents in Spi-1: homologues of dsbDG genes involved in disulphide bond formation; CV2638, encoding a compositionally biased 763-amino acid protein that has a single homologue, encoded in a type-III secretion gene cluster in Vibrio parahaemolyticus (i.e. both proteins might be candidate effectors); and one gene, CV2639, that encodes a short glycine-alanine-rich protein (a potential chaperone?) and another, CV2642, encoding a membrane-associated protein belonging to the protein family defined by Pfam domain UPF0005. Chromobacterium pathogenicity island 2 Cpi-2 encompasses 39 genes upstream of Cpi-1, and is defined by a reduction in GCC content that extends from CV2574 to CV2614 (Figure 1b). The overall Cpi-2 gene order and organization most closely resembles that of Spi-2, although the top BLAST hits from individual genes or proteins are often to components of a chromosomally encoded TTSS from Yersinia pestis. However, the Y. pestis system is uncharacterised, lacks translocation apparatus www.sciencedirect.com
genes, and might not even be functional. For this reason, we have restricted our analysis to comparisons with the well-characterised Spi-2 and LEE systems, and have adapted the Spi-2 nomenclature to suit Cpi-2 along similar lines as we have with Spi-1 and Cpi-1, so that ssaN becomes csaN and so on. Sitting at one end of the island, the arrangement of the csaMVNOQRSTU genes encoding the Cpi-2 secretion apparatus mirrors the organization of their equivalents in Spi-2, although CV2605, which is situated between CsaO and CsaQ, does not appear to be homologous to ssaP. The organization of other genes for the secretion apparatus, including the needle complex genes, is more divergent (Figure 1b). For example, the run of genes ssaGHIJSTM1410-ssaKL in Spi-2 is represented in Cpi-2 by csaGHIJ-CV2589-csaL (where the homology between SsaH and CsaH is only supported by PSI-BLAST and CV2589 is homologous to STM1410), with csaK separated from the rest of these genes by a three-gene insertion. The ssaBCDE genes from Spi-2 are represented only by csaCD. Cpi-2 resembles Spi-2 in possessing homologues of the regulatory genes ssrAB. In addition, in contrast to Spi-2, it possesses a transcriptional regulator CV2584, homologous to VirF from the Yersinia Ysc-Yop TTSS. The Cpi-2 translocation apparatus: a feast of surprises! The Cpi-2 genes for the translocation apparatus lie at the opposite end of the island to the main secretion-apparatus genes (Figure 1; see Box 1 for a brief introduction to the LEE-encoded translocation apparatus). When these genes are compared to their equivalents in Spi-2 and in the LEE, several surprises leap out. The Cpi-2 translocation cluster has the biggest surprise of all: an initial run of two pairs of sseB-espA homologues (cseB1B2; cseB3B4), separated by a single short orphan gene, CV2581. This is the first time multiple EspA homologues have been found in the same TTSS, and begs the question of how they might be deployed. Are they all expressed at the same time or do they show phase-variation? Do they all assemble into one single filament, or form two different filaments in pairs, or are there four distinct filaments? Scrutiny of a multiple alignment of EspA and SseB homologues (Figure 2) reveals another surprise: two of the CseB proteins contain
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CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
1 1 1 1 1 1 1 1 1
----MSSINAQGGVQPASITTDEVIDNDHIEYLRAARNYSLLGQAIATMEEVMLLFTELS MSTAISSISSFSNGQVDSVG--KAGDGDNIDYLRAARNYSLLGQAIATMEEVMLLFTELS ----MNTTVNQTTPGVNALTTGAADSSDNNDYLRAARNYSLLGQAITTMEEVMLLFTELS ----MNTAVNQPPSGVNVPTTGTADSSDNNDYLRAARNYSLLGQAITTMEEVMLLFTELS -----MTVNTDYHGGVNHVG---ANGYDNNLDRMAGQGDSIMSDGISVLYQFMTLFTDLA MDTSTTASVASANASTSTSMAYDLGSMSKDDVIDLFNKLGVFQAAILMFAYMYQAQSDLS MDTSTMTSVAGASASTSTSMTYDLGSMSKEKVIELFAKVGVFQAALLMFEYMFHAQSELS MDTSNATSVVNVSASSSTSTIYDLGNMSKDEVVKLFEELGVFQAAILMFSYMYQAQSNLS --MSSGNILWGSQNPIVFKNSFGVSNADTGSQDDLSQQ-NPFAEGYGVLLILLMVIQAIA
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
57 59 57 57 53 61 61 61 58
NAKFAQMSKKMEVSRDAQEMANKVEAVLAGITDPKDT---KKLPQDVLDYLKANGISVDS NAKFAQMSKKMEVSRDAQEMANKVEAVLAGITDPKDT---KKLPQDVLDYLKANGISVDS NAKFAQMSKKMEVSRDAAEMANKVEALLASITDPNGK---ASLPEDVIEYMRKNGVAING NAKFAQMSKKMEVSRDAQEKANVMEAVLASLTDPNSK---GQLPPDVIEYIRENGILVGN QGKYDQMKAKADRARNSQQVANQIDAIIAKFKKAGDK---GDLPPEVLKYLRDHNINITV IAKFADMNEASKESTTAQKMANLVDAKIADVQSSSDKNAKAQLPDEVISYINDPRNDITI IAKFADMNEASKASITAQKMANLVDAKIADVQSSSDKNAKAKLPQEVIDYISDSRNSITV IAKFADMNEASKASTTAQKMANLVDAKIADVQSSTDKNAKAKLPQDVIDYINDPRNDISV NNKFIEVQKNAERARNTQEKSNEMDEVIAKAAKGDAKT-KEEVPEDVIKYMRDNGILIDG
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
114 116 114 114 110 121 121 121 117
----------------------------------------------------------------------------------------------------------------------KSIDEFLRDDADLDSRWALNNIHSYTIGHTYFCLDSAQAIAKNLDDAGVKIDGQKASDWL QTIDDFIRENGQFVGCVSEGRFEKMDEYIKRLANLIESCDRTGSDLHPFERMKAFSDFMD Q----------------------------------------------------------S----------------------------------------------------------S----------------------------------------------------------T----------------------------------------------------------MT----------------------------------------------------------
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
114 116 174 174 111 122 122 122 119
-------------------------VENLEGDLS----------------QADLTAVKSA -------------------------VENLEGDLS----------------QADLTAVKSA -----------------------KNQENVDGSYSKEAMAKLFSNGRQLLGRADLTAVKSA ALGVKVDGKSVSDLINETLYIDENGYERVPVAVLEKMQNALEEAKMPVFNISSLTAVKSS -----------------------DDGKNATSDIDQYLKSIGHPDGKG-LDKGELDVIKGA ------------------------GIDNINAQLG----------------AGDLQTVKAA ------------------------GISDLAAELS----------------AGDLQTVKAS ------------------------GIRDLSGDLS----------------AGDLQTVKAA -------------------------IDDYMAKYG----------DHGKLDKGGLQAIKAA
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
133 135 211 234 147 142 142 142 144
LESFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLESFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLETFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLESFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLETDSGRSSDFVTQAQLQIQKTMQSYNVCVSLINSMQTLLAEMNKSIAQNIRISAKANNLTTTVNNSQLEIQQMSNTLNLLTSARSDMQSLQYRTISGISLGK-ISAKANNLTTTVDNSRLDIQQMTNTLNLLTSARSDIQSLQYRTVSAIPIGK-ISAKANNLTTVVNNSQLEIQQMSNTLNLLTSARSDVQSLQYRTISAISLGK-LDNDANRNTDLMSQGQITIQKMSQELNAVLTQLTGLISKWGEISSMIAQKTYS TRENDS in Microbiology
Figure 2. Multiple alignment of EspA homologues. The following are from Chromobacterium violaceum: CseB1 (CV2583; GenBank accession number AAQ60253), CesB2 (CV2582; GenBank accession number AAQ60252), CesB3 (CV2580; GenBank accession number AAQ60250) and CesB4 (CV2579; GenBank accession number AAQ60249). SseB_Edwta is an uncharacterised secreted protein from the fish pathogen Edwardsiella tarda (GenBank accession number AAN52733). EspA_EPEC is from enteropathogenic Escherichia coli strain E2348/69 (GenBank accession number AAC38394), EspA_EHEC is from enterohaemorrhagic E. coli O157:H7 Sakai strain (GenBank accession number AAB71083.1), EspA_Citro is from the mouse pathogen Citrobacter rodentium (GenBank accession number AAL06381). SseB_Salty is from Salmonella enterica serovar Typhimurium strain LT2 (GenBank accession number CAA12185). Alignment performed with ClustalW (http://www.ebi.ac.uk/clustalw/) and shaded using the Boxshade server (http://www.ch.embnet.org/software/BOX_form.html) to highlight identical or similar residues present at O50% of positions.
internal insertions between conserved N- and C-terminal regions. This is highly reminiscent of the variability observed in flagellins, where the central portion of the protein sequence, which in the assembled flagellum folds up into the surface-exposed D3 domains, is highly tolerant of sequence diversity and of natural, or even artificial, insertions [16,17]. This adds weight to the notion that the EspA filament and flagellum are homologous and are under similar selective pressure to show surface variability [17–19]. Curiously, the insertions in CseB2 and CseB4 are quite distinct from each other and show no significant similarity to any other sequences in the database, prompting the question of how did they arise? In the Cpi-2 translocation gene cluster, the cseB homologues are followed by cseC, two sseD-like genes www.sciencedirect.com
(presumably the result of gene duplication) and an sseE homologue. As with the CesB proteins, it is unclear why Cpi-2 needs two CseD proteins; is each associated with a separate translocation filament? SseE is of unknown function and was thought to be unique to Spi-2; the existence of CseE removes this orphan status and might assist in identifying crucial conserved residues. A salient difference between the LEE-encoded TTSS, Spi-2 and Cpi-2 is in their repertoires of TTSS chaperones. All three systems possess a tetratricopeptide-repeat chaperone, SscB, CscB or CesD, which presumably chaperone translocation proteins; more specifically in the LEE system, CesD chaperones EspD [15,20]. However, obvious homologues of other translocation-related chaperones from the LEE (CesD2, CesAB) or Spi-2 systems
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Box 2. Questions for future research Function † What are the secretion targets of Cpi-1 and Cpi-2? † What role do they play in virulence? † Is the shared ability to cause fulminating sepsis of Chromobacterium violaceum and Burkholderia pseudomallei dependent on a shared propensity for type-III secretion? † What role do they play in the usual niche of C. violaceum?
Distribution and evolution † How many strains of C. violaceum possess these systems and how often are they intact? Initial short PCR studies [5] suggest strain-tostrain variation, but full tiling-path PCR studies [8] have not been performed. † How did C. violaceum acquire these systems? † What is the significance of the lack of GCC deviation in Cpi-1? † Why are the two clusters next to one another?
† Why do type-III secretion systems often come in pairs (Spi-1/Spi-2; Cpi-1/Cpi-2; LEE/ETT2)? † How did the needle-complex genes become separated from Cpi-1?
Regulation † How are the Cpi-1 and Cpi-2 systems regulated? † Are they dependent on quorum-sensing? † Is there crosstalk between them? † Under what conditions are these systems competent for secretion? † What are the targets of the CivB effector-chaperone?
Translocation apparatus † Are the CseB proteins assembled into filaments, and if so, how? † How many CseB filaments are there? Are they phase-variable? † How many other examples of similar filaments exist? † Why two CseD proteins?
(SseA) are missing in the Cpi-2 system [21–23]. The coiledcoil protein encoded by CV2581 within the cseB cluster might play a similar role, but even if this is the case, this leaves unanswered the question of why chaperone repertoires vary so much among closely related systems.
Acknowledgements
C. violaceum type-III effectors Although neither Cpi-1 nor Cpi-2 encode any obvious effectors, aside from the translocator CipABC, homology searches using effectors from related systems have enabled the identification of several candidate C. violaceum type-III effectors encoded elsewhere in the chromosome (Table 1). Armed with this effector repertoire, one would anticipate that this strain of C. violaceum would, once in contact with mammalian cells, be able to manipulate vesicular trafficking, the actin cytoskeleton and apoptotic pathways to its own advantage.
References
Concluding remarks These two TTSSs raise many questions for future research (Box 2). An immediate issue for all those working on type-III secretion in other organisms is whether type-III secretion genes from C. violaceum can complement mutations in better characterised systems, and consequently help to unravel the links between protein sequence, structure and function (e.g. could cpaN complement mutations in spaN or spa32?). However, given the genetic tractability of this organism and its role as a deadly pathogen, more direct attempts to link the Cpi-1 and Cpi-2 systems and the associated effectors to virulence will also be attractive goals for future studies. Furthermore, the fact that an obscure inhabitant of the Amazon basin might shed light on the pathogenesis of important human infections is a testament to the surprising power of sequence homology in the post-genomic era. Supplementary material A GenBank-style sequence file, re-annotated with the new Cpi-1 and Cpi-2 gene names, is now available (http:// chromo.bham.ac.uk/supplement). Readers are invited to explore the Cpi islands using Chromobase (http://chromo. bham.ac.uk). www.sciencedirect.com
We would like to thank the BBSRC for funding Helen Betts through a studentship grant and Roy Chaudhuri through the University of Birmingham Exploiting Genomics project on E. coli (grant number EGA16107).
1 Buttner, D. and Bonas, U. (2003) Common infection strategies of plant and animal pathogenic bacteria. Curr. Opin. Plant Biol. 6, 312–319 2 Pallen, M.J. et al. (2003) Genomic analysis of secretion systems. Curr. Opin. Microbiol. 6, 519–527 3 Chattopadhyay, A. et al. (2002) Chromobacterium violaceum infection: A rare but frequently fatal disease. J. Pediatr. Surg. 37, 108–110 4 Chong, C.Y. and Lam, M.S. (1997) Case report and review of Chromobacterium sepsis – a gram-negative sepsis mimicking melioidosis. Singapore Med. J. 38, 263–265 5 Brazilian National Genome Project Consortium. (2003) The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci. U. S. A. 100, 11660–11665 6 Brito, C.F. et al. (2004) Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity. Genet. Mol. Res. 3, 148–161 7 Hayashi, T. et al. (2001) Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res. 8, 11–22 8 Ren, C.P. et al. (2004) The ETT2 Gene Cluster, Encoding a Second Type III Secretion System from Escherichia coli, Is Present in the Majority of Strains but Has Undergone Widespread Mutational Attrition. J. Bacteriol. 186, 3547–3560 9 Crago, A.M. and Koronakis, V. (1998) Salmonella InvG forms a ringlike multimer that requires the InvH lipoprotein for outer membrane localization. Mol. Microbiol. 30, 47–56 10 Pucciarelli, M.G. and Garcia-del Portillo, F. (2003) Protein-peptidoglycan interactions modulate the assembly of the needle complex in the Salmonella invasion-associated type III secretion system. Mol. Microbiol. 48, 573–585 11 Tamano, K. et al. (2002) Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length. J. Bacteriol. 184, 1244–1252 12 Sukhan, A. et al. (2003) Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ. J. Bacteriol. 185, 3480–3483 13 Day, J.B. and Lee, C.A. (2003) Secretion of the orgC gene product by Salmonella enterica serovar Typhimurium. Infect. Immun. 71, 6680–6685 14 Ehrbar, K. et al. (2004) InvB is required for type III-dependent secretion of SopA in Salmonella enterica serovar Typhimurium. J. Bacteriol. 186, 1215–1219
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15 Pallen, M.J. et al. (2003) Tetratricopeptide-like repeats in type-IIIsecretion chaperones and regulators. FEMS Microbiol. Lett. 223, 53–60 16 Wang, L. et al. (2003) Species-wide variation in the Escherichia coli flagellin (H-antigen) gene. J. Bacteriol. 185, 2936–2943 17 Yonekura, K. et al. (2003) Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 424, 643–650 18 Daniell, S.J. et al. (2003) 3D structure of EspA filaments from enteropathogenic Escherichia coli. Mol. Microbiol. 49, 301–308 19 Neves, B.C. et al. (2003) Polymorphisms within EspA filaments of enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 71, 2262–2265 20 Wainwright, L.A. and Kaper, J.B. (1998) EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli. Mol. Microbiol. 27, 1247–1260
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21 Creasey, E.A. et al. (2003) Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli. Microbiology 149, 2093–2106 22 Neves, B.C. et al. (2003) CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD. Infect. Immun. 71, 2130–2141 23 Ruiz-Albert, J. et al. (2003) SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2encoded type III secretion system. Microbiology 149, 1103–1111
0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.09.010
Microbial Genomics
Insights into the evolution of phytopathogens Claire M. Fraser The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
Although not as well-represented in genome sequencing projects as human pathogens, phytopathogens are extremely important because of their economic impact in the field of agriculture. Our understanding of the diversity and mechanisms of virulence in plant pathogens has increased following two recent reports on the sequencing and analysis of Erwinia carotovora subsp. atroseptica [1], the causative agent of soft rot and blackleg in potatoes, and Leifsonia xyli subsp. xyli [2], which causes ratoon stunting disease in sugarcane worldwide. E. carotovora is a member of the bacterial family Enterobacteriaceae, which contains several important human pathogens that include Salmonella, Yersinia, Shigella and Escherichia spp. as well as several plant pathogens. In temperate climates it causes blackleg in the field and soft rot of tubers after harvest. Other related Erwinia spp. also cause soft rot but they have broader host and climate ranges [3]. Although it has been known that soft rot results from the elaboration of extracellular enzymes that degrade plant cell-walls (PCWDEs) [3], the 5.06 Mbp genome sequence of E. carotovora has revealed other insights into the mechanisms by which this pathogen causes disease. Comparative analysis suggests the presence of 11 horizontally acquired genomic islands (HAIs) in the E. carotovora genome [1]. Not surprisingly, many genes encoding pathogenicity determinants were found within the predicted HAIs, suggesting that horizontal gene transfer has played a role in the evolution of E. carotovora as a plant pathogen. In several cases, these pathogenicity determinants appear to have been acquired from nonenterobacterial species, such as Pseudomonas syringae pv. tomato DC3000. The E. carotovora genome contains almost 400 putative pathogenicity genes [1]. A large repertoire of PCWDEs, which cause extensive tissue damage, was identified by Corresponding author: Claire M. Fraser ([email protected]). Available online 22 September 2004 www.sciencedirect.com
genome analysis. It was previously known that PCWDE production is linked to the production of siderophores that scavenge iron from the host. In addition to genes for achromobactin uptake and transport and enterobactin synthesis, two other iron-uptake systems related to the ferric citrate uptake system in E. coli and the HasA hemebinding protein in Serratia marcesens were identified, providing additional support to the importance of iron acquisition. E. carotovora contains all six protein secretion systems known in Gram-negative bacteria as well as a large number of genes that are probably involved in attachment to host cells. A cluster of genes within one of the HAIs in E. carotovora contains genes that appear to be related to a plasmid conjugation system in E. coli and the type IV secretion system (T4SS) in Agrobacterium tumifaciens [4]. Because similar loci have been identified in other phytopathogens [5,6] and previous studies have suggested that conjugation systems can also deliver virulence proteins, the role of this locus in virulence was assessed with transposon insertion mutants [1]. The reduced virulence of the T4SS mutant suggests that this system is involved in the delivery of virulence factors to the host cell. The soft rot Erwiniae can also live a non-pathogenic lifestyle as epiphytes or endophytes on plants or as saprophytes in the soil [3]. It is not surprising that genome analysis has revealed the presence of all major metabolic pathways and a diverse set of genes that enable the use of a range of nutrients. E. carotovora contains more methyl-accepting chemotaxis proteins than any other sequenced enterobacterial species and a large number of putative regulators, many of which might have been acquired through horizontal gene transfer. An unexpected finding was the presence of genes involved in nitrogen fixation and the catabolism of opines, which might enable survival in soil. The sugarcane pathogen, L. xyli, belongs to a group of fastidious phytopathogens that colonize the lumen of
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Genome Analysis
An analysis of type-III secretion gene clusters in Chromobacterium violaceum Helen J. Betts, Roy R. Chaudhuri and Mark J. Pallen Bacterial Pathogenesis and Genomics Unit, Division of Immunity and Infection, Institute for Biomedical Research, Medical School, University of Birmingham, Birmingham B15 2TT UK
Chromobacterium violaceum is an environmental Gramnegative bacterium that is common in soil and water in tropical and sub-tropical regions. It is also a model organism for studying quorum-sensing and is a rare but deadly human pathogen. Recent completion of the genome sequence of C. violaceum strain ATCC 12472 revealed the presence of genes associated with type-III secretion systems (TTSSs). One of these systems resembles the Spi-1 system found in Salmonella enterica, whereas another is similar to the Spi-2 system from the same organism. Here, we present a detailed analysis and a fresh annotation of the two gene clusters. Moreover, we highlight the presence of several genes encoding putative type-III effector proteins that lead us to predict that this organism can manipulate vesicular trafficking, the actin cytoskeleton and apoptotic pathways within mammalian cells to its own advantage. Type-III secretion is one of at least five different types of secretion systems that are used by Gram-negative bacteria to export proteins across the cell envelope [1]. During type-III secretion, a multi-protein complex spans the inner and outer membranes and delivers proteins to the exterior in an ATP-dependent manner. An additional key feature of virulence-associated type-III secretion systems (TTSSs) is their ability to deliver effector proteins into the cytoplasm of a target eukaryotic cell through a translocation apparatus that connects the bacterial and eukaryotic cells and facilitates passage of proteins through the target cell plasma membrane. Among animal and human pathogens, research has focused on a small group of intensively studied TTSSs, which include the Ysc-Yop system from Yersinia, the Mxi-Spa system from Shigella, the system encoded by the locus for enterocyte effacement (LEE) in Escherichia coli and Citrobacter rodentium (Box 1), and two systems in Salmonella enterica encoded by the two Salmonella pathogenicity islands Spi-1 and Spi-2. Studies on these systems and the gene clusters encoding them have clarified many of the mechanistic details of type-III secretion and identified numerous secreted effector proteins [1]. Genome sequencing efforts have recently delivered a dramatic increase in the number of known TTSSs [2]. Although many of these newly discovered systems have not been linked to virulence – or been shown to be functional – their Corresponding author: Mark J. Pallen ([email protected]). Available online 22 September 2004 www.sciencedirect.com
discovery prompts the question of how representative the better-characterised systems are of TTSSs in general. Chromobacterium violaceum: a rare but deadly pathogen Chromobacterium violaceum is a motile environmental b-proteobacterium that is common in soil and water in tropical and sub-tropical regions. In response to autoinducers, it produces a purple pigment, known as violacein, which has led to its adoption in the research laboratory as a tool for studying quorum-sensing. C. violaceum is also a cause of sporadic infection in humans and other mammals, typically presenting as a fulminant septicaemia that resembles melioidosis [3]. Despite its rarity, C. violaceum infection is associated with a high mortality rate [4]. However, very little is known about the molecular basis of virulence in this organism. The Brazilian National Genome Project Consortium recently reported the genome sequence of the C. violaceum type strain ATCC 12472. One unexpected finding was the presence of genes capable of encoding two TTSSs [5,6]. One of these systems resembles the Spi-1 system from S. enterica, whereas the other resembles systems encoded by the Spi-2 gene cluster in S. enterica and by the LEE in E. coli. Rather confusingly, in the C. violaceum genome sequence the second system has been annotated with designations taken haphazardly from both the Spi-2 and the LEE systems. Here, we present a more detailed analysis and a fresh annotation of the two gene clusters, which we have termed Chromobacterium pathogenicity islands 1 and 2 (or Cpi-1 and Cpi-2), and highlight some of the insights and questions they evoke. Chromobacterium pathogenicity island 1 Cpi-1 lies directly downstream of Cpi-2 and its homology with Spi-1 spans 26 genes from CV2615 to CV2642, although the island might extend upstream to encompass CV2613 and CV2614 (Figure 1a). The island extends 183 base pairs downstream of CV2642 to end at a tRNA-Leu gene. As type-III secretion gene clusters are commonly inserted into tRNA genes, this might suggest that Cpi-1 was acquired by horizontal gene transfer and inserted into the tRNA-Leu gene. However, the GCC content of Cpi-1 resembles the chromosomal average, suggesting that it has been resident in the C. violaceum genome for a long time and might even have originated in this species or in a close relative. We have renamed the genes in Cpi-1 using the approach that was adopted during the assignment of
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Box 1. The type-III secretion system (TTSS) encoded by the locus for enterocyte effacement (LEE) from Escherichia coli and Citrobacter rodentium similarity to EspB and EspD. However, to date, an equivalent to the EspA pilus has not been visualized on the surface of S. enterica cells; thus, despite compelling support from sequence homology, it remains an open question whether SseB polymerizes into a filamentous structure. An SseB homologue has been described in Edwardsiella tarda, but has not been characterised. EspA and its associated pilus show structural and sequence similarities to flagellins and to the flagellar filament; this probably indicates weak homology. Effector molecules subvert host cellular functions: † Tir acts as receptor for intimin. † Tir, EspH and Map modulate actin cytoskeleton. † EspF triggers apoptosis. † Map targets mitochondria. † Cif blocks cell cycle G2/M transition.
The basal secretion apparatus of highly conserved proteins spanning the inner and outer membranes and periplasmic space is shown in Figure I. Components of the needle and of the translocation apparatus are secreted through a molecular syringe into the host cell cytoplasm. Effectors and translocators require molecular chaperones for stabilization before secretion. The LEE-encoded system is unusual because it possesses a pilus (made from EspA) that connects the needle to the translocation pore – in most TTSSs, the needles and pore are in direct contact. Translocators EspB and EspD are required to form the pore in the host cell plasma membrane and to contribute to EspA pilus formation. Proteins from the Spi-2 system of Salmonella enterica show sequence homology to translocation-associated proteins encoded by the LEE: SseB resembles EspA, whereas SseD and SseC show weak
Translocation of effectors through a molecular syringe into host cell cytoplasm
Eukaryotic host cell plasma membrane
Translocation pore in plasma membrane Made from EspB and EspD EspA filament Made from EspA
Needle OM
Basal secretion apparatus spanning bacterial inner and outer membranes and periplasmic space
IM
Bacterial cytoplasm
Chaperones bind to translocators and effectors in bacterial cytoplasm TRENDS in Microbiology
Figure I. A schematic representation of the type-III secretion system encoded by the LEE (locus for enterocyte effacement) region.
gene names in the recently discovered cryptic Spi-1-like gene cluster in E. coli (known as ETT2 for E. coli type-III secretion system 2) [7]. Thus, where the Spi-1 genes begin with an initial to represent their origin within the genus Salmonella, we have in Cpi-1 substituted the corresponding initial from Chromobacterium (e.g. sipA becomes cipA, spaT becomes cpaT); elsewhere we have abbreviated the www.sciencedirect.com
three-letter gene name to two letters prefaced with a ‘c’ (e.g. invF becomes civF, prgK becomes cprK, orgABC becomes corABC; Figure 1a). Cpi-1 contains homologues of the inv-spa genes encoding the secretion apparatus in an arrangement identical to that in Spi-1. However, comparison of the Cpi-1 system with Spi-1, and with ETT2, raises several points. First,
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(a)
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Cpi-1A
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Cpi-1 cicA
iacP cor
cpr
cip A
C B A KJ I H
D
iag B cpa
C
B
civ
S RQ PO N M C B
A
E
arm dsb S R D G
G F
cilA
C. violaceum
Spi-1 avrA
hilC
hilD or g
C B A KJ I H
sprB
sip hilA
A
sptP
invH
sicA
iag B sicP iacP
pr g D
spa
C
B
inv
S RQ PO N M C B
A
E
G F
S. typhimurium LT2
ETT2
eip island
iag B eilA
D
eip X
B eicA
yge
or g
epr
G H
B A
K JI H
epa
eiv
S RQ PO J I C B
A
E
G
F
pkg A
E. coli EAEC 042
(b)
Cpi-2 slt
cse cscB cseB cseB csa D E 1 2 C 1 2 3 4 H J csaL GI
csa CV2593
csaK D C
csr B A
csa N OPQ RST U
M V
umuC
C. violaceum
Spi-2 B
ssr A
ssa HJ K L M V GI
ssa B C DE
ssa N OPQ RST U
S. typhimurium LT2
sepD escJ
LEE ler
esc RST U
cesD escC
sepZ esc sepQ V N
sepL cesD2 esp eae escD A D B espF
cesT tir
E. coli O157 Sakai
Type III apparatus protein
Chaperone
Other TTSS protein
Secreted effector/translocator protein
Regulator
Non-TTSS protein TRENDS in Microbiology
Figure 1. Chromobacterium pathogenicity islands. (a) Chromobacterium pathogenicity island 1 (Cpi-1) compared with homologous type-III secretion gene clusters from Salmonella enterica serovar Typhimurium strain LT2 and enteroaggregative Escherichia coli strain 042. (b) Chromobacterium pathogenicity island 2 (Cpi-2) compared with homologous type-III secretion gene clusters from S. enterica serovar Typhimurium strain LT2 and enterohaemorrhagic E. coli O157:H7 Sakai strain. Genes are coloured according to functional category. Abbreviations: LEE, locus for enterocyte effacement; TTSS, type-III secretion system.
Cpi-1 lacks an invH homologue (whereas, by comparison, ETT2 contains only an invH pseudogene fragment [8]). Notably, there are no homologues of InvH in any other known TTSS. Thus, in evolutionary terms, InvH appears to be a optional feature of type-III secretion confined to Spi-1; however, within the Spi-1 TTSS it appears to play a key role in secretion through interactions with InvG and peptidoglycan [9,10]. This prompts the question: how and why was invH recruited to this role in this one system? A second interesting observation is the absence of needle-complex genes within the Cpi-1 cluster. Instead, www.sciencedirect.com
homologues of the needle-complex genes prgHIJK and of the orgABC genes are found in a separate gene cluster (CV2417–2423), located more than 200 kilobases upstream of Cpi-1, which we have named Cpi-1a (Figure 1a). A third noteworthy feature of the Cpi-1 system is that it includes novel members of otherwise sparsely populated type-III secretion protein families. These include CpaN, a novel fourth member of the SpaN-EivJ-Spa32 family of proteins (implicated in the control of needle length in Salmonella and Shigella TTSSs [11,12]) and CorC, the only known homologue of the
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Table 1. Putative Chromobacterium violaceum effectora CV number CV0296 CV2292 CV0974
Homologue SopE SopD SptP
E value 2e-18 2e-09 7e-26
% ID 34% 31% 35%
CV2266
SseJ
6e-49
38%
CV2274
OspG
3e-04
28%
CipA
SipA
4e-39
26%
CipB CipC CV2638
SipB SipC Vpa1373
0.0 3e-73 4e-18
62% 44% 22%
Comments on function SopE is a guanine nucleotide exchange factor for host-cell Rho-GTPases SopD is required for fluid accumulation in host cells SptP contains GTPase-activating domain at the N-terminus and tyrosinephosphatase domain at the C-terminus. CV0974 possesses C-terminal tyrosine phosphatase domain, with N-terminal domain of unknown function SseJ induces large membranous conglomerations in host cells; assists in regulating dynamics of the vacuolar membrane in infected cells OspG is a putative Shigella effector containing an unusual protein kinase domain SipA induces host cytoskeletal reorganization by locally inhibiting ADF/cofilinand gelsolin-directed actin disassembly, and by stimulating pathogeninduced actin polymerization; works in concert with SipC SipB disrupts mitochondria, inducing autophagy and cell death SipC works with SipA in polymerising actin Hypothetical Vibrio parahaemolyticus protein, clusters with type-III secretion genes, putative effector?
a
Putative effectors from C. violaceum identified by homology with proven or suspected type-III secretion effectors from other organisms. E value is the expect value and %ID is the percentage identity reported by BLASTP searches.
recently characterised Salmonella protein OrgC (hypothesized to be a secreted repressor of the SPI1 virulence genes [13]). Similar to Spi-1, the Cpi-1 cluster contains genes for the translocation apparatus (cipABCD) and for a SicA-like molecular chaperone (cicA), which is likely to bind to the translocators. Some of the Cpi-1 translocators are, by analogy with Spi-1, likely to have additional functions as virulence effectors (Table 1). However, Cpi-1 contains no other obvious effector genes; more specifically, it lacks homologues of avrA and sptP, although interestingly, in contrast to ETT2, Cpi-1 does encode a homologue of InvB, which chaperones several effectors in S. enterica [14]. Cpi-1 also encodes a tetratricopeptide-repeat TTSS regulator [2], although the cilA gene in Cpi-1 is at the opposite end of the island to hilA in Spi-1 (Figure 1). CilA represents a novel member of the HilA family of TTSS regulators, joining HilA from Spi-1, YgeH from ETT2 and EilA from the ETT2-associated eip cluster [8,15]. Cpi-1 also encodes an InvF-EivF-like regulator, CivF, and a twocomponent regulator system (CV2634–5), which lacks orthologues in Spi-1. Additional Spi-1-like genes in Cpi-1 include homologues of iagB and iacP. There are six other Cpi-1 genes that lack equivalents in Spi-1: homologues of dsbDG genes involved in disulphide bond formation; CV2638, encoding a compositionally biased 763-amino acid protein that has a single homologue, encoded in a type-III secretion gene cluster in Vibrio parahaemolyticus (i.e. both proteins might be candidate effectors); and one gene, CV2639, that encodes a short glycine-alanine-rich protein (a potential chaperone?) and another, CV2642, encoding a membrane-associated protein belonging to the protein family defined by Pfam domain UPF0005. Chromobacterium pathogenicity island 2 Cpi-2 encompasses 39 genes upstream of Cpi-1, and is defined by a reduction in GCC content that extends from CV2574 to CV2614 (Figure 1b). The overall Cpi-2 gene order and organization most closely resembles that of Spi-2, although the top BLAST hits from individual genes or proteins are often to components of a chromosomally encoded TTSS from Yersinia pestis. However, the Y. pestis system is uncharacterised, lacks translocation apparatus www.sciencedirect.com
genes, and might not even be functional. For this reason, we have restricted our analysis to comparisons with the well-characterised Spi-2 and LEE systems, and have adapted the Spi-2 nomenclature to suit Cpi-2 along similar lines as we have with Spi-1 and Cpi-1, so that ssaN becomes csaN and so on. Sitting at one end of the island, the arrangement of the csaMVNOQRSTU genes encoding the Cpi-2 secretion apparatus mirrors the organization of their equivalents in Spi-2, although CV2605, which is situated between CsaO and CsaQ, does not appear to be homologous to ssaP. The organization of other genes for the secretion apparatus, including the needle complex genes, is more divergent (Figure 1b). For example, the run of genes ssaGHIJSTM1410-ssaKL in Spi-2 is represented in Cpi-2 by csaGHIJ-CV2589-csaL (where the homology between SsaH and CsaH is only supported by PSI-BLAST and CV2589 is homologous to STM1410), with csaK separated from the rest of these genes by a three-gene insertion. The ssaBCDE genes from Spi-2 are represented only by csaCD. Cpi-2 resembles Spi-2 in possessing homologues of the regulatory genes ssrAB. In addition, in contrast to Spi-2, it possesses a transcriptional regulator CV2584, homologous to VirF from the Yersinia Ysc-Yop TTSS. The Cpi-2 translocation apparatus: a feast of surprises! The Cpi-2 genes for the translocation apparatus lie at the opposite end of the island to the main secretion-apparatus genes (Figure 1; see Box 1 for a brief introduction to the LEE-encoded translocation apparatus). When these genes are compared to their equivalents in Spi-2 and in the LEE, several surprises leap out. The Cpi-2 translocation cluster has the biggest surprise of all: an initial run of two pairs of sseB-espA homologues (cseB1B2; cseB3B4), separated by a single short orphan gene, CV2581. This is the first time multiple EspA homologues have been found in the same TTSS, and begs the question of how they might be deployed. Are they all expressed at the same time or do they show phase-variation? Do they all assemble into one single filament, or form two different filaments in pairs, or are there four distinct filaments? Scrutiny of a multiple alignment of EspA and SseB homologues (Figure 2) reveals another surprise: two of the CseB proteins contain
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CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
1 1 1 1 1 1 1 1 1
----MSSINAQGGVQPASITTDEVIDNDHIEYLRAARNYSLLGQAIATMEEVMLLFTELS MSTAISSISSFSNGQVDSVG--KAGDGDNIDYLRAARNYSLLGQAIATMEEVMLLFTELS ----MNTTVNQTTPGVNALTTGAADSSDNNDYLRAARNYSLLGQAITTMEEVMLLFTELS ----MNTAVNQPPSGVNVPTTGTADSSDNNDYLRAARNYSLLGQAITTMEEVMLLFTELS -----MTVNTDYHGGVNHVG---ANGYDNNLDRMAGQGDSIMSDGISVLYQFMTLFTDLA MDTSTTASVASANASTSTSMAYDLGSMSKDDVIDLFNKLGVFQAAILMFAYMYQAQSDLS MDTSTMTSVAGASASTSTSMTYDLGSMSKEKVIELFAKVGVFQAALLMFEYMFHAQSELS MDTSNATSVVNVSASSSTSTIYDLGNMSKDEVVKLFEELGVFQAAILMFSYMYQAQSNLS --MSSGNILWGSQNPIVFKNSFGVSNADTGSQDDLSQQ-NPFAEGYGVLLILLMVIQAIA
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
57 59 57 57 53 61 61 61 58
NAKFAQMSKKMEVSRDAQEMANKVEAVLAGITDPKDT---KKLPQDVLDYLKANGISVDS NAKFAQMSKKMEVSRDAQEMANKVEAVLAGITDPKDT---KKLPQDVLDYLKANGISVDS NAKFAQMSKKMEVSRDAAEMANKVEALLASITDPNGK---ASLPEDVIEYMRKNGVAING NAKFAQMSKKMEVSRDAQEKANVMEAVLASLTDPNSK---GQLPPDVIEYIRENGILVGN QGKYDQMKAKADRARNSQQVANQIDAIIAKFKKAGDK---GDLPPEVLKYLRDHNINITV IAKFADMNEASKESTTAQKMANLVDAKIADVQSSSDKNAKAQLPDEVISYINDPRNDITI IAKFADMNEASKASITAQKMANLVDAKIADVQSSSDKNAKAKLPQEVIDYISDSRNSITV IAKFADMNEASKASTTAQKMANLVDAKIADVQSSTDKNAKAKLPQDVIDYINDPRNDISV NNKFIEVQKNAERARNTQEKSNEMDEVIAKAAKGDAKT-KEEVPEDVIKYMRDNGILIDG
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
114 116 114 114 110 121 121 121 117
----------------------------------------------------------------------------------------------------------------------KSIDEFLRDDADLDSRWALNNIHSYTIGHTYFCLDSAQAIAKNLDDAGVKIDGQKASDWL QTIDDFIRENGQFVGCVSEGRFEKMDEYIKRLANLIESCDRTGSDLHPFERMKAFSDFMD Q----------------------------------------------------------S----------------------------------------------------------S----------------------------------------------------------T----------------------------------------------------------MT----------------------------------------------------------
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
114 116 174 174 111 122 122 122 119
-------------------------VENLEGDLS----------------QADLTAVKSA -------------------------VENLEGDLS----------------QADLTAVKSA -----------------------KNQENVDGSYSKEAMAKLFSNGRQLLGRADLTAVKSA ALGVKVDGKSVSDLINETLYIDENGYERVPVAVLEKMQNALEEAKMPVFNISSLTAVKSS -----------------------DDGKNATSDIDQYLKSIGHPDGKG-LDKGELDVIKGA ------------------------GIDNINAQLG----------------AGDLQTVKAA ------------------------GISDLAAELS----------------AGDLQTVKAS ------------------------GIRDLSGDLS----------------AGDLQTVKAA -------------------------IDDYMAKYG----------DHGKLDKGGLQAIKAA
CseB1 CseB3 CseB2 CesB4 SseB_Edwta EspA_EPEC EspA_Citro EspA_EHEC SseB_SALTY
133 135 211 234 147 142 142 142 144
LESFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLESFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLETFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLESFSGRASDFVQQSQLKMQQLIQNFNTAVTMANSLQSMNAESTKSIAQAIRLETDSGRSSDFVTQAQLQIQKTMQSYNVCVSLINSMQTLLAEMNKSIAQNIRISAKANNLTTTVNNSQLEIQQMSNTLNLLTSARSDMQSLQYRTISGISLGK-ISAKANNLTTTVDNSRLDIQQMTNTLNLLTSARSDIQSLQYRTVSAIPIGK-ISAKANNLTTVVNNSQLEIQQMSNTLNLLTSARSDVQSLQYRTISAISLGK-LDNDANRNTDLMSQGQITIQKMSQELNAVLTQLTGLISKWGEISSMIAQKTYS TRENDS in Microbiology
Figure 2. Multiple alignment of EspA homologues. The following are from Chromobacterium violaceum: CseB1 (CV2583; GenBank accession number AAQ60253), CesB2 (CV2582; GenBank accession number AAQ60252), CesB3 (CV2580; GenBank accession number AAQ60250) and CesB4 (CV2579; GenBank accession number AAQ60249). SseB_Edwta is an uncharacterised secreted protein from the fish pathogen Edwardsiella tarda (GenBank accession number AAN52733). EspA_EPEC is from enteropathogenic Escherichia coli strain E2348/69 (GenBank accession number AAC38394), EspA_EHEC is from enterohaemorrhagic E. coli O157:H7 Sakai strain (GenBank accession number AAB71083.1), EspA_Citro is from the mouse pathogen Citrobacter rodentium (GenBank accession number AAL06381). SseB_Salty is from Salmonella enterica serovar Typhimurium strain LT2 (GenBank accession number CAA12185). Alignment performed with ClustalW (http://www.ebi.ac.uk/clustalw/) and shaded using the Boxshade server (http://www.ch.embnet.org/software/BOX_form.html) to highlight identical or similar residues present at O50% of positions.
internal insertions between conserved N- and C-terminal regions. This is highly reminiscent of the variability observed in flagellins, where the central portion of the protein sequence, which in the assembled flagellum folds up into the surface-exposed D3 domains, is highly tolerant of sequence diversity and of natural, or even artificial, insertions [16,17]. This adds weight to the notion that the EspA filament and flagellum are homologous and are under similar selective pressure to show surface variability [17–19]. Curiously, the insertions in CseB2 and CseB4 are quite distinct from each other and show no significant similarity to any other sequences in the database, prompting the question of how did they arise? In the Cpi-2 translocation gene cluster, the cseB homologues are followed by cseC, two sseD-like genes www.sciencedirect.com
(presumably the result of gene duplication) and an sseE homologue. As with the CesB proteins, it is unclear why Cpi-2 needs two CseD proteins; is each associated with a separate translocation filament? SseE is of unknown function and was thought to be unique to Spi-2; the existence of CseE removes this orphan status and might assist in identifying crucial conserved residues. A salient difference between the LEE-encoded TTSS, Spi-2 and Cpi-2 is in their repertoires of TTSS chaperones. All three systems possess a tetratricopeptide-repeat chaperone, SscB, CscB or CesD, which presumably chaperone translocation proteins; more specifically in the LEE system, CesD chaperones EspD [15,20]. However, obvious homologues of other translocation-related chaperones from the LEE (CesD2, CesAB) or Spi-2 systems
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Box 2. Questions for future research Function † What are the secretion targets of Cpi-1 and Cpi-2? † What role do they play in virulence? † Is the shared ability to cause fulminating sepsis of Chromobacterium violaceum and Burkholderia pseudomallei dependent on a shared propensity for type-III secretion? † What role do they play in the usual niche of C. violaceum?
Distribution and evolution † How many strains of C. violaceum possess these systems and how often are they intact? Initial short PCR studies [5] suggest strain-tostrain variation, but full tiling-path PCR studies [8] have not been performed. † How did C. violaceum acquire these systems? † What is the significance of the lack of GCC deviation in Cpi-1? † Why are the two clusters next to one another?
† Why do type-III secretion systems often come in pairs (Spi-1/Spi-2; Cpi-1/Cpi-2; LEE/ETT2)? † How did the needle-complex genes become separated from Cpi-1?
Regulation † How are the Cpi-1 and Cpi-2 systems regulated? † Are they dependent on quorum-sensing? † Is there crosstalk between them? † Under what conditions are these systems competent for secretion? † What are the targets of the CivB effector-chaperone?
Translocation apparatus † Are the CseB proteins assembled into filaments, and if so, how? † How many CseB filaments are there? Are they phase-variable? † How many other examples of similar filaments exist? † Why two CseD proteins?
(SseA) are missing in the Cpi-2 system [21–23]. The coiledcoil protein encoded by CV2581 within the cseB cluster might play a similar role, but even if this is the case, this leaves unanswered the question of why chaperone repertoires vary so much among closely related systems.
Acknowledgements
C. violaceum type-III effectors Although neither Cpi-1 nor Cpi-2 encode any obvious effectors, aside from the translocator CipABC, homology searches using effectors from related systems have enabled the identification of several candidate C. violaceum type-III effectors encoded elsewhere in the chromosome (Table 1). Armed with this effector repertoire, one would anticipate that this strain of C. violaceum would, once in contact with mammalian cells, be able to manipulate vesicular trafficking, the actin cytoskeleton and apoptotic pathways to its own advantage.
References
Concluding remarks These two TTSSs raise many questions for future research (Box 2). An immediate issue for all those working on type-III secretion in other organisms is whether type-III secretion genes from C. violaceum can complement mutations in better characterised systems, and consequently help to unravel the links between protein sequence, structure and function (e.g. could cpaN complement mutations in spaN or spa32?). However, given the genetic tractability of this organism and its role as a deadly pathogen, more direct attempts to link the Cpi-1 and Cpi-2 systems and the associated effectors to virulence will also be attractive goals for future studies. Furthermore, the fact that an obscure inhabitant of the Amazon basin might shed light on the pathogenesis of important human infections is a testament to the surprising power of sequence homology in the post-genomic era. Supplementary material A GenBank-style sequence file, re-annotated with the new Cpi-1 and Cpi-2 gene names, is now available (http:// chromo.bham.ac.uk/supplement). Readers are invited to explore the Cpi islands using Chromobase (http://chromo. bham.ac.uk). www.sciencedirect.com
We would like to thank the BBSRC for funding Helen Betts through a studentship grant and Roy Chaudhuri through the University of Birmingham Exploiting Genomics project on E. coli (grant number EGA16107).
1 Buttner, D. and Bonas, U. (2003) Common infection strategies of plant and animal pathogenic bacteria. Curr. Opin. Plant Biol. 6, 312–319 2 Pallen, M.J. et al. (2003) Genomic analysis of secretion systems. Curr. Opin. Microbiol. 6, 519–527 3 Chattopadhyay, A. et al. (2002) Chromobacterium violaceum infection: A rare but frequently fatal disease. J. Pediatr. Surg. 37, 108–110 4 Chong, C.Y. and Lam, M.S. (1997) Case report and review of Chromobacterium sepsis – a gram-negative sepsis mimicking melioidosis. Singapore Med. J. 38, 263–265 5 Brazilian National Genome Project Consortium. (2003) The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci. U. S. A. 100, 11660–11665 6 Brito, C.F. et al. (2004) Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity. Genet. Mol. Res. 3, 148–161 7 Hayashi, T. et al. (2001) Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res. 8, 11–22 8 Ren, C.P. et al. (2004) The ETT2 Gene Cluster, Encoding a Second Type III Secretion System from Escherichia coli, Is Present in the Majority of Strains but Has Undergone Widespread Mutational Attrition. J. Bacteriol. 186, 3547–3560 9 Crago, A.M. and Koronakis, V. (1998) Salmonella InvG forms a ringlike multimer that requires the InvH lipoprotein for outer membrane localization. Mol. Microbiol. 30, 47–56 10 Pucciarelli, M.G. and Garcia-del Portillo, F. (2003) Protein-peptidoglycan interactions modulate the assembly of the needle complex in the Salmonella invasion-associated type III secretion system. Mol. Microbiol. 48, 573–585 11 Tamano, K. et al. (2002) Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length. J. Bacteriol. 184, 1244–1252 12 Sukhan, A. et al. (2003) Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ. J. Bacteriol. 185, 3480–3483 13 Day, J.B. and Lee, C.A. (2003) Secretion of the orgC gene product by Salmonella enterica serovar Typhimurium. Infect. Immun. 71, 6680–6685 14 Ehrbar, K. et al. (2004) InvB is required for type III-dependent secretion of SopA in Salmonella enterica serovar Typhimurium. J. Bacteriol. 186, 1215–1219
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15 Pallen, M.J. et al. (2003) Tetratricopeptide-like repeats in type-IIIsecretion chaperones and regulators. FEMS Microbiol. Lett. 223, 53–60 16 Wang, L. et al. (2003) Species-wide variation in the Escherichia coli flagellin (H-antigen) gene. J. Bacteriol. 185, 2936–2943 17 Yonekura, K. et al. (2003) Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 424, 643–650 18 Daniell, S.J. et al. (2003) 3D structure of EspA filaments from enteropathogenic Escherichia coli. Mol. Microbiol. 49, 301–308 19 Neves, B.C. et al. (2003) Polymorphisms within EspA filaments of enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 71, 2262–2265 20 Wainwright, L.A. and Kaper, J.B. (1998) EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli. Mol. Microbiol. 27, 1247–1260
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21 Creasey, E.A. et al. (2003) Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli. Microbiology 149, 2093–2106 22 Neves, B.C. et al. (2003) CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD. Infect. Immun. 71, 2130–2141 23 Ruiz-Albert, J. et al. (2003) SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2encoded type III secretion system. Microbiology 149, 1103–1111
0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.09.010
Microbial Genomics
Insights into the evolution of phytopathogens Claire M. Fraser The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
Although not as well-represented in genome sequencing projects as human pathogens, phytopathogens are extremely important because of their economic impact in the field of agriculture. Our understanding of the diversity and mechanisms of virulence in plant pathogens has increased following two recent reports on the sequencing and analysis of Erwinia carotovora subsp. atroseptica [1], the causative agent of soft rot and blackleg in potatoes, and Leifsonia xyli subsp. xyli [2], which causes ratoon stunting disease in sugarcane worldwide. E. carotovora is a member of the bacterial family Enterobacteriaceae, which contains several important human pathogens that include Salmonella, Yersinia, Shigella and Escherichia spp. as well as several plant pathogens. In temperate climates it causes blackleg in the field and soft rot of tubers after harvest. Other related Erwinia spp. also cause soft rot but they have broader host and climate ranges [3]. Although it has been known that soft rot results from the elaboration of extracellular enzymes that degrade plant cell-walls (PCWDEs) [3], the 5.06 Mbp genome sequence of E. carotovora has revealed other insights into the mechanisms by which this pathogen causes disease. Comparative analysis suggests the presence of 11 horizontally acquired genomic islands (HAIs) in the E. carotovora genome [1]. Not surprisingly, many genes encoding pathogenicity determinants were found within the predicted HAIs, suggesting that horizontal gene transfer has played a role in the evolution of E. carotovora as a plant pathogen. In several cases, these pathogenicity determinants appear to have been acquired from nonenterobacterial species, such as Pseudomonas syringae pv. tomato DC3000. The E. carotovora genome contains almost 400 putative pathogenicity genes [1]. A large repertoire of PCWDEs, which cause extensive tissue damage, was identified by Corresponding author: Claire M. Fraser ([email protected]). Available online 22 September 2004 www.sciencedirect.com
genome analysis. It was previously known that PCWDE production is linked to the production of siderophores that scavenge iron from the host. In addition to genes for achromobactin uptake and transport and enterobactin synthesis, two other iron-uptake systems related to the ferric citrate uptake system in E. coli and the HasA hemebinding protein in Serratia marcesens were identified, providing additional support to the importance of iron acquisition. E. carotovora contains all six protein secretion systems known in Gram-negative bacteria as well as a large number of genes that are probably involved in attachment to host cells. A cluster of genes within one of the HAIs in E. carotovora contains genes that appear to be related to a plasmid conjugation system in E. coli and the type IV secretion system (T4SS) in Agrobacterium tumifaciens [4]. Because similar loci have been identified in other phytopathogens [5,6] and previous studies have suggested that conjugation systems can also deliver virulence proteins, the role of this locus in virulence was assessed with transposon insertion mutants [1]. The reduced virulence of the T4SS mutant suggests that this system is involved in the delivery of virulence factors to the host cell. The soft rot Erwiniae can also live a non-pathogenic lifestyle as epiphytes or endophytes on plants or as saprophytes in the soil [3]. It is not surprising that genome analysis has revealed the presence of all major metabolic pathways and a diverse set of genes that enable the use of a range of nutrients. E. carotovora contains more methyl-accepting chemotaxis proteins than any other sequenced enterobacterial species and a large number of putative regulators, many of which might have been acquired through horizontal gene transfer. An unexpected finding was the presence of genes involved in nitrogen fixation and the catabolism of opines, which might enable survival in soil. The sugarcane pathogen, L. xyli, belongs to a group of fastidious phytopathogens that colonize the lumen of