A Novel Pathogenicity Island Integrated Adjacent to the thrW tRNA Gene of Avian Pathogenic Escherichia coli Encodes a Vacuolating Autotransporter Toxin

V. R. Parreira; C. L. Gyles

doi:10.1128/IAI.71.9.5087-5096.2003

DOI: 10.1128/IAI.71.9.5087-5096.2003

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FIG. 1.
Genetic map of the VAT-PAI with 22,139 bp of unique DNA downstream of the tRNA gene thrW and upstream of the yagU gene of E. coli K-12 MG1655. Numbers indicate ORFs described in Table 2. ORFs have not been drawn to scale.
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FIG. 2.
Structure of the Vat precursor protein (not drawn to scale). The protein consists of 1,377 amino acids, with a signal sequence (SS) of 5.7 kDa, the Vat cytotoxin of 111.8 kDa, and a C-terminal outer membrane translocator of 30.5 kDa. A, signal sequence cleavage site; B, two cysteine residues; C, atypical serine protease motif; D, region that is deleted in vat mutant strain Ec222VPΔ2; E, outer membrane translocator cleavage site.
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FIG. 3.
Alignment of the sequence of the predicted Vat protein (accesion number AY151282) with those of its closest homologues, Tsh (AJ223631), SepA (Z48219), EspP (X97542), Pet (AF056581), and Sat (AF289092). Residues identical in all six sequences are shaded in dark grey and those residues conserved in at least three sequences are shaded in light grey. The asterisk at residue 56 indicates the first amino acid of the mature Vat protein. The serine protease motif is underlined.
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FIG. 4.
Vacuolation of CEF cells caused by culture supernatant of E. coli Ec222. (A) Vacuoles (arrows) were evident after 6 h of exposure of CEF cells to a 1/16 dilution of the filter-sterilized supernatant of an overnight culture of wild-type E. coli Ec222. The cells were stained with neutral red, which accumulated in the vacuoles. (B) Normal appearance of CEF cells 6 h after treatment as described for panel A except that the E. coli strain was Ec222ΔVP, a vat deletion mutant derivative of Ec222. Bars, 10 μm.

Tables

Figures

TABLE 1.

Oligonucleotide primers used in allelic exchange experiment

Primer	5′-3′ Sequence^a
Vat74up	`GC-GTCGAC-CTTACCTCTCTGGCACT (SalI)`
Vat74down	`CG-TCTAGA-GTCAGTGAACCGGCACC (XbaI)`
Vat73up	`GC-TCTAGA-TCTTCAACGGCACCGTC (XbaI)`
Vat73down	`CG-GAGCTC-GATGTGCATGAATGTGG (SacI)`

↵ a Recognition sites for restriction endonucleases are underlined, and restriction endonucleases are indicated. The reference for all primers is this study.

TABLE 2.

ORFs and mobile genetic elements within the VAT-PAI

ORF or feature	G + C content (%)	No. of aa^a encoded	Position^b (bp)	Gene or protein with similar sequence	% nt^c (protein) identity	Accession no.
			1-641	proA ( E. coli K-12 MG1655)	95	AE000132
			642-717	thrW ( E. coli K-12 MG1655)	100	AE000132
att			699-720	Bacteriophage SflI attachment site (S. flexneri)	100	AF021347
1	39.3	149	1818-2267	Phosphoserine aminotransferase (Spinacea oleracea)	(24)	D84061
2	40.4	60	3736-3918	No significant homology
3	33.8	208	4165-4791	Putative protein (A. thaliana)	(24)	AL049730
4	33.3	89	4801-5070	No significant homology
5	39.1	68	5342-5548	No significant homology
6	36.9	130	5727-6119	No significant homology
7	46.9	117	7362-7715	No significant homology
8	47.1	96	7708-7418	No similarity
9	46.5	164	7732-8226	No significant homology
10	48.2	169	8252-8761	No significant homology
11	47.7	173	8319-7798	No similarity
12	48.8	206	8725-8105	No similarity
13	36.9	65	8926-9123	No significant homology
14	44.5	190	9158-9730	No similarity
15	43.9	84	9654-9400	No similarity
16	44	424	9715-10989	gp22 Mycobacteriophage Bxb1	(30)	AF271693
17	43.6	90	10424-10152	No significant homology
18	41	225	11037-11714	No significant homology
19	46.4	60	11146-10964	No significant homology
20	41.4	61	11164-11349	No significant homology
21	48.3	68	12099-12305	No significant homology
22	44.2	159	12275-11796	Putative integrase (E. coli O157:H7 EDL933)	(33)	AE005658
23	43.2	290	13131-12259	Integrase (X. axonopodis 306)	(27)	AE011739
24	46	112	13389-13727	Integrase (E. coli)	(49)	AJ278144
25	41	130	13831-13439	Integrase (E. coli)	(50)	AJ278144
26	30.5	174	14685-14161	PapX regulatory protein (E. coli)	(48)	P42193
27^d	48.4	1377	18944-14811	Tsh and Hbp (E. coli)	80 (75)	AF218073
						AJ223631
28	48.3	71	19534-19749	iso-IS1 insA (S. flexneri 2a)	93 (89)	AF386526.1
29	52.8	64	19831-20025	iso-IS1 insB (S. flexneri 2a)	94 (88)	AF386526.1
30	52.3	64	20012-19818	iso-IS1 insB (S. flexneri 2a)	94 (88)	AF386526.1
31	51.1	74	20206-19982	iso-IS1 insB (S. flexneri 2a)	93 (65)	AF386526.1
att			20421-20571	Bacteriophage SflI attachment site (S. flexneri)	91	AF021347
32	56.7	22	20679-20745	Unknown function (pathogenic poultry E. coli)	86	AF524932.1
33	41.1	63	20709-20900	No similarity
		191	21045-21620	Protein YagU ( E. coli K-12 MG1655)	91	AE000136
		76	22138-21908	Ferredoxin ( E. coli K-12 MG1655)		AE000136

↵ a aa, amino acids.
↵ b Data for E. coli K-12 are in bold.
↵ c nt, nucleotide sequence; percent identity with the translated amino acid sequence is shown in parentheses.
↵ d ORF 27 encodes the Vat protein.

TABLE 3.

Mortality, lesion scores, and recovery of challenge E. coli from infected chickens

Challenge E. coli	Mortality (%)	Mean lesion score ± SEM	Recovery of E. coli
Challenge E. coli	Mortality (%)	Mean lesion score ± SEM	Direct^a	Total^b
Aerosol challenge
Wild-type Ec222	15	3.65 ± 0.65^c	43/60	56/60
Ec222 vat deletion mutant	0	0.11 ± 0.07	9/60	32/60
Cellulitis challenge
Wild-type Ec222	33	5.0 ± 0.25^c	12/12	ND^d
Ec222 vat deletion mutant	0	0.01 ± 0	0/12	ND

↵ a Number recovered by direct plating of swabs on MacConkey agar/total.
↵ b Number recovered by culture of swabs in Trypticase soy broth followed by subculture on MacConkey agar/total.
↵ c Significantly higher than that for lesions caused by infection with for Ec222 vat mutant. P = <0.0001. Maximum score was 7.0. Normal was scored as 0.01.
↵ d ND, not done.