Identification and prevalence of in vivo-induced genes in enterohaemorrhagic Escherichia coli

Figures & data

Figure 1. Efficiency of the RIVET system in EHEC. Indicated strains carrying tnpR gene under the control of chuA promoter were grown in LB supplemented with various concentrations of 2,2ʹ-dipyridyl. After 6 h of growth, cultures were enumerated on LB plates and on LB + sucrose plates to calculate the percentage of resolved CFU. Results are presented as mean and standard deviations from at least three independent experiments. Blue and red curves represent resolution obtained from the 3 tnpR variants inserted into EDL-RES and EDL-RES1 strains, respectively.

Figure 2. Resolution of selected ivi genes following in vitro or in vivo growth. EDL-RES strain carrying transcriptional fusion between indicated gene promoters and tnpR gene were either grown in vitro for 6 h in LB or were given to mice by oral gavage and feces were collected 16 h post-infection. Samples were then diluted and plated on LB plates or LB + sucrose plates to calculate the percentage of resolved CFU after in vitro growth (white bars) or following mouse challenge (black bars). Results are presented as mean and standard deviations from at least three independent experiments. Differences between in vitro and in vivo conditions were analyzed by a two-tailed unpaired t-test (*P < 0.05; **P < 0.01).

Table 1. List of in vivo-induced genes identified in EHEC O157:H7 strain EDL933.

	Gene
COG category	label	name	O-island	Role or putative function
Metabolism
[C]	Z0072	araB		L-Ribulokinase
[C]	Z4726	nirB		Large subunit of nitrite reductase
[E]	Z3960	gabT		4-Aminobutyrate aminotransferase
[E]	Z3500	glpB		sn-Glycerol-3-phosphate dehydrogenase
[E]	Z2551	trpA		α-Subunit of tryptophan synthase
[E]	Z0607	ybaT		Putative nitrogen-containing metabolite transporter
[E]	Z5746	yjeH		Member of the APC superfamily of amino acid transporters
[G]	Z4486	agaW	OI#126	Enzyme of the N-acetylgalactosamine phosphotransferase system
[H]	Z2540	btuR		Cobalamin adenolsyltransferase
[H]	Z0061	pdxA		4-Hydroxythreonine-4-phosphate dehydrogenase
[I]	Z2315	azoR		NADH-aZoreductase
[I]	Z5366	fadA		3-Ketoacyl-CoA thiolase
[P]	Z5611	yjbB		Putative transporter
Information storage and processing
[J]	Z4409	cca		Multifunctional CCA protein
[K]	Z0444	mhpR		Transcriptional activator of 3-hydroxyphenylpropionic acid catabolism
[K]	Z0073	araC		Transcriptional regulator of L-arabinose transport and catabolism
[K]	Z4022	ascG		Transcriptional repressor of arbutin and salicin transport
[K], [E]	Z5941	yjiR		Putative transcriptional regulator
[K], [T]	Z4017	norR		Transcriptional activator of nitric oxide reductase NorV
[L], [U]	Z4799	-	OI#134	Putative DNA processing protein
Cellular processes and signaling
[D]	Z5820	ytfE		Iron–sulphur cluster repair protein
[M]	Z4886	yhiI		Putative HlyD family secretion protein
[M]	Z4926	mdtE		Multidrug efflux system protein
[G], [E], [P], [R]	Z5939	mdtM (yjiO)		Multidrug efflux system protein
[O]	Z4519	yhbU		Putative collagenase
[O]	Z4343	yghU		Putative glutathione S-transferase
[O]	Z4593	degQ		Serine endoprotease
Poorly characterized
[R]	Z4070	-	OI#111	Uncharacterized protein
[R]	Z2757	ydjM		Uncharacterized protein; regulated by the SOS system regulator LexA
[R]	Z0964	-	OI#36	Putative DNA packaging protein of prophage CP-933K
No COG
-	Z3135	-	OI#80	Putative adhesin/invasin

COG category: [C] Energy production and conversion; [D] Cell cycle control, cell division, chromosome partitioning; [E] Amino acid transport and metabolism; [G] Carbohydrate transport and metabolism; [H] Coenzyme transport and metabolism; [I] Lipid transport and metabolism; [J] Translation, ribosomal structure and biogenesis; [K] Transcription; [L] Replication, recombination and repair; [M] Cell wall/membrane/envelope biogenesis; [O] Post-translational modification, protein turnover, and chaperones; [P] Inorganic ion transport and metabolism; [R] General function prediction only; [T] Signal transduction mechanisms; [U] Intracellular trafficking, secretion, and vesicular transport.

Table 2. Distribution of eight ivi genes in 51 E. coli strains grouped as commensals, InPEC or ExPEC.

	mhpR	ascG	mdtM	yjiR	Z0964	Z3135	Z4070	Z4799
commensal (n = 6)	0.83	0.83	0.83	0.67	0.33	0	0.33	0.33
InPEC (n = 31)	0.97	0.97	0.97	0.74	0.71	0.52	0.68	0.35
EAEC (n = 3)	1	1	1	0.67	0.67	0	1	0
EHEC (n = 18)	1	1	1	1	0.78	0.72	0.72	0.5
EPEC (n = 4)	0.75	0.75	0.75	0.5	0.75	0.25	0.5	0.5
ETEC (n = 6)	1	1	1	0.17	0.5	0.33	0.5	0
ExPEC (n = 14)	0.5	0.71	0.43	0.43	0.5	0.21	0.14	0.5

Values indicate the proportion of strains carrying ivi genes (columns) for each category (lines).

Table 3. Distribution of the eight ivi genes in 228 EHEC strains classified by serotypes.

	mhpR	ascG	mdtM	yjiR	Z0964	Z3135	Z4070	Z4799
TOP 7 priority serotypes (n = 97)	1	1	1	0.82	0.37	0.66	0.77	0.46
O157:H7 (n = 27)	1	1	1	1	1	0.96	0.96	1
O26:H11 (n = 31)	1	1	1	1	0	0	1	0
O103:H2 (n = 2)	1	1	1	1	0	0.5	1	1
O146:H21 (n = 12)	1	1	1	1	0.08	1	1	1
O91:H21 (n = 4)	1	1	1	1	1	1	1	1
O145:H28 (n = 4)	1	1	1	1	1	1	0	0
O80:H2 (n = 17)	1	1	1	0	0	1	0	0
Other serotypes (n = 131)	0.86	0.87	0.86	0.77	0.11	0.58	0.72	0.24

Values indicate the proportion of strains carrying ivi genes (columns) for each category (lines).

Figure 3. Distribution of the eight ivi genes in the EHEC strain collection. A phylogenetic tree was constructed after a core-genome multi-alignment with all 228 STEC strains and the O157 E. coli reference genome (NC_002695). Presence of each ivi gene is indicated by different coloured forms. Phylogroups are shown by colorizing the numbering of each strain and serotypes are written. All this information is indicated at the end of each branch of the phylogenetic tree.

Table 4. Co-distribution of the eight ivi genes with known virulence factors in the EHEC strain collection.

	mhpR	ascG	mdtM	yjiR	Z0964	Z3135	Z4070	Z4799
stx1 + stx2 (n = 52)	1	1	1	1	0.37	0.71	0.92	0.67
stx1a + stx2a (n = 13)	1	1	1	1	0.38	0.62	1	0.38
stx1a + stx2c (n = 7)	1	1	1	1	1	0.86	1	1
stx1a + stx2d (n = 7)	1	1	1	1	0.43	1	0.71	0.43
stx1c + stx2b (n = 23)	1	1	1	1	0.17	0.61	0.91	0.87
stx2 (n = 114)	0.99	1	0.99	0.76	0.23	0.75	0.75	0.3
stx2a (n = 39)	1	1	1	0.92	0.31	0.51	0.9	0.28
stx2b (n = 12)	0.92	1	0.92	1	0.17	0.5	0.92	0.83
stx2c (n = 17)	1	1	1	0.88	0.41	1	0.82	0.47
stx2d (n = 28)	1	1	1	0.46	0.07	0.96	0.32	0
stx2e (n = 3)	1	1	1	0	0.33	0.33	0.67	0
stx2g (n = 3)	1	1	1	0.67	0.33	1	1	0
stx1 (n = 62)	0.73	0.73	0.73	0.68	0.1	0.27	0.56	0.11
stx1a (n = 46)	0.63	0.63	0.63	0.61	0.07	0.17	0.59	0.07
stx1c (n = 15)	1	1	1	0.93	0.2	0.53	0.47	0.27
eae^+ (n = 102)	1	1	1	0.82	0.31	0.62	0.77	0.28
eae β (n = 42)	1	1	1	1	0	0.17	1	0
eae γ (n = 31)	1	1	1	1	1	0.97	0.84	0.87
eae ε (n = 6)	1	1	1	1	0.17	0.83	1	0.33
eae θ (n = 3)	1	1	1	0.67	0	0.33	0.67	0
eae ξ (n = 17)	1	1	1	0	0	1	0	0
eae^− (n = 126)	0.86	0.87	0.86	0.77	0.15	0.61	0.71	0.37

Values indicate the proportion of strains carrying ivi genes (columns) for each category (lines)

NB: only categories with a strain number ≥3 are shown.

Table 5. Distribution of stx, eae, and the eight ivi genes in EHEC strains with regards to the symptoms they have caused in patients.

	stx1	stx2	stx1 + stx2	eae	mhpR	ascG	mdtM	yjiR	Z0964	Z3135	Z4070	Z4799
HUS (n = 34)	0.03	0.79	0.18	0.71	1	1	1	0.85	0.29	0.71	0.85	0.32
bloody diarrhoea (n = 33)	0.15	0.55	0.3	0.49	0.94	0.94	0.94	0.82	0.3	0.61	0.85	0.42
watery diarrhoea (n = 47)	0.26	0.43	0.32	0.4	0.98	0.98	0.98	0.87	0.19	0.64	0.81	0.47

Values indicate the proportion of strains carrying indicated genes (columns) for each category (lines).

Figure 4. Impact of ivi gene deletion on the fitness of EDL933. Competition assays were performed by co-infecting mice with an equal mixture of EDL933 WT (Sm^R) and the indicated ivi gene mutant (Sm^R Kan^R). Eight days post-infection, faeces were sampled, homogenized in PBS, diluted and spotted on LB + Sm plates and LB + Sm + Kan plates to count, respectively, WT + ivi mutant and ivi mutant alone. The WT population was obtained by subtracting ivi mutant CFU from total EHEC CFU. Competitive indices (ratio WT/mutant) were calculated for each animal. Each dot represents one mouse and lines represent median values. Differences between each group and the control group agaF were analyzed by ANOVA with Fisher’s LSD test (** P < 0.01).