A highly conserved complete accessory Escherichia coli type III secretion system 2 is widespread in bloodstream isolates of the ST69 lineage

Abstract

Bacterial type III secretion systems (T3SSs) play an important role in pathogenesis of Gram-negative infections. Enteropathogenic and enterohemorrhagic Escherichia coli contain a well-defined T3SS but in addition a second T3SS termed E. coli T3SS 2 (ETT2) has been described in a number of strains of E. coli. The majority of pathogenic E. coli contain elements of a genetic locus encoding ETT2, but which has undergone significant mutational attrition rendering it without predicted function. Only a very few strains have been reported to contain an intact ETT2 locus. To investigate the occurrence of the ETT2 locus in strains of human pathogenic E. coli, we carried out genomic sequencing of 162 isolates obtained from patient blood cultures in Scotland. We found that 22 of 26 sequence type (ST) 69 isolates from this collection contained an intact ETT2 together with an associated eip locus which encodes putative secreted ETT2 effectors as well as eilA, a gene encoding a putative transcriptional regulator of ETT2 associated genes. Using a reporter gene for eilA activation, we defined conditions under which this gene was differentially activated. Analysis of published E. coli genomes with worldwide representation showed that ST69 contained an intact ETT2 in these strains as well. The conservation of the genes encoding ETT2 in human pathogenic ST69 strains strongly suggests it has importance in infection, although its exact functional role remains obscure.

Figure 1

Comparisons of the ETT2 locus between EAEC 042, ST69 (1#9) and O157:H7 Sakai. Degree of identity is shown by the level of grey shading as indicated. Genes are colour coded according to putative function as shown.

Figure 2

Comparison of the ETT2 operon in 24 ST69 strains. Degree of identity is shown by the level of grey shading as indicated. Genes are colour coded according to putative function as shown. The epaO gene is shown green.

Figure 3

Comparison of elements of the ETT2 locus found in non-ST69 strains. Degree of identity is shown by the level of grey shading as indicated. Genes are colour coded according to putative function as shown. Genes unrelated to the ETT2 locus genes are coloured grey.

Figure 4

Comparison of the selC operon in different strains. Degree of identity is shown by the level of grey shading as indicated. Genes are colour coded according to putative function as shown. *Shows the position of a frameshift mutation in the eilA gene of sample 1#47 (ST59).

Figure 5

Activity of the eilA reporter in different strains and media. (A,B) Graphs show growth (Optical Density, panels A) and reporter activity (GFP fluorescence, panels B) at the times indicated. The strains are: EC1#2 (A), EC1#19 (B), EC1#5 (C), EC1#21 (D), and EC1#9 (E), all grown in LB:DMEM mixture. Each point is the mean of a triplicate determination; error bars (sem) are contained within the points. (C,D) strain EC1#2 is grown in the different media as indicated.

Figure 6

Length conservation of the ETT and selC/Eip locus in different strains of E. coli compared to the reference strain, EAEC 042. Data from the local Scottish strains (panels A and C) and global data (panels B and D) are shown. (A,B) are the comparisons for the ETT2 locus and (C,D) are for the selC/Eip locus. STs with fewer than 4 representatives are classed as Other in panels A, B, and D.