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

The work was funded by the Scottish Executive via the Chief Scientists Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.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.Publisher PDFPeer reviewe

Distinct ecological fitness factors coordinated by a conserved Escherichia coli regulator during systemic bloodstream infection

The ability of bacterial pathogens to adapt to host niches is driven by the carriage and regulation of genes that benefit pathogenic lifestyles. Genes that encode virulence or fitness-enhancing factors must be regulated in response to changing host environments to allow rapid response to challenges presented by the host. Furthermore, this process can be controlled by preexisting transcription factors (TFs) that acquire new roles in tailoring regulatory networks, specifically in pathogens. However, the mechanisms underlying this process are poorly understood. The highly conserved Escherichia coli TF YhaJ exhibits distinct genome-binding dynamics and transcriptome control in pathotypes that occupy different host niches, such as uropathogenic E. coli (UPEC). Here, we report that this important regulator is required for UPEC systemic survival during murine bloodstream infection (BSI). This advantage is gained through the coordinated regulation of a small regulon comprised of both virulence and metabolic genes. YhaJ coordinates activation of both Type 1 and F1C fimbriae, as well as biosynthesis of the amino acid tryptophan, by both direct and indirect mechanisms. Deletion of yhaJ or the individual genes under its control leads to attenuated survival during BSI. Furthermore, all three systems are up-regulated in response to signals derived from serum or systemic host tissue, but not urine, suggesting a niche-specific regulatory trigger that enhances UPEC fitness via pleiotropic mechanisms. Collectively, our results identify YhaJ as a pathotype-specific regulatory aide, enhancing the expression of key genes that are collectively required for UPEC bloodstream pathogenesis

Ovine tracheal epithelial cell cultures display stable barrier function and junctional integrity.

<p>(A) Ovine tracheal epithelial cell cultures were grown at ALI for the indicated number of days (relative to establishment of the ALI) and tissue layers were fixed and immunostained using an anti-ZO1 antibody at the indicated time points (relative to establishment of the ALI). (B) Orthogonal representation of ALI culture at 24 days post-ALI. (C) 3-dimensional model of the Z-stack shown in panel B. (D) TEER measurements from four independent cell culture inserts at each time-point. Results for ALI cultures derived from three independent animals are shown (mean +/- standard deviation).</p

Submerged growth media have different effects on differentiation of ovine tracheal epithelial cells.

<p>Ovine tracheal epithelial cells were cultured to confluency in SGM or AEGM and at ALI for 21 days in ALI medium. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E, F and G). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks.</p

Ovine tracheal epithelial cells differentiate optimally at sub-ambient oxygen tension.

<p>Ovine tracheal epithelial cells were cultured in an atmosphere of 7, 14 or 21% O₂. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E and G) or one-way ANOVA with Dunnet’s post-test (F). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks.</p

Mucus production by differentiated ovine tracheal epithelial cell cultures.

<p>(A) Ovine tracheal epithelial cell cultures were grown at ALI for the indicated number of days (relative to establishment of the ALI), fixed and processed for SEM. (B) Ovine tracheal epithelial cell cultures were grown for the indicated number of days, fixed and stained with jacalin-FITC (green), rhodamine-phalloidin (red) and DAPI (blue). Mucus globules are indicated by white arrows, carpets of amorphous mucus are indicated by white arrowheads and jacalin-labelled mucin-positive cells are indicated by yellow arrows.</p

Retinoic acid is required for <i>in vitro</i> differentiation of ovine tracheal epithelial cells.

<p>Ovine tracheal epithelial cells were cultured at ALI for 21 days with the indicated concentrations of retinoic acid. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Cell layer thickness measured from three points per field in H&E-stained sections. (H) Cell layer thickness as determined by counting nuclei at three points per field in H&E-stained sections. (I) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G, H, I) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E, G, H and I) or one-way ANOVA with Dunnet’s post-test (F). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks. Black asterisks indicate all samples were significantly different to untreated control in panel F.</p

Summary of parameters tested, and degree of cellular differentiation observed.

<p>Summary of parameters tested, and degree of cellular differentiation observed.</p

Ultrastructural analysis of the apical surface of ovine tracheal epithelial cell cultures by SEM.

<p>Ovine tracheal epithelial cell cultures were grown at an ALI for the indicated number of days (relative to establishment of the ALI), fixed and processed for SEM. (A) Images were taken at 1500× magnification. (B) Images were taken at 5000× magnification. Ciliated epithelial cells were observed from day 12 onwards.</p