La cultura musical independiente entre 2013 y 2015

Cell surface proteins of enteropathogenic E. coli are involved with the formation of attaching- effacing (A/E) lesions on mammalian cells. Using FITC-phalloidin it was possible to stain the actin of the mammalian cells to visualise these pedestals. An individual bacterium was located at the centre of each pedestal. To determine whether the ability of E2348/69 to form aggregates affected the propensity of the bacteria to form A/E lesions, the FAS test was performed in the presence and absence of AHL. For the purposes of this study human colonic cell-line HT-29 was used to produce the eukaryotic monolayer. We hypothesised that more aggregative cultures would produce a higher number of A/E lesions on the human cells.

Figure 61 –Aggregation profile ofE2348/69 espA- _{in the presence and absence of N-(3-}

oxohexanoyl)-L-HSL and N-dodecanoyl-L-HSLat 25°C

Each profile is representative of the distribution of aggregate sizes produced by E2348/69 espA-_{in the presence and} absence of N-(3-oxohexanoyl)-L-HSL and N-dodecanoyl-L-HSL. AHLs were added at 4 hours growth to a final concentration of 5nM. The images analysed were represented in Figure 61.

(A) Culture A; and (B) Culture B (Blue) E2348/69 espA-_{with no AHL}

(Red) E2348/69 espA-_{in the presence of N-(3-oxohexanoyl)-L-HSL} (Green) E2348/69 espA- _{in the presence of N-dodecanoyl-L-HSL}

Table 32 – Statistical analysis of the effect of active AHL on E2348/69 espA- _aggregation

Biological variation compares profiles between cultures independent A and B; technical variation compares the profiles within a culture. Where D < 0.4 there is no significant difference between the profiles of the given treatments, but where D ≥ 0.4 the difference is deemed to be significant.

Biological Variation (A

vs B) Technical Variation

E2348/69 espA- _{0.22 0.11}

A B Biological Variation _{(A vs B)} Unsupplemented vs _Supplemented

N-(3-oxohexanoyl)-L-HSL (C6) 0.79 0.69 0.06 0.72 N-dodecanoyl-L-HSL (C12) 0.15 0.07 0.03 0.02 0 50000 100000 150000 200000 250000 300000 350000 Fr e que ncy x A re a (R A U )

Aggregate Area (Pixels2₎

0 50000 100000 150000 200000 250000 300000 350000 Fr eque ncy x A re a (RA U)

Aggregate Area (Pixels2₎

A

Figure 62 – Aggregation profile of E2348/69 espA- _{in the presence and absence of N-(3-}

oxo-hexanoyl)-D-HSL and N-(3-oxododecanoyl)-D-HSL at 25°C

Each profile is representative of the distribution of aggregate sizes produced by E2348/69 espA-_{in the presence and} absence of N-(3-oxo-hexanoyl)-D-HSL and N-(3-oxododecanoyl)-D-HSL. AHLs were added at 4 hours growth to a final concentration of 5nM. The images analysed were represented in Figure 61.

(A) Culture A; and (B) Culture B (Blue) E2348/69 espA- _{with no AHL}

(Red) E2348/69 espA- _{in the presence of N-(3-oxo-hexanoyl)-D-HSL} (Green) E2348/69 espA- _{in the presence of N-(3-oxododecanoyl)-D-HSL}

Table 33 – Statistical analysis of the effect of D-isomers on E2348/69 espA- _aggregation

Biological variation compares profiles between cultures independent A and B; technical variation compares the profiles within a culture. Where D < 0.4 there is no significant difference between the profiles of the given treatments, but where D ≥ 0.4 the difference is deemed to be significant.

Biological Variation

(A vs B) Technical Variation

E2348/69 espA- _{0.22 0.11}

A B Biological Variation _{(A vs B)} Unsupplemented vs _Supplemented

N-(3-oxo-hexanoyl)-D-HSL (C6) 0.23 0.07 0.07 0.04 0 50000 100000 150000 200000 250000 300000 350000 Fr e que ncy x A re a (R A U )

Aggregate Area (Pixels2₎

0 50000 100000 150000 200000 250000 300000 350000 Fr e que ncy x A re a (R A U )

Aggregate Area (Pixels2₎

A

Furthermore, we hypothesised that where addition of AHL changed the aggregative behaviour of the population, a corresponding change would be observed in the A/E lesion formation.

In the absence of bacterial culture, HT-29 cells were outlined faintly in green, with no intense areas of fluorescence (Figure 63). Addition of N-(3-oxohexanoyl)-L-HSL and N- dodecanoyl-L-HSL at a final concentration of 5nM, or ethyl acetate at 0.1% (v/v) had no effect on the fluorescence observed in the absence of bacteria (Figure 63).

E2348/69 produced a high number of A/E lesions on HT-29 cells in all biological replicates analysed (Figure 64). No obvious change in the quantity of A/E lesions formed was observed with the addition of N-(3-oxohexanoyl)-L-HSL and N-dodecanoyl-L-HSL (Figure 64).

Although important for localised adherence of EPEC to host epithelial cells, using the FAS test we showed that in the absence of BfpA, E2348/69 was able to form A/E lesions on HT- 29 cells (Figure 65). The absence of Bfp resulted in a significant decrease in the observed number of A/E lesions formed by E2348/69, which was in the order of 100 fold less (Figure 65).

AE2348/69 luxS- _{showed areas of intense fluorescence, but the characteristic pedestal structure}

was not visible. Previous work by S. Snape had shown AE2348/69 luxS-_{did produce pedestals,}

but these were often masked by high density micro-colonies [232]. Intense green fluorescence indicated actin cytoskeleton rearrangements in the HT-29 cells, showing AE2348/69 luxS-

successfully attached to the epithelial cells. When compared to the wildtype, a higher frequency of fluorescent colonies were observed (Figure 66A). AE2348/69 luxS-_{contained the EAF plasmid}

and therefore bfpA, so conclusive comparisons between the LuxS mutant and the wild type strain of E2348/69 used in this study cannot be drawn from this data. With addition of N-(3- oxohexanoyl)-L-HSL or N-dodecanoyl-L-HSL the number of intense fluorescent spots appeared to decrease (Figure 66B).

HT-29 only Ethyl acetate

N-(3-oxohexanoyl)-L-HSL (C6) N-dodecanoyl-L-HSL (C12)

Figure 63 – FITC-Phalloidin staining of HT-29 cells in the presence and absence of AHL and solvent

Representative images of the negative controls used for all assays investigating attaching/effacing lesions formation (Figures 64-67).

AHLs were added to the growth medium to a final concentration of 5nM, with an equivalent volume of ethyl acetate added to a separate sample to provide a negative control for the effect of AHL and the solvent used to dilute the signals. HT-29 cells were treated with FITC-Phalloidin to show the background staining of epithelial cells that do not have bacteria attached.

Scale bars are indicated in the corner of each image, and set to 10µm.

Using Microsoft Word the brightness of each image has been increased by 30%.

10µM 10µM

10µM 10µM

Culture 1 Culture 2