Effect of HosA and AHL on Aggregation by E2348/

Ferrándiz et al observed that in the absence of HosA the population of E2348/69 became less motile [68]. No changes in expression of flagella between the wild type strain and the mutant were found in that study, but clumping was observed in the HosA mutant broth cultures which formed non-motile aggregates that were absent in the wild type culture. To determine whether the changes observed in population expansion were in response to changes in cell aggregation, this section described the application of the method by Rowe et al to calculate the aggregation indices for both the wild type and mutant in the absence of AHL [79].

The aggregation index (AI) is a comparative measure of the proportion of bacteria within a culture that have aggregated. Growth was monitored during the course of the experiment to illustrate the equivalence of the optical density of both E2348/69 and E2348/69 hosA- _cultures

(Figure 22A). The AI values represent the proportion of the population within aggregates, for example an AI of 0.5 is equivalent to 50% of the population. At 25°C E2348/69 had a low

proportion of aggregated cells after 3 hours incubation. The AI increased exponentially after 4 hours until 8 hours (Figure 22B). Maximum aggregation was achieved at 8 hours with 77% (AI = 0.77) of the bacteria located in aggregates. Between 8 and 12 hours the aggregates began to disperse and the AI decreased to 0.43 (Figure 22B).

E2348/69 hosA-_{had a higher AI than the wild type strain of 0.79 after 3 hours incubation.}

Maximum aggregation was achieved at 6 hours incubation (AI = 0.98), but a decrease was observed by 8 hours (AI = 0.93). The aggregates began to disperse with 82% (AI = 0.82) remaining aggregated by 12 hours incubation (Figure 22B). No formal statistical confirmation of these observations was performed.

Figure 22 – Comparison of cell aggregation by E2348/69 and E2348/69 hosA- _using

aggregation indices and incubation at 25°C

The aggregation indices method uses differential centrifugation to determine the proportion of the bacterial population bound together to form aggregates. An AI of 1.0 indicates 100% of the population are aggregated. Four independent experiments are shown. Two different shades of colour are used for each strain to allow differentiation between the experiments.

(Dark/Light Blue) Wild type E2348/69 (Dark/Light Red) E2348/69 hosA- Two different conditions are described:

(A) Growth curve of the aggregating cultures determined using log10 optical density (Log10OD600)

(B) Aggregation indices of the cultures

0.1 1 10 0 2 4 6 8 10 12 Log 10 OD 600 Time (Hours) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 2 4 6 8 10 12 A gg re gation In d e x (AI) Time (Hours)

A

B

N-(3-oxohexanoyl)-L-HSL had significantly increased the population expansion of E2348/69 hosA- (Section 3.2.2). To determine whether this was as a result of N-(3-oxohexanoyl)-L-HSL affecting cell aggregation, the AI was calculated in the presence and absence of 5 nM N-(3- oxohexanoyl)-L-HSL supplemented into the culture after 4 hours incubation.

In the absence of AHL, the proportion of aggregating cells within the culture was high correlating with previous experiments. Peak aggregation was achieved at 6 hours (AI = 0.82) (Figure 23A). Addition of N-(3-oxohexanoyl)-L-HSL decreased the level of cell aggregation of E2348/69 hosA- to a maximum of 79% (AI = 0.79) at 7 hours incubation. Between 7 and 9 hours the aggregates began to disperse in both cultures regardless of supplementation, indicated by a decrease in AI indices (Figure 23A).

The maximum difference in the AI in the presence and absence of AHL was calculated at 0.048 after 6 hours incubation, suggesting N-(3-oxohexanoyl)-L-HSL had little effect on the aggregation of E2348/69 hosA-_{(Figure 23A).}

After 5 hours incubation, samples were centrifuged. 10µl samples were taken from the bottom 4ml of both the unsupplemented and N-(3-oxohexanoyl)-L-HSL -treated E2348/69 hosA-

cultures and analysed using phase contrast microscopy at 1000x magnification (Figures 23B(1)

and B(2)). In the absence of AHL, E2348/69 hosA- formed large complex aggregates which

spanned the fields of view (Figure 23B(1)). N-(3-oxohexanoyl)-L-HSL was added to technical

replicates of the same initial biological culture which had equivalent optical density to that of the unsupplemented culture. Addition of this AHL resulted in much smaller aggregates being formed, with a higher proportion of non-aggregated planktonic cells (Figure 23B(2)). The difference in

visual aggregation analysis appeared to be greater than the AI analysis indicated (Figure 23). Therefore, a new method was required to investigate the effect of AHL-dependent quorum sensing on the aggregation of E2348/69 and the HosA mutant and this is described in the following section.

Figure 23 – Effect of N-(3-oxohexanoyl)-L-HSL on cell aggregation by E2348/69 hosA- _at

25°C

Comparison of aggregation by E2348/69 hosA- _{in the presence and absence of N-(3-oxohexanoyl)-L-HSL.} Supplementation with N-(3-oxohexanoyl)-DL-HSL was performed at 4 hours incubation to a final concentration of 5nM. Effect of AHL was assessed using two different methods:

(A) Aggregation Indices. This method uses differential centrifugation to determine the proportion of the bacterial population bound together to form aggregates. An AI of 1.0 indicates 100% of the population are aggregated. (Blue) E2348/69 hosA-_{with no AHL supplement}

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

(B) Phase contrast microscopy. Images are representative of the aggregates formed by E2348/69 hosA-_{in the} presence and absence of N-(3-oxohexanoyl)-L-HSL after 5 hours incubation. Images were taken at 1000x magnification. Cell densities of the cultures were recorded using optical densities at OD600 = 0.6.

[B(1)] E2348/69 hosA-with no AHL supplement

[B(2)] E2348/69 hosA-in the presence of N-(3-oxohexanoyl)-L-HSL

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 1 2 3 4 5 6 7 8 9 10 A gg reg ati on Ind ex Time (Hours)

B(2)

B(1)

A