Functional amyloids promote retention of public goods in bacteria

Abstract

The growth and virulence of bacteria depends upon a number of factors that are secreted into the environment. These factors can diffuse away from the producing cells, to be either lost or used by cells that do not produce them (cheats). Mechanisms that act to reduce the loss of secreted factors through diffusion are expected to be favoured. One such mechanism may be the production of Fap fibrils, needle-like fibres on the cell surface observed in P. aeruginosa, which can transiently bind several secreted metabolites produced by cells. We test whether Fap fibrils help retain a secreted factor, the iron-scavenging molecule pyoverdine, and hence reduce the potential for exploitation by non-producing, cheating cells. We found that: (i) wild-type cells retain more iron-chelating metabolites than fibril non-producers; (ii) purified Fap fibrils can prevent the loss of the iron-chelators PQS ( Pseudomonas quinolone signal) and pyoverdine; and (iii) pyoverdine non-producers have higher fitness in competition with fibril non-producers than with wild-type cells. Our results suggest that by limiting the loss of a costly public good, Fap fibrils may play an important role in stabilizing cooperative production of secreted factors.

Keywords: Pseudomonas; cooperation; functional amyloid; pyoverdine; social evolution.

Figure 1.

Growth and pyoverdine production of Fap fibril producers (black lines, grey envelope) and non-producers (grey lines, pink envelope) over 24 h in iron-limited media. Mean and standard error envelope are plotted for each. (a) Growth of strains, measured by cell density at A₆₀₀. (b) Cumulative pyoverdine available, measured by RFU_400–460. (c) Pyoverdine available per cell, measured by RFU_400–460/A₆₀₀. (d) Rate of pyoverdine production, measured per cell per 30 min (((RFU_{400–4600(2)} − RFU_400–460(1))/30 min)/A₆₀₀₍₂₎). Cell density, available pyoverdine and pyoverdine available per cell were not significantly different between fibril producers and non-producers.

Figure 2.

Retention of iron-chelating metabolites by fibril producers and non-producers. Iron chelating activity was quantified using a CAS assay and standardized by cell density (iron-chelating activity per cell ((1 − A₆₃₀)/A₆₀₀)). Boxplots give the median value, boxes show the interquartile range and whiskers extend to the largest or smallest value respectively. Iron-chelating activity of fibril producers was significantly greater than that of non-producers.

Figure 3.

Retention of iron-chelating metabolites by Fap fibrils. (a) Iron-chelating activity, measured as (1 − A₆₃₀), of 0, 5 and 50 µg ml⁻¹ of untreated purified Fap fibrils (white) or purified Fap fibrils treated with PQS (cyan) or pyoverdine (green). Iron-chelating activity increases with increasing fibril concentration for both pyoverdine and PQS treatments. (b) Growth, measured as A_{600(24 h)} − A_{600(0 h)}, of a pyoverdine non-producer (PAO1ΔpvdD) in iron-limited media supplemented with 0, 5 and 50 µg ml⁻¹ of untreated purified Fap fibrils or purified Fap fibrils treated with PQS (cyan) or pyoverdine (green). Growth of a pyoverdine non-producer increases with increasing concentrations of fibrils treated with pyoverdine and decreases with increasing concentrations of fibrils treated with PQS.

Figure 4.

Relative fitness (RF) of a pyoverdine non-producer (PAO1ΔPvdD) in competition with fibril producers and non-producers in iron-limited media. Boxplots give the median value, boxes show the interquartile range and whiskers extend to either 1.5 times the interquartile range or to the largest or smallest value respectively. Outliers are shown as small circles. Pyoverdine non-producer significantly increase in frequency over 24 h in competition with both fibril producers and non-producers, but pyoverdine non-producer relative fitness is significantly higher when competed against fibril non-producers than against fibril producers.