Molecular chaperone function of three small heat-shock proteins from a model probiotic species

Abstract

Small heat-shock proteins (sHSP) are ubiquitous ATP-independent chaperones that prevent irreversible aggregation of heat-damaged denaturing proteins. Lactiplantibacillus plantarum is a widespread Gram-positive bacterium with probiotic claims and vast potential for agro-food, biotechnological and biomedical applications. L. plantarum possesses a family of three sHSP, which were previously demonstrated to be involved in its stress tolerance mechanisms. Here, the three L. plantarum sHSP were heterologously expressed, purified and shown to have a chaperone activity in vitro, measuring their capacity to suppress protein aggregation, as assayed spectrophotometrically by light scattering. Their anti-aggregative capacity was found to be differently influenced by pH. Differences were also found relative to their holdase function and their capacity to modulate liposome membrane fluidity, suggesting interplays between them and indicating diversified activities. This is the first study assessing the chaperone action of sHSP from a probiotic model. The different roles of the three sHSP can increase L. plantarum's capabilities to survive the various types of stress characterising the diverse habitats of this highly adaptable species. Reported evidence supports the interest in L. plantarum as one of the model species for bacteria that have three different sHSP-encoding genes in their genomes.

Keywords: Heat stress; Holdase; Lipochaperone; Membrane fluidity; Protein aggregation; sHSP.

Fig. 1

Dose-dependent inhibition of citrate synthase (CS) aggregation by L. plantarum sHSP. CS (0.3 µM) was incubated at 45 °C with increasing concentrations of HSP1, HSP2 or HSP3, and protein aggregation was monitored by the increase in light scattering at 360 nm. The saturation signal was normalised to 100. CS alone, solid square; CS + 1.2 µM sHSP (molar ratio 4:1, sHSP:CS), crosses; CS + 4.8 µM sHSP (molar ratio 16:1), grey circles; CS + 9.6 µM sHSP (molar ratio 32:1), open triangle. Mean and SD from at least 2 experiments

Fig. 2

The effect of pH on the chaperone activity of HSP1, HSP2 and HSP3. Thermal induced aggregation of 0.3 µM citrate synthase (CS), solid squares, was monitored in 50 mM Na-phosphate buffer at pH 5.5, pH 7.0 and pH 8.0 in the presence of 1.2 µM HSP1 (grey triangle), or HSP2 (open square), or HSP3 (open circle), (4:1 molar ratio, sHSP:CS). The cuvette was incubated at 45 °C, and the protein aggregation was monitored by detecting light scattering at 360 nm. The saturation signal was normalised to 100. Mean and SD from at least 2 experiments

Fig. 3

Holdase activity of L. plantarum sHSP. The solubility of a heat destabilised (45 °C, 60 min) CS in the absence and presence of HSP1, HSP2, HSP3 or all three sHSP (molar ratio sHSP: CS = 16:1) was determined by separation into supernatants and pellet fractions. Pellet (aggregated CS) was isolated from supernatants (soluble CS) by centrifugation, and both fractions were analysed by SDS-PAGE. A representative SDS-PAGE of the analysed fractions is reported. The same amount of CS not exposed to thermal stress (5 °C) represents the negative and positive control for pellet and supernatant, respectively. M, molecular weight markers. *p < 0.05 vs CS, CS + HSP2, CS + HSP3; **p < 0.05 vs CS, CS + HSP2, CS + HSP3; ***p < 0.05 vs CS + HSP3

Fig. 4

The effect of HSP1, HSP2 or HSP3 on membrane fluidity of lipid vesicles derived from L. plantarum. Fluorescence anisotropy of DPH probe inserted into vesicles was measured according to the temperature increase, in the absence (open circle) or presence of HSP1, HSP2 or HSP3 (solid symbols). Anisotropy was also measured in presence of lysozyme (solid circle), as a negative control. Mean and SD from at least 3 independent experiments. *p < 0.05, Mann–Whitney U test