Analysis of the FTIR absorption spectrum of Saccharomyces cerevisiae cells
We have chosen to describe S. cerevisiae cells growing in the presence or absence of inhibitory concentrations of lactic acid through their IR absorption spectrum. To exemplify a possible outcome of this analysis and the potential of the consequent observations, in Fig. 1 the measured IR absorption spectrum of S. cerevisiae intact cells, collected during the exponential phase of growth on minimal glucose medium (Additional file 1: Figure S1), is reported.
As illustrated, the spectrum is complex since it results from the absorption of the different biomolecules. In particular, the lipid hydrocarbon tails absorb between 3050 and 2800 cm−1 and between 1500 and 1350 cm−1, where also lipid head group absorption occurs, while around 1740 cm−1 the ester carbonyl IR response is observed [22, 24]. In addition, between 1700 and 1500 cm−1 the spectrum is dominated by the amide I and amide II bands, respectively due to the C=O stretching and the NH bending of the peptide bond. In particular, the amide I band gives information on the protein secondary structure and aggregation [19, 25–28]. Furthermore, the spectral range between 1250 and 900 cm−1 is dominated by the absorption of phosphate groups mainly from phospholipids and nucleic acids, as well as by the C–O absorption of carbohydrates [20–22].
To better evaluate possible spectral changes occurring under stressful conditions, often imposed by the fermentation processes, the second derivatives of the FTIR absorption spectra have been analysed, as they enable to resolve the overlapping components of the IR absorption bands [29]. Therefore, the results presented in the next sections will directly report the second derivatives spectra of S. cerevisiae cells grown in the different media and collected at different times after inoculation.
FTIR microspectroscopy analysis of Saccharomyces cerevisiae BY4741 strain under lactic acid stress
S. cerevisiae BY4741 cells were challenged with increasing concentrations of lactic acid, observing a gradual effect, from no perturbation of the kinetic of growth (data not shown) to detrimental effects, measured as a reduction in growth rate (see Fig. 2, closed symbols, minimal medium with 2 % w/v glucose in the absence -left- and in the presence -right- of 46 g/L lactic acid at pH 3). Independently from the media, cells reached the stationary phase of growth, but with a time delay and a reduced final biomass when treated with lactic acid. It is therefore relevant to analyse cellular response in this transition phase, especially in the view of a possible industrial process, where environmental fluctuations are inescapable, but undesirable if affecting microbial performances.
Samples collected at 18 and 40 h after inoculation, respectively corresponding to the exponential and the stationary phase of growth, were then analysed by FTIR microspectroscopy.
In Fig. 3 we reported the second derivative spectra of BY4741 S. cerevisiae cells grown for 18 h in the absence (pH3) and in the presence of 46 g/L of lactic acid at pH3 (pH3 + LA46), in the amide I band between 1700 and 1600 cm−1 (a), in the spectral ranges between 1500 and 1200 cm−1 (b) and between 3050 and 2800 cm−1 (c).
In the absence of the stressing agent the second derivative spectrum is characterized by a band at ~1657 cm−1, mainly due to alpha-helix and random-coil structures of the whole cell proteins, and by a band at ~1639 cm−1, assigned to intramolecular native beta-sheets [19, 26], (Fig. 3a). In the presence of lactic acid, an intensity reduction of the alpha helix/random coil and native beta-sheet components was observed, accompanied by the appearance of a new band at ~1629 cm−1, due to intermolecular beta-sheets, typical of protein aggregates [25, 27, 28, 30–32]. Interestingly, we found that the change in the intensity of the band assigned to protein aggregates is lactic acid dose-dependent (see Additional file 2: Figure S2a).
The spectral range between 1500 and 1200 cm−1 (Fig. 3b) is dominated by vibrational modes due to lipid hydrocarbon tails and head groups [22, 24]. In particular, the second derivative spectrum of cells grown in the absence of lactic acid is characterized by a number of well resolved bands mainly due to the CH2 and CH3 deformation modes: the ~1467 cm−1 band is due to the overlapping absorption of CH2 and CH3 [22, 24, 33], while the ~1455, 1440 and 1368 cm−1 bands are due to CH3 [22, 24], and the ~1414 cm−1 absorption to CH2 [34]. In addition, the component at ~1400 cm−1 is mainly assigned to the CH3 bending vibration of the N(CH3)3 head group of phosphatidylcholine (PC) and the absorption at ~1387 cm−1 can be assigned to the CH3 deformation mainly arising from ergosterol [22, 35, 36]. Finally, the component at ~1246 cm−1 is also observed, due to the PO2- stretching mode mainly from phospholipids and nucleic acids [20, 22].
In this study, we focused our attention on the bands that were found to significantly change after exposure to the stressing agent. In particular, the 1400 cm−1 and the 1246 cm−1 absorptions decreased in intensity concurrently when cells are in the presence of 46 g/L of lactic acid, indicating an overall reduction of PC component. Moreover, the ergosterol band at ~1387 cm−1 was found to become more resolved. We should also note that the variation of the PC marker band (~1400 cm−1) resulted again to be lactic acid dose-dependent (Additional file 2: Figure S2b).
Furthermore, in the spectral range between 3050 and 2800 cm−1 (Fig. 3c) the spectrum of cells grown at pH3 is characterized by four well resolved and intense bands due to the CH2 (at ~2921 and 2851 cm−1) and CH3 (at ~2959 and 2872 cm−1) stretching vibrations of lipid hydrocarbon tails [22, 24]. A shoulder around 2935 cm−1 is also present, that can be mainly assigned to the CH2 stretching of ergosterol [35].
Interestingly, in pH3 + LA46 cell spectrum, the CH2 stretching bands at ~2921 cm−1 and 2851 cm−1 were found to slightly decrease in intensity, likely suggesting a rearrangement of the hydrocarbon tails [37].
We investigated also the spectral range between 1200 and 900 cm−1 (see Additional file 3: Figure S3), dominated by the absorption of cell wall carbohydrates, including glucans and mannans [38]. As illustrated in Additional file 3: Figure S3a, compared to unchallenged cells, cells treated with lactic acid displayed a slight reduction in the intensity of the β1 → 3 glucan and mannan spectral components, accompanied by a weak but significant reduction of the low intensity band due to β1 → 6 glucans.
Overall, these results depict a change in the biochemical fingerprint of yeast cells exponentially growing in medium added with lactic acid. In particular, PC is not only one of the most abundant membrane phospholipids but it is also responsible for membrane fluidity [39, 40]. The decreasing in PC observed during the response to lactic acid exposure might therefore be a strategy adopted by the cells to make the membrane more compact and, consequently, less permeable to the lactic acid influx. As a consequence, this might also reflect in a general rearrangement of the transport rates. Moreover, if the plasma membrane is considered not only as a barrier between the extracellular and the intracellular environments, but also as a stress sensor [41], changes in its composition might additionally trigger a variety of intracellular events intended to rewire or adapt the cells to the different environment. As we will discuss in the next paragraph, the growth delay observed when cells are exposed to the stressing agent might be therefore related to the observed protein aggregation.
At 40 h after inoculation, corresponding to the stationary phase of growth, in the amide I band the spectral features of cells grown in the presence and in the absence of lactic acid resulted to be quite similar, with two main components at ~1657 cm−1 due to alpha helices and random coils, and at ~1637 cm−1 mainly due to intramolecular native beta-sheets (Fig. 4a). These results indicate that in this growth phase the lactic acid exposure does no more affect significantly the overall secondary structures of the whole cell proteins.
On the contrary, an important decrease of the PC marker band intensity at ~1402 cm−1 was still detected in pH3 + LA46 cells (Fig. 4b), accompanied by an increase of the ergosterol absorption at ~1387 cm−1 and a slight decrease of the PO2− band at ~1246 cm−1. Furthermore, dramatic changes in the spectral features between 3050 and 2800 cm−1 were found. In particular, a significant intensity decrease of the CH2 bands at 2921 and 2851 cm−1, consistent with a reduction of the lipid hydrocarbon tail length, took place in cells exposed to lactic acid (Fig. 4c). Moreover, in agreement with the ergosterol absorption at ~1387 cm−1, the shoulder around 2935 cm−1 became more evident compared to pH3 cells. The analysis of the cell wall carbohydrate absorption between 1200 and 900 cm−1 (see Additional file 3: Figure S3b) highlighted firstly a higher level of β1 → 6 glucans in unchallenged cells at the stationary phase of growth, compared to the exponential. In addition, at 40 h after inoculation, in lactic acid treated cells we observed a reduction in intensity of the spectral components mainly due to glucans. These spectral changes, which suggest again a rearrangement of the cell wall properties, were found to be more pronounced in the stationary phase compared to the exponential (Additional file 3: Figure S3a).
Effects of OPI1 deletion on lactic acid tolerance and on macromolecular fingerprint
As described above, in the yeast strain under investigation a correlation between lactic acid exposure and a decrease in PC levels exists. Opi1p is a transcription factor that acts as a repressor of the genes involved in the synthesis of PC [23]. Consequently, we have envisaged OPI1 as a useful target for further supporting this indication and, in particular, the effects of its overexpression and deletion were analysed under lactic acid stress. Since the OPI1 gene overexpression caused severe growth deficiencies both in the absence and in the presence of lactic acid (data not shown), we focused our attention on its deletion. Figure 2 (open symbols) shows the growth curves obtained for the OPI1-lacking in the absence and in the presence of lactic acid. No remarkable differences were observed between the control and the OPI1 deleted strain during growth without lactic acid at low pH (left panel), while lactic acid exerted a clear negative effect. Notably, in the limiting condition (right panel) a marked difference between the two strains was observed: the BY4741 opi1Δ rescued growth earlier than the parental strain, showing a faster growth rate (0.11 vs. 0.06 h−1) despite the two strains reached a similar final biomass value.
In Fig. 5, we reported the second derivative spectra of these cells collected in the exponential phase of growth (see also Additional file 4: Figure S4). In particular, in Fig. 5a the amide I band analysis indicates that—contrary to what observed for the parental strain (Fig. 3a)—the lactic acid exposure of the BY4741 opi1Δ cells did not dramatically affect the cell protein structures, just leading to a slight decrease in the intensity of the alpha-helix/random coil component at ~1657 cm−1, compared to unchallenged cells (Additional file 5: Figure S5a). Furthermore, a minor decrease of the PC marker band at ~1400 cm−1 and of the ~1246 cm−1 (PO2−) component occurred in pH3 + LA46 cells compared to pH3, accompanied by a slight increase of the ergosterol absorption at ~1387 cm−1 (Figs. 5b, 3b, Additional file 4: S4b and Additional file 5: S5b for comparison). In addition, a weak reduction in the intensity of the hydrocarbon tail CH2 absorption at ~2921 and ~2852 cm−1 (Fig. 5c) has been detected.
Moreover, for opi1Δ cells the spectral features mainly due to cell wall carbohydrates displayed in particular a slight decrease in the intensity of the β1 → 3 glucan bands upon LA treatment (see Additional file 3: Figure S3c). Indeed, the extent of these spectral variations was similar to that observed for the parental strain cells in the exponential phase (see Additional file 3: Figure S3a).
Overall, these results indicate that the OPI1 deletion has a direct effect on the levels of PC, as expected, and this in turn avoids the formation of protein aggregates, as indicated by the absence of the aggregate marker band around 1629 cm−1 in the presence of lactic acid (Additional file 4: Figures S4a, Additional file 5: S5a, Additional file 6: S6a). This finally correlates with an increased tolerance to the stressing agent (Fig. 2).
Moreover, as reported in Fig. 6a, when BY4741 opi1Δ cells collected in stationary phase were examined, it appeared evident how the exposure to 46 g/L of lactic acid led to a decrease in intensity of both alpha-helix/random coil (~1656 cm−1) and intramolecular beta-sheet (~1638 cm−1) bands, accompanied by the appearance of a shoulder around 1629 cm−1, due to protein aggregates. Surprisingly, compared to pH3 cells, a significant decrease of the ~1402 cm−1 band was found, indicating a PC reduction in pH3 + LA46 cells (Fig. 6b). We should, however, note that the PC reduction in opi1Δ cells was slightly lower compared to that monitored for the lactic acid treated parental cells (see Fig. 4b, Additional file 6: S6b, Additional file 7: S7). In addition, in this phase of growth a weak intensity reduction of the CH2 bands between 3050 and 2800 cm−1 (Fig. 6c) was still observed for lactic acid treated opi1Δ cells.
Concerning the cell wall carbohydrate components (see Additional file 3: Figure S3d), upon LA treatment we observed spectral changes quite similar to those observed for the parental strain cells in the exponential phase (Additional file 3: Figure S3a). In addition, interestingly, the intensity of the β1 → 6 glucan band was again found to be higher in the unchallenged cells at the stationary phase, compared to the LA treated cells.
Evaluation of unfolded protein response (UPR) under lactic acid exposure
As previously described, the growth advantage of BY4741 opi1Δ strain occurred during the exponential phase of growth (see Fig. 2). One of the main differences emerging from FTIR analysis is the phenomenon of protein aggregation, which in particular occurred at higher extent in the parental strain cells challenged with LA, compared to the opi1Δ strain (Figs. 3a, 5a, Additional file 5: S5a, Additional file 6: S6a) during this phase of growth.
Cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the so-called unfolded protein response (UPR). UPR is triggered by the presence of protein aggregates, and involves a signal transduction cascade from the endoplasmic reticulum to the nucleus [42]. It acts at different levels, by promoting the transcription of genes encoding for chaperones localized in this cellular compartment, such as BiP (Hsp70) and PDI (Protein Disulfide Isomerase), by accelerating the rate of degradation of misfolded proteins with the action of ERAD (Endoplasmic Reticulum Associated protein Degradation) and by decreasing protein synthesis [43].
Because of the protein aggregation observed in exponentially growing cells under lactic acid stress, the UPR activation was evaluated for all the strains by monitoring HAC1 mRNAs. Indeed, the transcription factor Hac1p is supposed to be the controller of the UPR in yeast. Cox and Walter [44] have identified two different forms of HAC1 mRNAs: the full length (969 base pairs), which is present in cells whether or not the UPR is induced; the shorter one (generated by the splicing of 251 base pairs from the full length mRNA form) that appears only when the UPR is induced by Ire1p.
Samples of BY4741 and BY4741 opi1Δ cells grown as previously described were collected 18 h after inoculation, mRNAs were isolated and treated for RT-PCR experiment with the specific amplification of the HAC1 cDNA (Fig. 7). In the presence of lactic acid (Fig. 7b), the full length and the spliced HAC1 mRNA are evident, indicating that the UPR is active in both strains. In the control condition, at pH3 without lactic acid (Fig. 7a), the shorter mRNA form is present only in the BY4741 opi1Δ strain, suggesting that in this strain the UPR mechanism is active even without the presence of the stressing agent.
Lactic acid and the triggering of lipid peroxidation
Lipid peroxidation is another of the reported effects of the weak organic counter-anions on S. cerevisiae cells [45], even if the triggering of this radical reaction was never reported for lactic acid exposure. Lipid peroxidation is a sudden molecular rearrangement that starts with the attack of a radical Reactive Oxygen Species (ROS) to a double bond of a polyunsaturated fatty acid, resulting in the formation of radical polyunsaturated fatty acids. These species, due to their high reactivity, can lead to the formation of several products including malondialdehyde (MDA), which can be, therefore, used as an index of lipid peroxidation level.
Here we were interested to determine if lipid peroxidation can occur after a sudden exposure to lactic acid. For this experiment, BY4741 and BY4741 opi1Δ cells were grown in minimal medium until the exponential phase was reached and then they were treated with a pulse of lactic acid (46 g/L at pH 3), and without the stressing agent at pH3 as control. After 30 min, cells were collected and the levels of MDA were evaluated (see “Methods”), (Fig. 8).
Unexpectedly, the presence of lactic acid correlates with a statistically significant decrease in peroxidized lipid content, phenomenon particularly pronounced in the deleted strain (13 and 37 % decrease for the BY4741 and BY4741 opi1Δ, respectively). In particular, in both tested conditions (with or without lactic acid stress) the peroxidized lipid content was statistically significant lower in the BY4741 opi1Δ strain compared to the parental strain (21 and 43 % decrease, respectively at pH3 and at pH3 with LA 46 g/L).