This work describes a coordinate and comprehensive view on the time course of the alterations occurring at the level of the cell wall during adaptation of a yeast cell population to sudden exposure to a sub-lethal stress induced by acetic acid. Acetic acid is a major inhibitory compound in industrial bioprocesses and a widely used preservative in foods and beverages. Results indicate that yeast cell wall resistance to lyticase activity increases during acetic acid-induced growth latency, corresponding to yeast population adaptation to sudden exposure to this stress. This response correlates with: (i) increased cell stiffness, assessed by atomic force microscopy (AFM); (ii) increased content of cell wall β-glucans, assessed by fluorescence microscopy, and (iii) slight increase of the transcription level of the GAS1 gene encoding a β-1,3-glucanosyltransferase that leads to elongation of (1→3)-β-d-glucan chains. Collectively, results reinforce the notion that the adaptive yeast response to acetic acid stress involves a coordinate alteration of the cell wall at the biophysical and molecular levels. These alterations guarantee a robust adaptive response essential to limit the futile cycle associated to the re-entry of the toxic acid form after the active expulsion of acetate from the cell interior.

Acetic acid is a major inhibitory compound in several industrial bioprocesses, in particular in lignocellulosic yeast biorefineries. Cell envelope remodeling, involving cell wall and plasma membrane composition, structure and function, is among the mechanisms behind yeast adaptation and tolerance to stress. Pdr18 is a plasma membrane ABC transporter of the pleiotropic drug resistance family and a reported determinant of acetic acid tolerance mediating ergosterol transport. This study provides evidence for the impact of Pdr18 expression in yeast cell wall during adaptation to acetic acid stress. The time-course of acetic-acid-induced transcriptional activation of cell wall biosynthetic genes (FKS1, BGL2, CHS3, GAS1) and of increased cell wall stiffness and cell wall polysaccharide content in cells with the PDR18 deleted, compared to parental cells, is reported. Despite the robust and more intense adaptive response of the pdr18Δ population, the stress-induced increase of cell wall resistance to lyticase activity was below parental strain levels, and the duration of the period required for intracellular pH recovery from acidification and growth resumption was higher in the less tolerant pdr18Δ population. The ergosterol content, critical for plasma membrane stabilization, suffered a drastic reduction in the first hour of cultivation under acetic acid stress, especially in pdr18Δ cells. Results revealed a crosstalk between plasma membrane ergosterol content and cell wall biophysical properties, suggesting a coordinated response to counteract the deleterious effects of acetic acid.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). A single recessive mutation, the deletion of phenylalanine 508 (F508del), causes severe CF and resides on 70% of mutant chromosomes. Disorganization of the actin cytoskeleton has been previously reported in relation to the CF phenotype. In this work, we aimed to understand this alteration by means of Atomic Force Microscopy and Force Feedback Microscopy investigation of mechanical properties of cystic fibrosis bronchial epithelial (CFBE) cells stably transduced with either wild type (wt-) or F508del-CFTR. We show here that the expression of mutant CFTR causes a decrease in the cell's apparent Young modulus as compared to the expression of the wt protein.

The influence that Burkholderia cenocepacia adaptive evolution during long-term infection in cystic fibrosis (CF) patients has on cell wall morphology and mechanical properties is poorly understood despite their crucial role in cell physiology, persistent infection and pathogenesis. Cell wall morphology and physical properties of three B. cenocepacia isolates collected from a CF patient over a period of 3.5 years were compared using atomic force microscopy (AFM). These serial clonal variants include the first isolate retrieved from the patient and two late isolates obtained after three years of infection and before the patient’s death with cepacia syndrome. A consistent and progressive decrease of cell height and a cell shape evolution during infection, from the typical rods to morphology closer to cocci, were observed. The images of cells grown in biofilms showed an identical cell size reduction pattern. Additionally, the apparent elasticity modulus significantly decreases from the early isolate to the last clonal variant retrieved from the patient but the intermediary highly antibiotic resistant clonal isolate showed the highest elasticity values. Concerning the adhesion of bacteria surface to the AFM tip, the first isolate was found to adhere better than the late isolates whose lipopolysaccharide (LPS) structure loss the O-antigen (OAg) during CF infection. The OAg is known to influence Gram-negative bacteria adhesion and be an important factor in B. cenocepacia adaptation to chronic infection. Results reinforce the concept of the occurrence of phenotypic heterogeneity and adaptive evolution, also at the level of cell size, form, envelope topography and physical properties during long-term infection.

Magnetite nanoparticles were synthesized by the co-precipitation method with and without the assistance of an additive, namely, gelatin, agar-agar or pectin, using eco-friendly conditions and materials embodying a green synthesis process. X-ray diffraction and transmission electron microscopy were used to analyze the structure and morphology of the nanoparticles. Magnetic properties were investigated by SQUID magnetometry and 57Fe Mössbauer spectroscopy. The results show that the presence of the additives implies a higher reproducibility of the morphological magnetic nanoparticle characteristics compared with synthesis without any additive, with small differences associated with different additives. To assess their potential for magnetic hyperthermia, water-based suspensions of these nanoparticles were prepared with and without citric acid. The stable solutions obtained were studied for their structural, magnetic and heating efficiency properties. The results indicate that the best additive for the stabilization of a water-based emulsion and better heating efficiency is pectin or a combination of pectin and agar-agar, attaining an intrinsic loss power of 3.6 nWg-1.