Growth In Acidic Environments Research Articles

Targeted Screening of Lactiplantibacillus plantarum Strains Isolated from Tomatoes and Its Application in Tomato Fermented Juice

This study explores the functional attributes of Lactiplantibacillus plantarum (L. plantarum) strains isolated from fermented tomato juice, focusing on their physiological, biochemical, and probiotic characteristics. The identified 66 gram-positive strains included 36 L. plantarum ones, which exhibited robust growth in acidic environments (pH 2.0–5.0) and utilization of various carbohydrates. Notably, seven strains outperformed a commercial strain in extreme acidic conditions. Antioxidant activity varied, with strain A24 showing the highest hydroxyl radical scavenging ability, while strains with high surface hydrophobicity had lower DPPH scavenging activity, indicating no direct correlation between these properties. Strains also showed strain-specific differences in carbohydrate utilization and antibiotic resistance, with some resistant to gentamicin and ciprofloxacin. Survival rates under simulated gastrointestinal conditions were strain-specific, with some strains demonstrating high survival rates, indicating their potential as probiotics. Furthermore, 13 strains used as fermentation starters in tomato juice significantly enhanced antioxidant activity and reduced pH and total soluble solids, indicating efficient sugar utilization and lactic acid production. These findings suggest that L. plantarum strains are well-suited for functional food fermentation and probiotic applications, with strain-specific traits offering versatility for use in acidic food products and probiotic formulations.

Molecular and Microscopic Methods of Quantifying Candida albicans Cell Wall PAMP Exposure.

The cell wall of Candida albicans is a multilayered structure consisting of polysaccharides and proteins. The inner cell wall layer is comprised of chitin and β1-3 and β1-6-glucan which contribute to the overall shape and structure of the cell, while the outer layer of highly glycosylated mannoproteins provides key functional traits such as cell adhesion required for virulence. However, the cell wall is not a static structure but is constantly being remodeled in response to the external environment. Given that all of the cell wall components act as pathogen-associated molecular patterns (PAMPs) that are recognized by a variety of receptors on the surface of innate immune cells, remodeling of the cell wall can have a dramatic impact on the host-pathogen interaction. For example, during growth in standard media, C. albicans shields its major cell wall PAMPs from the innate immune system, but during growth in acidic environments as encountered during colonization of the female reproductive tract, key PAMPs become exposed on the fungal cell surface initiating a strong pro-inflammatory innate immune response. The impact of environmental adaptation on fungal cell wall remodeling, and the subsequent impact this has on the host-pathogen interaction, has been the subject of much research. In this chapter, we outline techniques to assess cell wall components in both resting and environmentally adapted C. albicans cells.

Candida albicans.

Pathogens often face zinc restriction due to the action of nutritional immunity - host processes which restrict microbial access to key micronutrients such as zinc and iron. Candida albicans scavenges environmental zinc via two pathways. The plasma membrane transporter Zrt2 is essential for zinc uptake and growth in acidic environments. Neutralisation to pH 7 severely decreases the solubility of ionic Zn2+; this increase in pH triggers expression and activity of a second zinc scavenging system, the zincophore. This fungus-specific system consists of a secreted zinc-binding protein, Pra1, which captures zinc and returns to the cell via a syntenically expressed receptor, Zrt1. If present in excess, zinc is detoxified via a Zrc1-dependent mechanism. In C. albicans Zrc1 plays an important role in the generation of zincosomes. C. albicans faces both low and high zinc bottlenecks in vivo as Zrt2 and Zrc1 are required for kidney and liver colonisation, respectively, in a murine infection model.

Zwitterionic Polymer-Gated Au@TiO2 Core-Shell Nanoparticles for Imaging-Guided Combined Cancer Therapy.

With advances in nanoparticle (NP) synthesis and engineering, nanoscale agents with both therapeutic and diagnostic functions have been increasingly exploited for cancer management. Herein, we synthesized a new type of zwitterionic polymer-gated Au@TiO2 core-shell nanoparticles, which showed that they could selectively target and efficiently eliminate cancer cells via photothermal therapy (PTT), photodynamic therapy (PDT), pH/NIR-induced drug release, and cationic therapy.Methods: In the present study, the multifunctional therapeutic agent [Mn@P(CitAPDMAEMA)@Au@TiO2@DOX] was prepared to treat cancer with imaging-guided combination method. Firstly, Au@TiO2 core-shell nanoparticles (NPs) were synthesized. Taking advantage of broad and strong photoabsorption and reactive oxygen species (ROS) generation, Au@TiO2 core-shell NPs facilitated the single light-induced PTT and PDT. Next, a chemotherapy drug doxorubicin (DOX) was loaded into Au@TiO2 core-shell NPs. Then, a biocompatible zwitterionic polymer P(CitAPDMAEMA) was grafted to improve the hemocompatibility of NPs and prolong the circulation time. The polymer also served as a capping or switching material for pH-triggered drug release. In addition, the cationic nature of P(CitAPDMAEMA) eased the binding to human cervical cancer (HeLa) cells and effectively inhibited their growth in acidic environments (termed cationic therapy). Moreover, with Mn2+ ions immanently chelated, Mn@P(CitAPDMAEMA)@Au@TiO2@DOX NPs were able to provide enhanced contrast under T1- or T2-weighted magnetic resonance imaging (MRI).Results: The in vitro and in vivo anticancer experiments demonstrated the tumor was effectively inhibited with minimal side effects by the multifunctional NPs.Conclusions: As far as we know, this is the first presentation of four therapeutic methods into one nanomaterial, which will open up a new dimension for the design of combined treatment.

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt

Escherichia coli produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption.IMPORTANCE Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by Escherichia coli during aerobic growth on sugars. In E. coli, acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.

Chitin Increases Mycobacterium ulcerans Growth in Acidic Environments.

Species with a chitinous exoskeleton are overrepresented among the aquatic organisms carrying Mycobacterium ulcerans (MU) in nature and laboratory experiments have demonstrated the enhancing effects of chitin on the growth of MU. Field surveys identified pH as one of the key parameters delineating the distribution of MU in tropical regions. The present study investigated the relationship between chitin and pH in MU growth. By focusing on pH variations in the field, our results revealed that chitin enhanced MU growth in acidic environments. The present study provides new information on the ecological conditions favoring the development of this mycobacterium in nature.

Adaptation of Candida albicans to environmental pH induces cell wall remodelling and enhances innate immune recognition.

Candida albicans is able to proliferate in environments that vary dramatically in ambient pH, a trait required for colonising niches such as the stomach, vaginal mucosal and the GI tract. Here we show that growth in acidic environments involves cell wall remodelling which results in enhanced chitin and β-glucan exposure at the cell wall periphery. Unmasking of the underlying immuno-stimulatory β-glucan in acidic environments enhanced innate immune recognition of C. albicans by macrophages and neutrophils, and induced a stronger proinflammatory cytokine response, driven through the C-type lectin-like receptor, Dectin-1. This enhanced inflammatory response resulted in significant recruitment of neutrophils in an intraperitoneal model of infection, a hallmark of symptomatic vaginal colonisation. Enhanced chitin exposure resulted from reduced expression of the cell wall chitinase Cht2, via a Bcr1-Rim101 dependent signalling cascade, while increased β-glucan exposure was regulated via a non-canonical signalling pathway. We propose that this “unmasking” of the cell wall may induce non-protective hyper activation of the immune system during growth in acidic niches, and may attribute to symptomatic vaginal infection.

Improved Antimicrobial Potency through Synergistic Action of Chitosan Microparticles and Low Electric Field

Techniques to inhibit gram-negative bacteria such as Shiga toxin-producing Escherichia coli are valuable as the prevalence of large-scale industrial food preparation increases the likelihood of contamination. Chitosan, the deacetylated derivative of chitin, has been demonstrated to inhibit bacteria growth in acidic environments, but is significantly less effective in preventing bacteria grown at pH >7.0. Pulsed electric fields, constituting another method of bacteria inhibition, are difficult to generate at sufficient strength due to the high electric potentials required. This study utilizes adsorption of particulate chitosan in a very low electric field for an increased inhibition of gram-negative bacteria in neutral or alkaline pH conditions. Chitosan microparticles are demonstrated to flocculate E. coli, inhibit growth, and exhibit increased efficacy when combined with a low voltage electric field applied over 2-min intervals. Using sustained pulses of approximately 100 V/cm, it is demonstrated that bacteria viability is reduced by several orders of magnitude. The degree of bacterial inhibition is increased when chitosan microparticles are introduced to the system prior to imposing a small electric field.

Molecular Structure and Peptidoglycan Recognition of Mycobacterium tuberculosis ArfA (Rv0899)

Mycobacterium tuberculosis ArfA (Rv0899) is a membrane protein encoded by an operon that is required for supporting bacterial growth in acidic environments. Its C-terminal domain (C domain) shares significant sequence homology with the OmpA-like family of peptidoglycan-binding domains, suggesting that its physiological function in acid stress protection may be related to its interaction with the mycobacterial cell wall. Previously, we showed that ArfA forms three independently structured modules, and we reported the structure of its central domain (B domain). Here, we describe the high-resolution structure and dynamics of the C domain, we identify ArfA as a peptidoglycan-binding protein and we elucidate the molecular basis for its specific recognition of diaminopimelate-type peptidoglycan. The C domain of ArfA adopts the characteristic fold of the OmpA-like family. It exhibits pH-dependent conformational dynamics (with significant heterogeneity at neutral pH and a more ordered structure at acidic pH), which could be related to its acid stress response. The C domain associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved ArfA residues, including two key arginines, establish the specificity for diaminopimelate- but not Lys-type peptidoglycan. ArfA is the first peptidoglycan-binding protein to be identified in M. tuberculosis. Its functions in acid stress protection and peptidoglycan binding suggest a link between the acid stress response and the physicochemical properties of the mycobacterial cell wall.

Draft Genome of the Psychrotolerant Acidophile Acidithiobacillus ferrivorans SS3

Acidithiobacillus ferrivorans SS3 is a psychrotolerant acidophile capable of growth in the range of 5° to 30°C (optimum, ≈25°C). It gains energy from the oxidation of ferrous iron and inorganic sulfur compounds and obtains organic carbon from carbon dioxide. Here, we present the draft genome sequence of A. ferrivorans SS3 that will permit investigation of genes involved in growth in acidic environments at low temperatures.

AprABC: a Mycobacterium tuberculosis complex‐specific locus that modulates pH‐driven adaptation to the macrophage phagosome

Following phagocytosis by macrophages, Mycobacterium tuberculosis (Mtb) senses the intracellular environment and remodels its gene expression for growth in the phagosome. We have identified an acid and phagosome regulated (aprABC) locus that is unique to the Mtb complex and whose gene expression is induced during growth in acidic environments in vitro and in macrophages. Using the aprA promoter, we generated a strain that exhibits high levels of inducible fluorescence in response to growth in acidic medium in vitro and in macrophages. aprABC expression is dependent on the two-component regulator phoPR, linking phoPR signalling to pH sensing. Deletion of the aprABC locus causes defects in gene expression that impact aggregation, intracellular growth, and the relative levels of storage and cell wall lipids. We propose a model where phoPR senses the acidic pH of the phagosome and induces aprABC expression to fine-tune processes unique for intracellular adaptation of Mtb complex bacteria.

Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.

Acute extracellular acidification reduces intracellular pH, 42°C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42°C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42°C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42°C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42°C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42°C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.

Bicarbonate-dependent proton extrusion in Chinese hamster ovary (CHO) cells adapted to growth at pH 6.7

A CHO cell model is described for in vitro studies of the mechanisms underlying heat resistance in cells adapted to growth in acidic environments. Adaptation is defined as a loss of pH 6.7-induced sensitization to 42.0°C cytotoxicity and it is accompanied with an elevation of steady-state intracellular pH (pH). CHO cells cultured between 75 and 100 days at pH 6.7 became fully adapted (6.7G cells), and the adapted phenotype was maintained for at least 100 additional days of culture at pH 6.7. The surviving fraction (SF) of 6.7G cells heated (42.0°C) at pH 6.7 was comparable with that of cells cultured at pH 7.3 (7.3G cells) and heated at pH 7.3, while the SF of 7.3G cells acutely acidified to pH 6.7 and heated was an order of magnitude less. Although this resistance of 6.7G cells to killing was observed at 42.0°C, it was not observed at 43.0 and 45.0°C. Both 6.7G and 7.3G cells were able to develop comparable levels of thermotolerance during 42.0°C at their growth pHs. However, in agreement with the literature, development of thermotolerance was reduced in acutely acidified 7.3G cells. An acute acidification of only 0.2 pH unit from pH 6.7 to 6.5 also reduced the ability of 6.7G cells to develop thermotolerance during heating at 42.0°C. The acquired 6.7G phenotype reverted to the 7.3G phenotype following 17 days of culture at pH 7.3. Amiloride (0.5 mM), an inhibitor of the Na+/H+ exchanger (NHE), did not sensitize 7.3G and 6.7G cells to 42.0° at their growth pHs. However, sensitization was observed for acutely acidified 7.3G cells. This is consistent with the hypothesis that extracellular acute acidification causes a decrease in pHi, and that the recovery from that decrease is achieved in part by activation of the NHE. An elevation of steady-state pHi, measured by analysing intracellular BCECF excitation spectra, was documented in a suspension assay for cells grown at pH 6.7 for 180 days. The elevation was bicarbonate-dependent (negligible in the absence of HCO−3, +0.17 pH units in the presence of HCO−3). These results suggest that the altered regulation of pHi in CHO cells adapted to pHe 6.7 is maintained by bicarbonate-dependent processes.

Adaptation by Streptococcus mutans to acid tolerance.

Our previous continuous culture studies with strains of Streptococcus mutans have indicated that the organism has the capacity of adapt to growth in acidic environments. This study was undertaken to examine this question in more detail. S. mutans Ingbritt and the phosphotransferase system (PTS)-defective mutant, S. mutans DR0001/6, were grown in continuous culture at pH 7.5 and 5.5 or 5.1, and the pH optimum for glucose uptake and glycolysis and the capacity of the cells to generate pH gradients were determined over the pH range 4.5 to 8.0 with steady state, washed cells. In addition, the proton permeability of the cells was measured over the pH range by an acid pulse technique. The results indicate that the pH optimum for glucose uptake by S. mutans Ingbritt grown at pH 7.5 was 7.5 and this optimum shifted to 7.0 and 6.0 for cells grown at pH 5.5 and 5.1, whereas with the S. mutans DR0001/6, the optimum shifted from 7.5 to 7.0 for the pH 5.5 cells. A similar shift in the pH optimum for glycolysis was observed for the 2 strains, and this was particularly pronounced for cells incubated with glucose in the presence of gramicidin to dissipate proton gradients. The capacity of the cells to generate pH gradients was related to their metabolic activity, and although larger gradients were not formed by the pH 5.5 cells, these cells were nevertheless capable of maintaining gradients at a lower pH; S. mutans DR0001/6 generated 2-fold larger pH gradients at pH 5.5 than S. mutans Ingbritt.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture

Streptococcus mutans GS-5 and IB1600 adapted to growth in acidic environments in continuous culture at slow (generation time = 8.3 h) or fast (generation time = 2.4 h) rates of growth in complex medium with a restricted glucose supply. The extent of adaptation was indicated by changes in minimum pH values attained by harvested cells suspended in dense suspensions with excess glucose and by increased levels of ATPase activity assayed in permeabilized cells. Also, adapted cells better withstood potentially lethal acidification. Cells harvested from cultures growing at pH values close to 5 reduced suspension pH to lower values than cells from cultures maintained at pH 7. Cells from pH 6 cultures were intermediate. The IB1600 strain had a higher level of constitutive acid resistance than the GS-5 strain and also was better able to adapt to growth in acidified media. Both had less adaptive capacity than Enterococcus hirae ATCC 9790. Adaptation occurred rapidly, mainly within a single generation in continuous culture, while deadaptation occurred more slowly over multiple generations. The capacity of S. mutans to adapt to acid conditions is likely to be important in the ecology of dental plaque and also for the cariogenicity of the organism.