Acid-adapted Cells Research Articles

Acid adaptation increased the resistance of Escherichia coli O157:H7 in bok choy (Brassica rapa subsp. chinensis) juice to high-pressure processing.

Based on the U.S. Food and Drug Administration regulations, E. coli O157:H7 is a pertinent pathogen in high acid juices that needs to be inactivated during the pasteurization process. The results of this study suggest that the effect of acid adaptation should be considered in the selection of HPP parameters for E. coli O157:H7 inactivation to ensure that pasteurization objectives are achieved.

Chronic acidosis rewires cancer cell metabolism through PPARα signaling.

The mechanisms linking tumor microenvironment acidosis to disease progression are not understood. Here, we used mammary, pancreatic, and colon cancer cells to show that adaptation to growth at an extracellular pH (pHe ) mimicking acidic tumor niches is associated with upregulated net acid extrusion capacity and elevated intracellular pH at physiological pHe , but not at acidic pHe . Using metabolic profiling, shotgun lipidomics, imaging and biochemical analyses, we show that the acid adaptation-induced phenotype is characterized by a shift toward oxidative metabolism, increased lipid droplet-, triacylglycerol-, peroxisome content and mitochondrial hyperfusion. Peroxisome proliferator-activated receptor-α (PPARA, PPARα) expression and activity are upregulated, at least in part by increased fatty acid uptake. PPARα upregulates genes driving increased mitochondrial and peroxisomal mass and β-oxidation capacity, including mitochondrial lipid import proteins CPT1A, CPT2 and SLC25A20, electron transport chain components, peroxisomal proteins PEX11A and ACOX1, and thioredoxin-interacting protein (TXNIP), a negative regulator of glycolysis. This endows acid-adapted cancer cells with increased capacity for utilizing fatty acids for metabolic needs, while limiting glycolysis. As a consequence, the acid-adapted cells exhibit increased sensitivity to PPARα inhibition. We conclude that PPARα is a key upstream regulator of metabolic changes favoring cancer cell survival in acidic tumor niches.

Transcriptome changes and polymyxin resistance of acid-adapted Escherichia coli O157:H7 ATCC 43889

BackgroundAcid treatment is commonly used for controlling or killing pathogenic microorganisms on medical devices and environments; however, inadequate acid treatment may cause acid tolerance response (ATR) and offer cross-protection against environmental stresses, including antimicrobials. This study aimed to characterise an Escherichia coli strain that can survive in the acidic gastrointestinal environment.ResultsWe developed an acid-tolerant E. coli O157:H7 ATCC 43889 (ATCC 43889) strain that can survive at pH 2.75 via cell adaptation in low pH conditions. We also performed RNA sequencing and qRT-PCR to compare differentially expressed transcripts between acid-adapted and non-adapted cells. Genes related to stress resistance, including kdpA and bshA were upregulated in the acid-adapted ATCC 43889 strain. Furthermore, the polymyxin resistance gene arnA was upregulated in the acid-adapted cells, and resistance against polymyxin B and colistin (polymyxin E) was observed. As polymyxins are important antibiotics, effective against multidrug-resistant gram-negative bacterial infections, the emergence of polymyxin resistance in acid-adapted E. coli is a serious public health concern.ConclusionThe transcriptomic and phenotypic changes analysed in this study during the adaptation of E. coli to acid environments can provide useful information for developing intervention technologies and mitigating the risk associated with the emergence and spread of antimicrobial resistance.

Collagen production and niche engineering: A novel strategy for cancer cells to survive acidosis in DCIS and evolve.

Growing tumors are dynamic and nonlinear ecosystems, wherein cancer cells adapt to their local microenvironment, and these adaptations further modify the environment, inducing more changes. From nascent intraductal neoplasms to disseminated metastatic disease, several levels of evolutionary adaptations and selections occur. Here, we focus on one example of such an adaptation mechanism, namely, “niche construction” promoted by adaptation to acidosis, which is a metabolic adaptation to the early harsh environment in intraductal neoplasms. The avascular characteristics of ductal carcinoma in situ (DCIS) make the periluminal volume profoundly acidic, and cancer cells must adapt to this to survive. Based on discovery proteomics, we hypothesized that a component of acid adaptation involves production of collagen by pre‐cancer cells that remodels the extracellular matrix (ECM) and stabilizes cells under acid stress. The proteomic data were surprising as collagen production and deposition are commonly believed to be the responsibility of mesenchymally derived fibroblasts, and not cells of epithelial origin. Subsequent experiments in 3D culture, spinning disk and second harmonic generation microscopy of DCIS lesions in patients’ samples are concordant. Collagen production assay by acid‐adapted cells in vitro demonstrated that the mechanism of induction involves the RAS and SMAD pathways. Secretome analyses show upregulation of ECM remodeling enzymes such as TGM2 and LOXL2 that are collagen crosslinkers. These data strongly indicate that acidosis in incipient cancers induces collagen production by cancer cells and support the hypothesis that this adaptation initiates a tumor‐permissive microenvironment promoting survival and growth of nascent cancers.

Effect of acid adaptation on the resistance of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium to X-ray irradiation in apple juice

This study aimed to investigate the effect of acid adaptation on the resistance of Escherichia coli O157:H7 and Salmonella Typhimurium to X-ray irradiation. The non-acid-adapted and acid-adapted E. coli O157:H7 cell counts decreased 8.02 and 5.48 log CFU/mL in apple juice, respectively, whereas the S. Typhimurium cell counts reduced by 3.87 and 3.98 log CFU/mL, respectively, after treatment with 1.0 kGy X-ray. In apple juice, the D5d (dose required for 5-log reduction in cell count) values for non-acid-adapted and acid-adapted E. coli O157:H7 cells were 0.66 and 0.92 kGy, respectively, whereas those for non-acid-adapted and acid-adapted S. Typhimurium cells were 1.40 and 1.30 kGy, respectively. The acid-adapted E. coli O157:H7 cells exhibited higher X-ray resistance than the non-acid-adapted cells in apple juice. However, the acid-adapted and non-acid-adapted S. Typhimurium cells exhibited similar resistance to X-ray irradiation in apple juice. Additionally, the effect of X-ray irradiation on apple juice quality attributes was evaluated. The color, pH, and total phenolic contents of apple juice were not significantly affected (P > 0.05) at all treatment doses of X-ray. The results of this study suggest that the inactivation of acid-adapted cells of E. coli O157:H7, which is the predominant pathogen implicated in apple juice-related disease outbreaks, must be considered for the application of X-ray irradiation in apple juice sterilization process.

Viability of acid-adapted and non-adapted Bacillus cereus during the lactic fermentation of skim milk and product storage at 5°C

In this study, Bacillus cereus was subjected to acid adaptation at pH 6.3 for 40 min. It was found that acid-adapted B. cereus exhibited a higher survival percentage than non-adapted cells after exposure to an acidic condition of pH 4.6. The viable population of both acid-adapted and non-adapted B. cereus increased during the first 12-18 hr of lactic fermentation of skim milk by Streptococcus thermophilus or Lactobacillus bulgaricus, and then declined as the fermentation extended. However, the acid-adapted cells exhibited a lower population reduction rate than the non-adapted B. cereus. A higher viable population of acid-adapted B. cereus than the non-adapted cells was also noted in Str. thermophilus-fermented skim milk and the two commercial lactic fermented products kept at 5°C. Additionally, the acid adaptation could reduce the susceptibility of B. cereus to acidity, refrigerated temperature and other detrimental principles present in lactic fermented milk products.

Acidosis-induced metabolic reprogramming in tumor cells enhances the anti-proliferative activity of the PDK inhibitor dichloroacetate.

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness.

Phenotypic transition of tumor cells between epithelial- and mesenchymal-like state during adaptation to acidosis

ABSTRACT Acidic microenvironment is an important feature of solid tumors that contributes to malignant transformation. Low extracellular pH could promote epithelial-mesenchymal transition (EMT) thereby endowing tumor cells with higher invasive capability. However, the relation between EMT and tumor cell proliferation under long-term acidic condition is still not fully understood. Here, we show that tumor cells have undergone a phenotypic transition from EMT to mesenchymal-epithelial transition (MET) during adaptation to acidosis, and is closely related with cell proliferative state. Under early stage of acidic stress, tumor cells entered a non-cycling quiescent state with mesenchymal phenotype and expressed high level of stemness genes. Whereas, after long-term acid culture (2 months), acid-adapted cells resumed proliferating but lost mesenchymal phenotype. Further, our results show that the acid-adapted cells have distinct proliferative mechanism from non-acid cells, as the G1-S transcriptional factor E2F1 protein was not recovered in the adapted cells. Meanwhile, mini-chromosome maintenance 7 (MCM7) is shown to regulate the EMT to MET phenotypic transition, and is required for proliferation of the adapted cells under acidic condition. MCM7 Knockdown promoted mesenchymal phenotype and inhibited proliferation of the acid-adapted cells. While, MCM7 overexpression inhibited acid-induced EMT and supported tumor cell proliferation under acidic condition. Thus, our study provides evidence that tumor cells display phenotypic plasticity that allows them to survive acid stress.

Increased Resistance of Salmonella enterica Serovar Typhimurium and Escherichia coli O157:H7 to 222-Nanometer Krypton-Chlorine Excilamp Treatment by Acid Adaptation.

In this study, we examined the change in resistance of Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 to 222-nm krypton-chlorine (KrCl) excilamp treatment as influenced by acid adaptation and identified a mechanism of resistance change. In addition, we measured changes in apple juice quality indicators, such as color, total phenols, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, during treatment. Non-acid-adapted and acid-adapted pathogens were induced by growing the cells in tryptic soy broth without dextrose (TSB w/o D) at pH 7.3 and in TSB w/o D at pH 5.0 (adjusted with HCl), respectively. For the KrCl excilamp treatment, acid-adapted pathogens exhibited significantly (P < 0.05) higher D5d values, which indicate dosages required to achieve a 5-log reduction, than those for non-acid-adapted pathogens in both commercially clarified apple juice and phosphate-buffered saline (PBS), and the pathogens in the juice showed significantly (P < 0.05) higher D5d values than those for pathogens in PBS because of the UV-absorbing characteristics of apple juice. Through mechanism identification, it was found that the generation of lipid peroxidation in the cell membrane, inducing cell membrane destruction, was significantly (P < 0.05) lower in acid-adapted cells than in non-acid-adapted cells for the same amount of reactive oxygen species (ROS) generated at the same dose because the ratio of unsaturated to saturated fatty acids (USFA/SFA) in the cell membrane was significantly (P < 0.05) decreased as a result of acid adaptation. Treated apple juice showed no significant (P > 0.05) difference in quality indicators compared to those of untreated controls during treatment at 1,773 mJ/cm2IMPORTANCE There is a need for novel, mercury-free UV lamp technology to replace germicidal lamps containing harmful mercury, which are routinely utilized for UV pasteurization of apple juice. In addition, consideration of the changes in response to antimicrobial treatments that may occur when pathogens are adapted to the acid in an apple juice matrix is critical to the practical application of this technology. Based on this, an investigation using 222-nm KrCl excilamp technology, an attractive alternative to mercury lamps, was conducted. Our study demonstrated increased resistance to 222-nm KrCl excilamp treatment as pathogens adapted to acids, and this was due to changes in reactivity to ROS with changes in the fatty acid composition of the cell membrane. Despite increased resistance, the 222-nm KrCl excilamp achieved pathogen reductions of 5 log or more at laboratory scale without affecting apple juice quality. These results provide valuable baseline data for application of 222-nm KrCl excilamps in the apple juice industry.

A drug screening assay on cancer cells chronically adapted to acidosis

BackgroundDrug screening for the identification of compounds with anticancer activity is commonly performed using cell lines cultured under normal oxygen pressure and physiological pH. However, solid tumors are characterized by a microenvironment with limited access to nutrients, reduced oxygen supply and acidosis. Tumor hypoxia and acidosis have been identified as important drivers of malignant progression and contribute to multicellular resistance to different forms of therapy. Tumor acidosis represents an important mechanism mediating drug resistance thus the identification of drugs active on acid-adapted cells may improve the efficacy of cancer therapy.MethodsHere, we characterized human colon carcinoma cells (HCT116) chronically adapted to grow at pH 6.8 and used them to screen the Prestwick drug library for cytotoxic compounds. Analysis of gene expression profiles in parental and low pH-adapted cells showed several differences relating to cell cycle, metabolism and autophagy.ResultsThe screen led to the identification of several compounds which were further selected for their preferential cytotoxicity towards acid-adapted cells. Amongst 11 confirmed hits, we primarily focused our investigation on the benzoporphyrin derivative Verteporfin (VP). VP significantly reduced viability in low pH-adapted HCT116 cells as compared to parental HCT116 cells and normal immortalized epithelial cells. The cytotoxic activity of VP was enhanced by light activation and acidic pH culture conditions, likely via increased acid-dependent drug uptake. VP displayed the unique property to cause light-dependent cross-linking of proteins and resulted in accumulation of polyubiquitinated proteins without inducing inhibition of the proteasome.ConclusionsOur study provides an example and a tool to identify anticancer drugs targeting acid-adapted cancer cells.

Influence of acid and low-temperature adaptation on pulsed electric fields resistance of Enterococcus faecium in media of different pH

Abstract This investigation was undertaken to study the inactivation of Enterococcus faecium ATCC 49624 by pulsed electric fields (PEF) treatments after exposure to sublethal acid and cold stresses. Cells were grown in Brain Heart Infusion (BHI) buffered to pH 7.4 (non-acid-adapted cells) and BHI acidified to pH 5.4 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids (acid-adapted cells). Fresh and refrigerated cultures (kept at 4 °C for up to 1 month) were treated by PEF at 37 kV/cm in citrate-phosphate buffer of different pH (4.0, 5.5 and 7.0), at specific energies ranging from 79 to 1580 kJ/kg. Results demonstrated that acid adaptation exerted a different effect on the PEF resistance of E. faecium depending on the pH of the treatment medium. Thus, when treated in a medium of pH 7.0, previous acid adaptation, regardless of the type of acidulant used, reduced the calculated δ values (specific energies required to inactivate the first log10 cycle of the population) by approximately two-fold. By contrast, in a medium of pH 4.0, acid-adapted cells showed a higher PEF resistance than non-adapted cells. On the other hand, exposure to 4 °C reduced the PEF resistance of acid-adapted cells, but not that of non-acid-adapted cells. Results obtained in this study suggested that, after the same PEF treatments, the number of electroporated cells was lower for non-acid-adapted cells, but that acid-adapted cells displayed a superior ability to repair sublethal damages caused by PEF. Nevertheless, exposure to refrigeration conditions would significantly reduce this damage repair ability. Finally, FT-IR spectroscopy measurements indicated that growth of E. faecium cells under acid conditions caused an increase in membrane fluidity. It was also observed that refrigerated storage resulted in a significant (p Industrial relevance Given the inability of PEF to inactivate spores, pasteurization of acid products such as fruit juices appears as one of the more feasible applications for this technology. Microbial cells present in these products would have, very likely, undergone a process of adaptation to this acid pH; however, the impact of this phenomenon on microbial PEF resistance has only been scarcely investigated. Thus, the objective of this work was to study the inactivation of Enterococcus faecium by PEF treatments after sequential exposure to sublethal acid and cold stresses. This work will provide very valuable data for process design to further develop PEF application and improve understanding of microbial inactivation.

Abstract 3538: Enhanced dependence on lipid metabolism is a cellular adaptation to acidic microenvironment

Abstract Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in highly acidic microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and thrive and to out-compete the stroma into which they invade. Acid adaptation is associated with chronic activation of autophagy as well as redistribution of the lysosomal proteins to the plasma membrane. These processes are major survival mechanisms adopted by tumor cells under acidic conditions. We have also observed that under acidic conditions, there is a rapid, reversible and robust increase in the accumulation of cytoplasmic lipid droplets (adiposomes) in a panel of breast cancer cells. Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins and a phospholipid monolayer. Breast cancer cells when grown in acidic media accumulated adiposomes as revealed by nile red and perilipin-2 staining followed by confocal microscopy. The acid-induced lipogenic phenotype persists even when the cells are grown in de-lipidated serum, indicating that the source of lipids is de-novo and endogenous. When cells were treated with inhibitors of fatty acid synthesis such as TOFA, an inhibitor of Acetyl CoA Carboxylase or FAS inhibitor C75, adiposome formation at low pH was attenuated. Inhibition of either lipid anabolic or catabolic pathways was specifically cytotoxic in acid-adapted cells, but not in control cells where the FAS inhibitor, C75, is selectively toxic under acidic conditions. Additionally, when treated with etomoxir, an inhibitor of carnitine palmitoyltransferase 1, the rate limiting step in βeta-oxidation, acid adapted cells showed increased sensitivity. To investigate the role of de novo fatty acid synthesis further, we employed high resolution NMR spectroscopy to measure 13C enriched lactate isotopomers following metabolism of D-[1,2-13C] glucose. These analyses showed that glucose flux through the pentose phosphate pathway (PPP) was significantly (&amp;gt;2.5 fold) higher in low pH exposed cells, compared to controls, representing a major shift in glucose metabolism from Embden Meyerhof to PPP, which results in increased production of NADPH, necessary for de novo lipid synthesis. Additional metabolic profiling using the Seahorse XF revealed that cells at low pH had higher rates of oxygen consumption (OCR) and that this was reversible. Further, we investigated the role of various acid sensing G-protein coupled receptors such as OGR1, TDAG8 and GPR4 in transducing the acid signal that results in the accumulation of lipid droplets. CRISPR/Cas9 mediated depletion of these receptors indicates that they might play a major role in inducing adiposome accumulation under acidic conditions. Taken together, increased dependence on lipid metabolism by cancer cells under acidic conditions reveals novel therapeutic vulnerabilities. Citation Format: Smitha Pillai, Jonathan W. Wojtkowiak, Mehdi Damaghi, Robert Gatenby, Robert Gillies. Enhanced dependence on lipid metabolism is a cellular adaptation to acidic microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3538. doi:10.1158/1538-7445.AM2017-3538

Amino acid decarboxylase-dependent acid tolerance, selected phenotypic, and virulence gene expression responses of Salmonella enterica serovar Heidelberg

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the pathogens most frequently detected in recent years in food products from animals; here, we examine the acid tolerance of this strain. Mild acid (pH5.5–5.0) induced a strong acid resistance in S. Heidelberg; the induced acid resistance improved within 0.5–6h and resulted in >95% cell survival following challenge in pH3.0 medium. Addition of lysine or arginine to pH2.5 acid challenge medium significantly improved the survival of S. Heidelberg; lysine induced the largest increase in survival. The lysine and arginine decarboxylase-related genes (i.e., cadA, cadB, adiA, and adiY) are acid induced genes, and they play an important role in S. Heidelberg acid resistance. RT-PCR showed that the genes expression levels increased as acid adaptation pH decreased. The increased expression was maintained for at least 4h during adaptation. Moreover, acid adaptation may have increased the production of cellulose and swimming in S. Heidelberg; pH5.5 acid-adapted cells showed a red, dry, and rough morphotype on Congo red plates. The transcriptional levels of Enterotoxin gene (stn) increased three times following acid adaptation; however, the expression of Salmonella pathogenicity island 1 virulence genes significantly decreased.

Phytic Acid and Sodium Chloride Show Marked Synergistic Bactericidal Effects against Nonadapted and Acid-Adapted Escherichia coli O157:H7 Strains.

The synergistic antimicrobial effects of phytic acid (PA), a natural extract from rice bran, plus sodium chloride against Escherichia coli O157:H7 were examined. Exposure to NaCl alone at concentrations up to 36% (wt/wt) for 5 min did not reduce bacterial populations. The bactericidal effects of PA alone were much greater than those of other organic acids (acetic, citric, lactic, and malic acids) under the same experimental conditions (P < 0.05). Combining PA and NaCl under conditions that yielded negligible effects when each was used alone led to marked synergistic effects. For example, whereas 0.4% PA or 3 or 4% NaCl alone had little or no effect on cell viability, combining the two completely inactivated both nonadapted and acid-adapted cells, reducing their numbers to unrecoverable levels (>7-log CFU/ml reduction). Flow cytometry confirmed that PA disrupted the cell membrane to a greater extent than did other organic acids, although the cells remained viable. The combination of PA and NaCl induced complete disintegration of the cell membrane. By comparison, none of the other organic acids acted synergistically with NaCl, and neither did NaCl-HCl solutions at the same pH values as the test solutions of PA plus NaCl. These results suggest that PA has great potential as an effective bacterial membrane-permeabilizing agent, and we show that the combination is a promising alternative to conventional chemical disinfectants. These findings provide new insight into the utility of natural compounds as novel antimicrobial agents and increase our understanding of the mechanisms underlying the antibacterial activity of PA.

Physiological and transcriptional responses and cross protection of Lactobacillus plantarum ZDY2013 under acid stress

Acid tolerance responses (ATR) in Lactobacillus plantarum ZDY2013 were investigated at physiological and molecular levels. A comparison of composition of cell membrane fatty acids (CMFA) between acid-challenged and unchallenged cells showed that acid adaptation evoked a significantly higher percentage of saturated fatty acids and cyclopropane fatty acids in acid-challenged than in unchallenged cells. In addition, reverse transcription-quantitative PCR analysis in acid-adapted cells at different pH values (ranging from 3.0 to 4.0) indicated that several genes were differently regulated, including those related to proton pumps, amino acid metabolism, sugar metabolism, and class I and class III stress response pathways. Expression of genes involved in fatty acid synthesis and production of alkali was significantly upregulated. Upon exposure to pH 4.5 for 2 h, a higher survival rate (higher viable cell count) of Lactobacillus plantarum ZDY2013 was achieved following an additional challenge to 40 mM hydrogen peroxide for 60min, but no difference in survival rate of cells was found with further challenge to heat, ethanol, or salt. Therefore, we concluded that the physiological and metabolic changes of acid-treated cells of Lactobacillus plantarum ZDY2013 help the cells resist damage caused by acid, and further initiated global response signals to bring the whole cell into a state of defense to other stress factors, especially hydrogen peroxide.

Abstract 1265: LAMP2 overexpression in the plasma membrane of breast cancer cells in response of chronic acidosis as a new imaging and therapeutic target

Abstract A combination of poor vasculature perfusion, hypoxia, and increased flux of carbons through fermentative glycolysis lead to extracellular acidosis in solid tumors; with extracellular pH values as low as 6.5. The proton concentration increases within the lumen due to diffusion limitations and increased production of acid from hypoxic-glycolytic cells, causing the interior of the lumen to become highly acidic. The glycolytic phenotype can become “hardwired” as Warburg proposed, leading to the continued generation of metabolic acids even in well-oxygenated conditions. This acidified habitat is constant and imparts a Darwinian selection pressure that favors cells that have adapted mechanisms to be resistant to acid mediated apoptosis, and this likely increases with increasing cancer stage. In order to understand tumor acidity, we investigated protein profiles of acid-adapted and non-adapted breast cancer cells (MCF-7) using SILAC-proteomics followed by LC-MRM confirmation. A large number of lysosomal proteins were observed to be upregulated in acid-adapted cells and, among these was the lysosome associated membrane protein 2 (LAMP2) confirmed by MRM. This protein is of particular interest because it is heavily glycosylated and functions to protect lysosomal membrane from acid proteolysis. Using independent in vitro as well as in vivo approaches such as intravital fluorescent microscopy of dorsal window chamber mouse xenografts, we observed that LAMP2 was associated with acid-adaptation. In vivo, treatment of animals with bicarbonate to reduce tumor acidity led to reductions in LAMP2. Further, mRNA-expression profiling (microarray) of patient tumor samples and IHC of Tissue Microarray (TMA) of breast cancer patient tissue samples, revealed a high correlation of LAMP2 expression with progression and metastasis of breast tumors. We also applied LAMP2 staining on the whole breast tumor samples of different stages and observed that LAMP2 was associated with the peri-luminal regions of DCIS, as well as regions of local invasion, which are expected to be acidic. Finally, we observed both in tumors by IHC and subsequently in acid adapted cells by ICC that, while LAMP2 was localized in lysosomes and late endosomes under normal pH conditions, it was redistributed to the plasma membrane under acidic pH conditions. To further investigate this cell membrane localization we carried out western blot on membrane extract of acid adapted cells. Based on these observations, we propose a novel cell defense mechanism wherein cells chronically exposed to an acidic environment translocate LAMP2 to their surface, which protects the plasmalemma via an unstirred layer effect. To prove the role of LAMP2 on acid adaptation we did shRNA and siRNA knockdown followed by in vitro and in vivo acid exposure of the cells. We further propose that this pathway may provide a novel therapeutic target against breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Mehdi Damaghi, Robert Sprung, Narges Tafreshi, Veronica Estrella, John Koomen, David Morse, Robert Gillies. LAMP2 overexpression in the plasma membrane of breast cancer cells in response of chronic acidosis as a new imaging and therapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1265. doi:10.1158/1538-7445.AM2015-1265

Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH.

This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells.

Effect of Acid Adaptation and Acid Shock on Thermal Tolerance and Survival of Escherichia coli O157:H7 and O111 in Apple Juice

Gradual exposure to moderate acidic environments may enhance the thermal tolerance and survival of Escherichia coli O157:H7 in acid and acidified foods. Limited studies comparing methodologies to induce this phenomenon have been performed. The effects of strain and physiological state on thermal tolerance and survival of E. coli in apple juice were studied. The decimal reduction time (D-value) at 56°C [D56°C] was determined for E. coli O157:H7 strains C7927 and ATCC 43895 and E. coli O111 at four physiological states: unadapted, acid-shocked (two methodologies used), and acid-adapted cells. The effect of acidulant was also evaluated by determining the D56°C for the O157:H7 strains subjected to acid shock during 18 h in Trypticase soy broth (TSB), with pH 5 adjusted with hydrochloric, lactic, and malic acids. Survival of the three strains at four physiological states was determined at 1 ± 1°C and 24 ± 2°C. Experiments were performed in triplicate. For thermal inactivation, a significant interaction was found between strain and physiological state (P < 0.0001). Highest thermal tolerance was observed for the 43895 strain subjected to acid shock during 18 h in TSB acidified with HCl (D56°C of 3.0 ± 0.1 min) and the lowest for the acid-shocked C7927 strain treated for 4 h in TSB acidified with HCl (D56°C of 0.45 ± 0.06 min). Acidulants did not alter the heat tolerance of strain C7927 (D56°C of 1.9 ± 0.1 min; P > 0.05) but significantly affected strain 43895 (P < 0.05), showing the greatest tolerance when malic acid was used (D56°C of 3.7 ± 0.3 min). A significant interaction between strain, storage temperature, and physiological state was noted during the survival experiments (P < 0.05). E. coli O111 was the most resistant strain, surviving 6 and 23 days at 24 and 1°C, respectively. Our findings may assist in designing challenge studies for juices and other pH-controlled products, where Shiga toxin-producing E. coli represents the pathogen of concern.

Physiological and proteomic analysis of Lactobacillus casei in response to acid adaptation.

The aim of this study was to investigate the acid tolerance response (ATR) in Lactobacillus casei by a combined physiological and proteomic analysis. To optimize the ATR induction, cells were acid adapted for 1h at different pHs, and then acid challenged at pH 3.5. The result showed that acid adaptation improved acid tolerance, and the highest survival was observed in cells adapted at pH 4.5 for 1h. Analysis of the physiological data showed that the acid-adapted cells exhibited higher intracellular pH (pHi), intracellular NH4 (+) content, and lower inner permeability compared with the cells without adaptation. Proteomic analysis was performed upon acid adaptation to different pHs (pH 6.5 vs. pH 4.5) using two-dimensional electrophoresis. A total of 24 proteins that exhibited at least 1.5-fold differential expression were identified. Four proteins (Pgk, LacD, Hpr, and Galm) involved in carbohydrate catabolism and five classic stress response proteins (GroEL, GrpE, Dnak, Hspl, and LCAZH_2811) were up-regulated after acid adaptation at pH 4.5 for 1h. Validation of the proteomic data was performed by quantitative RT-PCR, and transcriptional regulation of all selected genes showed a positive correlation with the proteomic patterns of the identified proteins. Results presented in this study may be useful for further elucidating the acid tolerance mechanisms and may help in formulating new strategies to improve the industrial performance of this species during acid stress.

Thermal Resistance Parameters of Acid-Adapted and Unadapted Escherichia coli O157:H7 in Apple-Carrot Juice Blends: Effect of Organic Acids and pH

Numerous outbreaks involving fresh juices contaminated with Escherichia coli O157:H7 have occurred in the United States and around the world, raising concern for the safety of these products. Until now, only a few studies regarding the thermal tolerance of this pathogen in acidic juices over a wide range of pH values have been published. Therefore, the effect of varying the pH with different organic acids on the thermal inactivation of non-acid-adapted and acid-adapted E. coli O157:H7 (strain C7927) was determined. The decimal reduction times (D-values) and the change in temperature required for the thermal destruction curve to traverse 1 log cycle (z-values) were calculated for non-acid-adapted E. coli in an apple-carrot juice blend (80:20) adjusted to three pH values (3.3, 3.5, and 3.7) by the addition of lactic, malic, or acetic acid and at a pH of 4.5 adjusted with NaOH. Thermal parameters were also determined for acid-adapted cells in juices acidified with malic acid. The effect of the soluble solids content on the thermal tolerance was studied in samples with a pH of 3.7 at 9.4 to 11.5 °Brix. The D-values were determined at 54, 56, and 58 °C, and trials were conducted in triplicate. Non-acid-adapted E. coli exhibited the highest thermal tolerance at pH 4.5 (D-value at 54 °C [D54 °C] of 20 ± 4 min and z-value of 6.2 °C), although on average, the D-values increased significantly (P < 0.01) due to acid adaptation. In acidified juices, the highest tolerance was observed in acid-adapted E. coli in samples adjusted to pH 3.7 with malic acid (D54 °C of 9 ± 2 min and z-value of 5.4 °C) and the lowest in unadapted E. coli at pH 3.3 acidified with acetic acid (D58 °C of 0.03 ± 0.01 min and z-value of 10.4 °C). For juices acidified to the same endpoint pH with different acids, E. coli was found to be more tolerant in samples acidified with malic acid, followed by lactic and acetic acids. Increasing the soluble solids content from 9.4 to 11.5 °Brix showed no significant effect on the thermal tolerance of E. coli (P > 0.01). The data from this study will be useful for establishing critical limits for safe thermal processing of pH-controlled juices and similar products.