Metabolic reprogramming in the food-borne pathogen Listeria monocytogenes as a critical defence against acid stress.

The ability to sense and respond effectively to acidic stress is important for microorganisms to survive and proliferate in fluctuating environments. As specific metabolic activities can serve to buffer the cytoplasmic pH, microorganisms re-wire their metabolism to favour these reactions and thereby mitigate acid stress. The orally-acquired pathogen Listeria monocytogenes exploits alternative metabolic activities to overcome the acidic stress encountered in the human stomach or food products. In this minireview, we discuss the metabolic processes in L. monocytogenes that mitigate acid stress, with an emphasis on the proton-depleting reactions including glutamate decarboxylation, arginine/agmatine deimination, and fermentative acetoin production. We also summarize the recent findings on regulatory mechanisms that control the expression of genes that are responsible for these metabolic activities, including the general stress response regulator SigB, arginine repressor ArgR, and the recently discovered RofA-like transcriptional regulatory GadR. We further discuss the importance of this metabolic reprogramming in the context of food products and within the host. Finally, we highlight some outstanding challenges in the field including an understanding of acid-sensing mechanisms, the role of intra-species heterogeneity in acid resistance, and how a fundamental understanding of acid stress response can be exploited for food formulation to improve food safety and reduce food waste.

Gain-of-function variants in CLCN7 cause hypopigmentation and lysosomal storage disease.

Together with its ß-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.

Alkalimonas mucilaginosa sp. nov. and Alkalimonas cellulosilytica sp. nov. isolated from alkaline Lonar lake, India.

Two alkaliphilic, Gram-stain-negative bacterial strains (MEB004T and MEB108T) were isolated from water samples collected from Lonar lake, India. The phylogenetic analysis of their 16S rRNA gene sequences showed the highest similarity to A. delamerensis DSM 18314T (98.4%), followed by A. amylolytica DSM 18337T and A. collagenimarina JCM 14267T (97.9%). The genome sizes of strains MEB004T and MEB108T were determined to be 3,858,702 and 4,029,814 bp, respectively, with genomic DNA G + C contents of 51.4 and 51.9%. Average Nucleotide Identity, DNA-DNA Hybridization and Amino Acid Identity values between strains (MEB004T and MEB108T) and A. amylolytica DSM 18337T were (82.3 and 85.5), (25.0 and 29.2) and (86.7 and 90.2%). Both novel strains produced industrially important enzymes, such as amylase, lipase, cellulase, caseinase, and chitinase at pH 10 evidenced by the genomic presence of carbohydrate-active enzymes encoding genes. Genomic analyses further identified pH tolerance genes, affirming their adaptation to alkaline Lonar Lake. Dominant fatty acids were Summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, Summed feature 3, Sum In Feature 2 and C12:0 3OH. The prevalent polar lipids included phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. The major respiratory quinone was ubiquinone-8. Based on the polyphasic data, we propose the classification of strains MEB004T and MEB108T as novel species within the genus Alkalimonas assigning the names Alkalimonas mucilaginosa sp. nov. and Alkalimonas cellulosilytica sp. nov., respectively. The type strains are MEB004T (= MCC 5208T = JCM 35954T = NCIMB 15460T) and MEB108T (= MCC 5330T = JCM 35955T = NCIMB 15461T).

Evaluation of Pyrophosphate-Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions.

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L-1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

Rho-dependent termination enables cellular pH homeostasis.

The termination factor Rho, an ATP-dependent RNA translocase, preempts pervasive transcription processes, thereby rendering genome integrity in bacteria. Here, we show that the loss of Rho function raised the intracellular pH to >8.0 in Escherichia coli. The loss of Rho function upregulates tryptophanase-A (TnaA), an enzyme that catabolizes tryptophan to produce indole, pyruvate, and ammonia. We demonstrate that the enhanced TnaA function had produced the conjugate base ammonia, raising the cellular pH in the Rho-dependent termination defective strains. On the other hand, the constitutively overexpressed Rho lowered the cellular pH to about 6.2, independent of cellular ammonia levels. Since Rho overexpression may increase termination activities, the decrease in cellular pH could result from an excess H+ ion production during ATP hydrolysis by overproduced Rho. Furthermore, we performed in vivo termination assays to show that the efficiency of Rho-dependent termination was increased at both acidic and basic pH ranges. Given that the Rho level remained unchanged, the alkaline pH increases the termination efficiency by stimulating Rho's catalytic activity. We conducted the Rho-mediated RNA release assay from a stalled elongation complex to show an efficient RNA release at alkaline pH, compared to the neutral or acidic pH, that supports our in vivo observation. Whereas acidic pH appeared to increase the termination function by elevating the cellular level of Rho. This study is the first to link Rho function to the cellular pH homeostasis in bacteria. IMPORTANCE The current study shows that the loss or gain of Rho-dependent termination alkalizes or acidifies the cytoplasm, respectively. In the case of loss of Rho function, the tryptophanase-A enzyme is upregulated, and degrades tryptophan, producing ammonia to alkalize cytoplasm. We hypothesize that Rho overproduction by deleting its autoregulatory DNA portion increases termination function, causing excessive ATP hydrolysis to produce H+ ions and cytoplasmic acidification. Therefore, this study is the first to unravel a relationship between Rho function and intrinsic cellular pH homeostasis. Furthermore, the Rho level increases in the absence of autoregulation, causing cytoplasmic acidification. As intracellular pH plays a critical role in enzyme function, such a connection between Rho function and alkalization will have far-reaching implications for bacterial physiology.

How do plants maintain pH and ion homeostasis under saline-alkali stress?

Salt and alkaline stresses often occur together, severely threatening plant growth and crop yields. Salt stress induces osmotic stress, ionic stress, and secondary stresses, such as oxidative stress. Plants under saline-alkali stress must develop suitable mechanisms for adapting to the combined stress. Sustained plant growth requires maintenance of ion and pH homeostasis. In this review, we focus on the mechanisms of ion and pH homeostasis in plant cells under saline-alkali stress, including regulation of ion sensing, ion uptake, ion exclusion, ion sequestration, and ion redistribution among organs by long-distance transport. We also discuss outstanding questions in this field.

pH sensing at the intersection of tissue homeostasis and inflammation.

pH is tightly maintained at cellular, tissue, and systemic levels, and altered pH - particularly in the acidic range - is associated with infection, injury, solid tumors, and physiological and pathological inflammation. However, how pH is sensed and regulated and how it influences immune responses remain poorly understood at the tissue level. Applying conceptual frameworks of homeostatic and inflammatory circuitries, we categorize cellular and tissue components engaged in pH regulation, drawing parallels from established cases in physiology. By expressing various intracellular (pHi) and extracellular pH (pHe)-sensing receptors, the immune system may integrate information on tissue and cellular states into the regulation of homeostatic and inflammatory programs. We introduce the novel concept of resistance and adaptation responses to rationalize pH-dependent immunomodulation intertwined with homeostatic equilibrium and inflammatory control. We discuss emerging challenges and opportunities in understanding the immunological roles of pH sensing, which might reveal new strategies to combat inflammation and restore tissue homeostasis.

Taxogenomics of Alkalihalobacterium chitinilyticum sp. nov.: an alkaliphilic chitin degrading bacterial strain isolated from Lonar Lake, India, with potential biotechnological applications.

A novel chitin degrading alkaliphilic bacterial strain (MEB 203 T) was isolated from sediment collected from Lonar lake, India. The strain exhibited its maximum growth at a temperature of 37 °C, with an optimal pH of 10 and a NaCl concentration of 2%. 16S rRNA gene based phylogenetic tree showed that strain was closely related to Alkalihalobacterium elongatum MCC 2982 T (98.64% similarity) followed by A. alkalinitrilicum DSM 22532 T (97.84% similarity). The genome size was 4.9 Mb with DNA G + C content of 37.7%. The dDDH value between strain MEB 203 T and A. elongatum MCC 2982 T was 26.4 ± 2.4% while OrthoANI value was 82.1%. Genome analysis revealed the presence of genes responsible for L-ectoine and cation/proton antiporter which may facilitate growth of strain in alkaline-saline habitat of Lonar lake. Strain MEB 203 T was able to utilize complex sugars such as chitin, cellulose, and starch as a carbon source at alkaline conditions which was also corroborated from the genomic presence of carbohydrate active enzymes (CAZymes). It was also able to produce biotechnologically important enzymes such as lipases and proteases which were stable at pH (9-10). The bacterium is majorly composed of C15:0 iso, C16:0 iso, and C17:0 iso (> 10%) fatty acids while diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipid (PL3) were identified as the predominant polar lipids. Based on differential physiological, biochemical, and genomic features of strain MEB 203 T, a novel species Alkalihalobacterium chitinilyticum sp. nov. (Type strain MEB 203 T = MCC 3920 T = NCIMB 15407 T = JCM 35078 T) is proposed.

Significance of the plasma membrane H+-ATPase and V-ATPase for growth and pathogenicity in pathogenic fungi.

Pathogenic fungi are widespread and cause a variety of diseases in human beings and other organisms. At present, limited classes of antifungal agents are available to treat invasive fungal diseases. With the wide use of the commercial antifungal agents, drug resistance of pathogenic fungi are continuously increasing. Therefore, exploring effective antifungal agents with novel drug targets is urgently needed to cope with the challenges that the antifungal area faces. pH homeostasis is vital for multiple cellular processes, revealing the potential for defining novel drug targets. Fungi have evolved a number of strategies to maintain a stable pH internal environment in response to rapid metabolism and a dramatically changing extracellular environment. Among them, plasma membrane H+-ATPase (PMA) and vacuolar H+-ATPase (V-ATPase) play a central role in the regulation of pH homeostasis system. In this chapter, we will summarize the current knowledge about pH homeostasis and its regulation mechanisms in pathogenic fungi, especially for the recent advances in PMA and V-ATPase, which would help in revealing the regulating mechanism of pH on cell growth and pathogenicity, and further designing effective drugs and identify new targets for combating fungal diseases.

Alkalinity modulates a unique suite of genes to recalibrate growth and pH homeostasis.

Alkaline soils pose a conglomerate of constraints to plants, restricting the growth and fitness of non-adapted species in habitats with low active proton concentrations. To thrive under such conditions, plants have to compensate for a potential increase in cytosolic pH and restricted softening of the cell wall to invigorate cell elongation in a proton-depleted environment. To discern mechanisms that aid in the adaptation to external pH, we grew plants on media with pH values ranging from 5.5 to 8.5. Growth was severely restricted above pH 6.5 and associated with decreasing chlorophyll levels at alkaline pH. Bicarbonate treatment worsened plant performance, suggesting effects that differ from those exerted by pH as such. Transcriptional profiling of roots subjected to short-term transfer from optimal (pH 5.5) to alkaline (pH 7.5) media unveiled a large set of differentially expressed genes that were partially congruent with genes affected by low pH, bicarbonate, and nitrate, but showed only a very small overlap with genes responsive to the availability of iron. Further analysis of selected genes disclosed pronounced responsiveness of their expression over a wide range of external pH values. Alkalinity altered the expression of various proton/anion co-transporters, possibly to recalibrate cellular proton homeostasis. Co-expression analysis of pH-responsive genes identified a module of genes encoding proteins with putative functions in the regulation of root growth, which appears to be conserved in plants subjected to low pH or bicarbonate. Our analysis provides an inventory of pH-sensitive genes and allows comprehensive insights into processes that are orchestrated by external pH.

A membrane protein of the rice pathogen Burkholderia glumae required for oxalic acid secretion and quorum sensing.

Bacterial panicle blight is caused by Burkholderia glumae and results in damage to rice crops worldwide. Virulence of B. glumae requires quorum sensing (QS)-dependent synthesis and export of toxoflavin, responsible for much of the damage to rice. The DedA family is a conserved membrane protein family found in all bacterial species. B. glumae possesses a member of the DedA family, named DbcA, which we previously showed is required for toxoflavin secretion and virulence in a rice model of infection. B. glumae secretes oxalic acid as a "common good" in a QS-dependent manner to combat toxic alkalinization of the growth medium during the stationary phase. Here, we show that B. glumae ΔdbcA fails to secrete oxalic acid, leading to alkaline toxicity and sensitivity to divalent cations, suggesting a role for DbcA in oxalic acid secretion. B. glumae ΔdbcA accumulated less acyl-homoserine lactone (AHL) QS signalling molecules as the bacteria entered the stationary phase, probably due to nonenzymatic inactivation of AHL at alkaline pH. Transcription of toxoflavin and oxalic acid operons was down-regulated in ΔdbcA. Alteration of the proton motive force with sodium bicarbonate also reduced oxalic acid secretion and expression of QS-dependent genes. Overall, the data show that DbcA is required for oxalic acid secretion in a proton motive force-dependent manner, which is critical for QS of B. glumae. Moreover, this study supports the idea that sodium bicarbonate may serve as a chemical for treatment of bacterial panicle blight.

Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel.

Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.

Transcriptomic and Metabolomic Profiling Uncovers Response Mechanisms of Alicyclobacillus acidoterrestris DSM 3922T to Acid Stress.

Alicyclobacillus acidoterrestris, which has strong acidophilic and heat-resistant properties, can cause spoilage of pasteurized acidic juice. The current study determined the physiological performance of A. acidoterrestris under acidic stress (pH 3.0) for 1 h. Metabolomic analysis was carried out to investigate the metabolic responses of A. acidoterrestris to acid stress, and integrative analysis with transcriptome data was also performed. Acid stress inhibited the growth of A. acidoterrestris and altered its metabolic profiles. In total, 63 differential metabolites, mainly enriched in amino acid metabolism, nucleotide metabolism, and energy metabolism, were identified between acid-stressed cells and the control. Integrated transcriptomic and metabolomic analysis revealed that A. acidoterrestris maintains intracellular pH (pHi) homeostasis by enhancing amino acids decarboxylation, urea hydrolysis, and energy supply, which was verified using real-time quantitative PCR and pHi measurement. Additionally, two-component systems, ABC transporters, and unsaturated fatty acid synthesis also play crucial roles in resisting acid stress. Finally, a model of the responses of A. acidoterrestris to acid stress was proposed. IMPORTANCE Fruit juice spoilage caused by A. acidoterrestris contamination has become a major concern and challenge in the food industry, and this bacterium has been suggested as a target microbe in the design of the pasteurization process. However, the response mechanisms of A. acidoterrestris to acid stress still remain unknown. In this study, integrative transcriptomic, metabolomic, and physiological approaches were used to uncover the global responses of A. acidoterrestris to acid stress for the first time. The obtained results can provide new insights into the acid stress responses of A. acidoterrestris, which will point out future possible directions for the effective control and application of A. acidoterrestris.

Nicotine activates HIF-1α and regulates acid extruders through the nicotinic acetylcholine receptor to promote the Warburg effect in non-small cell lung cancer cells.

Cigarette smoking is the greatest risk factor for lung cancer, accounting for approximately 90% of all lung cancer-related deaths. Moreover, nicotine is associated with lung cancer onset and progression. Hypoxia-inducible factor 1α (HIF-1α) is involved in the metabolic reprogramming of cancer cells and accelerates cancer progression via regulation of pH and acid-base homeostasis. Previous studies have reported that nicotine upregulates HIF-1α expression. Therefore, we hypothesized that nicotine-mediated activation of HIF-1α regulates metabolic reprogramming and pH homeostasis in non-small cell lung cancer A549 cells and could potentially play a role in the progression of lung cancer. We examined the effects of nicotine on metabolic reprogramming and intracellular pH (pHi) homeostasis, which are critical for cancer progression. A549 cells were exposed to nicotine in the absence and presence of the nicotinic acetylcholine receptor antagonist, mecamylamine (MEC). We then analyzed glycolytic stress and the activity and expression of acid-extruder proteins, including the Na+-H+ exchanger 1 (NHE1) and monocarboxylate cotransporters 1 & 4 (MCT1 and MCT4, respectively). Nicotine promoted the Warburg effect, which is associated with accelerated migration of A549 cells through the activation of nicotinic acetylcholine receptors. Furthermore, nicotine upregulated the activities and expression of acid-extruder proteins, namely NHE1 and MCT4, and facilitated glycolysis. To the best of our knowledge, this is the first study to demonstrate that nicotine plays a pivotal regulatory role in metabolic reprogramming as well as regulation of pHi homeostasis in A549 cells via activation of nicotinic acetylcholine receptors and can therefore aggravate lung cancer progression.

Contribution of amino acids to Alicyclobacillus acidoterrestris DSM 3922T resistance towards acid stress.

Spoilage of juice and beverages by a thermo-acidophilic bacterium, Alicyclobacillus acidoterrestris, has been considered to be a major and widespread concern for juice industry. Acid-resistant property of A. acidoterrestris supports its survival and multiplication in acidic juice and challenges the development of corresponding control measures. In this study, intracellular amino acid differences caused by acid stress (pH 3.0, 1 h) were determined by targeted metabolomics. The effect of exogenous amino acids on acid resistance of A. acidoterrestris and the related mechanisms were also investigated. The results showed that acid stress affected the amino acid metabolism of A. acidoterrestris, and the selected glutamate, arginine, and lysine contributed to its survival under acid stress. Exogenous glutamate, arginine, and lysine significantly increased the intracellular pH and ATP level, alleviated cell membrane damage, reduced surface roughness, and suppressed deformation caused by acid stress. Additionally, the up-regulated gadA and speA genes and the enhanced enzymatic activity confirmed that glutamate and arginine decarboxylase systems played a crucial role in maintaining pH homeostasis of A. acidoterrestris under acid stress. Our research reveals an important factor contributing to acid resistance of A. acidoterrestris, which provides an alternative target for effectively controlling this contaminant in fruit juices.

Analyzing mechanisms of action of antimicrobial peptides on bacterial membranes requires multiple complimentary assays and different bacterial strains.

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs.

Cytosolic pH Controls Fungal MAPK Signaling and Pathogenicity.

Mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes in eukaryotes. In fungal pathogens, conserved MAPK pathways control key virulence functions such as infection-related development, invasive hyphal growth, or cell wall remodeling. Recent findings suggest that ambient pH acts as a key regulator of MAPK-mediated pathogenicity, but the underlying molecular events are unknown. Here, we found that in the fungal pathogen Fusarium oxysporum, pH controls another infection-related process, hyphal chemotropism. Using the ratiometric pH sensor pHluorin we show that fluctuations in cytosolic pH (pHc) induce rapid reprogramming of the three conserved MAPKs in F. oxysporum, and that this response is conserved in the fungal model organism Saccharomyces cerevisiae. Screening of a subset of S. cerevisiae mutants identified the sphingolipid-regulated AGC kinase Ypk1/2 as a key upstream component of pHc-modulated MAPK responses. We further show that acidification of the cytosol in F. oxysporum leads to an increase of the long-chain base (LCB) sphingolipid dihydrosphingosine (dhSph) and that exogenous addition of dhSph activates Mpk1 phosphorylation and chemotropic growth. Our results reveal a pivotal role of pHc in the regulation of MAPK signaling and suggest new ways to target fungal growth and pathogenicity. IMPORTANCE Fungal phytopathogens cause devastating losses in global agriculture. All plant-infecting fungi use conserved MAPK signaling pathways to successfully locate, enter, and colonize their hosts. In addition, many pathogens also manipulate the pH of the host tissue to increase their virulence. Here, we establish a functional link between cytosolic pH (pHc) and MAPK signaling in the control of pathogenicity in the vascular wilt fungal pathogen Fusarium oxysporum. We demonstrate that fluctuations in pHc cause rapid reprogramming of MAPK phosphorylation, which directly impacts key processes required for infection, such as hyphal chemotropism and invasive growth. Targeting pHc homeostasis and MAPK signaling can thus open new ways to combat fungal infection.

The yeast Gdt1 protein mediates the exchange of H+ for Ca2+ and Mn2+ influencing the Golgi pH.

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photosynthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca2+ or Mn2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H+ ions are transported in exchange for Ca2+ and Mn2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca2+, Mn2+ or H+ gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H+ ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H+ transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts.