Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella‐containing vacuole

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high‐throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.     

WASH‐driven actin polymerization is required for efficient mycobacterial phagosome maturation arrest

Pathogenic mycobacteria survive in phagocytic host cells primarily as a result of their ability to prevent fusion of their vacuole with lysosomes, thereby avoiding a bactericidal environment. The molecular mechanisms to establish and maintain this replication compartment are not well understood. By combining molecular and microscopical approaches we show here that after phagocytosis the actin nucleation‐promoting factor WASH associates and generates F‐actin on the mycobacterial vacuole. Disruption of WASH or depolymerization of F‐actin leads to the accumulation of the proton‐pumping V‐ATPase around the mycobacterial vacuole, its acidification and reduces the viability of intracellular mycobacteria. This effect is observed for M. marinum in the model phagocyte Dictyostelium but also for M. marinum and M. tuberculosis in mammalian phagocytes. This demonstrates an evolutionarily conserved mechanism by which pathogenic mycobacteria subvert the actin‐polymerization activity of WASH to prevent phagosome acidification and maturation, as a prerequisite to generate and maintain a replicative niche.     

Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance

Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo ³¹P and ¹³C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism. In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate. Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H⁺. With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added. Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action. Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where [¹³C]ethanol was added and formation of labeled acetic acid was detected. The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) [¹³C]ethanol was added.Abbreviations P_i inorganic phosphate - P_ic inorganic phosphate in the cytoplasm - P_iv inorganic phosphate in the vacuole - tP terminal phosphate in polyphosphate     

Secretion of an acid phosphatase provides a possible mechanism to acquire host nutrients by Plasmodium falciparum

As an intracellular proliferating parasite, Plasmodium falciparum exploits the human host to acquire nutrients. However, nutrients such as nucleotides and cofactors are mostly phosphorylated in the host cell cytosol and thus have to be dephosphorylated in order to be taken up by the parasite. Here we report the functional characterization of a unique secreted phosphatase in P. falciparum, which is expressed throughout the developmental stages in the red blood cell. We show that this enzyme, formerly described as anchoring glideosome‐associated protein 50 (GAP50), reveals a broad substrate profile with preference for di‐ and triphosphates at pH 5–7. Bioinformatic studies of the protein sequence identified an N‐terminal signal anchor (SA) as well as a C‐terminal transmembrane domain. By means of live microscopy of parasites transfected with GFP‐fusions of this secreted acid phosphatase (PfSAP), we demonstrate that PfSAP enters the secretory pathway en route to the parasite periphery – mediated by SA – and is subsequently engulfed into the food vacuole. We corroborate this with independent data where acid phosphatase activity is visualized in close proximity to hemozoin. The biochemical as well as the trafficking results support the proposed role of PfSAP in the acquisition of host nutrients by dephosphorylation.     

The pH sensitivity of histidine‐contain‐ ing lytic peptides

Many bioactive peptides are featured by their unique amino acid compositions such as argine/lysine‐rich peptides. However, histidine‐rich bioactive peptides are hardly found. In this study, histidine‐containing peptides were constructed by selectively replacing the corresponded lysine residues in a lytic peptide LL‐1 with histidines. Interestingly, all resulting peptides demonstrated pH‐dependent activities. The cell lysis activities of these peptides could be increased up to four times as the solution pHs dropped from pH = 7.4 to pH = 5.5. The pH sensitivity of a histidine‐containing peptide was determined by histidine substitution numbers. Peptide derivatives with more histidines were associated with increased pH sensitivity. Results showed that not the secondary structures but pH‐affected cell affinity changes were responsible for the pH‐dependent activities of histidine‐containing peptides. The histidine substitution approach demonstrated here may present a general strategy to construct bioactive peptides with desired pH sensitivity for various applications. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)

Zinc activates a specific Zn²⁺-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca²⁺ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na⁺/H⁺ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn²⁺ binding site, His¹⁷ or His¹⁹, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp³¹³ with alanine resulted in similar Ca²⁺ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na⁺/H⁺ exchange at pH 7.4 and pH 6.5. Substitution of Asp³¹³ to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp³¹³, which was shown to modulate Zn²⁺ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.     

Role of Lys281 in the <Emphasis Type="Italic">Dunaliella salina</Emphasis> (6-4) Photolyase Reaction

His³⁵⁴ and His³⁵⁸, two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His⁴⁰¹ and His⁴⁰⁵, in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys²⁸¹ might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys²⁸¹, which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His⁴⁰¹ when the environmental pH is higher than the pKa value of histidine.     

The Correlation of Digestive Vacuole pH and Size with the Digestive Cycle in Paramecium caudatum1

ABSTRACT. The temporal changes in the size and pH of digestive vacuoles (DV) in Paramecium caudatum were reevaluated. Cells were pulsed briefly with polystyrene latex spheres or heat-killed yeast stained with three sulfonphthalein indicator dyes. Within 5 min of formation the intravacuolar pH declined from ～7 to 3. With the exception of a transient and early increase in vacuolar size, vacuole condensation occurred rapidly and paralleled the acidification so that vacuoles reached their lowest pH and minimal size simultaneously. Neutralization and expansion of vacuole size began when vacuoles were GT8 min old. No labeled vacuoles were defecated prior to 21 min after formation but almost all DV were defecated within 1 h so that the digestive cycle of individual vacuoles ranged from 21 to 60 min. Based on these size and pH changes, the presence of acid phosphatase activity, and membrane morphology, digestive vacuoles can be grouped into four stages of digestion. The DV-I are GT6 min old and undergo rapid condensation and acidification. The DV-II are between 4 to 10 min old and are the most condensed and acidic vacuoles. The DV-III range in age from 8 to ～20 min and include the expanding or expanded vacuoles that result from lysosomes fusing with DV-II. The DV-IV are GD21 min old, and since digestion is presumably completed, they can be defecated. The rise in intravacuolar pH that accompanies vacuole expansion suggests that lysosomes play a role in vacuole neutralization in addition to their degradative functions. The acidification and condensation processes in DV-I appear to be unrelated to lysosomal function, as no acid phosphaiase activity has been detected at this stage, but may be related to phagosomal functions important in killing food organisms, denaturing proteins prior to digestion, and preparing vacuole membrane for fusion with lysosomes.     

Crystal structure of the sensor domain of BaeS from Serratia marcescens FS14

The sensor histidine kinases of two‐component signal‐transduction systems (TCSs) are essential for bacteria to adapt to variable environmental conditions. The two‐component regulatory system BaeS/R increases multidrug and metal resistance in Salmonella and Escherichia coli. In this study, we report the X‐ray structure of the periplasmic sensor domain of BaeS from Serratia marcescens FS14. The BaeS sensor domain (34–160) adopts a mixed α/β‐fold containing a central four‐stranded antiparallel β‐sheet flanked by a long N‐terminal α‐helix and additional loops and a short C‐terminal α‐helix on each side. Structural comparisons revealed that it belongs to the PDC family with a remarkable difference in the orientation of the helix α2. In the BaeS sensor domain, this helix is situated perpendicular to the long helix α1 and holds helix α1 in the middle with the beta sheet, whereas in other PDC domains, helix α2 is parallel to helix α1. Because the helices α1 and α2 is involved in the dimeric interface, this difference implies that BaeS uses a different dimeric interface compared with other PDC domains. Proteins 2017; 85:1784–1790. © 2017 Wiley Periodicals, Inc.     

Ultracytochemical characterization of non-specific acid phosphatase activities in Saccharomyces cerevisiae.

Glutaraldehyde prefixation causes a considerable inactivation of the acid phosphatase of yeast protoplasts in dependence on the duration of aldehyde influence. Lead ions necessary for ultracytochemical demonstration effect a still stronger inhibition of enzymatic activity. Prefixation, however, protects the enzyme from further inhibition by lead. At pH 4.4 in intact cells acid phosphatase activities are mainly localized in the periplasmic space and in vesicles fused with the plasma membrane. The cell wall and cytoplasm usually remain free of reaction products. On the cell surface activities are found in form of globular lead deposits. At pH 5.2 and 6.3 the periplasmic activity appears decreased compared to that at lower pH values and the intracellular activity is increased. The plasma membrane of protoplasts is completely free of precipitates. The intracellular activity sites of protoplasts (cisternae of endoplasmic reticulum and/or Golgi-like system, small vesicles, central vacuole, nuclear envelope) are the same as for intact cells. The occurrence of at least two forms of acid phosphatase in S. cerevisiae id deduced.     

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER‐associated compartment termed the Legionella‐containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule‐resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant‐negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P‐positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1‐dependent aggregation of purified, ER‐positive LCVs in vitro. Thus, Sey1/Atl3‐dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.     

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella‐containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane‐bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP‐bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63‐containing late endosomes. Nischarin is recruited to the SCV in a Rab14‐dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners—Rac1, Rab14 and Rab9 GTPases—reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.     

Defects in intracellular trafficking and endocytic/vacuolar acidification determine the efficiency of endocytotic DNA uptake in yeast

The yeast Saccharomyces cerevisiae is a standard model system to study endocytosis. Here we describe the examination of a representative subset of deletion mutants to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases in non‐viral transfection processes. When transport in late endocytosis is inhibited, transfection efficiency is significantly enhanced. Similarly, transfection efficiency is enhanced when the pH‐value of the endosomal/vacuolar system is modified. Transfection efficiency is furthermore elevated when the Na⁺/K⁺ transport in the endosomal system is disturbed. Finally, we observe enhanced transfection efficiency in mutants disturbed in the CVT/autophagy pathway and in hydrolase transport to the vacuole. In summary, non‐viral transfection efficiency can be significantly increased by either (i) inhibiting the transport of endocytosed material before it enters the vacuole, or (ii) inducing a non‐natural pH‐value of the endosomal/vacuolar system, or (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole. J. Cell. Biochem. 106: 327–336, 2009. © 2008 Wiley‐Liss, Inc.     

A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA

Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metabolism of citrate in response to its extracellular concentration. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor.     

Role of the ESCRT‐III complex in controlling integrity of the Salmonella‐containing vacuole

Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2‐encoded type III secretion system (SPI2‐T3SS). Fusion events of endosomal compartments with the Salmonella‐containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella‐modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT‐III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT‐III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2‐T3SS‐deficient mutant strain was partially abrogated. ESCRT‐dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT‐III recruitment in Salmonella‐infected host cells.     

Neuronal intracellular pH directly mediates nitric oxide‐induced programmed cell death

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH_i) regulation during nitric oxide (NO)‐induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick‐end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 μM), 3‐morpholinosydnonimine (300 μM), or 6‐(2‐hyrdroxy‐1‐methyl‐2‐nitrosohydrazino)‐N‐methyl‐1‐hexanamine (300 μM). The pH_i was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH_i 7.36 ± 0.01 to approximately 7.00 (p < .0001). Within 45 min, a biphasic alkaline response was evident, with pH_i reaching 7.40 ± 0.02, that was persistent for a 6‐h period. To mimic the effect of NO‐induced acidification, neurons were acid‐loaded with ammonium ions to yield a pH_i of 7.09 ± 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 ± 2% (24 h) and DNA fragmentation increased to 58 ± 8% (24 h) (p < .0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH_i of 7.60 ± 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO‐induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 171–184, 1999     

Contribution of individual histidines to the global stability of human prolactin

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500‐fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH‐dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH‐dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2–3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function.