Extracellular Vesicles and Renal Endothelial Cells: A Fatal Attraction in Hemolytic Uremic Syndrome.

This review focuses on typical hemolytic uremic syndrome (HUS), a life-threatening sequela of human infections caused, particularly in children, by Shiga toxin-producing Escherichia coli strains. Thrombotic microangiopathy of the brain and the kidney is the end point of toxin action, resulting in the hallmarks of HUS (ie, thrombocytopenia, anemia, and acute renal failure). A growing body of evidence points to the role of extracellular vesicles released in the blood of patients by toxin-challenged circulating cells (monocytes, neutrophils, and erythrocytes) and platelets, as a key factor in the pathogenesis of HUS. This review provides i) an updated description of the pathogenesis of Shiga toxin-producing E. coli infections; ii) an analysis of blood cell-derived extracellular vesicles, and of their parent cells, as triggering factors in HUS; and iii) a model explaining why Shiga toxin-containing vesicles dock preferentially to the endothelia of target organs.

Bloody Diarrhea and Shiga Toxin-Producing Escherichia coli Hemolytic Uremic Syndrome in Children: Data from the ItalKid-HUS Network.

OBJECTIVE: To analyze the results of an enhanced laboratory-surveillance protocol for bloody diarrhea aimed at identifying children with Shiga toxin-producing Escherichia coli (STEC) infection early in the course of the disease toward the early identification and management of patients with hemolytic uremic syndrome (HUS). STUDY DESIGN: The study (2010-2019) involved a referral population of 2.3 million children. Stool samples of patients with bloody diarrhea were screened for Shiga toxin (Stx) genes. Positive patients were rehydrated and monitored for hemoglobinuria until diarrhea resolved or STEC-HUS was diagnosed. RESULTS: A total of 4767 children were screened; 214 (4.5%) were positive for either Stx1 (29.0%) or Stx2 (45.3%) or both Stx1+2 (25.7%); 34 patients (15.9%) developed STEC-HUS (0.71% of bloody diarrheas). Hemoglobinuria was present in all patients with HUS. Patients with Stx2 alone showed a greater risk of STEC-HUS (23.7% vs 12.7%) and none of the patients with Stx1 alone developed HUS. During the same period of time, 95 other patients were diagnosed STEC-HUS but were not captured by the screening program (26 had nonbloody diarrhea, 11 came from areas not covered by the screening program, and 58 had not been referred to the screening program, although they did meet the inclusion criteria). At HUS presentation, serum creatinine of patients identified by screening was significantly lower compared with that of the remaining patients (median 0.9 vs 1.51 mg/dL). CONCLUSIONS: Nearly 1% of children with bloody diarrhea developed STEC-HUS, and its diagnosis was anticipated by the screening program for Stx. The screening of bloody diarrhea for Stx is recommended, and monitoring patients carrying Stx2 with urine dipstick for hemoglobinuria is suggested to identify the renal complication as early as possible.

The structure of the Shiga toxin 2a A-subunit dictates the interactions of the toxin with blood components.

Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)-producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A-subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re-evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS-pathogenesis and to develop therapeutic approaches.

Is Shigatoxin 1 protective for the development of Shigatoxin 2-related hemolytic uremic syndrome in children? Data from the ItalKid-HUS Network.

BACKGROUND: Shigatoxin (Stx)-producing Escherichia coli (STEC) are the most common causes of hemolytic uremic syndrome (STEC-HUS). The aim of our study is to compare the risk of developing STEC-HUS in relation to the type of Stx genes (Stx1, Stx2, or both). METHODS: This is a prospective, observational, multicenter study involving 63 pediatric units in Northern Italy (ItalKid-HUS Network). STEC-infected children were identified within a screening program for bloody diarrhea during a 10-year period (2010-2019). Stx genes were detected by reverse dot blot or real-time PCR. After the identification of STEC infection, children were followed until diarrhea complete recovery for the possible development of STEC-HUS. RESULTS: Of the 214 Stx-positive patients, 34 (15.9%) developed STEC-HUS. The risk of HUS in STEC-infected children with Stx1 (n: 62; 29.0%) and Stx2 (n: 97; 45.3%) was respectively 0% and 23.7%, while in patients carrying both Stx1 and Stx2 (n: 55; 25.7%), the risk was 12.7% (p: 0.001). CONCLUSIONS: Our data confirm that Stx1 is a very rare cause of STEC-HUS and demonstrate that the risk of STEC-HUS halves in the case of Stx1+2-producing Escherichia coli infection compared with infections where Stx2 is present alone. This observation is helpful in assessing the risk of individual STEC-infected patients for the development of HUS and suggests that Stx1, in the presence of Stx2, might exert a protective role possibly by receptor competition.

Human monocytes stimulated by Shiga toxin 1a via globotriaosylceramide release proinflammatory molecules associated with hemolytic uremic syndrome.

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1ß, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1ß) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1ß, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.

The Antibiotic Polymyxin B Impairs the Interactions between Shiga Toxins and Human Neutrophils.

Hemolytic uremic syndrome (HUS) is the life-threatenig sequela of intestinal infections by Shiga toxin (Stx)-producing Escherichia coli (STEC) in children. Human neutrophils specifically bind Stx through TLR4, the receptor of LPS. The binding could be considered protective (Stx sequestration) or harmful (toxin delivery to target organs). The amount of Stx on neutrophils is in equilibrium with the amount of Stx present in the gut, and it is also related to renal and neurologic symptoms. The TLR4-mediated interaction of LPS with innate immune cells is hampered by the well-known antibiotic polymyxin B. In this study, we show that the same antibiotic impairs the binding of Stx to neutrophils, also blocking their functional effects (release of CXCL8, formation of neutrophil/platelet aggregates) involved in HUS pathogenesis. Controls for contaminating LPS in Stx-induced neutrophil responses inhibited by polymyxin B were performed. Stx interact with human neutrophils through their A chain, since these leukocytes do not express globotriaosylceramide, the specific receptor for Stx B chains. Consistently, polymyxin B blocked the enzymatic activity of Stx1, Stx2, Stx1 A chain, and the analogous plant protein gelonin, whereas the antibiotic did not show any protective effect on Stx-induced cytotoxicity in globotriaosylceramide-expressing Raji cells. Antibiotic administration is not recommended in human STEC infections during the prodromal intestinal phase, and the toxicity of polymyxin B could further discourage its therapeutic use. However, nontoxic, nonbactericidal polymyxin derivatives have been developed and might be used in animal models of STEC infection to study their efficacy in preventing the onset of HUS during the systemic blood phase of Stx.

The inhibition of lactate dehydrogenase A hinders the transcription of histone 2B gene independently from the block of aerobic glycolysis.

Most cancer cells use aerobic glycolysis to fuel their growth and many efforts are made to selectively block this metabolic pathway in cancer cells by inhibiting lactate dehydrogenase A (LDHA). However, LDHA is a moonlighting protein which exerts functions also in the nucleus as a factor associated to transcriptional complexes. Here we found that two small molecules which inhibit the enzymatic activity of LDHA hinder the transcription of histone 2B gene independently from the block of aerobic glycolysis. Moreover, we observed that silencing this gene reduces cell replication, hence suggesting that the inhibition of LDHA can also affect the proliferation of normal non-glycolysing dividing cells.

Cap-independent protein synthesis is enhanced by betaine under hypertonic conditions.

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged. In the presence of betaine, CrPV IRES- and sodium-dependent neutral amino acid transporter-2 (SNAT2) 5'-UTR-driven translation is 2- and 1.5-fold stimulated in MCF7 cells, respectively. Thus, betaine could provide an advantage in translation of messengers coding for proteins implicated in the response of cells to different stressors, which are often recognized by ribosomal 40 S subunit through simplified cap-independent mechanisms.

RiboAbacus: a model trained on polyribosome images predicts ribosome density and translational efficiency from mammalian transcriptomes.

Fluctuations in mRNA levels only partially contribute to determine variations in mRNA availability for translation, producing the well-known poor correlation between transcriptome and proteome data. Recent advances in microscopy now enable researchers to obtain high resolution images of ribosomes on transcripts, providing precious snapshots of translation in vivo. Here we propose RiboAbacus, a mathematical model that for the first time incorporates imaging data in a predictive model of transcript-specific ribosome densities and translational efficiencies. RiboAbacus uses a mechanistic model of ribosome dynamics, enabling the quantification of the relative importance of different features (such as codon usage and the 5' ramp effect) in determining the accuracy of predictions. The model has been optimized in the human Hek-293 cell line to fit thousands of images of human polysomes obtained by atomic force microscopy, from which we could get a reference distribution of the number of ribosomes per mRNA with unmatched resolution. After validation, we applied RiboAbacus to three case studies of known transcriptome-proteome datasets for estimating the translational efficiencies, resulting in an increased correlation with corresponding proteomes. RiboAbacus is an intuitive tool that allows an immediate estimation of crucial translation properties for entire transcriptomes, based on easily obtainable transcript expression levels.

Human ribosomes from cells with reduced dyskerin levels are intrinsically altered in translation.

Dyskerin is a pseudouridine (ψ) synthase involved in fundamental cellular processes including uridine modification in rRNA and small nuclear RNA and telomere stabilization. Dyskerin functions are altered in X-linked dyskeratosis congenita (X-DC) and cancer. Dyskerin's role in rRNA pseudouridylation has been suggested to underlie the alterations in mRNA translation described in cells lacking dyskerin function, although relevant direct evidences are currently lacking. Our purpose was to establish definitely whether defective dyskerin function might determine an intrinsic ribosomal defect leading to an altered synthetic activity. Therefore, ribosomes from dyskerin-depleted human cells were purified and 1) added to a controlled reticulocyte cell-free system devoid of ribosomes to study mRNA translation; 2) analyzed for protein contamination and composition by mass spectrometry, 3) analyzed for global pseudouridylation levels. Ribosomes purified from dyskerin-depleted cells showed altered translational fidelity and internal ribosome entry site (IRES)-mediated translation. These ribosomes displayed reduced uridine modification, whereas they were not different in terms of protein contamination or ribosomal protein composition with respect to ribosomes from matched control cells with full dyskerin activity. In conclusion, lack of dyskerin function in human cells induces a defect in rRNA uridine modification, which is sufficient to alter ribosome activity.

Identification of TLR4 as the receptor that recognizes Shiga toxins in human neutrophils.

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin-producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.

Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation.

Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.

Serum Shiga toxin 2 values in patients during acute phase of diarrhoea-associated haemolytic uraemic syndrome.

AIM: Shiga toxins are delivered via systemic circulation and are considered to be the cause of diarrhoea-associated haemolytic uraemic syndrome (HUS), as they injure endothelial cells, particularly in the glomeruli. This study measured Shiga toxin 2 (Stx2) in the serum of children affected in by HUS due to Stx2 producing Escherichia coli. METHODS: The concentration of free Stx2 was measured in the serum of 16 children, collected immediately after admission to the clinic in the acute phase of HUS, using a sandwich enzyme-linked immunosorbent assay. The family members of two children were also investigated, with the relative toxicity of Stx2 assessed by a Vero cell-based fluorescent assay. RESULTS: Stx2 was found in the serum of eight of the 16 children who were investigated. It was also detected in four of the six family members not showing symptomatic HUS, with an extremely high level in two. CONCLUSION: An absent or rather low concentration of Stx2 was found in the serum of children admitted to the clinic with diarrhoea-associated HUS. The high concentration of Stx2 in family members without HUS, but mostly with watery diarrhoea and raised functional activity, was in line with the concept of early injury by Stx2.

Peptides inhibiting the assembly of monomeric human l-lactate dehydrogenase into catalytically active homotetramer decrease the synthesis of lactate in cultured cells.

The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A). However, to identify these peptides, tetrameric hLDH-A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH-A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH-A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296-303 portion of hLDH-A C-terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH-A and hLDH-B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C-terminal region of hLDH-A effectively interfere with protein-protein interactions responsible for the assembly of the tetrameric enzyme.

5'-Untranslated region of heat shock protein 70 mRNA drives translation under hypertonic conditions.

In mammalian cells, adaptation to hypertonic conditions leads to the activation of an array of early (cell shrinkage, regulatory volume increase) and late (accumulation of compatible osmolytes) responses and increased level of HSPs (heat shock proteins). Protein synthesis is strongly inhibited few minutes after the hypertonic challenge as demonstrated in whole cells and as reproduced under controlled conditions in cell-free systems. Different mechanisms known to mediate the accumulation of HSP70, such as mRNA transcription and stabilization, require fully active protein synthesis. We show that the 5'-untranslated region of HSP70 messenger drives a hypertonicity-resistant translation (up to 0.425 osmol/kg of water), whereas cap-dependent protein synthesis is almost totally blocked under the same conditions. The results, obtained in cell-free systems and in whole cells, might help to explain why HSP70 is accumulated in cells when total protein synthesis is impaired. We also observed that translation initiated by viral IRES (from Cricket paralysis virus) is highly efficient in cells exposed to hyperosmolarity, suggesting that the resistance to hypertonic conditions is a more general feature of cap-independent translation. The described mechanism may also play a role in the control of translation of other messengers encoding for proteins involved in the adaptation to hypertonicity.

Galloflavin prevents the binding of lactate dehydrogenase A to single stranded DNA and inhibits RNA synthesis in cultured cells.

Lactate dehydrogenase A (LDH-A) binds single stranded DNA (ssDNA) and stimulates cell transcription. Binding is prevented by NADH, suggesting that the coenzyme site is involved in the interaction LDH-A/ssDNA. We recently identified an inhibitor of LDH-A enzymatic activity (Galloflavin, GF) which occupies the NADH site. In the experiments reported here we studied whether GF can also hinder the binding of LDH-A to ssDNA and investigated its effects on RNA synthesis in cultured cells. Using a filter binding assay we observed that 4 µM GF inhibited the binding of human LDH-A to a single stranded [(3)H]DNA sample by 50%. After only 0.5-1h, 50-100 µM GF inhibited RNA synthesis in SW620 cells maintained in a medium in which galactose substituted glucose. In these culture conditions, SW620 cells did not produce lactic acid and effects caused by the inhibition of the enzymatic activity of LDH-A could be excluded. Novel LDH-A inhibitors which hinder aerobic glycolysis of cancer cells are at present actively searched. Our results suggest that: (i) inhibitors which bind the NADH site can exert their antiproliferative activity not only by blocking aerobic glycolysis but also by causing an inhibition of RNA synthesis independent from the effect on glycolysis; (ii) GF can be a useful tool to study the biological role of LDH-A binding to ssDNA.

Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host's protease profile could affect disease development by changing the toxin's biological features.

Detection of Cleaved Stx2a in the Blood of STEC-Infected Patients.

Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing Escherichia coli (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently. The outcome of STEC infections and the development of HUS could be influenced by the relative amounts of uncleaved or cleaved Stx2 circulating in patients' blood. Cleaved Stx2 was identified and quantified for the first time in four out of eight STEC-infected patients' sera by a method based on the inhibition of cell-free translation. Cleaved Stx2 was present in the sera of patients with toxins bound to neutrophils and in two out of three patients developing HUS, suggesting its involvement in HUS pathogenesis, although in association with other bacterial or host factors.

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1.

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis-Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.