Distinct Interactions with Cellular E-Cadherin of the Two Virulent Metalloproteinases Encoded by a Bacteroides fragilis Pathogenicity Island

Bacteroides fragilis causes the majority of Gram-negative anaerobic infections in the humans. The presence of a short, 6-kb, pathogenicity island in the genome is linked to enterotoxigenic B. fragilis (ETBF). The role of the enterotoxin in B. fragilis virulence, however, remains to be determined, as the majority of clinical isolates lack ETBF genes and healthy individuals carry enterotoxin-positive B. fragilis. The island encodes secretory metalloproteinase II (MPII) and one of three homologous enterotoxigenic fragilysin isoenzymes (FRA; also termed B. fragilis toxin or BFT). The secretory metalloproteinases expressed from the genes on the B. fragilis pathogenicity island may have pathological importance within the gut, not linked to diarrhea. MPII and FRA are counter-transcribed in the bacterial genome, implying that regardless of their structural similarity and overlapping cleavage preferences these proteases perform distinct and highly specialized functions in the course of B. fragilis infection. The earlier data by us and others have demonstrated that FRA cleaves cellular E-cadherin, an important adherens junction protein, and weakens cell-to-cell contacts. Using E-cadherin-positive and E-cadherin–deficient cancer cells, and the immunostaining, direct cell binding and pull-down approaches, we, however, demonstrated that MPII via its catalytic domain efficiently binds, rather than cleaves, E-cadherin. According to our results, E-cadherin is an adherens junction cellular receptor, rather than a proteolytic target, of the B. fragilis secretory MPII enzyme. As a result of the combined FRA and MPII proteolysis, cell-to-cell contacts and adherens junctions are likely to weaken further.     

Evaluation of the Pathogenicity of the Bacteroides fragilis Toxin Gene Subtypes in Gnotobiotic Mice

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a metalloprotease toxin (BFT) related to diarrheal disease in animals, young children, and adults. Three different isoforms of the enterotoxin, designated BFT-1, BFT-2, and BFT-3, have been identified and sequenced. In the present study, the pathogenicity of the ETBF strains carrying bft-1 or bft-2 was evaluated. Each toxin gene subtype of ETBF (bft-1 or bft-2) was intragastrically monoassociated to germ-free mice during 10 days and histopathological data from intestines and liver compared with those from mice monoassociated to a non-enterotoxigenic B. fragilis. Histopathological alterations were observed in all groups of animals related to ETBF. These alterations were characterized mainly by ulceration, edema, and inflammatory infiltration in intestine. However, these lesions were slightly more severe in mice monoassociated with bft-2 subtype. No alteration or lesion was observed in animals associated with the non-enterotoxigenic B. fragilis. In conclusion, strains harboring bft-1 or bft-2 gene subtypes were able to induce histopathological alterations in intestine of a gnotobiotic mice model and it could explain the effect produced for the enterotoxin.     

Structural basis for substrate specificity of l‐methionine decarboxylase

l ‐Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B₆‐dependent enzyme and catalyzes the non‐oxidative decarboxylation of l ‐methionine to produce 3‐methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand‐free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.     

Case-Control Study on the Role of Enterotoxigenic Bacteroides fragilis as a Cause of Diarrhea among Children in Kolkata,India

A total of 874 fecal specimens (446 diarrheal cases and 428 controls) from diarrheal children admitted in the Infectious Diseases Hospital, Kolkata and age and sex matched asymptomatic subjects from an urban community were assessed for the prevalence of enterotoxigenic Bacteroides fragilis (ETBF). Isolates of B. fragilis were tested for the presence of enterotoxin gene (bft) by PCR. The detection rate of ETBF was 7.2% (63 of 874 specimens) that prevailed equally in diarrheal cases and controls (7.2% each; 32 of 446 cases and 31 of 428 controls). Male children up to one year age group was significantly (p<0.05) associated with ETBF infection as compared to children > 2 years of age in cases and controls. In 25 ETBF isolates, the bft gene was genotyped using PCR-RFLP and only two alleles were identified with prevalence rate of 40% and 60% for bft-1 and bft-3, respectively. All the ETBF isolates were susceptible for chloramphenicol and imipenem but resistant to clindamycin (48%), moxifloxacin (44%) and metronidazole (32%). Resistance of ETBF to moxifloxacin (44%) and metronidazole is an emerging trend. Pulsed-field gel electrophoresis (PFGE) revealed that majority of the ETBF isolates are genetically diverse. In the dendrogram analysis, two clusters were identified, one with ETBF resistant to 5–8 antimicrobials and the other cluster with metronidazole and moxifloxacin susceptible isolates from diarrheal cases. To our knowledge, this is the first detailed report on ETBF from India indicating its clinical importance and molecular characteristics.     

Frontotemporal dementia‐linked P112H mutation of TDP‐43 induces protein structural change and impairs its RNA binding function

TDP‐43 forms the primary constituents of the cytoplasmic inclusions contributing to various neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Over 60 TDP‐43 mutations have been identified in patients suffering from these two diseases, but most variations are located in the protein''s disordered C‐terminal glycine‐rich region. P112H mutation of TDP‐43 has been uniquely linked to FTD, and is located in the first RNA recognition motif (RRM1). This mutation is thought to be pathogenic, but its impact on TDP‐43 at the protein level remains unclear. Here, we compare the biochemical and biophysical properties of TDP‐43 truncated proteins with or without P112H mutation. We show that P112H‐mutated TDP‐43 proteins exhibit higher thermal stability, impaired RNA‐binding activity, and a reduced tendency to aggregate relative to wild‐type proteins. Near‐UV CD, 2D‐nuclear‐magnetic resonance, and intrinsic fluorescence spectrometry further reveal that the P112H mutation in RRM1 generates local conformational changes surrounding the mutational site that disrupt the stacking interactions of the W113 side chain with nucleic acids. Together, these results support the notion that P112H mutation of TDP‐43 contributes to FTD through functional impairment of RNA metabolism and/or structural changes that curtail protein clearance.     

Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo‐EM and by imaging toxin import, we uncover the mechanism by which the Tol‐dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9’s disordered N‐terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton‐motive force, which is delivered by the TolQ‐TolR‐TolA‐TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol‐ and Ton‐dependent bacteriocins cross the bacterial outer membrane.     

3,4‐Dihydroxyphenylethanol and 3,4‐dihydroxyphenylacetic acid affect the aggregation process of E46K variant of α‐synuclein at different extent: Insights into the interplay between protein dynamics and catechol effect

Parkinson''s disease (PD) is a chronic multifactorial disease, whose etiology is not completely understood. The amyloid aggregation of α‐synuclein (Syn) is considered a major cause in the development of the disease. The presence of genetic mutations can boost the aggregation of the protein and the likelihood to develop PD. These mutations can lead to early onset (A30P, E46K, and A53T) or late‐onset (H50Q) forms of PD. The disease is also linked to an increase in oxidative stress and altered levels of dopamine metabolites. The molecular interaction of these molecules with Syn has been previously studied, while their effect on the pathological mutant structure and function is not completely clarified. By using biochemical and biophysical approaches, here we have studied the interaction of the familial variant E46K with two dopamine‐derived catechols, 3,4‐dihydroxyphenylacetic acid and 3,4‐dihydroxyphenylethanol. We show that the presence of these catechols causes a decrease in the formation of amyloid fibrils in a dose‐dependent manner. Native‐ and Hydrogen/deuterium exchange‐mass spectrometry (HDX‐MS) provide evidence that this effect is strongly conformation dependent. Indeed, these molecules interact differently with the interconverting conformers of Syn and its familial variant E46K in solution, selecting the most prone‐to‐aggregation one, confining it into an off‐pathway oligomer. These findings suggest that catechols could be a molecular scaffold for the design of compounds potentially useful in the treatment of Parkinson''s disease and related conditions.     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.     

PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE‐cadherin‐Y731. Here, we reveal the underlying mechanism. Leukocyte‐induced stimulation of PECAM‐1 triggers dissociation of the phosphatase SHP2 which then directly targets VE‐cadherin‐Y731. The binding site of PECAM‐1 for SHP2 is needed for VE‐cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM‐1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE‐cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca²⁺‐signaling, non‐muscle myosin II activation, and endothelial cell tension. Since we found that β‐catenin/plakoglobin mask VE‐cadherin‐Y731 and leukocyte docking to endothelial cells exert force on the VE‐cadherin–catenin complex, we propose that leukocytes destabilize junctions by PECAM‐1‐SHP2‐triggered dephosphorylation of VE‐cadherin‐Y731 which becomes accessible by actomyosin‐mediated mechanical force exerted on the VE‐cadherin–catenin complex.     

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C

Mycobacterium tuberculosis virulence is highly metal‐dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu²⁺ ≥ Zn²⁺ > Ni²⁺ > Co²⁺) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k _cat rather than K _M. Removal of the artificial N‐terminal 6xHis‐tag did not change the metal‐dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn²⁺ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal‐dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.     

Quantification of noradrenergic‐, dopaminergic‐, and tectal‐neurons during aging in the short‐lived killifish Nothobranchius furzeri

Parkinson''s disease (PD) is characterized by phosphorylation and aggregation of the protein α‐Synuclein and ensuing neuronal death progressing from the noradrenergic locus coeruleus to midbrain dopaminergic neurons. In 2019, Matsui and colleagues reported a spontaneous age‐dependent degeneration of dopaminergic neurons and an even greater neurodegeneration of the noradrenergic neurons in the short‐lived killifish Nothobranchius furzeri. Given the great possible relevance of a spontaneous model for PD, we assessed neurodegeneration of noradrenergic and dopaminergic neurons in two further laboratory strains of N. furzeri. We implemented, for the first time in N. furzeri, a whole‐brain clarification technique and proceeded to entire 3D nuclei reconstruction to quantify total cell numbers in two different stains of N. furzeri. In both strains, we observed that age‐dependent neurodegeneration is limited to the locus coeruleus and does not involve the posterior tuberculum. We also applied 3D counting to the optic tectum, an area of active adult neurogenesis, and detected an increase of neurons with age. Our results confirm age‐dependent neurodegeneration of noradrenergic neurons, a condition reminiscent of the presymptomatic stage of PD indicating that N. furzeri could be used in the future to identify modifying factors for age‐dependent neurodegeneration and open the intriguing possibility that natural genetic variation may influence the susceptibility of dopaminergic neurons.     

Cryo‐EM structure of the octameric pore of Clostridium perfringens β‐toxin

Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens β‐toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo‐electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo‐octameric pores like the hetero‐oligomeric pores of the bi‐component leukocidins, with important differences in the receptor binding region and the N‐terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16‐stranded β‐barrel protrusion atop of the cap domain that is formed by the N‐termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin‐like family. In addition, we show that the β‐barrel protrusion domain can be modified without affecting the pore‐forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo‐EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.     

A new subtype of the metalloprotease toxin gene and the incidence of the three bft subtypes among Bacteroides fragilis isolates in Japan

The bft gene encoding Bacteroides fragilis toxin (BFT) has been devided into two subtypes, bft-1 and bft-2. We found a novel subtype by sequencing a segment of the bft gene from 64 enterotoxigenic B. fragilis (ETBF) strains isolated in Japan. The 1548-bp nucleotide sequences of the new bft, the bft-1, and bft-2 genes were determined for five, four, and four ETBF strains, respectively; the nucleotide sequence was identical among each bft subtype and the degree of identity between each subtype was between 89 and 94%. Most of the variations between the three subtypes were detected in the region encoding mature toxin. A multiplex PCR was developed with a four-primer mix to subtype the bft sequences. The subtyping of 143 ETBF isolates from extraintestinal and stool specimens of humans and cows showed that the bft-1 was the most prevalent subtype, followed by bft-2 and a new bft subtype. No other subtype was found among the strains tested.     

BNIP3 promotes HIF‐1α‐driven melanoma growth by curbing intracellular iron homeostasis

BNIP3 is a mitophagy receptor with context‐dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient’s survival and depletion of BNIP3 in B16‐F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2‐mediated downregulation of HIF‐1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3‐deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4‐mediated ferritinophagy, which fostered PHD2‐mediated HIF‐1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF‐1α levels in BNIP3‐depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF‐1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro‐tumorigenic HIF‐1α glycolytic program in melanoma cells.     

Effects of SGLT2 inhibitors on haematocrit and haemoglobin levels and the associated cardiorenal benefits in T2DM patients: A meta‐analysis

To explore the effect and magnitude of effect of sodium‐glucose cotransporter‐2 (SGLT2) inhibitors on haematocrit and haemoglobin and the related cardiorenal benefits in patients with type 2 diabetes mellitus (T2DM), PubMed, Web of Science, CENTRAL and EMBASE were searched to identify eligible trials. Weighted mean differences (WMDs) with 95% confidence intervals (CIs) were calculated using a random‐effects model. Seventy‐eight studies were included in the meta‐analysis. SGLT2 inhibitors significantly increased haematocrit and haemoglobin levels compared with control (total WMD 2.27% [95% CI 2.08, 2.47] and 6.20 g/L [95% CI 5.68, 6.73], respectively). Except for dapagliflozin (p = 0.000), no notable dose‐dependent relationship was revealed for other SGLT2 inhibitors. The effect could be sustained or even slightly increased with long‐term therapy (coef. =0.009, 95% CI [0.005, 0.013], p = 0.000). In subgroup analyses, haematocrit elevation increased with higher body mass index (BMI). A greater haematocrit elevation could be observed in white patients or when compared with active controls. In conclusion, SGLT2 inhibitors increased haematocrit and haemoglobin levels in T2DM patients. Changes in haematocrit and haemoglobin seem to be surrogate markers of improvement in renal metabolic stress, and important mediators involved in cardiorenal protection.     

A plant reovirus hijacks the DNAJB12–Hsc70 chaperone complex to promote viral spread in its planthopper vector

Many viruses usurp the functions of endoplasmic reticulum (ER) for virus‐encoded membrane proteins proper functional folding or assembly to promote virus spread. Southern rice black‐streaked dwarf virus (SRBSDV), a plant reovirus, exploits virus‐containing tubules composed of nonstructural membrane protein P7‐1 to spread in its planthopper vector Sogatella furcifera. Here, we report that two factors of the ER‐associated degradation (ERAD) machinery, the ER chaperone DNAJB12 and its cytosolic co‐chaperone Hsc70, are activated by SRBSDV to facilitate ER‐to‐cytosol export of P7‐1 tubules in S. furcifera. Both P7‐1 of SRBSDV and Hsc70 directly bind to the J‐domain of DNAJB12. DNAJB12 overexpression induces ER retention of P7‐1, but Hsc70 overexpression promotes the transport of P7‐1 from the ER to the cytosol to initiate tubule assembly. Thus, P7‐1 is initially retained in the ER by interaction with DNAJB12 and then delivered to Hsc70. Furthermore, the inhibitors of the ATPase activity of Hsc70 reduce P7‐1 tubule assembly, suggesting that the proper folding and assembly of P7‐1 tubules is dependent on the ATPase activity of Hsc70. The DNAJB12–Hsc70 chaperone complex is recruited to P7‐1 tubules in virus‐infected midgut epithelial cells in S. furcifera. The knockdown of DNAJB12 or Hsc70 strongly inhibits P7‐1 tubule assembly in vivo, finally suppressing effective viral spread in S. furcifera. Taken together, our results indicate that the DNAJB12–Hsc70 chaperone complex in the ERAD machinery facilitates the ER‐to‐cytosol transport of P7‐1 for proper assembly of tubules, enabling viral spread in insect vectors in a manner dependent on ATPase activity of Hsc70.     

The genotype‐phenotype landscape of an allosteric protein

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype‐phenotype relationships remains elusive. Here, we report the large‐scale measurement of the genotype‐phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long‐read and short‐read DNA sequencing, we quantitatively measure the dose‐response curves for nearly 10⁵ variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence‐structure‐function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural‐network models, but unpredictable changes also occur. For example, we were surprised to discover a new band‐stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.