Toxoplasma gondii UBL-UBA Shuttle Protein DSK2s Are Important for Parasite Intracellular Replication

Toxoplasma gondii (T. gondii) is an important human and veterinary pathogen causing life-threatening disease in immunocompromised patients. The UBL-UBA shuttle protein family are important components of the ubiquitin–proteasome system. Here, we identified a novel UBL-UBA shuttle protein DSK2b that is charactered by an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). DSK2b was localized in the cytoplasm and nucleus. The deletion of dsk2b did not affect the degradation of ubiquitinated proteins, parasite growth in vitro or virulence in mice. The double-gene knockout of dsk2b and its paralogs dsk2a (ΔΔdsk2adsk2b) results in a significant accumulation of ubiquitinated proteins and the asynchronous division of T. gondii. The growth of ΔΔdsk2adsk2b was significantly inhibited in vitro, while virulence in mice was not attenuated. In addition, autophagy occurred in the ΔΔdsk2adsk2b, which was speculated to degrade the accumulated ubiquitinated proteins in the parasites. Overall, DSK2b is a novel UBL-UBA shuttle protein contributing to the degradation of ubiquitinated proteins and is important for the synchronous cell division of T. gondii.     

The Knockout of Enterobactin-Related Gene in Pectobacterium atrosepticum Results in Reduced Stress Resistance and Virulence towards the Primed Plants

Siderophores produced by microorganisms to scavenge iron from the environment have been shown to contribute to virulence and/or stress resistance of some plant pathogenic bacteria. Phytopathogenic bacteria of Pectobacterium genus possess genes for the synthesis of siderophore enterobactin, which role in plant-pathogen interactions has not been elucidated. In the present study we characterized the phenotype of the mutant strain of Pba deficient for the enterobactin-biosynthetic gene entA. We showed that enterobactin may be considered as a conditionally beneficial virulence factor of Pba. The entA knockout did not reduce Pba virulence on non-primed plants; however, salicylic acid-primed plants were more resistant to ΔentA mutant than to the wild type Pba. The reduced virulence of ΔentA mutant towards the primed plants is likely explained by its compromised resistance to oxidative stress.     

A Highlight on the Inhibition of Fungal Carbonic Anhydrases as Drug Targets for the Antifungal Armamentarium

Carbon dioxide (CO₂), a vital molecule of the carbon cycle, is a critical component in living organisms’ metabolism, performing functions that lead to the building of compounds fundamental for the life cycle. In all living organisms, the CO₂/bicarbonate (HCO₃⁻) balancing is governed by a superfamily of enzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the pivotal physiological reaction, consisting of the reversible hydration of the CO₂ to HCO₃⁻ and protons. Opportunistic and pathogenic fungi can sense the environmental CO₂ levels, which influence their virulence or environmental subsistence traits. The fungal CO₂-sensing is directly stimulated by HCO₃⁻ produced in a CA-dependent manner, which directly activates adenylyl cyclase (AC) involved in the fungal spore formation. The interference with CA activity may impair fungal growth and virulence, making this approach interesting for designing antifungal drugs with a novel mechanism of action: the inhibition of CAs linked to the CO₂/HCO₃⁻/pH chemosensing and signaling. This review reports that sulfonamides and their bioisosteres as well as inorganic anions can inhibit in vitro the β- and α-CAs from the fungi, suggesting how CAs may be considered as a novel “pathogen protein” target of many opportunistic, pathogenic fungi.     

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh^Δex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh^Δex5−6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh^Δex5−6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh^Δex5−6 mutant larvae in a context-dependent manner. We expect the sgsh^Δex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.     

Aryl Hydrocarbon Receptor Repressor and TiPARP (ARTD14) Use Similar,but also Distinct Mechanisms to Repress Aryl Hydrocarbon Receptor Signaling

The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP^−/− mouse embryonic fibroblasts (MEFs) and AHRR^−/− MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP^−/− MEFs, AHRR^−/− MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR^−/− MEFs or AHRRΔ8, the active isoform of AHRR, in TiPARP^−/− MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRRΔ8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRRΔ8_Δ1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRRΔ8_Δ1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRRΔ8_Δ1-49 nor GFP-AHRRΔ8_Δ1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.     

Metabolic,Organoleptic and Transcriptomic Impact of Saccharomyces cerevisiae Genes Involved in the Biosynthesis of Linear and Substituted Esters

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.     

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains

Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.     

The Environmental Effects on Virulence Factors and the Antifungal Susceptibility of Cryptococcus neoformans

Cryptococcus neoformans is a facultative intracellular pathogen responsible for fungal meningoencephalitis primarily in immunocompromised individuals. It has become evident the pathogenicity of C. neoformans is dependent on the fungal cell’s environment. The differential expression of virulence factors, based on the cell’s environmental conditions, is one mechanism allowing for the environmental control of the pathogenic ability of C. neoformans. Here, we discuss how these virulence factors (including melanin, the polysaccharide capsule, and Antiphagocytic protein 1) have been shown to be differentially expressed dependent on the cell’s environment. The genetics and signaling pathways leading to the environmental-dependent regulation of virulence factors will also be examined. Susceptibility to antifungal therapeutics is also regulated by the environment, and thus affects the pathogenic abilities of C. neoformans and disease outcomes. This review will also examine the role of the C. neoformans’s environment on antifungal susceptibilities, and the genetics and signaling pathways responsible for these susceptibility alterations. By examining the complex interplay between the environment and the pathogenicity of C. neoformans, we have a better understanding of the intricacies of the pathogen–environment interaction and how to exploit this interaction to develop the most effective treatment protocols.     

Molecular Basis of Essentiality of Early Critical Steps in the Lipopolysaccharide Biogenesis in Escherichia coli K-12: Requirement of MsbA,Cardiolipin, LpxL,LpxM and GcvB

To identify the physiological factors that limit the growth of Escherichia coli K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire waa locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IV_A derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ(gmhD-waaA) identified two independent single-amino-acid substitutions—P50S and R310S—in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene clsA was found to be essential for the growth of ΔlpxLMP, ΔlpxL, ΔwaaA, and Δ(gmhD-waaA) bacteria, with a conditional lethal phenotype of Δ(clsA lpxM), which could be overcome by suppressor mutations in MsbA. Suppressor mutations basS A20D or basR G53V, causing a constitutive incorporation of phosphoethanolamine (P-EtN) in the lipid A, could abolish the Ca⁺⁺ sensitivity of Δ(waaC eptB), thereby compensating for P-EtN absence on the second Kdo. A single-amino-acid OppA S273G substitution is shown to overcome the synthetic lethality of Δ(waaC surA) bacteria, consistent with the chaperone-like function of the OppA oligopeptide-binding protein. Furthermore, overexpression of GcvB sRNA was found to repress the accumulation of LpxC and suppress the lethality of LapAB absence. Thus, this study identifies new and limiting factors in regulating LPS biosynthesis.     

Glitazone Treatment Rescues Phenotypic Deficits in a Fly Model of Gaucher/Parkinson’s Disease

Parkinson’s Disease (PD) is the most common movement disorder, and the strongest genetic risk factor for PD is mutations in the glucocerebrosidase gene (GBA). Mutations in GBA also lead to the development of Gaucher Disease (GD), the most common type of lysosomal storage disorder. Current therapeutic approaches fail to address neurological GD symptoms. Therefore, identifying therapeutic strategies that improve the phenotypic traits associated with GD/PD in animal models may provide an opportunity for treating neurological manifestations of GD/PD. Thiazolidinediones (TZDs, also called glitazones) are a class of compounds targeted for the treatment of type 2 diabetes, and have also shown promise for the treatment of neurodegenerative disease, including PD. Here, we tested the efficacy of glitazone administration during development in a fly GD model with deletions in the GBA homolog, dGBA1b (GBA1^ΔTT/ΔTT). We observed an optimal dose of pioglitazone (PGZ) at a concentration of 1 μM that reduced sleep deficits, locomotor impairments, climbing defects, and restoration of normal protein levels of Ref(2)P, a marker of autophagic flux, in GBA1^ΔTT/ΔTT mutant flies, compared to GBA1^+/+ control flies. These data suggest that PGZ may represent a potential compound with which to treat GD/PD by improving function of lysosomal-autophagy pathways, a cellular process that removes misfolded or aggregated proteins.     

Targeting Copper Homeostasis Improves Functioning of vps13Δ Yeast Mutant Cells,a Model of VPS13-Related Diseases

Ion homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson’s diseases, respectively, and iron imbalance is observed in Parkinson’s and Alzheimer’s diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A–D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases.     

Computer-Aided Design of Orally Bioavailable Pyrrolidine Carboxamide Inhibitors of Enoyl-Acyl Carrier Protein Reductase of Mycobacterium tuberculosis with Favorable Pharmacokinetic Profiles

We have carried out a computational structure-based design of new potent pyrrolidine carboxamide (PCAMs) inhibitors of enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTb). Three-dimensional (3D) models of InhA-PCAMx complexes were prepared by in situ modification of the crystal structure of InhA-PCAM1 (Protein Data Bank (PDB) entry code: 4U0J), the reference compound of a training set of 20 PCAMs with known experimental inhibitory potencies (IC₅₀^exp). First, we built a gas phase quantitative structure-activity relationships (QSAR) model, linearly correlating the computed enthalpy of the InhA-PCAM complex formation and the IC₅₀^exp. Further, taking into account the solvent effect and loss of inhibitor entropy upon enzyme binding led to a QSAR model with a superior linear correlation between computed Gibbs free energies (ΔΔG_com) of InhA-PCAM complex formation and IC₅₀^exp (pIC₅₀^exp = −0.1552·ΔΔG_com + 5.0448, R² = 0.94), which was further validated with a 3D-QSAR pharmacophore model generation (PH4). Structural information from the models guided us in designing of a virtual combinatorial library (VL) of more than 17 million PCAMs. The VL was adsorption, distribution, metabolism and excretion (ADME) focused and reduced down to 1.6 million drug like orally bioavailable analogues and PH4 in silico screened to identify new potent PCAMs with predicted IC₅₀^pre reaching up to 5 nM. Combining molecular modeling and PH4 in silico screening of the VL resulted in the proposed novel potent antituberculotic agent candidates with favorable pharmacokinetic profiles.     

Cyclophilin BcCyp2 Regulates Infection-Related Development to Facilitate Virulence of the Gray Mold Fungus Botrytis cinerea

Cyclophilin (Cyp) and Ca²⁺/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca²⁺/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.     

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3^Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3^Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein–protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0–C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3^Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.     

A Polymorphism at the Translation Start Site of the Vitamin D Receptor Gene Is Associated with the Response to Anti-Osteoporotic Therapy in Postmenopausal Women from Southern Italy

The present study investigated the effect of two single nucleotide polymorphisms (SNPs) of the vitamin D receptor (VDR) gene, rs1544410 A/G and rs2228570 C/T, in modulating bone mineral density (BMD) and the response to treatment with bisphosphonates or strontium ranelate in postmenopausal osteoporosis (PMO). Four hundred eighteen postmenopausal women from Southern Italy treated with bisphosphonates or strontium ranelate for three years were enrolled and stratified according to their genotype. Changes in BMD were expressed as the delta t-score (Δt-score). Allelic frequencies for rs1544410 A/GSNP were 11.2% AA, 50.0% GA and 38.8% GG; for rs2228570 C/TSNP were 54.8% CC, 39.5% TC and 5.7% TT. TT carriers showed a lower t-score than TC and CC (both p < 0.02) genotypes and were more responsive to the therapy when compared to both TC (p < 0.02) and CC (p < 0.05) carriers. Specifically, TT carriers receiving alendronate demonstrated a significant improvement of the Δt-score compared to TC and CC (both p < 0.0001) carriers. After adjustment for confounders, the Δt-score showed evidence of a statistically significant positive association with TT in all treatments considered. Therapy response was independent of rs1544410 A/G SNP; instead, rs2228570 C/TSNP was associated with a better response to antiresorptive treatment, thus suggesting that the therapy for PMO should be personalized.