Homozygous variant in TKFC abolishing triokinase activities is associated with isolated immunodeficiency.

BACKGROUND: Triokinase and FMN cyclase (TKFC) is a bifunctional enzyme involved in fructose metabolism. Triokinase catalyses the phosphorylation of fructose-derived glyceraldehyde (GA) and exogenous dihydroxyacetone (DHA), while FMN cyclase generates cyclic FMN. TKFC regulates the antiviral immune response by interacting with IFIH1 (MDA5). Previously reported pathogenic variants in TKFC are associated with either a multisystemic disease or isolated hypotrichosis with loose anagen hairs. METHODS: Whole-exome sequencing identified a homozygous novel variant in TKFC (c.1624G>A; p.Gly542Arg) in an individual with a complex primary immunodeficiency disorder. The variant was characterised using enzymatic assays and yeast studies of mutant recombinant proteins. RESULTS: The individual presented with chronic active Epstein-Barr virus disease and multiple bacterial and viral infections. Clinical investigations revealed hypogammaglobulinaemia, near absent natural killer cells and decreased memory B cells. Enzymatic assays showed that this variant displayed defective DHA and GA kinase activity while maintaining FMN cyclase activity. An allogenic bone marrow transplantation corrected the patient's immunodeficiency. CONCLUSION: Our report suggests that TKFC may have a role in the immunological system. The pathological features associated with this variant are possibly linked with DHA/GA kinase inactivation through a yet an unknown mechanism. This report thus adds a possible new pathway of immunometabolism to explore further.

Starkeya nomas sp. nov., a prosthecate and budding bacterium isolated from an immunocompromized patient.

Strain HF14-78462T is an environmental bacterium found in clinical samples from an immunocompromized patient in 2014 at Hospital Universitari i Politècnic La Fe (Valencia, Spain). Phenotypically, strain HF14-78462T cells were Gram-stain-negative, aerobic, non-spore forming and non-motile small rods which formed mucous and whitish-translucent colonies when incubated at 20-36 °C. Phylogenetic analyses based on the 16S rRNA genes and the whole genomes of closest sequenced relatives confirmed that strain HF14-78462T is affiliated with the genus Starkeya. The strain was oxidase, catalase and urease positive; but indole, lysine decarboxylase, ornithine decarboxylase and DNase negative, did not produce H2S and was able to utilize a wide variety of carbon sources including acetamide, adonitol, amygdalin, l-arabinose, citric acid, glucose, mannitol and melibiose. Unlike Starkeya novella and Starkeya koreensis, strain HF14-78462T failed to grow in thiosulphate-oxidizing media and had a narrower temperature growth range. Its genome was characterized by a size of 4.83 Mbp and a C+G content of 67.75 mol%. Major fatty acids were C18:1 ω7c, cyclo C19 : 0 and C16 : 0, its polar acids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and an aminophospholipid; while the ubiquinones were Q9 (1.8 %) and Q10 (98.2 %). Digital DNA-DNA hybridization values were 41 and 41.4 against S. novella and S. koreensis, respectively, while average nucleotide identity values were around 84 %. Phenotypic, average nucleotide identity and phylogenomic comparative studies suggest that strain HF14-78462T is a new representative of the genus Starkeya and the name Starkeya nomas sp. nov. is proposed. The type strain is HF14-78462T (=CECT 30124T=LMG 31874T).

Activity of imipenem/relebactam against Enterobacterales and Pseudomonas aeruginosa in Spain. SMART 2016-2020

Objectives: To determine susceptibility to the novel β-lactam/β-lactamase inhibitor combination imipenem/relebactam in clinical isolates recovered from intra-abdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream (BSI) infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study in SPAIN during 2016 – 2020.Methods: Broth microdilution MICs for imipenem/relebactam and comparators were determined by a central laboratory against isolates of Enterobacterales and Pseudomonas aeruginosa. MICs were interpreted using EUCAST-2021 breakpoints.Results: In total, 5,210 Enterobacterales and 1,418 P. aeruginosa clinical isolates were analyzed. Imipenem/relebactam inhibited 98.8% of Enterobacterales. Distinguishing by source of infection susceptibility was 99.1% in BSI, 99.2% in IAI, 97.9% in RTI, and 99.2% in UTI. Of intensive care unit isolates (ICU) 97.4% were susceptible and of non-ICU isolates 99.2% were susceptible. In Enterobacterales, activity against Class A, Class B and Class D carbapenemases was 96.2%, 15.4% and 73.2%, respectively. In P. aeruginosa, imipenem/relebactam was active in 92.2% of isolates. By source of infection it was 94.8% in BSI, 92.9% in IAI, 91.7% in RTI, and 93.1% in UTI. An 88.7% of ICU isolates and 93.6% of non-ICU isolates were susceptible to imipenem/relebactam. Imipenem/relebactam remained active against P. aeruginosa ceftazidime-resistant (76.3%), cefepime-resistant (73.6%), imipenem-resistant (71.5%) and piperacillin-resistant (78.7%) isolates. Of all multidrug-resistant or difficult-to-treat resistance P. aeruginosa isolates, 75.1% and 46.2%, respectively, were susceptible to imipenem/relebactam. (AU)

Objetivos: Determinar la sensibilidad a la nueva combinación de β-lactámico e inhibidor de β-lactamasas imipenem/relebactam en aislados clínicos procedentes de infecciones intraabdominales (IIA), urinarias (ITU), respiratorias (ITR) y bacteriemias del estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) en ESPAÑA durante 2016 - 2020. Métodos. Se determinó la CMI mediante microdilución en caldo de imipenem/relebactam y antibióticos comparadores frente a aislados de Enterobacterales y Pseudomonas aeruginosa. Las CMI se analizaron empleando los puntos de corte EUCAST-2021. Resultados: En total, se incluyeron 5.210 aislados de Enterobacterales y 1.418 aislados de P. aeruginosa. Imipenem/ relebactam fue activo frente al 98,8% de los Enterobacterales. Distinguiendo por foco de infección, la sensibilidad fue del 99,1% en bacteriemia, del 99,2% en IIA, del 97,9% en ITR y del 99,2% en ITU. El 97,4% de los aislados procedentes de unidades de cuidados intensivos (UCI) fueron sensibles, y el 99,2% de los aislados no procedentes de UCI. En Enterobacterales, la sensibilidad frente a carbapenemasas de clase A, clase B y clase D fue del 96,2%, 15,4% y 73,2%, respectivamente. En P. aeruginosa,imipenem/relebactam fue activo en el 92,2% de los aislados. Distinguiendo por foco de infección, la sensibilidad frente a P. aeruginosa fue del 94,8% en bacteriemia, 92,9% en IIA, 91,7% en ITR y 93,1% en ITU. El 88,7% de los aislados de la UCI y el 93,6% de los aislados no procedentes de UCI fueron sensibles a imipenem/relebactam. Imipenem/relebactam fue activo frente a aislados de P. aeruginosa resistentes a ceftazidima (76,3%), cefepima (73,6%), imipenem (71,5%) y piperacilina/tazobactam (78,7%). Frente a los aislados de P. aeruginosa clasificados como MDR o DTR, el 75,1% y el 46,2%, respectivamente, fueron sensibles a imipenem/relebactam. (AU)

Genomic Distribution of Pro-Virulent cpdB-like Genes in Eubacteria and Comparison of the Enzyme Specificity of CpdB-like Proteins from Salmonella enterica, Escherichia coli and Streptococcus suis.

The cpdB gene is pro-virulent in avian pathogenic Escherichia coli and in Salmonella enterica, where it encodes a periplasmic protein named CpdB. It is structurally related to cell wall-anchored proteins, CdnP and SntA, encoded by the also pro-virulent cdnP and sntA genes of Streptococcus agalactiae and Streptococcus suis, respectively. CdnP and SntA effects are due to extrabacterial hydrolysis of cyclic-di-AMP, and to complement action interference. The mechanism of CpdB pro-virulence is unknown, although the protein from non-pathogenic E. coli hydrolyzes cyclic dinucleotides. Considering that the pro-virulence of streptococcal CpdB-like proteins is mediated by c-di-AMP hydrolysis, S. enterica CpdB activity was tested as a phosphohydrolase of 3'-nucleotides, 2',3'-cyclic mononucleotides, linear and cyclic dinucleotides, and cyclic tetra- and hexanucleotides. The results help to understand cpdB pro-virulence in S. enterica and are compared with E. coli CpdB and S. suis SntA, including the activity of the latter on cyclic-tetra- and hexanucleotides reported here for the first time. On the other hand, since CpdB-like proteins are relevant to host-pathogen interactions, the presence of cpdB-like genes was probed in eubacterial taxa by TblastN analysis. The non-homogeneous genomic distribution revealed taxa with cpdB-like genes present or absent, identifying eubacteria and plasmids where they can be relevant.

Enzyme Characterization of Pro-virulent SntA, a Cell Wall-Anchored Protein of Streptococcus suis, With Phosphodiesterase Activity on cyclic-di-AMP at a Level Suited to Limit the Innate Immune System.

Streptococcus suis and Streptococcus agalactiae evade the innate immune system of the infected host by mechanisms mediated by cell wall-anchored proteins: SntA and CdnP, respectively. The former has been reported to interfere with complement responses, and the latter dampens STING-dependent type-I interferon (IFN) response by hydrolysis of bacterial cyclic-di-AMP (c-di-AMP). Both proteins are homologous but, while CdnP has been studied as a phosphohydrolase, the enzyme activities of SntA have not been investigated. The core structure of SntA was expressed in Escherichia coli as a GST-tagged protein that, after affinity purification, was characterized as phosphohydrolase with a large series of substrates. This included 3'-nucleotides, 2',3'-cyclic nucleotides, cyclic and linear dinucleotides, and a variety of phosphoanhydride or phosphodiester compounds, most of them previously considered as substrates of E. coli CpdB, a periplasmic protein homologous to SntA and CdnP. Catalytic efficiency was determined for each SntA substrate, either by dividing parameters k cat /K M obtained from saturation curves or directly from initial rates at low substrate concentrations when saturation curves could not be obtained. SntA is concluded to act as phosphohydrolase on two groups of substrates with efficiencies higher or lower than ≈ 105 M-1 s-1 (average value of the enzyme universe). The group with k cat /K M ≥ 105 M-1 s-1 (good substrates) includes 3'-nucleotides, 2',3'-cyclic nucleotides, and linear and cyclic dinucleotides (notably c-di-AMP). Compounds showing efficiencies <104 M-1 s-1 are considered poor substrates. Compared with CpdB, SntA is more efficient with its good substrates and less efficient with its poor substrates; therefore, the specificity of SntA is more restrictive. The efficiency of the SntA activity on c-di-AMP is comparable with the activity of CdnP that dampens type-I IFN response, suggesting that this virulence mechanism is also functional in S. suis. SntA modeling revealed that Y530 and Y633 form a sandwich with the nitrogen base of nucleotidic ligands in the substrate-binding site. Mutants Y530A-SntA, Y633A-SntA, and Y530A+Y633A-SntA were obtained and kinetically characterized. For orientation toward the catalytic site, one tyrosine is enough, although this may depend on the substrate being attacked. On the other hand, both tyrosines are required for the efficient binding of good SntA substrates.

The TKFC Ala185Thr variant, reported as 'null' for fructose metabolism, is fully active as triokinase.

TKFC-encoded triokinase catalyses glyceraldehyde phosphorylation in fructose metabolism and favours lipogenesis in mice. In Tkfc knockouts or knockdowns, fructose oxidation predominates over lipogenesis. The highly prevalent human variant Ala185Thr-Triokinase/FMN cyclase (TKFC) has been reported to be 'null' for fructose metabolism, since Ala185-TKFC rescues the mouse TKFC-deficient phenotype, whereas Ala185Thr-TKFC does not. Such report implies that most humans would display a noncanonical fructose metabolism, but it ignores the well-characterized triokinase activity of Ala185Thr-TKFC. Here, earlier evidence is summarized, along with new evidence that both human variants are equally active in yeast. Therefore, future research on triokinase in the context of human fructose metabolism should consider that Ala185Thr-TKFC is not biochemically 'null'.

Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5'-Nucleotidase UshA and the 3'-Nucleotidase CpdB.

The 5'-nucleotidase UshA and the 3'-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom-CpdB_Cdom and CpdB_Ndom-UshA_Cdom were constructed and tested on substrates specific to UshA (5'-AMP, CDP-choline, UDP-glucose) or to CpdB (3'-AMP), as well as on 2',3'-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5'-nucleotidase nor 3'-nucleotidase activity. When compared to UshA, UshA_Ndom-CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2',3'-cAMP. When compared to CpdB, CpdB_Ndom-UshA_Cdom conserved phosphodiesterase activities on 2',3'-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.

Molecular Dissection of Escherichia coli CpdB: Roles of the N Domain in Catalysis and Phosphate Inhibition, and of the C Domain in Substrate Specificity and Adenosine Inhibition.

CpdB is a 3'-nucleotidase/2'3'-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP (3',5'-cyclic diadenosine monophosphate) and c-di-GMP (3',5'-cyclic diadenosine monophosphate). These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-ß response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplasmic or cell-wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3'-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2',3'-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.

Streptococcus agalactiae in childbearing age immigrant women in Comunitat Valenciana (Spain).

Streptococcus agalactiae (GBS) remains the leading cause of meningitis and neonatal sepsis in the world, and causes disease in pregnant and puerperal women. This is a retrospective study of GBS infections on women of childbearing age living in Comunitat Valenciana, Spain (years 2009-2014) and GBS colonization rate on pregnant women attending Hospital La Fe (years 2013-2015) according to their origin. An aggregated total of 6,641,960 women exposed during the study period had an average GBS isolation rate of 5.19‰ (5.14-5.25‰), geographical group rates being: Western Europe (2.2‰), North America (2.1‰), Australia (3.7‰), Spain (4.6‰), Latin America II (4.5‰), Eastern Europe (5.3‰), Asia (6.7‰), Latin America I (7.7‰), Middle East (7.9‰), Indian Subcontinent (17.2‰), North Africa (17.8‰), Sub-Saharan Africa (22.7‰). The 4532 pregnant women studied had an average GBS colonization rate of 12.47% (11.51-13.43) and geographical group rates varied similar to geographical isolation rates. Low GDP and high temperatures of the birth country were associated with higher colonization rates. Thus, differences in GBS colonization depend on the country of origin; Africa and the Indian subcontinent presented the highest, while Western Europe and North America had the lowest. This variability portrays a geographical pattern influenced by temperature and GDP.

Closure of the Human TKFC Active Site: Comparison of the Apoenzyme and the Complexes Formed with Either Triokinase or FMN Cyclase Substrates.

Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.

Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase.

Cyclic ADP-ribose (cADPR) is a messenger for Ca2+ mobilization. Its turnover is believed to occur by glycohydrolysis to ADP-ribose. However, ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) acts as cADPR phosphohydrolase with much lower efficiency than on its major substrates. Recently, we showed that mutagenesis of human ADPRibase-Mn at Phe37, Leu196 and Cys253 alters its specificity: the best substrate of the mutant F37A + L196F + C253A is cADPR by a short difference, Cys253 mutation being essential for cADPR preference. Its proximity to the 'northern' ribose of cADPR in docking models indicates Cys253 is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp250, Val252, Cys253 and Thr279, all near the 'northern' ribose. First, the mutant F37A + L196F + C253G, with a smaller residue 253 (Ala > Gly), showed increased cADPR specificity. Then, the mutant F37A + L196F + V252A + C253G, with another residue made smaller (Val > Ala), displayed the desired specificity, with cADPR kcat/KM ≈20-200-fold larger than for any other substrate. When tested in nucleotide mixtures, cADPR was exhausted while others remained unaltered. We suggest that the specific cADPR phosphohydrolase, by cell or organism transgenesis, or the designed mutations, by genome editing, provide opportunities to study the effect of cADPR depletion on the many systems where it intervenes.

Influence of environmental conditions and pollution on the incidence of Streptococcus pneumoniae infections.

Fossil fuel derived pollutants (SO2, NO), dry air and cold increase the incidence of S. pneumoniae infections http://ow.ly/RnLW30gogb1.

Nosocomial fungemia by Candida auris: first four reported cases in continental Europe / Fungemia nosocomial por Candida auris: primeros cuatro casos en Europa continental

Background. Candida auris is an emerging multidrug-resistant yeast that can cause invasive infections and is associated with high mortality. It is typically resistant to fluconazole and voriconazole and, some cases, also to echinocandins and amphotericin B. This species, phylogenetically related to Candida haemulonii, is frequently misidentified by commercial identification techniques in clinical laboratories; therefore, the real prevalence of C. auris infections may be underestimated. Aims. To describe the clinical and microbiological features of the first four cases of C. auris fungemia episodes observed in the European continent. Methods. The four patients were hospitalized in the adult surgical intensive care unit. A total of 8 isolates (two per patient) from blood and catheter tip were analyzed. Results. All isolates were misidentified as Saccharomyces cerevisiae by AuxaColor 2, and as Candida sake by API ID20C. VITEK MS technology misidentified one isolate as Candida lusitaniae, another as C. haemulonii and could not identify the other six. C. auris identification was confirmed by ITS rDNA sequencing. All isolates were fluconazole (MIC >256mg/l) and voriconazole (MIC 2mg/l) resistant and susceptible to posaconazole, itraconazole, echinocandins and amphotericin B. Conclusions. C. auris should be regarded as an emerging pathogen, which requires molecular methods for definitive identification. Our isolates were highly resistant to fluconazole and resistant to voriconazole, but susceptible to the other antifungals tested, which emphasizes the importance of accurately identifying this species to avoid therapeutic failures (AU)

Antecedentes. Candida auris es una levadura multirresistente de reciente aparición que puede causar infecciones invasivas asociadas con una elevada mortalidad. Habitualmente, C. auris es resistente al fluconazol y el voriconazol, y en algunos casos, también a las equinocandinas y la anfotericina B. Esta especie, relacionada filogenéticamente con Candida haemulonii, no se identifica por las técnicas comerciales habitualmente disponibles en los laboratorios clínicos, por lo que la prevalencia real de las infecciones causadas por C. auris puede estar subestimada. Objetivos. Describir las características clínicas y microbiológicas de los cuatro primeros casos de fungemia por C. auris observados en el continente europeo. Métodos. Los cuatro pacientes eran adultos y estaban en la unidad de cuidados intensivos quirúrgicos. Se analizaron un total de 8 aislamientos (dos por paciente), obtenidos a partir de un hemocultivo y de punta de catéter. Resultados. Todos los aislamientos se identificaron erróneamente como Saccharomyces cerevisiae por AuxaColor 2 y como Candida sake por API ID20C. El sistema VITEK MS identificó erróneamente un aislamiento como Candida lusitaniae, otro como C. haemulonii y no pudo identificar los seis aislamientos restantes. La identificación de C. auris se confirmó mediante secuenciación de la región ITS del ADNr. Todos los aislamientos fueron resistentes al fluconazol (CMI>256mg/l) y el voriconazol (CMI 2mg/l) y sensibles al posaconazol, el itraconazol, las equinocandinas y la anfotericina B. Conclusiones. C. auris es un agente patógeno de reciente aparición que actualmente solo puede ser identificado mediante secuenciación molecular. Nuestros aislamientos fueron muy resistentes al fluconazol y resistentes al voriconazol, pero sensibles a los otros antifúngicos ensayados, lo cual destaca la importancia de identificar correctamente esta especie en la práctica asistencial para evitar fracasos terapéuticos (AU)

Nosocomial fungemia by Candida auris: First four reported cases in continental Europe.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast that can cause invasive infections and is associated with high mortality. It is typically resistant to fluconazole and voriconazole and, some cases, also to echinocandins and amphotericin B. This species, phylogenetically related to Candida haemulonii, is frequently misidentified by commercial identification techniques in clinical laboratories; therefore, the real prevalence of C. auris infections may be underestimated. AIMS: To describe the clinical and microbiological features of the first four cases of C. auris fungemia episodes observed in the European continent. METHODS: The four patients were hospitalized in the adult surgical intensive care unit. A total of 8 isolates (two per patient) from blood and catheter tip were analyzed. RESULTS: All isolates were misidentified as Saccharomyces cerevisiae by AuxaColor 2, and as Candida sake by API ID20C. VITEK MS technology misidentified one isolate as Candida lusitaniae, another as C. haemulonii and could not identify the other six. C. auris identification was confirmed by ITS rDNA sequencing. All isolates were fluconazole (MIC >256mg/l) and voriconazole (MIC 2mg/l) resistant and susceptible to posaconazole, itraconazole, echinocandins and amphotericin B. CONCLUSIONS: C. auris should be regarded as an emerging pathogen, which requires molecular methods for definitive identification. Our isolates were highly resistant to fluconazole and resistant to voriconazole, but susceptible to the other antifungals tested, which emphasizes the importance of accurately identifying this species to avoid therapeutic failures.

The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase.

Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the 'average' enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.

Molecular bases of catalysis and ADP-ribose preference of human Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase and conversion by mutagenesis to a preferential cyclic ADP-ribose phosphohydrolase.

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.

Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.

Characterization of Danio rerio Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase, the structural prototype of the ADPRibase-Mn-like protein family.

The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn(2+)-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg(2+) and active with low micromolar Mn(2+) in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR) in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn(2+), significant (≈25%) Mg(2+)-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart). The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.

CGDEase, a Pseudomonas fluorescens protein of the PLC/APase superfamily with CDP-ethanolamine and (dihexanoyl)glycerophosphoethanolamine hydrolase activity induced by osmoprotectants under phosphate-deficient conditions.

A novel enzyme, induced by choline, ethanolamine, glycine betaine or dimethylglycine, was released at low temperature and phosphate from Pseudomonas fluorescens (CECT 7229) suspensions at low cell densities. It is a CDP-ethanolamine pyrophosphatase/(dihexanoyl)glycerophosphoethanolamine phosphodiesterase (CGDEase) less active on choline derivatives, and inactive on long-chain phospholipids, CDP-glycerol and other NDP-X compounds. The reaction pattern was typical of phospholipase C (PLC), as either phosphoethanolamine or phosphocholine was produced. Peptide-mass analyses, gene cloning and expression provided a molecular identity for CGDEase. Bioinformatic studies assigned it to the PLC branch of the phospholipase C/acid phosphatase (PLC/APase) superfamily, revealed an irregular phylogenetic distribution of close CGDEase relatives, and suggested their genes are not in operons or conserved contexts. A theoretical CGDEase structure was supported by mutagenesis of two predicted active-site residues, which yielded essentially inactive mutants. Biological relevance is supported by comparisons with CGDEase relatives, induction by osmoprotectants (not by osmotic stress itself) and repression by micromolar phosphate. The low bacterial density requirement was related to phosphate liberation from lysed bacteria in denser populations, rather than to a classical quorum-sensing effect. The results fit better a CGDEase role in phosphate scavenging than in osmoprotection.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn(2+)-dependent ADP-ribose/CDP-alcohol pyrophosphatase.

Cyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N(1)-glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N(1)-(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.