The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. DATABASE: Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively.

• Klíčová slova
• GMP, IMP, Trypanosoma brucei, XMP, 6-oxopurine phosphoribosyltransferase, purine salvage,
• MeSH
• inosinmonofosfát chemie   metabolismus   MeSH
• konformace proteinů MeSH
• kyselina 5'-guanylová chemie   metabolismus   MeSH
• pentosyltransferasy chemie   metabolismus   MeSH
• ribonukleotidy chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• Trypanosoma brucei brucei enzymologie   MeSH
• xanthin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inosinmonofosfát MeSH
• kyselina 5'-guanylová MeSH
• pentosyltransferasy MeSH
• ribonukleotidy MeSH
• xanthin MeSH
• xanthine phosphoribosyltransferase MeSH Prohlížeč
• xanthosine monophosphate MeSH Prohlížeč

Structural details of changes accompanying interaction between insulin-related hormones and their binding partners are often enigmatic. Here, cross-linking/mass spectrometry could complement structural techniques and reveal details of these protein-protein interfaces. We used such approach to clarify missing structural description of the interface in human insulin-like growth factor (IGF-1): Drosophila melanogaster imaginal morphogenesis protein-late 2 protein (Imp-L2) complex which we studied previously by X-ray crystallography. We crosslinked these proteins by heterobifunctional cross-linker sulfosuccinimidyl 4,4'-azidopentanoate (Sulfo-SDA) for the subsequent mass spectrometry (MS) analysis. The MS analysis revealed IGF-1:Imp-L2 interactions which were not resolved in the crystal structure of this assembly, and they converged with X-ray results, indicating the importance of the IGF-1 N-terminus interaction with the C-terminal (185-242) part of the Imp-L2 for stability of this complex. Here, we also showed the advantage and reliability of MS approach in solving details of protein-protein interactions that are too flexible for solid state structural methods.

• Klíčová slova
• IGF-1, Imp-L2, cross-linking, diazirine ring, mass spectrometry,
• Publikační typ
• časopisecké články MeSH

Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• alosterická regulace MeSH
• bakteriální proteiny metabolismus   chemie   genetika   MeSH
• elektronová kryomikroskopie MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintrifosfát * metabolismus   MeSH
• IMP-dehydrogenasa * metabolismus   chemie   antagonisté a inhibitory   MeSH
• inosinmonofosfát metabolismus   chemie   MeSH
• katalytická doména MeSH
• molekulární modely MeSH
• Mycobacterium smegmatis enzymologie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• bakteriální proteiny MeSH
• guanosinpentafosfát MeSH
• guanosintrifosfát * MeSH
• IMP-dehydrogenasa * MeSH
• inosinmonofosfát MeSH

Toxic effects of both main colicin types, i.e. of porin and nuclease types, involve the direct contact of their molecules with the plasma membrane of sensitive cells. In the present study, it was tested whether this contact provokes a lateral or vertical movement of intramembrane protein particles (IMP) or a direct cleavage of the proteins. IMP were visualized by freeze-fracturing and electron microscopy on the protoplasmic fracture face (PF) of colicin-treated cells of Escherichia coli. Possible changes in distribution and in density of IMP due to treatment with colicins E1-E7 and K were followed. As a control, the bacteria were equilibrated at 0 degrees C before quenching, which caused a reversible formation of smooth areas and a decrease in the mean density of IMP on the PF. Colicins E1-E7 had no clear-cut effect on the disposition of IMP. Only colicin K decreased the IMP density, by 10% in E. coli strain 58-161 and by 17% in strain C6; the distribution of IMP remained homogeneous. Trypsin reactivation of colicin-K-inactivated bacteria was not reflected by restoration of the original density of IMP; on the contrary, it led to a further decrease, of 1-13%, in IMP density, presumably by proteolytic cleavage. Varying densities of IMP in different strains of the same bacterial species (under standard conditions) were confirmed.

• MeSH
• buněčná membrána účinky léků   ultrastruktura   MeSH
• elektronová mikroskopie MeSH
• Escherichia coli účinky léků   MeSH
• koliciny farmakologie   MeSH
• mrazové lámání MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• koliciny MeSH

Mycobacteria express enzymes from both the de novo and purine-salvage pathways. However, the regulation of these processes and the roles of individual metabolic enzymes have not been sufficiently detailed. Both Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm) possess three guaB genes, but information is only available on guaB2, which encodes an essential inosine 5'-monophosphate dehydrogenase (IMPDH) involved in de novo purine biosynthesis. This study shows that guaB1, annotated in databases as a putative IMPDH, encodes a guanosine 5'-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine-salvage pathway and contains a cystathionine-β-synthase domain (CBS), which is essential for enzyme activity. GMPR activity is allosterically regulated by the ATP/GTP ratio in a pH-dependent manner. Bioinformatic analysis has indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.

• Klíčová slova
• CBS domain, GMPR, Mycobacterium, guaB1, purine biosynthesis,
• MeSH
• adenosintrifosfát MeSH
• cystathionin-beta-synthasa genetika   MeSH
• cystathionin * MeSH
• GMP-reduktasa genetika   metabolismus   MeSH
• guanosintrifosfát MeSH
• IMP-dehydrogenasa genetika   metabolismus   MeSH
• inosin MeSH
• inosinmonofosfát metabolismus   MeSH
• kyselina 5'-guanylová metabolismus   MeSH
• Mycobacterium tuberculosis * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• cystathionin-beta-synthasa MeSH
• cystathionin * MeSH
• GMP-reduktasa MeSH
• guanosintrifosfát MeSH
• IMP-dehydrogenasa MeSH
• inosin MeSH
• inosinmonofosfát MeSH
• kyselina 5'-guanylová MeSH

The objective of this study was to perform molecular surveillance for assessing the spread of carbapenemase-producing Pseudomonas aeruginosa in Czech hospitals. One hundred thirty-six carbapenemase-producing isolates were recovered from 22 hospitals located throughout the country. Sequence type 357 (ST357) dominated (n = 120) among carbapenemase producers. One hundred seventeen isolates produced IMP-type (IMP-7 [n = 116] and IMP-1 [n = 1]) metallo-β-lactamases (MβLs), 15 produced the VIM-2 MβL, and the remaining isolates expressed the GES-5 enzyme. The blaIMP-like genes were located in three main integron types, with In-p110-like being the most prevalent (n = 115). The two other IMP-encoding integrons (In1392 and In1393) have not been described previously. blaVIM-2-carrying integrons included In59-like, In56, and a novel element (In1391). blaGES-5 was carried by In717. Sequencing data showed that In-p110-like was associated with a Tn4380-like transposon inserted in genomic island LESGI-3 in the P. aeruginosa chromosome. The other integrons were also integrated into the P. aeruginosa chromosome. These findings indicated the clonal spread of ST357 P. aeruginosa, carrying the IMP-7-encoding integron In-p110, in Czech hospitals. Additionally, the sporadic emergence of P. aeruginosa producing different carbapenemase types, associated with divergent or novel integrons, punctuated the ongoing evolution of these bacteria.

• Klíčová slova
• GES, Illumina sequencing, ST111, ST235, VIM, class 1 integrons, genomic islands (GIs), integrative conjugative element (ICE),
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální chromozomy chemie   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• epidemiologické monitorování MeSH
• exprese genu MeSH
• genomové ostrovy MeSH
• genotyp MeSH
• incidence MeSH
• integrony MeSH
• izoenzymy genetika   metabolismus   MeSH
• karbapenemy farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• multilokusová sekvenční typizace MeSH
• nemocnice MeSH
• pseudomonádové infekce epidemiologie   mikrobiologie   MeSH
• Pseudomonas aeruginosa účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-lactamase IMP-7 protein, Pseudomonas aeruginosa MeSH Prohlížeč
• beta-laktamasy MeSH
• izoenzymy MeSH
• karbapenemy MeSH

IMP-8 metallo-β-lactamase was identified in Klebsiella pneumoniae sequence type 252 (ST252), isolated in a Portuguese hospital in 2009. blaIMP-8 was the first gene cassette of a novel class 3 integron, In1144, also carrying the blaGES-5, blaBEL-1, and aacA4 cassettes. In1144 was located on a ColE1-like plasmid, pKP-M1144 (12,029 bp), with a replication region of limited nucleotide similarity to those of other RNA-priming plasmids, such as pJHCMW1. In1144 and pKP-M1144 represent an interesting case of evolution of resistance determinants in Gram-negative bacteria.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   MeSH
• DNA bakterií genetika   MeSH
• infekce bakteriemi rodu Klebsiella mikrobiologie   MeSH
• karbapenemy farmakologie   MeSH
• Klebsiella pneumoniae účinky léků   genetika   izolace a purifikace   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• molekulární sekvence - údaje MeSH
• plazmidy účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Portugalsko MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-lactamase BEL-1, Klebsiella pneumoniae MeSH Prohlížeč
• beta-lactamase GES-5, Klebsiella pneumoniae MeSH Prohlížeč
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• DNA bakterií MeSH
• IMP-8 enzyme MeSH Prohlížeč
• karbapenemy MeSH

Inosine-5'-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed "Rods and Rings" (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.

• Klíčová slova
• Inosine-5′-monophosphate dehydrogenase, Rods and Rings, correlative light and electron microscopy, electron tomography, inclusions, inhibitors of IMPDH, ultrastructure,
• MeSH
• aktivní transport - buněčné jádro účinky léků   MeSH
• buněčné jádro enzymologie   metabolismus   ultrastruktura   MeSH
• cytoplazma enzymologie   ultrastruktura   MeSH
• HeLa buňky MeSH
• IMP-dehydrogenasa antagonisté a inhibitory   chemie   metabolismus   ultrastruktura   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• IMP-dehydrogenasa MeSH
• IMPDH2 protein, human MeSH Prohlížeč
• inhibitory enzymů MeSH

The complete nucleotide sequences of six IMP-4-encoding plasmids recovered from Enterobacteriaceae isolates of wildlife origin were characterized. Sequencing data showed that plasmids of different incompatibility groups (IncM, IncI1, IncF, and nontypeable [including an IncX5_2 and two pPrY2001-like]) carried the blaIMP-4-carrying integrons In809 or In1460. Most of the plasmids carried an mph(A) region, and chrA-like, aac(3)-IId, and blaTEM-1b genes. Finally, plasmid analysis revealed the involvement of two different IS26- and Tn1696-associated mechanisms in the mobilization of IMP-4-encoding integrons.

• Klíčová slova
• Enterobacter aerogenes, Escherichia coli, In1460, In809, IncF, IncI1, IncM, IncX5_2, Proteus mirabilis, Proteus penneri,
• MeSH
• antibakteriální látky farmakologie   MeSH
• Enterobacteriaceae genetika   patogenita   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• plazmidy genetika   MeSH
• přenos genů horizontální genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH

OBJECTIVES: The objective of this study was to investigate the silver gull as an indicator of environmental contamination by salmonellae and carbapenemase-producing Enterobacteriaceae (CPE) in south-east Australia. METHODS: A total of 504 cloacal samples were collected from gull chicks at three nesting colonies in New South Wales, Australia [White Bay (n = 144), Five Islands (n = 200) and Montague Island (n = 160)] and were examined for salmonellae and CPE. Isolates were tested for carbapenemase genes and susceptibility to 14 antibiotics. Clonality was determined by PFGE and MLST. Genetic context and conjugative transfer of the carbapenemase gene were determined. RESULTS: A total of 120 CPE of 10 species, mainly Escherichia coli (n = 85), carrying the gene blaIMP-4, blaIMP-38 or blaIMP-26 were obtained from 80 (40%) gulls from Five Islands. Thirty percent of birds from this colony were colonized by salmonellae. Most isolates contained the gene within a class 1 integron showing a blaIMP-4-qacG-aacA4-catB3 array. The blaIMP gene was carried by conjugative plasmids of variable sizes (80-400 kb) and diverse replicons, including HI2-N (n = 30), HI2 (11), A/C (17), A/C-Y (2), L/M (5), I1 (1) and non-typeable (6). Despite the overall high genetic variability, common clones and plasmid types were shared by different birds and bacterial isolates, respectively. CONCLUSIONS: Our data demonstrate a large-scale transmission of carbapenemase-producing bacteria into wildlife, likely as a result of the feeding habits of the birds at a local waste depot. The isolates from gulls showed significant similarities with clinical isolates from Australia, suggesting the human origin of the isolates. The sources of CPE for gulls on Five Islands should be explored and proper measures applied to stop the transmission into the environment.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny analýza   genetika   MeSH
• beta-laktamasy analýza   genetika   MeSH
• Charadriiformes mikrobiologie   MeSH
• Enterobacteriaceae enzymologie   izolace a purifikace   MeSH
• enterobakteriální infekce epidemiologie   mikrobiologie   veterinární   MeSH
• genotyp MeSH
• kloaka mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární epidemiologie MeSH
• molekulární typizace MeSH
• nemoci ptáků epidemiologie   mikrobiologie   MeSH
• ostrovy epidemiologie   MeSH
• prevalence MeSH
• ptáci MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Nový Jižní Wales epidemiologie   MeSH
• ostrovy epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč