Identification of IncA Plasmid, Harboring blaVIM-1 Gene, in S. enterica Goldcoast ST358 and C. freundii ST62 Isolated in a Hospitalized Patient.

In the present study, we analyzed the genome of two S. enterica strains TS1 and TS2 from stool and blood cultures, respectively, and one strain of C. freundii TS3, isolated from a single hospitalized patient with acute myeloid leukemia. The S. enterica Goldcoast ST358 (O:8 (C2-C3) serogroup), sequenced by the MiSeq Illumina system, showed the presence of ß-lactamase genes (blaVIM-1, blaSHV-12 and blaOXA-10), aadA1, ant(2″)-Ia, aac(6')-Iaa, aac(6')-Ib3, aac(6')-Ib-cr, qnrVC6, parC(T57S), and several incompatibility plasmids. A wide variety of insertion sequences (ISs) and transposon elements were identified. In C. freundii TS3, these were the blaVIM-1, blaCMY-150, and blaSHV-12, aadA1, aac(6')-Ib3, aac(6')-Ib-cr, mph(A), sul1, dfrA14, ARR-2, qnrVC6, and qnrB38. IncA plasmid isolated from E.coli/K12 transconjugant and C. freundii exhibited a sequence identity >99.9%. The transfer of IncA plasmid was evaluated by conjugation experiments.

Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators.

The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CLpro. The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CLpro. The compounds are slow-binding inactivators of 3CLpro with a Ki of 3.95 µM and 3.77 µM for protocetraric and salazinic acid, respectively, and inhibitory efficiency kinact/Ki at about 3 × 10-5 s-1µM-1. The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 µM, which is well below the Ki values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CLpro. The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors.

Transient disappearance of CD19+/CD5+ B-lymphocyte clone in peripheral blood in a patient with CLL during SARS-CoV-2-related mild disease.

Although lymphopenia is currently considered a good predictor for the prognosis of COVID-19, it must be critically evaluated in patients with CLL, where other clinical markers should be considered to define the prognosis and treatment.

Molecular characterization of carbapenem-resistant Klebsiella pneumoniae ST14 and ST512 causing bloodstream infections in ICU and surgery wards of a tertiary university hospital of Verona (northern Italy): co-production of KPC-3, OXA-48, and CTX-M-15 ß-lactamases.

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae, isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-ß-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that blaKPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas blaCTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.

Interaction of carbapenems and ß-lactamase inhibitors towards CTX-M-15 and CTX-M-15G238C mutant.

OBJECTIVES: The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various ß-lactamase inhibitors. METHODS: A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. RESULTS: In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. CONCLUSIONS: Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

BACKGROUND: RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. PURPOSE: Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. METHODS: The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. RESULTS: Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. CONCLUSION: In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity.

Protolichesterinic acid enhances doxorubicin-induced apoptosis in HeLa cells in vitro.

AIM: The aim of this study was to investigate the effect of protolichesterinic acid, a lichen secondary metabolite, on anti-proliferative activity of doxorubicin in three human cancer cell lines, HeLa, SH-SY5Y and K562 cells. MAIN METHODS: The data obtained from MTT assays, performed on cells treated with protolichesterinic acid and doxorubicin alone and in combination, were analysed by the median-effect method as proposed by Chou and Talalay and the Bliss independence model. Apoptosis rate was evaluated by fluorescence microscopy, caspase-3, 8 and 9 activities were detected by spectrofluorimetric analysis and protein expression of Bim, Bid, Bax and Mcl-2 was analysed by Western blotting. The interaction of protolichesterinic acid with thioesterase domain of human fatty acid synthase (hFAS) was investigated by a molecular docking study. KEY FINDINGS: The in vitro activity of doxorubicin against HeLa cancer cell line, but not against SH-SY5Y and K562 cells, was synergically increased by protolichesterinic acid. The increased cytotoxicity caused by protolichesterinic acid in HeLa cells was due to a pro-apoptotic effect and was associated to caspase-3, 8 and 9 activation. The simultaneous treatment for 24h with protolichesterinic acid plus doxorubicin caused an increase of Bim protein expression and the appearance of cleaved form of Bid protein. The molecular modelling analysis showed that protolichesterinic acid seemed to behave as a competitive inhibitor of hFAS. SIGNIFICANCE: These results suggest that protolichesterinic acid could be envisaged as an useful tool against certain types of tumor cells in combination with anticancer drugs.

Cytotoxic activity and antioxidant capacity of purified lichen metabolites: an in vitro study.

The purpose of this study was to investigate the effects of six lichen metabolites (diffractaic acid, lobaric acid, usnic acid, vicanicin, variolaric acid, protolichesterinic acid) on proliferation, viability and reactive oxygen species (ROS) level towards three human cancer cell lines, MCF-7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and HCT-116 (colon carcinoma). Cells were treated with different concentrations (2.5-100 µM) of these compounds for 48 h. In this comparative study, our lichen metabolites showed various cytotoxic effects in a concentration-dependent manner, and usnic acid was the most potent cytotoxic agent, while variolaric acid did not inhibit the proliferation of any of the three cell lines used. All tested lichen compounds did not exhibit free radical scavenging activity using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The lichen metabolites did not significantly increase the intracellular ROS level and did not prevent oxidative injury induced by t-butylhydroperoxide in HeLa cells. To better clarify the mechanism(s) of cytotoxic effect induced by protolichesterinic acid in HeLa cells, we investigated apoptotic markers such as condensation and fragmentation of nuclear chromatin and activation of caspase-3, 8 and 9. Our results revealed that the antiproliferative activity of 40 µM protolichesterinic acid in HeLa cells is related to its ability to induce programmed cell death involving caspase-3, 8 and 9 activation.

ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites.

PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status.

Parp1 localizes within the Dnmt1 promoter and protects its unmethylated state by its enzymatic activity.

BACKGROUND: Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found. METHODOLOGY/PRINCIPAL FINDINGS: Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation. CONCLUSIONS/SIGNIFICANCE: In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression.

Class I integron-borne bla(VIM-1) carbapenemase in a strain of Enterobacter cloacae responsible for a case of fatal pneumonia.

We report here a fatal case of nosocomial pneumonia due to a multidrug-resistant (MDR) strain of Enterobacter cloacae, which was also resistant to carbapenems, in a patient with a diagnosis of Churg-Strauss syndrome. The microorganism was investigated for the presence of metallo- and extended-spectrum beta-lactamases. The strain produced two class 1 integrons carrying bla(VIM-1) and aadA2 gene cassettes located on chromosomal and plasmidic DNA, respectively, and a plasmid-encoded SHV-12 extended-spectrum beta-lactamase. Our results clearly demonstrate the transfer of this kind of resistance in E. cloacae with potentially serious clinical implications.