2-Sulfonylpyrimidines as Privileged Warheads for the Development of S. aureus Sortase A Inhibitors

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with emerging multiresistant isolates causing a significant burden to public health systems. We identified 2-sulfonylpyrimidines as a new class of potent inhibitors against S. aureus sortase A acting by covalent modification of the active site cysteine 184. Series of derivatives were synthesized to derive structure-activity relationship (SAR) with the most potent compounds displaying low micromolar K(I) values. Studies on the inhibition selectivity of homologous cysteine proteases showed that 2-sulfonylpyrimidines reacted efficiently with protonated cysteine residues as found in sortase A, though surprisingly, no reaction occurred with the more nucleophilic cysteine residue from imidazolinium-thiolate dyads of cathepsin-like proteases. By means of enzymatic and chemical kinetics as well as quantum chemical calculations, it could be rationalized that the S ( N )Ar reaction between protonated cysteine residues and 2-sulfonylpyrimidines proceeds in a concerted fashion, and the mechanism involves a ternary transition state with a conjugated base. Molecular docking and enzyme inhibition at variable pH values allowed us to hypothesize that in sortase A this base is represented by the catalytic histidine 120, which could be substantiated by QM model calculation with 4-methylimidazole as histidine analog

An Insect Nidovirus Emerging from a Primary Tropical Rainforest

Tropical rainforests show the highest level of terrestrial biodiversity and may be an important contributor to microbial diversity. Exploitation of these ecosystems may foster the emergence of novel pathogens. We report the discovery of the first insect-associated nidovirus, tentatively named Cavally virus (CAVV). CAVV was found with a prevalence of 9.3% during a survey of mosquito-associated viruses along an anthropogenic disturbance gradient in Côte d’Ivoire. Analysis of habitat-specific virus diversity and ancestral state reconstruction demonstrated an origin of CAVV in a pristine rainforest with subsequent spread into agriculture and human settlements. Virus extension from the forest was associated with a decrease in virus diversity (P < 0.01) and an increase in virus prevalence (P < 0.00001). CAVV is an enveloped virus with large surface projections. The RNA genome comprises 20,108 nucleotides with seven major open reading frames (ORFs). ORF1a and -1b encode two large proteins that share essential features with phylogenetically higher representatives of the order Nidovirales, including the families Coronavirinae and Torovirinae, but also with families in a basal phylogenetic relationship, including the families Roniviridae and Arteriviridae. Genetic markers uniquely conserved in nidoviruses, such as an endoribonuclease- and helicase-associated zinc-binding domain, are conserved in CAVV. ORF2a and -2b are predicted to code for structural proteins S and N, respectively, while ORF3a and -3b encode proteins with membrane-spanning regions. CAVV produces three subgenomic mRNAs with 5′ leader sequences (of different lengths) derived from the 5′ end of the genome. This novel cluster of mosquito-associated nidoviruses is likely to represent a novel family within the order Nidovirales

Biofilm-inhibiting effect and anti-infective activity of N,C-linked aryl isoquinolines and the use thereof

Anti-infective and biofilm-inhibiting activities of aryl isoquinoline-derivatives of the general formulae 1 to 3 Figure US08173673-20120508-C00001 are described

Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names

Binding of the 5′-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription

Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5′-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem–loop I of IBV 5′-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis

recA mediated spontaneous deletions of the icaADBC operon of clinical Staphylococcus epidermidis isolates: a new mechanism of phenotypic variations

Phenotypic variation of Staphylococcus epidermidis involving the slime related ica operon results in heterogeneity in surface characteristics of individual bacteria in axenic cultures. Five clinical S. epidermidis isolates demonstrated phenotypic variation, i.e. both black and red colonies on Congo Red agar. Black colonies displayed bi-modal electrophoretic mobility distributions at pH 2, but such phenotypic variation was absent in red colonies of the same strain as well as in control strains without phenotypic variation. All red colonies had lost ica and the ability to form biofilms, in contrast to black colonies of the same strain. Real time PCR targeting icaA indicated a reduction in gene copy number within cultures exhibiting phenotypic variation, which correlated with phenotypic variations in biofilm formation and electrophoretic mobility distribution of cells within a culture. Loss of ica was irreversible and independent of the mobile element IS256. Instead, in high frequency switching strains, spontaneous mutations in lexA were found which resulted in deregulation of recA expression, as shown by real time PCR. RecA is involved in genetic deletions and rearrangements and we postulate a model representing a new mechanism of phenotypic variation in clinical isolates of S. epidermidis. This is the first report of S. epidermidis strains irreversibly switching from biofilm-positive to biofilm-negative phenotype by spontaneous deletion of icaADBC

Antibiotic resistance and biofilm formation ability among coagulase-negative staphylococci in healthy individuals from Portugal

In the past few years the interest in coagulase-negative staphylococci (CoNS) species has significantly increased due to their impact on human health and disease. CoNS are common bacterial colonizers of the normal human microflora and usually have a benign relationship with the host.This work was funded by Fundacao para a Ciencia e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5'-cap-structures of higher eukaryote mRNAs are ribose 2'-O-methylated. Likewise, a number of viruses replicating in the cytoplasm of eukayotes have evolved 2'-O-methyltransferases to modify autonomously their mRNAs. However, a defined biological role of mRNA 2'-O-methylation remains elusive. Here we show that viral mRNA 2'-O-methylation is critically involved in subversion of type-I-interferon (IFN-I) induction. We demonstrate that human and murine coronavirus 2'-O-methyltransferase mutants induce increased IFN-I expression, and are highly IFN-I sensitive. Importantly, IFN-I induction by 2'-O-methyltransferase-deficient viruses is dependent on the cytoplasmic RNA sensor melanoma differentiation-associated gene 5 (MDA5). This link between MDA5-mediated sensing of viral RNA and mRNA 2'-O-methylation suggests that RNA modifications, such as 2'-O-methylation, provide a molecular signature for the discrimination of self and non-self mRNA

Effect of farnesol on structure and composition of staphylococcus epidermidis biofilm matrix

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections in which biofilm formation is considered to be one of the main virulence mechanisms. Moreover, their increased resistance to conventional antibiotic therapy enhances the need to develop new therapeutical agents. Farnesol, a natural sesquiterpenoid present in many essential oils, has been described as impairing bacterial growth. The aim of this study was to evaluate the effect of farnesol on the structure and composition of biofilm matrix of S. epidermidis. Biofilms formed in the presence of farnesol (300 μM) contained less biomass, and displayed notable changes in the composition of the biofilm matrix. Changes in the spacial structure were also verified by confocal scanning laser microscopy (CSLM). The results obtained by the quantification of extracellular polymers and by wheat germ agglutinin (WGA) fluorescent detection of glycoproteins containing β(1→4)-N-acetyl-d-glucosamine support the hypothesis that farnesol causes disruption of the cytoplasmic membrane and consequently release of cellular content.Fernanda Gomes and Pilar Teixeira fully acknowledge the financial support of Fundacao para a Ciencia e Tecnologia (FCT) through the grants SFRH/BD/32126/2006 and SFRH/BPD/26803/2006, respectively