Chemogenomic profiling to understand the antifungal action of a bioactive aurone compound.

Every year, more than 250,000 invasive candidiasis infections are reported with 50,000 deaths worldwide. The limited number of antifungal agents necessitates the need for alternative antifungals with potential novel targets. The 2-benzylidenebenzofuran-3-(2H)-ones have become an attractive scaffold for antifungal drug design. This study aimed to determine the antifungal activity of a synthetic aurone compound and characterize its mode of action. Using the broth microdilution method, aurone SH1009 exhibited inhibition against C. albicans, including resistant isolates, as well as C. glabrata, and C. tropicalis with IC50 values of 4-29 μM. Cytotoxicity assays using human THP-1, HepG2, and A549 human cell lines showed selective toxicity toward fungal cells. The mode of action for SH1009 was characterized using chemical-genetic interaction via haploinsufficiency (HIP) and homozygous (HOP) profiling of a uniquely barcoded Saccharomyces cerevisiae mutant collection. Approximately 5300 mutants were competitively treated with SH1009 followed by DNA extraction, amplification of unique barcodes, and quantification of each mutant using multiplexed next-generation sequencing. Barcode post-sequencing analysis revealed 238 sensitive and resistant mutants that significantly (FDR P values ≤ 0.05) responded to aurone SH1009. The enrichment analysis of KEGG pathways and gene ontology demonstrated the cell cycle pathway as the most significantly enriched pathway along with DNA replication, cell division, actin cytoskeleton organization, and endocytosis. Phenotypic studies of these significantly enriched responses were validated in C. albicans. Flow cytometric analysis of SH1009-treated C. albicans revealed a significant accumulation of cells in G1 phase, indicating cell cycle arrest. Fluorescence microscopy detected abnormally interrupted actin dynamics, resulting in enlarged, unbudded cells. RT-qPCR confirmed the effects of SH1009 in differentially expressed cell cycle, actin polymerization, and signal transduction genes. These findings indicate the target of SH1009 as a cell cycle-dependent organization of the actin cytoskeleton, suggesting a novel mode of action of the aurone compound as an antifungal inhibitor

Role of Acinetobactin-mediated iron acquisition functions in the interaction of Acinetobacter baumannii strain ATCC 19606T with human lung epithelial cells, Galleria mellonella caterpillars, and mice

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acquisition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial interaction of the ATCC 19606T type strain with A549 human alveolar epithelial cells is independent of the production of BasD and BauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for this strain to persist within epithelial cells and cause their apoptotic death. Infection assays using Galleria mellonella larvae showed that impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606T cells to persist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with these ex vivo and in vivo approaches were validated using a mouse sepsis model, which showed that expression of the acinetobactinmediated iron acquisition system is critical for ATCC 19606T to establish an infection and kill this vertebrate host. These observations demonstrate that the virulence of the ATCC 19606T strain depends on the expression of a fully active acinetobactinmediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediate virulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactin intermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of mature acinetobactin. © 2012, American Society for Microbiology.This work was supported by funds from Public Health AI070174 and NSF 0420479 grants and Miami University research fundsPeer Reviewe

The inside scoop: Comparative genomics of two intranuclear bacteria, "Candidatus Berkiella cookevillensis" and "Candidatus Berkiella aquae".

"Candidatus Berkiella cookevillensis" (strain CC99) and "Candidatus Berkiella aquae" (strain HT99), belonging to the Coxiellaceae family, are gram-negative bacteria isolated from amoebae in biofilms present in human-constructed water systems. Both bacteria are obligately intracellular, requiring host cells for growth and replication. The intracellular bacteria-containing vacuoles of both bacteria closely associate with or enter the nuclei of their host cells. In this study, we analyzed the genome sequences of CC99 and HT99 to better understand their biology and intracellular lifestyles. The CC99 genome has a size of 2.9Mb (37.9% GC) and contains 2,651 protein-encoding genes (PEGs) while the HT99 genome has a size of 3.6Mb (39.4% GC) and contains 3,238 PEGs. Both bacteria encode high proportions of hypothetical proteins (CC99: 46.5%; HT99: 51.3%). The central metabolic pathways of both bacteria appear largely intact. Genes for enzymes involved in the glycolytic pathway, the non-oxidative branch of the phosphate pathway, the tricarboxylic acid pathway, and the respiratory chain were present. Both bacteria, however, are missing genes for the synthesis of several amino acids, suggesting reliance on their host for amino acids and intermediates. Genes for type I and type IV (dot/icm) secretion systems as well as type IV pili were identified in both bacteria. Moreover, both bacteria contain genes encoding large numbers of putative effector proteins, including several with eukaryotic-like domains such as, ankyrin repeats, tetratricopeptide repeats, and leucine-rich repeats, characteristic of other intracellular bacteria

Identification of a Small Molecule Inhibitor of the Aminoglycoside 6\u27- N -acetyltransferase Type Ib [AAC(6\u27)-Ib] Using Mixture-Based Combinatorial Libraries

The aminoglycoside, 6′-N-acetyltransferase type Ib [AAC(6\u27)-Ib] is the most widely distributed enzyme among AAC(6\u27)-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides, including amikacin. This enzyme is therefore an ideal target for enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R–group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin it potentiated the inhibition of growth of three resistant bacteria in culture, and it improved survival when used as treatment of Galleria mellonella infected with aac(6\u27)-Ib -harboring Klebsiella pneumoniae and Acinetobacter baumannii strains