Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites.

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 µM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 µM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a cysteine-dependent manner with relatively low mammalian cytotoxicity.

Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 µM), DHA (100 µM), or siamycin I (2.5 µM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis.

Rhizobium puerariae sp. nov., an endophytic bacterium from the root nodules of the medicinal plant Pueraria candollei var. candollei.

A Gram-stain-negative, rod-shaped, motile bacterium, PC004T, was isolated from root nodules of the Thai medicinal plant Pueraria candollei var. candollei. 16S rRNA gene sequence analysis indicated that the strain is phylogenetically related to species in the genus Rhizobium, showing highest similarity (96.6 %) with Rhizobium mesosinicum HAMBI 3194T. The phylogenetic tree reconstructed based on 16S rRNA gene sequences showed that strain PC004T forms a cluster with Rhizobium petrolearium KCTC 23288T. Based on atpD, gyrB and recA gene sequences, strain PC004T also showed low similarity ( < 90 %) to reference strains. These phylogenetic data indicate that PC004T may represent a novel species. Strain PC004T also exhibited low DNA-DNA relatedness with R. mesosinicum HAMBI 3194T (8.2 %) and R. petrolearium KCTC 23288T (26.3 %). The DNA G+C content of strain PC004T was 64 mol%, which is within the range reported for the genus Rhizobium. The major fatty acid of PC004T was C18 : 1ω7c with minor amounts of C16 : 0, C16 : 0 3-OH, C18 : 0 3-OH, C18 : 1 2-OH, C19 : 0 cyclo ω8c and summed feature 2. The strain was able to grow at pH 12 and with up to 2 % (w/v) NaCl. Strain PC004T did not nodulate five tested legumes and the nifH and nodC genes were not detected by PCR. Based on the physiological, chemotaxonomic and phenotypic data from this study, strain PC004T represents a novel species of the genus Rhizobium, for which the name Rhizobium puerariae sp. nov. is proposed; the type strain is PC004T ( = BCC 73740T = NBRC 110722T).

A small-molecule inhibitor of the bacterial type III secretion system protects against in vivo infection with Citrobacter rodentium.

The type III secretion system (T3SS) is highly conserved in many Gram-negative pathogenic bacteria and functions as an injector of bacterial proteins (effectors) into host cells. T3SSs are involved in establishing disease processes, but this machinery is not essential for bacterial growth or homeostasis. Thus, T3SS is expected to be a candidate therapeutic target, and inhibitors of T3SSs could potentially reduce virulence without causing bacterial death, thereby avoiding any subsequent development of resistance. We identified a linear polyketide compound, aurodox, as a specific T3SS inhibitor from the culture broth of Streptomyces sp. using a screening system for the T3SS-mediated hemolysis of enteropathogenic Escherichia coli (EPEC) established by our group. Aurodox strongly inhibited T3SS-mediated hemolysis with an IC(50) value of 1.5 µg ml(-1) without affecting bacterial growth in liquid media. We also demonstrated that aurodox specifically inhibits the secretion of type III-secreted proteins such as EspB, EspF and Map, without affecting the expression of the housekeeping protein GroEL. Furthermore, an in vivo infection study using mice clearly indicated that the administration of aurodox allowed the mice to survive a lethal dose of Citrobactor rodentium, a model bacterium for human pathogens such as EPEC. Thus, our in vivo study directly demonstrated for the first time that this putative T3SS inhibitor can be applied as a novel class of anti-infective agents.

Demequina salsinemoris sp. nov., isolated on agar media supplemented with ascorbic acid or rutin.

Three strains, KV-810(T), KV-811 and KV-816, were isolated from mangrove soil from a southern island in Japan on media supplemented with ascorbic acid or rutin. These strains contained l-ornithine as the diagnostic diamino acid in the cell-wall peptidoglycan and DMK-9(H(4)) as the predominant menaquinone. The G+C content of the DNA was 70-72 mol%. These characteristics in combination with 16S rRNA gene sequence analysis revealed that the novel strains belonged to the genus Demequina. The DNA-DNA hybridization values showed that the three new strains belonged to the same species, a novel species of the genus Demequina. Therefore strains KV-810(T), KV-811 and KV-816 are proposed as representing a novel species, Demequina salsinemoris sp. nov. The type strain is KV-810(T) (=DSM 22060(T)=NBRC 105323(T)).

Kineococcus gynurae sp. nov., isolated from a Thai medicinal plant.

A novel, Gram-positive, motile, coccus-shaped, orange-pigmented organism, designated strain KKD096(T), was isolated from the roots of a Thai medicinal plant, Gynura pseudochina DC. var. hispida Thwaites. Growth of strain KKD096(T) occurred at temperatures of 14-34 degrees C, at pH 5.0-9.0 and at NaCl concentrations up to 7 % (w/v). Whole-cell hydrolysates contained arabinose and galactose as the characteristic sugars. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The glycan moiety of the murein contained acetyl residues. The predominant menaquinone was MK-9(H2); mycolic acids were not detected. The genomic DNA G+C content was 73.3 mol%. The major cellular fatty acid was anteiso-C(15 : 0) (81.42 % of the total). Strain KKD096(T) was assigned to the genus Kineococcus on the basis of 16S rRNA gene sequence analysis; it was most closely related to Kineococcus radiotolerans DSM 14245(T) (97.1 % similarity). DNA-DNA hybridization revealed 39.4 % relatedness between these two taxa. On the basis of the genotypic and phenotypic data presented, strain KKD096(T) is considered to represent a novel species of the genus Kineococcus, for which the name Kineococcus gynurae sp. nov. is proposed. The type strain is KKD096(T) (=TISTR 1856(T)=NRRL B-24568(T)=BCC 26245(T)=NBRC 103943(T)).

Patulibacter minatonensis gen. nov., sp. nov., a novel actinobacterium isolated using an agar medium supplemented with superoxide dismutase, and proposal of Patulibacteraceae fam. nov.

A novel Gram-positive bacterial strain, designated KV-614T, was isolated from a soil sample using an agar medium supplemented with superoxide dismutase. Based on 16S rRNA gene sequence analysis, it was found that the strain represented a novel deep-rooting lineage within the class Actinobacteria and clustered with yet-uncultivated bacteria from terrestrial environments and some unidentified strains isolated by unique methods. The most closely related established genus was Conexibacter (92.4% sequence similarity to Conexibacter woesei DSM 14684T). Cells of strain KV-614T were rod-shaped and motile with long flagella. The strain was catalase-positive, oxidase-negative and grew under aerobic conditions. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid and alanine and glutamic acid. The peptidoglycan acyl type was acetyl. The only detected isoprenoid quinone was demethylmenaquinone with seven isoprene units (DMK-7). Mycolic acids were not detected. The predominant cellular fatty acid was omega9c-octadecenoic acid (C18:1omega9c). Minor components were 12-methyl tetradecanoic acid (anteiso-C15:0) and 14-methyl hexadecanoic acid (anteiso-C17:0). The DNA G+C content was 72 mol%. On the basis of phenotypic and genotypic characteristics, it is proposed that strain KV-614T represents a new genus and a novel species, Patulibacter minatonensis gen. nov., sp. nov., in the class Actinobacteria. The type strain is KV-614T (=NRRL B-24346T=JCM 12834T=NBRC 100761T). The creation of the family Patulibacteraceae fam. nov. is proposed to encompass the genus Patulibacter gen. nov.