A Choline-Recognizing Monomeric Lysin, ClyJ-3m, Shows Elevated Activity against Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading pathogen for bacterial pneumonia, which can be treated with bacteriophage lysins harboring a conserved choline binding module (CBM). Such lysins regularly function as choline-recognizing dimers. Previously, we reported a pneumococcus-specific lysin ClyJ comprising the binding domain from the putative endolysin gp20 from the Streptococcus phage SPSL1 and the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain from the PlyC lysin. A variant of ClyJ with a shortened linker, i.e., ClyJ-3, shows improved activity and reduced cytotoxicity. Resembling typical CBM-containing lysins, ClyJ-3 dimerized upon binding with choline. Herein, we further report a choline-recognizing variant of ClyJ-3, i.e., ClyJ-3m, constructed by deleting its C-terminal tail. Biochemical characterization showed that ClyJ-3m remains a monomer after it binds to choline yet exhibits improved bactericidal activity against multiple pneumococcal strains with different serotypes. In an S. pneumoniae-infected bacteremia model, a single intraperitoneal administration of 2.32 µg/mouse of ClyJ-3m showed 70% protection, while only 20% of mice survived in the group receiving an equal dose of ClyJ-3 (P < 0.05). A pharmacokinetic analysis following single intravenously doses of 0.29 and 1.16 mg/kg of ClyJ-3 or ClyJ-3m in BALB/c mice revealed that ClyJ-3m shows a similar half-life but less clearance and a greater area under curve than ClyJ-3. Taken together, the choline-recognizing monomer ClyJ-3m exhibited enhanced bactericidal activity and improved pharmacokinetic proprieties compared to those of its parental ClyJ-3 lysin. Our study also provides a new way for rational design and programmed engineering of lysins targeting S. pneumoniae.

Stereochemical determination of four 10-membered ring resorcylic acid lactones from the desert plant endophytic fungus Chaetosphaeronema hispidulum.

Four 10-membered ring resorcylic acid lactones (RALs) including a new compound hispidulactone F (1) and three known analogs hispidulactone B (2), 2 R, 4R-sonnerlactone (3), and 2 R, 4S-sonnerlactone (4) were isolated from the special bioenvironmental desert plant endophytic fungus Chaetosphaeronema hispidulum. The structure of the new compound hispidulactone F (1) was determined by extensive spectra analysis including HR-ESI-MS, NMR (1H, 13C, 1H-1H COSY, HSQC, and HMBC). Hispidulactone F (1) and hispidulactone B (2) were a pair of stereoisomers at C-3, whereas 2 R, 4R-sonnerlactone (3) and 2 R, 4S-sonnerlactone (4) were another pair of stereoisomers at C-4. The stereochemistries of the hydroxyl groups at C-3 in 1 and 2, and at C-4 in 3 and 4 were first determined by modified Mosher's reactions. Thus, the absolute configuration C-3 in hispidulactone B (2) was not right in our previous report, and was rectified to be R. Compounds 1 and 4 were evaluated for their cytotoxic effects on the proliferation of HepG2. The possible biosynthetic pathway of compounds 1-4 was also presented.

Construction and characterization of a chimeric lysin ClyV with improved bactericidal activity against Streptococcus agalactiae in vitro and in vivo.

The emergence of antibiotic-resistant beta-hemolytic Streptococcus agalactiae strains poses increasing threat to human beings globally. As an attempt to create a novel lysin with improved activity against S. agalactiae, a chimeric lysin, ClyV, was constructed by fusing the enzymatically active domain (EAD) from PlyGBS lysin (GBS180) and the cell wall binding domain (CBD) from PlyV12 lysin (V12CBD). Plate lysis assay combined with lytic kinetic analysis demonstrated that ClyV has improved activity than its parental enzymatic domain GBS180 against multiple streptococci. Biochemical characterization showed that ClyV is active from pH 7 to 10, with the optimum pH of 9, and is stable under NaCl concentration of < 500 mM. In a S. agalactiae infection model, a single intraperitoneally administration of 0.1 mg/mouse of ClyV protected 100% mice, while it was observed that ~ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180. Moreover, a high dose of 0.8 mg/mouse ClyV did not show any adverse effects to the health or survival rate of the mice. Considering the robust bactericidal activity and good safety profile of ClyV, it represents a potential candidate for the treatment of S. agalactiae infections.

Influence of Lactobacillus buchneri on soybean curd residue co-conversion by black soldier fly larvae (Hermetia illucens) for food and feedstock production.

Black soldier fly larvae (BSFL), Hermetia illucens (Diptera: Stratiomyidae) can reduce environmental pollution and convert organic wastes into biomass that is rich in protein and fat. The influence of the nutritional characteristics of organic waste on BSFL characteristics relevant for food and feed safety remains poorly understood. To evaluate the conversion of soybean curd residues (SCR) into high-quality animal-derived proteins and fats for human and livestock consumption, this study assessed the co-conversion efficacy, nutrient composition, safety, and anti-nutritional factor concentrations in BSFL after the development on SCR with Lactobacillus buchneri (L3-9). SCR was pretreated with L. buchneri (108 cfu/ml), and then BSFL was employed for conversion. BSFL fed with SCR and L. buchneri had a significantly higher dry mass reduction (55.7 ±â€¯0.9%), bioconversion rate (6.9 ±â€¯0.3%), crude protein content (55.3 ±â€¯0.6%), and fat content (30.0 ±â€¯0.6%) than SCR (49.0 ±â€¯0.7%, 5.0 ±â€¯0.3%, 52.8 ±â€¯0.3%, and 26.1 ±â€¯0.8%, respectively) and artificial feed (43.9 ±â€¯0.8%, 3.9 ±â€¯0.1%, 50.3 ±â€¯0.4%, and 24.3 ±â€¯0.4%, respectively). However, the feed conversion ratio (8.0 ±â€¯0.3), of BSFL fed with SCR and L. buchneri was lower than that of the BSFL fed with SCR (9.8 ±â€¯0.1) and artificial feed (11.1 ±â€¯0.5). In addition, BSFL had satisfactory concentrations of all essential amino acids and fatty acids required for human consumption as recommended by WHO/FAO/UNU. The heavy metals and anti-nutritional factor concentrations were within the safety intake levels for food and feedstock. Therefore, the addition of L. buchneri with BSFL on SCR did not only increase co-conversion performance but also enhanced the nutritional value of BSFL.

PrfA-like transcription factor gene lmo0753 contributes to L-rhamnose utilization in Listeria monocytogenes strains associated with human food-borne infections.

Listeria monocytogenes is a food-borne bacterial pathogen and the causative agent of human and animal listeriosis. Among the three major genetic lineages of L. monocytogenes (i.e., LI, LII, and LIII), LI and LII are predominantly associated with food-borne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor gene, lmo0753, that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains, including a DNA binding domain, with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, we constructed lmo0753 deletion and complementation mutants in two fully sequenced L. monocytogenes LII strains, 10403S and EGDe, and compared the flagellar motility, phospholipase C production, hemolysis, and intracellular growth of the mutants and their respective wild types. Our results suggested that lmo0753 plays a role in hemolytic activity in both EGDe and 10403S. More interestingly, we found that deletion of lmo0753 led to the loss of l-rhamnose utilization in EGDe, but not in 10403S. RNA-seq analysis of EGDe Δ0753 incubated in phenol red medium containing l-rhamnose as the sole carbon source revealed that 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome were up- and downregulated more than 2-fold, respectively, compared to the wild-type strain. Genes related to biotin biosynthesis, general stress response, and rhamnose metabolism were shown to be differentially regulated. Findings from this study collectively suggested varied functional roles of lmo0753 in different LII L. monocytogenes strain backgrounds associated with human listeriosis outbreaks.

[16S rDNA diversity analysis of 30 Streptomycetes isolates displaying significant cytotoxic activity against B16 cell from near-shore sediments of Hainan Island].

A total of 354 isolates of actinomycetes, of which 76 were detected cytotoxic activity was isolated from near-shore marine samples collected at Wenchang mangrove, DanZhou harbor and YanPu harbor. Four isolation methods were employed, which are SDS pretreatment, phenol pretreatment, heating pretreatment and potassium dichromate selection culture, and media such as'Yeast extract-Malt extract (YE), Glucose-Asprine (GA), Starch-Casin (SC), Starch-KNO3 (Gause) were used. It was showed that heating pretreatment and potassium dichromate selection culture were more considerable methods for extensive isolation of actinomycetes. Medium YE and Gause showed best results in both the total number of actinomycetes and the number of active isolates against tumor cell B16. The genotypic diversity of 30 strains of Streptomycetes possessing strong cytotoxic activity against B16 cell (ID50 > or =200) was analyzed by 16S ARDRA, which resulted in 17 RFLP types, and indicated relatively rich genotypic diversity among these Streptomycetes. 16S rDNA sequence analysis of three strains, 050642, 060386 and 060524 (ID50 > or = 1200) further confirmed that they all belong to Streptomyces genus and strain 050642 was suggested a novel Streptomyces. Spp with the highest similarity of 95% to Streptomyces cattleya.