Proteomic analysis of crocodile white blood cells reveals insights into the mechanism of the innate immune system.

Crocodiles have a particularly powerful innate immune system because their blood contains high levels of antimicrobial peptides. They can survive injuries that would be fatal to other animals, and they are rarely afflicted with diseases. To better understand the crocodile's innate immune response, proteomic analysis was performed on the white blood cells (WBC) of an Aeromonas hydrophila-infected crocodile. Levels of WBC and red blood cells (RBC) rapidly increased within 1 h. In WBC, there were 109 up-regulated differentially expressed proteins (DEP) that were up-regulated. Fifty-nine DEPs dramatically increased expression from 1 h after inoculation, whereas 50 up-regulated DEPs rose after 24 h. The most abundant DEPs mainly had two biological functions, 1) gene expression regulators, for example, zinc finger proteins and histone H1 family, and 2) cell mechanical forces such as actin cytoskeleton proteins and microtubule-binding proteins. This finding illustrates the characteristic effective innate immune response mechanism of crocodiles that might occur via boosted transcription machinery proteins to accelerate cytoskeletal protein production for induction of phagocytosis, along with the increment of trafficking proteins to transport essential molecules for combating pathogens. The findings of this study provide new insights into the mechanisms of the crocodile's innate immune system.

Emergence of NDM-1-producing Raoultella ornithinolytica from reservoir water in Northeast Thailand.

Background and Aim: Antibiotic resistance is a major global health threat. The increasing prevalence of drug-resistant bacteria poses a serious challenge to the effective treatment of infections in both humans and animals. Water is a major source of human and animal exposure to bacteria, and the presence of drug-resistant bacteria in water could present a severe threat to public health and animal production. This study investigated the presence of drug-resistant bacteria in Lam Pao Dam (LPD) water in Kalasin, Thailand. Materials and Methods: Ampicillin-resistant strains were obtained from LPD water and identified using 16s rDNA sequencing. Antibiotic resistance genes were detected by polymerase chain reaction using specific primers. The presence of antibiotic-resistant bacteria was evaluated using 16s amplicon analysis. The minimum inhibitory concentration (MIC) of Raoultella ornithinolytica strains against antibiotics was determined. Results: A total of 12 R. ornithinolytica, 4 Bacillus cereus, and 4 Enterococcus faecalis isolates were resistant to ampicillin. Almost all R. ornithinolytica strains harbored blaSHV and blaOXA genes, and two strains also harbored the blaNDM-1 gene. All four E. faecalis strains harbored the blaIMP gene. The most abundant species in the LPD sample was Exiguobacterium indicum, followed by E. faecalis and R. ornithinolytica. The MICs of 10 R. ornithinolytica strains against five antibiotics revealed that all strains were resistant to ampicillin but susceptible to meropenem, doripenem, ertapenem, and imipenem. Conclusion: These findings suggest a high prevalence of drug-resistant bacteria in LPD water. This is a cause for concern, as it could spread antibiotic-resistant infections in the community.

Rational design and characterization of cell-selective antimicrobial peptides based on a bioactive peptide from Crocodylus siamensis hemoglobin.

Antimicrobial resistance is a growing health concern. Antimicrobial peptides are a potential solution because they bypass conventional drug resistance mechanisms. Previously, we isolated a peptide from Crocodylus siamensis hemoglobin hydrolysate, which has antimicrobial activity and identified the main peptide from this mixture (QL17). The objective of this work was to evaluate and rationally modify QL17 in order to: (1) control its mechanism of action through bacterial membrane disruption; (2) improve its antimicrobial activity; and (3) ensure it has low cytotoxicity against normal eukaryotic cells. QL17 was rationally designed using physicochemical and template-based methods. These new peptide variants were assessed for: (1) their in vitro inhibition of microbial growth, (2) their cytotoxicity against normal cells, (3) their selectivity for microbes, and (4) the mode of action against bacteria using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopy. The results indicate that all designed peptides have more potent antimicrobial efficacy than QL17 and IL15 peptides. However, only the most rationally modified peptides showed strong antimicrobial activity and minimal toxicity against normal cells. In particular, IL15.3 (hydrophobicity of 47% and net charge of + 6) was a potent antimicrobial agent (MIC = 4-12 µg/mL; MBC = 6-25 µg/mL) and displayed excellent selectivity for microbes (cf. human cells) via FACS assays. Microscopy confirmed that IL15.3 acts against bacteria by disrupting the cell membrane integrity and penetrating into the membrane. This causes the release of intracellular content into the outer environment leading to the death of bacteria. Moreover, IL15.3 can also interact with DNA suggesting it could have dual mode of action. Overall, a novel variant of QL17 is described that increases antimicrobial activity by over 1000-fold (~ 5 µg/mL MIC) and has minimal cytotoxicity. It may have applications in clinical use to treat and safeguard against bacteria.

Rational Design of RN15m4 Cathelin Domain-Based Peptides from Siamese Crocodile Cathelicidin Improves Antimicrobial Activity.

Antimicrobial peptides are becoming a new generation of antibiotics due to their therapeutic potential and ability to decrease drug-resistant bacteria development. Cathelicidins are known as effective peptides of vertebrate immunity that play crucial roles in the defensive strategy against pathogens. To improve its potency, the RN15 antibacterial peptide derived from the cathelin domain of Crocodylus siamensis cathelicidin has been modified and its antimicrobial properties investigated. Peptides were derived by template-based and physicochemical designation. The RN15 derivative peptides were predicted through their structure modeling, antimicrobial potency, and peptide-membrane calculation. The antimicrobial and cytotoxic activities of candidate peptides were investigated. Simultaneous consideration of physicochemical characteristics, secondary structure modeling, and the result of antimicrobial peptide tools prediction indicated that RN15m4 peptide was a candidate derivative antimicrobial peptide. The RN15m4 peptide expresses antimicrobial activity against most Gram-positive and Gram-negative bacteria and fungi with a lower minimum inhibition concentration (MIC) than the parent peptide. Besides, the time-killing assay shows that the designed peptide performed its ability to quickly kill bacteria better than the original peptide. Scanning electron microscopy (SEM) displayed the destruction of the bacterial cell membrane caused by the RN15m4 peptide. In addition, the RN15m4 peptide exhibits low hemolytic activity and low cytotoxic activity as good as the template peptide. The RN15m4 peptide performs a range of antimicrobial activities with low cell toxicity. Our study has illustrated the combination approach to peptide design for potent antibiotic peptide discovery.

Prevalence of gastrointestinal parasites in cattle in Kalasin Province, Thailand.

BACKGROUND AND AIM: Parasitic infections are one of the major problems to the production of cattle in Thailand. The study was conducted to determine the prevalence of gastrointestinal (GI) parasites of cattle in Kalasin Province, Thailand. MATERIALS AND METHODS: A total of 333 fecal samples of cattle were collected directly from the rectum. The fecal samples were subjected to formalin-ethyl acetate concentration methods for examination. The eggs or oocysts were identified based on the morphology and size of the eggs or oocysts. RESULTS: Out of 333 fecal samples examined, 320 were found positive for GI parasitic infections with a prevalence of 96.09%. Overall, among the prevalence of nematodes, trematodes, and protozoa, the most prevalent parasites were Strongyle-type 278 (84.24%), followed by Strongyloides spp. 54 (16.36%) and Trichuris spp. 75 (22.73%), while Protozoan oocyst recorded Eimeria spp. amounted to 131 (39.7%). Fasciola spp. and Paramphistomum spp. were 67 (20.30%) and 81 (24.55%), respectively. Most of the positive fecal samples were infected with the double infection which has the highest prevalence rate of about 40.24%, followed by single, three, and 4-5 types of parasites 30.63, 16.82, and 7.21%, respectively. CONCLUSION: This study suggests that Kalasin Province is highly endemic for GI parasites and this area may be an important source for an outbreak. Therefore, every household should deworm its cattle and eliminate and control snails as intermediate hosts. Findings from this study provide information that will assist in improving the cattle in Kalasin Province for better production and higher profitability.

Comparison of polymerase chain reaction and microscopy for the detection of Fasciola spp. in the fecal matter of domestic bovines in Kalasin Province, Thailand.

BACKGROUND AND AIM: Fasciola spp. are important foodborne trematodes and waterborne zoonotic parasites that cause health problems and economic losses worldwide, including in Thailand. Fasciola spp. are usually detected by sedimentation or the formalin-ethyl acetate concentration technique (FECT) under microscopy, which is less specific and sensitive. Accurate detection is important to detect real incidence for protection against and elimination of fasciolosis in the area. This study aimed to determine the distribution of Fasciola spp. and compare the specificity and sensitivity of FECT under microscopy to that of polymerase chain reaction (PCR) in cattle feces. MATERIALS AND METHODS: The study was conducted in Kalasin Province, Thailand. Feces of 46 cattle were investigated for infection with Fasciola spp. To detect infection, FECT under microscopy and PCR amplification of the 28S rRNA gene of Fasciola spp. were used to identify egg parasites. RESULTS: Feces of 16 of 46 (34.78%) cattle were positive for Fasciola spp. using FECT under microscopy, whereas PCR showed that 67.39% (31 of 46) were positive for Fasciola spp. False-negative results were as high as 32.61% when diagnosed under microscopy. CONCLUSION: This study confirmed the infection of cattle with Fasciola spp. in Kalasin Province, indicating that PCR demonstrated higher sensitivity and specificity when diagnosing infection. FECT under microscopy can still be used as a primary and traditional method for diagnosis. However, relapse cases of Fasciola spp. and Paramphistomum spp. should be diagnosed by microscopy combined with PCR. This is the first report on the molecular distribution of fecal samples in cattle in Kalasin Province.

Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7.

ß-Glucosidases and ß-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 ß-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the -1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant ß-glycosidases.

Evaluation of tyrosinase inhibitory activity and mechanism of Leucrocin I and its modified peptides.

This research first reports the tyrosinase inhibition and mechanism of Leucrocin I and its modified peptides (TILI-1 and TILI-2). Docking simulation showed that these peptides were predicted to bind and interact to active site of tyrosinase and exhibited the possibility to promote tyrosinase inhibition. Therefore, these peptides were synthesized, and their inhibitory activity was investigated. The results showed that the highest tyrosinase inhibition was achieved by TILI-2 followed by TILI-1 and Leucrocin I. A Lineweaver-Burk plot indicated that Leucrocin I exhibited mixed type characteristics, while its modified peptides exhibited competitive inhibition. Based on the greatest tyrosinase inhibition, TILI-2 was selected for further study. TILI-2 showed irreversible inhibition with two-step inactivation. Additionally, Leucrocin I and its modified peptides showed no toxicity toward B16F1 and HaCaT cells and decreased melanin and tyrosinase content in B16F1 cells. These results suggest that these peptides are promising peptides for the treatment of hyperpigmentation.

Identification of the first Crocodylus siamensis cathelicidin gene and RN15 peptide derived from cathelin domain exhibiting antibacterial activity.

Cathelicidins are effector molecules of vertebrate immunity that play vital roles against microbial invasion. They are widely identified in mammals, but few have been reported in Crocodilians, which are considered to be species with a powerful immune system. In the present study, we identified and characterized a novel cathelicidin from the blood of the Siamese crocodile, Crocodylus siamensis. A cDNA sequence (501 base pair) encoded a predicted 166-residue prepropeptide of C. siamensis cathelicidin (Cs-CATH), which comprised a 21-residue signal peptide, a 109-residue cathelin domain, and a 36-residue mature cathelicidin peptide. Multiple sequence alignment and phylogenetic analysis demonstrated that Cs-CATH shared a high degree of similarity with other crocodilian cathelicidins. Joint consideration of elastase cleavage site, physicochemical properties, and predicted secondary structure demonstrated that RN15 peptide is a candidate antimicrobial peptide derived from Cs-CATH. The synthetic RN15 peptide demonstrates antimicrobial activity against Gram-positive and Gram-negative bacteria. Scanning electron microscopy illustrated RN15-peptide-induced bacteria cells exhibited morphological change. Besides, RN15 peptide demonstrates low hemolytic activity against human erythrocytes and low cytotoxic activity against normal human dermal fibroblasts. This is the first cathelicidin identified from C. siamensis, and it is highlighted that its derived peptide from cathelin domain promises potent novel peptide antibiotics templates.

Bacterial ß-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2).

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 ß-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 ß-D-mannosidase.

Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km value for 4-nitrophenyl ß-D-mannoside (4NPMan) compared with 4-nitrophenyl ß-D-glucoside (4NPGlc). To investigate its selectivity for ß-D-mannoside and ß-D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Šand of Os7BGlu26 with mannose at a resolution of 2.45 Šwere elucidated from isomorphous crystals in space group P212121. The (ß/α)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with D-mannose corresponds to a product complex, with ß-D-mannose in the (1)S5 skew-boat conformation. Docking of the (1)S3, (1)S5, (2)SO and (3)S1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the (1)S5 and (1)S3 skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without ß-D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar kcat/Km values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher kcat/Km for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for ß-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.

Purification process for the preparation and characterizations of hen egg white ovalbumin, lysozyme, ovotransferrin, and ovomucoid.

In this study, four major egg white proteins were purified by two step ion exchange chromatography followed by precipitation. Lysozyme and ovalbumin were separated with Q Sepharose Fast Flow anion exchange chromatography in the first step, resulting in two peaks of lysozyme and three peaks of ovalbumin with 87% and 70% purity by HPLC, respectively. Ovotransferrin was separated with CM-Toyopearl 650 M cation exchange chromatography in the second step, giving 80% purity. Ovomucoid was precipitated from the partial purified protein fraction from the first two steps, and concentrated in the final step to yield 90% purity. Protein recoveries were estimated to be 55, 21, 54, and 21% for lysozyme, ovotransferrin, ovalbumin, and ovomucoid, respectively. Lysozyme was identified from the purified peaks using zymogram refolding gel, whereas ovalbumin was identified by western blotting. Purified ovotransferrin and ovomucoid was identified by mass spectrometry. The results indicate that this purification process is adequate for preparation of lysozyme, ovalbumin, ovotransferrin, and ovomucoid, at least on a laboratory scale.