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Molecules (Basel, Switzerland) Jun 2024Secondary metabolites, bioactive compounds produced by living organisms, can unveil symbiotic relationships in nature. In this study, soilborne entomopathogenic...
Preliminary Screening on Antibacterial Crude Secondary Metabolites Extracted from Bacterial Symbionts and Identification of Functional Bioactive Compounds by FTIR, HPLC and Gas Chromatography-Mass Spectrometry.
Secondary metabolites, bioactive compounds produced by living organisms, can unveil symbiotic relationships in nature. In this study, soilborne entomopathogenic nematodes associated with symbiotic bacteria ( and ) were extracted from solvent supernatant containing secondary metabolites, demonstrating significant inhibitory effects against , , , , , and . The characterization of these secondary metabolites by Fourier transforms infrared spectroscopy revealed amine groups of proteins, hydroxyl and carboxyl groups of polyphenols, hydroxyl groups of polysaccharides, and carboxyl groups of organic acids. Furthermore, the obtained crude extracts were analyzed by high-performance liquid chromatography for the basic identification of potential bioactive peptides. Gas chromatography-mass spectrometry analysis of ethyl acetate extracts from identified major compounds including nonanoic acid derivatives, proline, paromycin, octodecanal derivatives, trioxa-5-aza-1-silabicyclo, 4-octadecenal, methyl ester, oleic acid, and 1,2-benzenedicarboxylicacid. Additional extraction from yielded functional compounds such as indole-3-acetic acid, phthalic acid, 1-tetradecanol, nemorosonol, 1-eicosanol, and unsaturated fatty acids. These findings support the potential development of novel natural antimicrobial agents for future pathogen suppression.
Topics: Chromatography, High Pressure Liquid; Anti-Bacterial Agents; Spectroscopy, Fourier Transform Infrared; Gas Chromatography-Mass Spectrometry; Symbiosis; Secondary Metabolism; Photorhabdus; Xenorhabdus; Microbial Sensitivity Tests; Animals
PubMed: 38930979
DOI: 10.3390/molecules29122914 -
Current Microbiology Jun 2024One Gram-negative, rod-shaped bacterial strain, isolated from an undescribed Heterorhabditis entomopathogenic nematode species was characterized to determine its...
One Gram-negative, rod-shaped bacterial strain, isolated from an undescribed Heterorhabditis entomopathogenic nematode species was characterized to determine its taxonomic position. The 16S rRNA gene sequences indicate that it belongs to the class Gammaproteobacteria, to the family Morganellaceae, to the genus Photorhabdus, and likely represents a novel bacterial species. This strain, designated here as CRI-LC, was therefore molecularly, biochemically, and morphologically characterized to describe the novel bacterial species. Phylogenetic reconstructions using 16S rRNA gene sequences show that CRI-LC is closely related to P. laumondii subsp. laumondii TT01 and to P. laumondii subsp. clarkei BOJ-47. The 16rRNA gene sequences between CRI-LC and P. laumondii subsp. laumondii TT01 are 99.1% identical, and between CRI-LC and P. laumondii subsp. clarkei BOJ-47 are 99.2% identical. Phylogenetic reconstructions using whole genome sequences show that CRI-LC is closely related to P. laumondii subsp. laumondii TT01 and to P. laumondii subsp. clarkei BOJ-47. Moreover, digital DNA-DNA hybridization (dDDH) values between CRI-LC and its two relative species P. laumondii subsp. laumondii TT01 and P. laumondii subsp. clarkei BOJ-47 are 65% and 63%, respectively. In addition, we observed that average nucleotide identity (ANI) values between CRI-LC and its two relative species P. laumondii subsp. laumondii TT01 and P. laumondii subsp. clarkei BOJ-47 are 95.8% and 95.5%, respectively. These values are below the 70% dDDH and the 95-96% ANI divergence thresholds that delimits prokaryotic species. Based on these genomic divergence values, and the phylogenomic separation, we conclude that CRI-LC represents a novel bacterial species, for which we propose the name Photorhabdus africana sp. nov. with CRI-LC (= CCM 9390 = CCOS 2112) as the type strain. The following biochemical tests allow to differentiate P. africana sp. nov. CRI-LC from other species of the genus, including its more closely related taxa: β-Galactosidase, citrate utilization, urease and tryptophan deaminase activities, indole and acetoin production, and glucose and inositol oxidation. Our study contributes to a better understanding of the taxonomy and biodiversity of this important bacterial group with great biotechnological and agricultural potential.
Topics: Phylogeny; Photorhabdus; Animals; RNA, Ribosomal, 16S; DNA, Bacterial; Rhabditoidea; Sequence Analysis, DNA; Bacterial Typing Techniques
PubMed: 38910178
DOI: 10.1007/s00284-024-03744-3 -
Journal of Applied Microbiology Jun 2024This study aimed to overproduce industrially relevant and safe bio-compound trans-cinnamic acid (tCA) from Photorhabdus luminescens with deletion strategies and...
AIM
This study aimed to overproduce industrially relevant and safe bio-compound trans-cinnamic acid (tCA) from Photorhabdus luminescens with deletion strategies and homologous expression strategies that had not been applied before for tCA production.
METHODS AND RESULTS
The overproduction of the industrially relevant compound tCA was successfully performed in Photorhabdus luminescens by deleting stlB (TTO1ΔstlB) encoding a cinnamic acid CoA ligase in the isopropylstilbene pathway and the hcaE insertion (knockout) mutation (hcaE:: cat) in the phenylpropionate catabolic pathway, responsible for tCA degradation. A double mutant of both stlB deletion and hcaE insertion mutation (TTO1DM ΔstlB-hcaE:: cat) was also generated. These deletion strategies and the phenylalanine ammonium lyase-producing (PI-PAL from Photorhabdus luminescens) plasmid, pBAD30C, carrying stlA (homologous expression mutants) are utilized together in the same strain using different media, a variety of cultivation conditions, and efficient anion exchange resin (Amberlite IRA402) for enhanced tCA synthesis. At the end of the 120-hour shake flask cultivation, the maximum tCA production was recorded as 1281 mg L-1 in the TTO1pBAD30C mutant cultivated in TB medium, with the IRA402 resin keeping 793 mg L-1 and the remaining 488 mg L-1 found in the supernatant.
CONCLUSION AND IMPACT OF THE STUDY
tCA production was successfully achieved with homologous expression, coupled with deletion and insertion strategies. 1281 mg L-1is the highest tCA concentration that achieved by bacterial tCA production in flask cultivation, according to our knowledge. IRA402 resin adsorbers seem useful for enhancing tCA acquisition in bacterial cultures. Mutations on the hcaE and stlB genes clearly increased the amount of tCA. P. luminescens is an effective bacterial candidate to produce tCA in industrial applications with the implemented strategies.
PubMed: 38906846
DOI: 10.1093/jambio/lxae149 -
Animals : An Open Access Journal From... May 2024Acute hepatopancreatic necrosis disease (AHPND) poses a significant threat to shrimp aquaculture worldwide, necessitating the accurate and rapid detection of the...
Acute hepatopancreatic necrosis disease (AHPND) poses a significant threat to shrimp aquaculture worldwide, necessitating the accurate and rapid detection of the pathogens. However, the increasing number of species that cause the disease makes diagnosis and control more difficult. This study focuses on developing a monoclonal antibody against the insect-related (Pir) toxin B (PirB), a pivotal virulence factor in AHPND-causing , and establishing a colloidal gold immunochromatographic assay for the enhanced early diagnosis and monitoring of AHPND. Monoclonal antibodies targeting PirB were developed and utilized in the preparation of colloidal-gold-labeled antibodies for the immunochromatographic assay. The specificity and sensitivity of the assay were evaluated through various tests, including antibody subclass detection, affinity detection, and optimal labeling efficiency assessment. The developed PirB immunochromatographic test strips exhibited a good specificity, as demonstrated by the positive detection of AHPND-causing and negative results for non-AHPND-causing . The study highlights the potential of the developed monoclonal antibody and immunochromatographic assay for the effective detection of AHPND-causing . Further optimization is needed to enhance the sensitivity of the test strips for improved practical applications in disease prevention and control in shrimp aquaculture.
PubMed: 38891648
DOI: 10.3390/ani14111600 -
Molecular Biology Reports Jun 2024Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the...
BACKGROUND
Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts.
METHODS AND RESULTS
Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph.
CONCLUSIONS
The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.
Topics: Animals; Photorhabdus; Moths; Peptide Hydrolases; Bacterial Proteins; Hemolymph; Proteomics; Recombinant Proteins; Escherichia coli
PubMed: 38824247
DOI: 10.1007/s11033-024-09654-8 -
Acta Tropica Aug 2024Chagas disease is a zoonosis caused by the protozoan Trypanosoma cruzi and transmitted through the feces of triatomines, mainly in Latin America. Since the 1950s,...
Chagas disease is a zoonosis caused by the protozoan Trypanosoma cruzi and transmitted through the feces of triatomines, mainly in Latin America. Since the 1950s, chemical insecticides have been the primary method for controlling these triatomines, yet resistance has emerged, prompting the exploration of alternative approaches. The objective of this research was to test the capacity of the entomopathogenic nematodes Heterorhabditis indica and its symbiotic bacteria Photorhabdus luminescens, to produce mortality of Triatoma dimidiata a key vector of T. cruzi in Mexico under laboratory conditions. Two bioassays were conducted. In the first bioassay, the experimental unit was a 250 ml plastic jar with 100 g of sterile soil and three adult T. dimidiata. Three nematode quantities were tested: 2250, 4500, and 9000 nematodes per 100 g of sterile soil (n/100 g) per jar, with 3 replicates for each concentration and 1 control per concentration (1 jar with 100 g of sterile soil and 3 T. dimidiata without nematodes). The experimental unit of the second bioassay was a 500 ml plastic jar with 100 g of sterile soil and 4 adult T. dimidiata. This bioassay included 5, 50, 500, and 5000 n/100 g of sterile soil per jar, with 3 replicates of each quantity and 1 control per quantity. Data were analyzed using Kaplan-Meyer survival analysis. Electron microscopy was used to assess the presence of nematodes and tissue damage in T. dimidiata. The results of the first bioassay demonstrated that the nematode induced an accumulated average mortality ranging from 55.5 % (2250 n/100 g) to 100 % (4500 and 9000 n/100 g) within 144 h. In the second bioassay, the 5000 n/100 g concentration yielded 87.5 % mortality at 86 h, but a concentration as small as 500 n/100 g caused 75 % mortality from 84 h onwards. Survival analysis indicated higher T. dimidiata mortality with increased nematode quantities, with significant differences between the 4500, 5000, and 9000 n/100 g and controls. Electron microscopy revealed the presence of nematodes and its presumably symbiotic bacteria in the digestive system of T. dimidiata. Based on these analyses, we assert that the H. indica and P. luminescens complex causes mortality in adult T. dimidiata under laboratory conditions.
Topics: Animals; Chagas Disease; Photorhabdus; Triatoma; Mexico; Survival Analysis; Rhabditida; Biological Control Agents; Pest Control, Biological; Rhabditoidea; Disease Vectors; Trypanosoma cruzi
PubMed: 38801912
DOI: 10.1016/j.actatropica.2024.107262 -
Journal of Invertebrate Pathology Jul 2024Aedes-transmitted arboviral infections such as Dengue, Yellow Fever, Zika and Chikungunya are increasing public health problems. Xenorhabdus and Photorhabdus bacteria...
Aedes-transmitted arboviral infections such as Dengue, Yellow Fever, Zika and Chikungunya are increasing public health problems. Xenorhabdus and Photorhabdus bacteria are promising sources of effective compounds with important biological activities. This study investigated the effects of cell-free supernatants of X. szentirmaii, X. cabanillasii and P. kayaii against Ae. aegypti eggs and larvae and identified the bioactive larvicidal compound in X. szentirmaii using The EasyPACId method. Among the three tested bacterial species, X. cabanillasii exhibited the highest (96%) egg hatching inhibition and larvicidal activity (100% mortality), whereas P. kayaii was the least effective species in our study. EasyPACId method revealed that bioactive larvicidal compound in the bacterial supernatant was fabclavine. Fabclavines obtained from promoter exchange mutants of different bacterial species such as X. cabanillasii, X. budapestensis, X. indica, X. szentirmaii, X. hominckii and X. stockiae were effective against mosquito larvae. Results show that these bacterial metabolites have potential to be used in integrated pest management (IPM) programmes of mosquitoes.
Topics: Animals; Aedes; Photorhabdus; Larva; Xenorhabdus; Ovum; Mosquito Control; Mosquito Vectors; Pest Control, Biological; Insecticides
PubMed: 38734162
DOI: 10.1016/j.jip.2024.108126 -
The Journal of Biological Chemistry May 2024Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host...
Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.
PubMed: 38703997
DOI: 10.1016/j.jbc.2024.107331 -
Pest Management Science Apr 2024In the perpetual struggle to manage mosquito populations, there has been increasing demand for the development of biopesticides to supplant/complement current products....
BACKGROUND
In the perpetual struggle to manage mosquito populations, there has been increasing demand for the development of biopesticides to supplant/complement current products. The insecticidal potential of Xenorhabdus and Photorhabdus has long been recognized and is of interest for the control of important mosquitoes like Aedes albopictus which vectors over 20 different arboviruses of global public health concern.
RESULTS
The larvicidal effects of cell-free supernatants, cell growth cultures and cell mass of an extensive list of Xenorhabdus and Photorhabdus spp. was investigated. They were quite effective against Ae. albopictus causing larval mortality ranging between 52-100%. Three Photorhabdus spp. and 13 Xenorhabdus spp. release larvicidal compounds in cell-free supernatants. Cell growth culture of all tested species exhibited larvicidal activity, except for Xenorhabdus sp. TS4. Twenty-one Xenorhabdus and Photorhabdus bacterial cells (pellet) exhibited oral toxicity (59-91%) against exposed larvae. The effect of bacterial supernatants on the mosquito eggs were also assessed. Bacterial supernatants inhibited the hatching of mosquito eggs; when unhatched eggs were transferred to clean water, they all hatched. Using the easyPACId approach, the larvicidal compounds in bacterial supernatant were identified as fabclavine from X. szentirmaii and xencoumacin from X. nematophila (causing 98 and 70% mortality, respectively, after 48 h). Xenorhabdus cabanillasii and X. hominickii fabclavines were as effective as commercial Bacillus thuringiensis subsp. israelensis and spinosad products within 5 days post-application (dpa).
CONCLUSION
Fabclavine and xenocoumacin can be developed into novel biolarvicides, can be used as a model to synthesize other compounds or/and can be combined with other commercial biolarvicides. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
PubMed: 38619291
DOI: 10.1002/ps.8127 -
Bio-protocol Apr 2024Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse...
Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from . The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.
PubMed: 38618175
DOI: 10.21769/BioProtoc.4966