Infective Larvae Research Articles

Evaluation of Anti-bacterial Activity Induced by Penicillium mallochii in the Hemolymph of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae)

Anti-microbial peptides (AMPs) exhibit anti-bacterial, anti-fungal and anti-parasite activity and are essential effectors for the immune response of insects. Insect hemolymph contains AMPs, which are one of the sources of antibiotics effective on drug-resistant microorganisms. This study was conducted to induce antimicrobial activity in hemolymph by topical application of different doses of Penicillium mallochii conidia and its metabolite to Ephestia kuehniella larvae. Tetracycline antibiotic disks (TE-10 µg, Sigma), Sulfametaxozole trimethoprim (SXT-25 µg, Sigma), PBS, sterile water, and non-induced hemolymphs of larvae were used as control groups. In total hemolymph induced with metabolite extract, 24-h application was determined to be more effective on test bacteria than 48-h application. The largest zone diameter was observed against Escherichia coli (20mm) in hemolymph collected 24 h after metabolite application. Antimicrobial activity was highly increased (24h and 48h) when larvae were induced with P. mallochii conidial suspension. The largest zone diameter was observed against Proteus vulgaris and Klebsiella pneumonia (20 and 24 mm) in hemolymph collected 24 h after conidial suspension application. When larvae were induced with fungus metabolite and conidia, the zone of inhibition was 1.5-2.5-fold larger than that of the control hemolymph, indicating a higher antimicrobial activity after application. In general, this study provides a novel contribution to the knowledge regarding enhancement of antimicrobial activity in response to fungal infections in larvae.

Aureispira anguillae sp. nov., isolated from Japanese eel Anguilla japonica leptocephali.

A novel filamentous eel-leptocephalus pathogenic marine bacterium, designated strain EL160426T, was isolated from Japanese eel, Anguilla japonica, leptocephali reared at a laboratory in Mie, Japan. In experimental infection studies on eel larvae, the strain EL160426T caused massive larval mortality and was reisolated from moribund leptocephali. Characteristically, observations of infected larvae found that EL160426T forms columnar colonies on the cranial surface of larvae. The novel isolate exhibited growth at 15-30°C, pH 7-9, and seawater concentrations of 60-150% (W/V). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EL160426T was most closely related to Aureispira maritima 59SAT with 97.7% sequence similarity. The whole genome sequence analysis of the strain EL160426T showed that the strain maintained a circular chromosome with a size of approximately 7.58Mbp and the DNA G + C content was 36.2%. The major respiratory quinone was MK-7 and the predominant cellular fatty acids were 16:0, 20:4 w6c (arachidonic acid), 17:0 iso and 16:0 N alcohol. DNA relatedness between the closest phylogenetic neighbor strain EL160426T and A. maritima (JCM23207T) was less than 13%. On the basis of the polyphasic taxonomic data, the strain represents a novel species of the genus Aureispira, for which the name Aureispira anguillae sp. nov. is proposed. The type strain is EL160426T (= JCM 35024T = TSD-286T).

Differential induction of NF-κB pathways by non-pathogenic and pathogenic bacteria in Helicoverpa armigera is critical for an efficient immune response and survival

Following pathogen infection in a host, extensive changes occur in the host’s gene expression pattern to suppress infection and increase the chance of host survival. Likewise, many pathogens have evolved to evade/suppress host immunity and increase their survival within the host. In this study, we assessed the NF-κB (Imd and Toll) essential gene expression response of Helicoverpa armigera to an entomopathogenic Serratia marcescens and non-pathogenic Escherichia coli. Bacterial cells of S. marcescens or E. coli were injected into the haemocoel of fifth-instar larvae of H. armigera, whereas distilled water was injected into control insects. Our results showed that the expression levels of the Imd and Toll pathway genes (i.e., Relish, imd, spätzle and dif) and the antimicrobial peptides (i.e., gloverin, transferin, gallerimycin, and galiomicin) were differentially expressed following the bacterial injections while control larvae showed no differences. The E. coli injection induced higher and longer-lasted gene expression than the S. marcescens injected larvae, in which the gene expressions were diminished from 24 h post injection. Transcript Knockdown of Relish increased the replication rates of S. marcescens and E. coli, and lowered the infected larvae survival rates. These results showed that H. armigera NF-κB immunity pathways (particularly Imd pathway) play a vital role in immunity against bacterial infections, and S. marcescens might modulate these pathways to survive and replicate in the host.

Improved pharmacokinetics and enhanced efficacy of the vancomycin derivative FU002 using a liposomal nanocarrier

Antibiotic resistance still represents a global health concern which diminishes the pool of effective antibiotics. With the vancomycin derivative FU002, we recently reported a highly potent substance active against Gram-positive bacteria with the potential to overcome vancomycin resistance. However, the translation of its excellent antimicrobial activity into clinical efficiency could be hampered by its rapid elimination from the blood stream. To improve its pharmacokinetics, we encapsulated FU002 in PEGylated liposomes. For PEG-liposomal FU002, no relevant cytotoxicity on liver, kidney and red blood cells was observed. Studies in Wistar rats revealed a significantly prolonged blood circulation of the liposomal antibiotic. In microdilution assays it could be demonstrated that encapsulation does not diminish the antimicrobial activity against staphylococci and enterococci. Highlighting its great potency, liposomal FU002 exhibited a superior therapeutic efficacy when compared to the free form in a Galleria mellonella larvae infection model.

Combining with domiphen bromide restores colistin efficacy against colistin-resistant Gram-negative bacteria in vitro and in vivo

Today, colistin is considered a last-resort antibiotic for treating multidrug-resistant (MDR) Gram-negative bacteria (GNB). However, the increased and improper use of colistin has led to the emergence of colistin-resistant (Col-R) GNB. Thus, it is urgent to develop new drugs and therapies in response to the ongoing emergence of colistin resistance. In this study, we investigated the antibacterial and antibiofilm activities of the quaternary ammonium compound domiphen bromide (DB) in combination with colistin against clinical Col-R GNB both in vitro and in vivo. Checkerboard assay and time-kill analysis demonstrated significant synergistic antibacterial effects of the colistin/DB combination. The synergistic antibiofilm activity was confirmed through crystal violet staining and scanning electron microscopy (SEM). Furthermore, the colistin/DB combination exhibited increased survival rates in infected larvae and reduced bacterial loads in a mouse thigh infection model. The cytotoxicity measurement and hemolysis test showed that the combination did not adversely affect cell viability at synergistic concentrations. The alkaline phosphatase (ALP) leak test and propidium iodide (PI) staining analysis further revealed that the colistin/DB combination enhanced the therapeutic effect of colistin by altering bacterial membrane permeability. The ROS assays revealed that the combination induced the accumulation of bacterial ROS, leading to bacterial death. In conclusion, our study is the first to identify DB as a colistin potentiator, effectively restoring the sensitivity of bacteria to colistin. It provides a promising alternative approach for combating Col-R GNB infections.

Modelling assessment of how small-scale vertical movements of infectious sea lice larvae can affect their large-scale distribution in fjordic systems

Sea lice are ectoparasites that can be found in high numbers in and around salmon farms, where they are a threat to fish health and can induce high aquacultural costs. Large numbers of suitable hosts facilitate the infection and subsequent release of their planktonic larvae in the surrounding environment where they can potentially infect wild salmonids, including migrating juvenile fish (smolts). Investigating sea lice spatial distribution is generally done using coupled hydrodynamic and particle tracking models. The quality of these numerical tools is critical to identify areas of higher infection risk to valuable wild salmon populations and thus support sustainable salmon aquaculture. While the transport of salmon lice is mainly affected by physical processes, biological behaviours such as vertical swimming can also play an important role. However, a review of previous sea lice studies shows no clear consensus on their swimming abilities and the parameters implemented in sea lice dispersion modelling. Here, we focus on the Diel Vertical Migration (DVM) behaviour, a vertical migration that sea lice perform within a daily cycle. Our sensitivity study of the infectious copepodid phase of sea lice highlights how their retention potential and spatial distribution within a water body are affected by their vertical swimming velocity and maximum swimming depth. In a fjordic system (Loch Linnhe on the West Coast of Scotland), the vertical position of sea lice can affect their horizontal trajectory due to the two-layer exchange flow of the estuarine circulation. At the surface, transport is mainly due to the wind driven circulation, and the residual seaward current. Lower in the water column, the saltier shelf water is drawn into the sea loch. This can potentially increase the retention of sea lice if they dive deep enough to reach those opposing subtidal currents. Therefore, lack of confidence in sea lice DVM parameterisation may introduce inaccuracy in modelled sea lice distribution. Effective sea lice management in aquaculture would benefit from more observational data like farm sea lice count, sea lice swimming laboratory observations, field observation of sea lice vertical distribution and accurate sea lice quantification in the field. This would help to reduce uncertainties derived from sea lice vertical swimming behaviour parameterisation in lice dispersal models.

Polystyrene Microplastics Exacerbate Candida albicans Infection Ability In Vitro and In Vivo.

Plastic pollution is an important environmental problem, and microplastics have been shown to have harmful effects on human and animal health, affecting immune and metabolic physiological functions. Further, microplastics can interfere with commensal microorganisms and exert deleterious effects on exposure to pathogens. Here, we compared the effects of 1 µm diameter polystyrene microplastic (PSMPs) on Candida albicans infection in both in vitro and in vivo models by using HT29 cells and Galleria mellonella larvae, respectively. The results demonstrated that PSMPs could promote Candida infection in HT29 cells and larvae of G. mellonella, which show immune responses similar to vertebrates. In this study, we provide new experimental evidence for the risk to human health posed by PSMPs in conjunction with Candida infections.

Bacterial diversity associated with the Hatay yellow strain silkworm (Bombyx mori L.): Isolation, identification and characterization

The Hatay yellow strain silkworm (Bombyx mori L.), which is in danger of extinction, is one of the most important local cultural heritages of Türkiye. Bacterial pathogens of silkworm are highly destructive and cause mostly acute diseases. The aim of this study was to determine the bacterial diversity and potential pathogenic bacterial species in infected and dead larvae of Hatay yellow race. A total of 16 bacterial isolates from Hatay yellow race were identified according to their morphological, biochemical and molecular characteristics. The bacterial isolates isolated from infected and dead larvae of Hatay yellow race were Staphylococcus sp. (BM-1), Staphylococcus xylosus (BM-5), Staphylococcus succinus (BM-7), Bacillus thuringiensis (BM-8), Bacillus subtilis (BM-9), Bacillus sp. (BM-10), Staphylococcus saprophyticus (BM-16, BM-19), Klebsiella sp. (BM-17), Staphylococcus arlettae (BM-18), Pseudomonas aeruginosa (BM-20), Enterococcus mundtii (BM-21), Pantoea agglomerans (BM-22), Kluyvera intermedia (BM-23), Serratia sp. (BM-24), Mammaliicoccus sciuri (BM-25). The high bacterial density and number of species indicate that Hatay yellow race is highly susceptible to bacterial diseases. Insecticidal activity studies revealed that species belonging to Bacillus and Staphylococcus genera are important pathogens of hybrid silkworm culture and Hatay yellow race.

SEROPREVALENCE OF IgG ANTIBODIES OF HUMAN TOXOCARIASIS AND ASOCIATED RISK FACTORS IN KHANH HOA PROVINCE

Background: Human toxocariasis is a zoonotic parasitic disease caused by Toxocara canis and T. cati roundworm larvae. While many studies have shown that dog and cat owners are at a higher risk of acquiring Toxocara spp. infection, there is no available evidence regarding the seroprevalence of Toxocara spp. infection among dog and cat owners in Khanh Hoa. Therefore, this study aims to investigate the prevalence of anti-IgG of Toxocara spp. infection and associated risk factors for visceral larval migrans among dog owners in three different areas of Khanh Hoa province..Subjects and methods: A descriptive cross-sectional study was conducted from 2022 to 2024 in three localities representing different socio-geographic areas (urban, rural, and mountainous) of Khanh Hoa province. A total of 1502 blood samples were collected from local individuals aged 5 to 75 for this study. Serum anti-T.canis IgG antibodies were detected using a commercial enzyme-linked immunosorbent assay (ELISA) kit: NovaLisaTM, Toxocara canis IgG ELISA. Simultaneously, a survey using structured questionnaire about information on risk factors associated with Toxocariasis was conducted among 720 people (aged 18 and over) from 1,502 individuals whose bloods were taken for testing.Results and conclusions: A total of 1,502 blood samples were examined for the presence of antibodies to dog and cat roundworm larval infection. Results revealed an overall positive rate of 57.66% across the three localities, with variations depending on geography. In Khanh Vinh, a mountainous district, the infection rate was 75.44%, and in its commune Khanh Trung, it was 81.20%. For the two urban wards, the rates were significantly lower (Phuoc Long Ward: 44.40% and Van Thanh Ward: 54.18%). A structured questionnaire was used to investigate risk factors among 720 individuals (aged 18 years old and over) out of the 1,502 individuals whose bloods were tested. Results indicated that 389 of them tested positive for the ELISA test. Infection rates varied among age groups: under 25 years old (6.94%), 25-50 years old (66.78%), and over 50 years old (37.28%). Notably, 49.87% of infected individuals owned dogs or cats, 93.32% regularly consumed raw vegetables, and 25.71% frequently handled dogs or cats. The high infection rate of dog and cat roundworm larvae in Khanh Hoa underscores the presence of several identified high-risk factors.

The streptococcal phase-variable type I restriction modification system SsuCC20p dictates the methylome of Streptococcus suis impacting the transcriptome and virulence in a zebrafish larvae infection model.

Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.

New insights into changing honey bee (Apis mellifera) immunity molecules pattern and fatty acid esters, in responses to Ascosphaera apis infection

Monitoring of metabolite changes could provide valuable insights into disturbances caused by an infection and furthermore, could be used to define the status of an organism as healthy or diseased and define what could be defensive elements against the infection. The present investigation conducted a gas chromatography–mass spectrometry (GC/MS) for haemolymph of larval honey bees (Apis mellifera L.) infected with the fungal pathogen Ascosphaera apis in comparison with control haemolymph non-infected insects. Results revealed that the pathogen caused a general disturbance of metabolites detected in the haemolymph of the honey bee. The majority of metabolites identified before and after infection were fatty acid esters. The disease caused an elevation in levels of methyl oleate, methyl palmitate, and methyl stearate, respectively. Further, the disease drove to the disappearance of methyl palmitoleate, and methyl laurate. Conversely, methyl linolelaidate, and ethyl oleate were identified only in infected larvae. A high reduction in diisooctyl phthalate was recorded after the infection. Interestingly, antimicrobial activities were confirmed for haemolymph of infected honey bee larvae. In spite of the presence of some previously known bioactive compounds in healthy larvae there were no antimicrobial activities.

First record of Aspergillus caelatus as a pathogen of Heterotrigona itama and Geniotrigona thoracica in Malaysia

Heterotrigona itama and Geniotrigona thoracica become popular in Malaysia as honey producer in the 21st century. Like other bee species, stingless bees can be infected by pathogens. Disease-related population declines may affect honey production, agricultural productivity, and biodiversity. Thus, this study aimed to evaluate the health status of stingless bee colonies in selected bee farms in Malaysia. Adults Heterotrigona itama and Geniotrigona thoracica were collected from 79 colonies in 9 farms and screened for known pathogens of honey bees and stingless bees using 15 primer sets. All results were negative, indicating the absence of these pathogens in the bee specimens. During the bee sampling at Infoport Halal Hub UPM, we encountered a collapsed colony of H. itama. A fungal isolate was isolated and confirmed to be Aspergillus caelatus. Virulence tests revealed that A. caelatus exhibited high virulence to both H. itama and G. thoracica. Infected larvae became lethargic after 1–2 day after treatment (DAT) with white mycelium developed on their integument. The fungus caused 100 % larval mortality in both H. itama and G. thoracica after 8 DAT, with LC50 values of 7.58 × 103 conidia/µl and 6.24 × 103 conidia/µl respectively. Yellow-green conidia were observed on the cadavers. A. caelatus demonstrated high virulence towards both H. itama and G. thoracica, leading to severe larval mortality. This study represents the first documented case of a disease infecting stingless bees in Malaysia. Future research is needed to assess the prevalence of A. caelatus in various stingless bee species and other pollinators within the country.

Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.

Protective efficacy of short-term infection with Necator americanus hookworm larvae in healthy volunteers in the Netherlands: a single-centre, placebo-controlled, randomised, controlled, phase 1 trial

Vaccine development against hookworm is hampered by the absence of the development of protective immunity in populations repeatedly exposed to hookworm, limiting identification of mechanisms of protective immunity and new vaccine targets. Immunisation with attenuated larvae has proven effective in dogs and partial immunity has been achieved using an irradiated larvae model in healthy volunteers. We aimed to investigate the protective efficacy of immunisation with short-term larval infection against hookworm challenge. We did a single-centre, placebo-controlled, randomised, controlled, phase 1 trial at Leiden University Medical Center (Leiden, Netherlands). Healthy volunteers (aged 18-45 years) were recruited using advertisements on social media and in publicly accessible areas. Volunteers were randomly assigned (2:1) to receive three short-term infections with 50 infectious Necator americanus third-stage filariform larvae (50L3) or placebo. Infection was abrogated with a 3-day course of albendazole 400 mg, 2 weeks after each exposure. Subsequently all volunteers were challenged with two doses of 50L3 at a 2-week interval. The primary endpoint was egg load (geometric mean per g faeces) measured weekly between weeks 12 and 16 after first challenge, assessed in the per-protocol population, which included all randomly assigned volunteers with available data on egg counts at week 12-16 after challenge. This study is registered with ClinicalTrials.gov, NCT03702530. Between Nov 8 and Dec 14, 2018, 26 volunteers were screened, of whom 23 enrolled in the trial. The first immunisation was conducted on Dec 18, 2018. 23 volunteers were randomly assigned (15 to the intervention group and eight to the placebo group). Egg load after challenge was lower in the intervention group than the placebo group (geometric mean 571 eggs per g [range 372-992] vs 873 eggs per g [268-1484]); however, this difference was not statistically significant (p=0·10). Five volunteers in the intervention group developed a severe skin rash, which was associated with 40% reduction in egg counts after challenge (geometric mean 742 eggs per g [range 268-1484] vs 441 eggs per g [range 380-520] after challenge; p=0·0025) and associated with higher peak IgG1 titres. To our knowledge, this is the first study to describe a protective effect of short-term exposure to hookworm larvae and show an association with skin response, eosinophilic response, and IgG1. These findings could inform future hookworm vaccine development. Dioraphte Foundation.

Antimicrobial Activity of Black Soldier Fly, Hermetia illucens (Diptera: Stratiomyidae) Larval Hemolymph against Various Pathogenic Bacteria

The larvae of the black soldier fly (BSFL), Hermetia illucens (Linnaeus, 1758) (Diptera: Stratiomyidae), can survive in environments contaminated with various bacteria by producing antimicrobial compounds. This study, for the very first time, investigated the potential antibacterial activity of hemolymph extracted from BSFL in Malaysia using diffusion and dilution methods. Prior to extraction, the larvae were infected with either Methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli. Then, the hemolymph was collected. Serial dilutions from 200 to 12.5 mg/ml of the hemolymph extracts were screened against ten different bacteria. The results showed inhibition of eight out of ten tested bacteria (i.e., MRSA, Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae and Acinetobacter sp.). We found that immunological-challenge larvae have stronger antimicrobial activity than the control groups. The Minimum Inhibitory Concentrations (MIC) for bacteria against for infected larvae were 12.5 mg/ml for MRSA, S. pyogenes, B. subtilis, M. luteus, E. coli, and K. pneumoniae. As for bactericidal activity, the MBC of E. coli infected larvae was 25mg/ml against S. pyogenes and B. subtilis. In conclusion, BSFL hemolymph has antibacterial activity against a range of bacteria and could be a candidate for novel antimicrobial development.

A Technique to Quantify Fungal Infection in Zebrafish Larvae

A Technique to Quantify Fungal Infection in Zebrafish Larvae

A Technique to Quantify Fungal Infection in Zebrafish Larvae

A Technique to Quantify Fungal Infection in Zebrafish Larvae

Invade or die: behaviours and biochemical mechanisms that drive skin penetration in Strongyloides and other skin-penetrating nematodes.

Skin-penetrating nematodes, including the human threadworm Strongyloides stercoralis and hookworms in the genera Necator and Ancylostoma, are gastrointestinal parasites that are a major cause of neglected tropical disease in low-resource settings worldwide. These parasites infect hosts as soil-dwelling infective larvae that navigate towards hosts using host-emitted sensory cues such as odorants and body heat. Upon host contact, they invade the host by penetrating through the skin. The process of skin penetration is critical for successful parasitism but remains poorly understood and understudied. Here, we review current knowledge of skin-penetration behaviour and its underlying mechanisms in the human parasite S. stercoralis, the closely related rat parasite Strongyloides ratti, and other skin-penetrating nematodes such as hookworms. We also highlight important directions for future investigations into this underexplored process and discuss how recent advances in molecular genetic and genomic tools for Strongyloides species will enable mechanistic investigations of skin penetration and other essential parasitic behaviours in future studies. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

Pathogenetic detection, retrospective and pathogenicity analysis of a fatal case of Vibrio vulnificus in Shenzhen, China

We report a 36-year-old male patient died of V. vulnificus-induced septicaemia and multiple organ failure syndrome after oyster consumption at a restaurant. We isolated and identified V. vulnificus vv16015 from the patient’s blood sample and antibiotic susceptibility tests indicated sensitivity to all 21 antibiotics. Oyster samples were subsequently collected from the restaurant’s supplier and three strains of V. vulnificus were isolated. Whole genome sequencing and analysis revealed vv16015 to be distantly related to these strains and confirmed that V. vulnificus contamination was present in the seafood of the restaurant and supplier. Using a Galleria mellonella larvae infection model, the virulence of vv16015 was determined to be higher than that of comparison strains isolated from a surviving patient (vv15018) and an oyster (vv220015). The human and environment distribution of V. vulnificus in Shenzhen is sporadic and heterogeneous, and vv16015 is highly virulent compared to other strains.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.