Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana.

Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana's pathogenic mechanisms.

Knockdown of Thitarodes host genes influences dimorphic transition of Ophiocordyceps sinensis in the host hemolymph.

The Chinese cordyceps, a unique parasitic complex of Thitarodes/Hepialus ghost moths and Ophiocordyceps sinensis fungus in the Tibetan Plateau, is a highly valuable biological resource for medicine and health foods in Asian countries. Efficient system for artificial cultivation of Chinese cordyceps relies on understanding the gene functions involved in the induction of growing blastospores into hyphae in the larval hemolymph of insect host, during O. sinensis infection. Transcriptome analysis and ribonucleic acid interference (RNA interference) method were employed to identify the key differentially expressed genes and to demonstrate their functions in Thitarodes xiaojinensis. Key larval genes critical for O. sinensis blastospore development or filamentation were identified. Nine of the 20 top upregulated genes encoded cuticles proteins, indicating that these proteins highly activated when the larval hemolymph was full of blastospores. Small interfering RNA (siRNA) knockdown of five larval genes such as Flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3 significantly stimulated the dimorphic transition from blastospores to prehyphae in O. sinensis in the larval hemolymph after 120 h after injection. The expressions of these genes determined by quantitative real-time PCR were suppressed in various levels from 38.64% to 91.54%, compared to the controls. These results demonstrated that injection of the siRNAs of key upregulated genes into the larval hemolymph containing high load of blastospores caused the gene silence in T. xiaojinensis larvae and induced the fungal transition from blastospores to prehyphae, providing novel knowledge on the regulation of O. sinensis fungal dimorphism by Thitarodes host and cues for further study of Thitarodes biology and commercial cultivation of Chinese cordyceps.

Activity of polymyxin B combined with cefepime-avibactam against the biofilms of polymyxin B-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae in in vitro and in vivo models.

Bacterial biofilms, often forming on medical devices, can lead to treatment failure due to their increased antimicrobial resistance. Cefepime-avibactam (CFP-AVI) exhibits potent activities against Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) when used with polymyxin B (PMB). However, its efficacy in biofilm-related infections is unknown. The present study aimed to evaluate the activity of PMB combined with CFP-AVI against the biofilms of PMB-resistant Gram-negative bacteria. Five K. pneumoniae strains and three P. aeruginosa strains known to be PMB-resistant and prone to biofilm formation were selected and evaluated. Antimicrobial susceptibility assays demonstrated that the minimal biofilm inhibitory and eradication concentrations of PMB and CFP-AVI for biofilms formed by the eight strains were significantly higher than the minimal inhibitory concentrations of the antibiotics for planktonic cells. The biofilm formation inhibition and eradication assays showed that PMB combined with CFP-AVI cannot only suppress the formation of biofilm but also effectively eradicate the preformed mature biofilms. In a modified in vitro pharmacokinetic/pharmacodynamic biofilm model, CFP-AVI monotherapy exhibited a bacteriostatic or effective activity against the biofilms of seven strains, whereas PMB monotherapy did not have any activity at 72 h. However, PMB combined with CFP-AVI demonstrated bactericidal activity against the biofilms of all strains at 72 h. In an in vivo Galleria mellonella infection model, the 7-day survival rates of larvae infected with biofilm implants of K. pneumoniae or P. aeruginosa were 0-6.7%, 40.0-63.3%, and 46.7-90.0%, respectively, for PMB alone, CFP-AVI alone, and PMB combined with CFP-AVI; the combination therapy increased the rate by 6.7-33.3% (P < 0.05, n = 6), compared to CFP-AVI monotherapy. It is concluded that PMB combined with CFP-AVI exhibits effective anti-biofilm activities against PMB-resistant K. pneumoniae and P. aeruginosa both in vitro and in vivo, and thus may be a promising therapeutic strategy to treat biofilm-related infections.

Intraspecific cooperation allows the survival of Staphylococcus aureus staff: a novel strategy for disease relapse.

BACKGROUND: The contribution of interspecies interactions between coinfecting pathogens to chronic refractory infection by affecting pathogenicity is well established. However, little is known about the impact of intraspecific interactions on infection relapse, despite the cross-talk of different strains within one species is more common in clinical infection. We reported a case of chronic refractory pulmonary infection relapse, caused by two methicillin-sensitive S. aureus (MSSA) strains (SA01 and SA02) and revealed a novel strategy for relapse via intraspecific cooperation. METHODS: The hemolytic ability, growth curve, biofilm formation, virulence genes and response of G. mellonella larvae to S. aureus infection were analysed to confirm this hypothesis. RESULTS: SA02 hemolytic activity was inhibited by SA01, along with the expression of hemolysin genes and the virulence factor Hla. Additionally, SA01 significantly enhanced the biofilm formation of SA02. AIP-RNAIII may be a possible pathway for this interaction. Compared with mono-infection, a worse outcome (decreased larval survival and increased microbial burden) of the two MSSA strains coinfected with G. mellonella confirmed that intraspecific interactions indeed enhanced bacterial survival in vivo. CONCLUSION: The intraspecific interaction of S. aureus could lead to chronic refractory infection via pathogenicity changes.

Virulence plasmid with IroBCDN deletion promoted cross-regional transmission of ST11-KL64 carbapenem-resistant hypervirulent Klebsiella pneumoniae in central China.

BACKGROUND: Carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) caused infections of high mortality and brought a serious impact on public health. This study aims to evaluate the epidemiology, resistance and virulence characteristics of CR-hvKP and to identify potential drivers of cross-regional transmission in different regions of China, in order to provide a basis for developing targeted prevention measures. METHODS: Clinical K. pneumoniae strains were collected from Jiujiang and Nanchang in Jiangxi province between November 2021 to June 2022. Clinical data of patients (age, sex, source of infection, and diagnosis) were also gathered. We characterized these strains for their genetic relatedness using PFGE, antimicrobial and virulence plasmid structures using whole-genome sequencing, and toxicity using Galleria mellonella infection model. RESULTS: Among 609 strains, 45 (7.4%) CR-hvKP were identified, while the strains. isolated from Nanchang and Jiujiang accounted for 10.05% (36/358) and 3.59% (9/251). We observed that ST11-KL64 CR-hvKP had an overwhelming epidemic dominance in these two regions. Significant genetic diversity was identified among all ST11-KL64 CR-hvKP cross-regional transmission between Nanchang and Jiujiang and this diversity served as the primary driver of the dissemination of clonal groups. Virulence genes profile revealed that ST11-KL64 CR-hvKP might harbour incomplete pLVPK-like plasmids and primarily evolved from CRKP by acquiring the hypervirulence plasmid. We found the predominance of truncated-IncFIB/IncHI1B type virulence plasmids with a 25 kb fragment deletion that encoded iroBCDN clusters. CONCLUSION: ST11-KL64 is the most cross-regional prevalent type CR-hvKPs in Jiangxi province, which mainly evolved from CRKPs by acquiring a truncated-IncHI1B/IncFIB virulence plasmid with the deletion of iroBCDN. Stricter surveillance and control measures are urgently needed to prevent the epidemic transmission of ST11-KL64 CR-hvKP.

In vitro and in vivo activities of scutellarein, a novel polyphosphate kinase 1 inhibitor against Acinetobacter baumannii infection.

BACKGROUND: Inorganic polyphosphate (polyP)-targeted polyphosphate kinase 1 (PPK1) has attracted much attention by virtue of its importance in bacterial pathogenicity and persistence, as well as its exclusive presence in microorganisms. However, only very few drugs have been found to be efficacious in inhibiting the Acinetobacter baumannii (A. baumannii) PPK1 protein. RESULTS: In this study, we identified Scutellarein (Scu), a potent PPK1 inhibitor that could significantly influence PPK1-regulated motility, biofilm formation, and bacterial persistence, which was further validated by the results of transcriptome analysis. Mechanistic explorations revealed that Scu achieved its enzyme inhibitory activity predominantly through direct engagement with the active center of PPK1. Moreover, the survival rate of Galleria mellonella larvae was increased by about 35% with 20 mg/kg of Scu treatment. The remarkable therapeutic benefits of Scu were also observed in the mouse pneumonia model, shown mainly by reduced bacterial colonization, pathological lesions, and inflammatory factors. CONCLUSION: Our results revealed that Scu could attenuate the pathogenicity and persistence of A. baumannii by interfering with its important kinase PPK1.

Knockdown of the glucosamine-6-phosphate N-acetyltransferase gene by RNA interference enhances the virulence of entomopathogenic fungi against rice leaffolder Cnaphalocrocis medinalis.

Insect cuticle acts as a first line of defense and a physical protective barrier against entomopathogens. Chitin biosynthesis pathway plays a crucial role in chitin formation in the cuticle of insects. Glucosamine-6-phosphate N-acetyltransferase (GNA) is a key enzyme in insect chitin biosynthesis that regulate the chitin formation. However, how GNA-mediated cuticle metabolism influences virulence of entomopathogenic fungi is still unknown. In this study, CmGNA gene was cloned and characterized from the rice leaffolder Cnaphalocrocis medinalis. The CmGNA contains an open read frame (ORF) 600 nucleotides, encoding 199 amino acids with an isoelectric point of 8.65 and a molecular weight of 22.30 kDa. The expression profile showed that CmGNA was highly expressed in 4th instar larvae and in the cuticle. Here, we also reported the impact of CmGNA gene and entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, on expression pattern of chitin biosynthesis genes, feeding behavior, survival rate and average body weight of infected larvae, phenotypic deformities, rate of pupation, and adult emergence. Our results showed that knockdown of CmGNA and application of M. anisopliae and B. bassiana three days after RNA interference (RNAi) significantly decreased the expression of CmGNA and other associated genes, reduced feeding efficiency and survival rate, and caused loss of average body weight, less rate of pupation and adult emergence of infected larvae. Knockdown of CmGNA gene also increased the lethality of larvae caused by M. anisopliae and B. bassiana and resulted in significantly phenotypic deformities of infected larvae. Our findings illustrated that RNAi-mediated CmGNA knockdown disturbed the chitin synthesis genes that led to enhancing the virulence of M. anisopliae and B. bassiana, which can provide us new insights to develop novel biocontrol strategies against C. medinalis.

Galleria mellonella as an Antimicrobial Screening Model.

To combat the rising global issue of antibiotic resistance, the accelerated development of novel antibiotics is essential. Current preclinical antimicrobial development yields a significant number of leads that prove unsuitable either prior to or during clinical trials. To increase the efficiency of preclinical development, relevant, standardized, accessible, and cost-effective models must be developed. Galleria mellonella (greater wax moth) larvae are widely used as an infection model to assess microbial virulence, conduct drug toxicity testing, and serve as a preliminary means of evaluating the in vivo efficacy of novel antimicrobial compounds. These infection models have greater biological relevance than many in vitro screens of comparable throughput and decrease reliance on mammalian models when used as a pre-screen for antimicrobial testing. This protocol describes a standardized methodology for the optimization of G. mellonella infection models, which can be applied to bacterial species and antimicrobial therapeutics of choice. Using the WHO priority pathogen Pseudomonas aeruginosa as an exemplar, we outline steps that can be undertaken to develop a reproducible model of infection and therapeutic testing. This includes recommendations on experimental setup, sample preparation, and infection and treatment protocols. Integration of this model within preclinical antimicrobial development pipelines would decrease reliance on mammalian models, reduce the number of ineffective compounds reaching clinical trials, and ultimately increase the efficiency of preclinical antimicrobial development.

Transcriptomic Analysis of the Response of the Dioryctria abietella Larva Midgut to Bacillus thuringiensis 2913 Infection.

Dioryctria abietella Denis Schiffermuller (Lepidoptera: Pyralidae) is an oligophagous pest that mainly damages Pinaceae plants. Here, we investigated the effects of the Bacillus thuringiensis 2913 strain (Bt 2913), which carries the Cry1Ac, Cry2Ab, and Vip3Aa genes, on the D. abietella midgut transcriptome at 6, 12, and 24 h after infection. In total, 7497 differentially expressed genes (DEGs) were identified from the midgut transcriptome of D. abietella larvae infected with Bt 2913. Among these DEGs, we identified genes possibly involved in Bt 2913-induced perforation of the larval midgut. For example, the DEGs included 67 genes encoding midgut proteases involved in Cry/Vip toxin activation, 74 genes encoding potential receptor proteins that bind to insecticidal proteins, and 19 genes encoding receptor NADH dehydrogenases that may bind to Cry1Ac. Among the three transcriptomes, 88 genes related to metabolic detoxification and 98 genes related to immune defense against Bt 2913 infection were identified. Interestingly, 145 genes related to the 60S ribosomal protein were among the DEGs identified in the three transcriptomes. Furthermore, we performed bioinformatic analysis of zonadhesin, GST, CYP450, and CarE in the D. abietella midgut to determine their possible associations with Bt 2913. On the basis of the results of this analysis, we speculated that trypsin and other serine proteases in the D. abietella larval midgut began to activate Cry/Vip prototoxin at 6 h to 12 h after Bt 2913 ingestion. At 12 h after Bt 2913 ingestion, chymotrypsin was potentially involved in degrading the active core fragment of Vip3Aa toxin, and the detoxification enzymes in the larvae contributed to the metabolic detoxification of the Bt toxin. The ABC transporter and several other receptor-protein-related genes were also downregulated to increase resistance to Bt 2913. However, the upregulation of 60S ribosomal protein and heat shock protein expression weakened the resistance of larvae to Bt 2913, thereby enhancing the expression of NADH dehydrogenase and other receptor proteins that are highly expressed in the larval midgut and bind to activating toxins, including Cry1Ac. At 24 h after Bt 2913 ingestion, many activated toxins were bound to receptor proteins such as APN in the larval midgut, resulting in membrane perforation. Here, we clarified the mechanism of Bt 2913 infection in D. abietella larvae, as well as the larval immune defense response to Bt 2913, which provides a theoretical basis for the subsequent control of D. abietella using B. thuringiensis.

Human breast milk isolated lactic acid bacteria: antimicrobial and immunomodulatory activity on the Galleria mellonella burn wound model.

Introduction: Managing burn injuries is a challenge in healthcare. Due to the alarming increase in antibiotic resistance, new prophylactic and therapeutic strategies are being sought. This study aimed to evaluate the potential of live Lactic Acid Bacteria for managing burn infections, using Galleria mellonella larvae as an alternative preclinical animal model and comparing the outcomes with a common antibiotic. Methods: The antimicrobial activity of LAB isolated from human breast milk was assessed in vitro against Pseudomonas aeruginosa ATCC 27853. Additionally, the immunomodulatory effects of LAB were evaluated in vivo using the G. mellonella burn wound infection model. Results and discussion: In vitro results demonstrated the antimicrobial activity of Lactic Acid Bacteria against P. aeruginosa. In vivo results show that their prophylactic treatment improves, statistically significant, larval survival and modulates the expression of immunity-related genes, Gallerimycin and Relish/NF-κB, strain-dependently. These findings lay the foundation and suggest a promising alternative for burn wound prevention and management, reducing the risk of antibiotic resistance, enhancing immune modulation, and validating the potential G. mellonella as a skin burn wound model.

A gene regulating ergot alkaloid biosynthesis in Metarhizium brunneum.

Ergot alkaloid synthesis (eas) gene clusters found in several fungi encode biosynthesis of agriculturally and pharmaceutically important ergot alkaloids. Although the biosynthetic genes of the ergot alkaloid pathway have been well characterized, regulation of those genes is unknown. We characterized a gene with sequence similarity to a putative transcription factor and that was found adjacent to the eas cluster of Metarhizium brunneum, a plant symbiont and insect pathogen. Function of the novel gene, easR, was explored by CRISPR-Cas9-derived gene knockouts. To maximize potential for ergot alkaloid accumulation, strains of M. brunneum were injected into larvae of the insect Galleria mellonella. Larvae infected with the wild type contained abundant ergot alkaloids, but those infected with easR knockouts lacked detectable ergot alkaloids. The easR knockout strains had significantly reduced or no detectable mRNA from eas cluster genes in RNAseq and qualitative RT-PCR analyses, whereas the wild-type strain contained abundant mRNA from all eas genes. These data demonstrate that the product of easR is required for ergot alkaloid accumulation and provide evidence that it has a role in the expression of ergot alkaloid biosynthesis genes. Larvae infected with an easR knockout survived significantly longer than those infected with the wild type (P < 0.0001), indicating a role for EasR, and indirectly confirming a role for ergot alkaloids, in the virulence of M. brunneum to insects. Homologs of easR were found associated with eas clusters of at least 15 other ergot alkaloid-producing fungi, indicating that EasR homologs may contribute to regulation of ergot alkaloid synthesis in additional fungi. IMPORTANCE: Ergot alkaloids produced by several species of fungi are important as contaminants of food and feed in agriculture and also as the foundation of numerous pharmaceuticals prescribed for dementia, migraines, hyperprolactinemia, and several other disorders. Information on control of the ergot alkaloid pathway may contribute to strategies to limit their production in agricultural settings or increase their yield for pharmaceutical production. Our results demonstrate that a previously uncharacterized gene clustered with the ergot alkaloid synthesis genes is required for the sufficient transcription of the ergot alkaloid biosynthesis genes. This observation suggests the gene encodes a factor regulating transcription of those biosynthetic genes.

Clinical, phenotypic characterization and genomic analysis of the mucoid Acinetobacter baumannii from a teaching hospital.

BACKGROUND: Acinetobacter baumannii (A. baumannii) has become a significant nosocomial pathogen globally over the past decade due to the increasing prevalence of antibiotic-resistant isolates. The formation of the mucoid phenotype is a crucial adaptive defense response to external pressure, but the clinical, phenotypic and genotypic characteristics and their relationship with sequence types (ST) and K locus (KL) types remain unclear. METHODS: In this study, we screened a total of 736 A. baumannii isolates, from which we identified and characterized 13 mucoid isolates. The study explored the clinical characteristics of patients with mucoid isolates, investigated the mucoid phenotype, performed capsule observation, quantified capsule production, and assessed antimicrobial susceptibility. Subsequently, whole-genome sequencing (WGS) was used to analyze the sequence types (ST), loci for capsular polysaccharide (KL), antibiotic resistance genes, virulence genes, and core-genome single-nucleotide polymorphisms (SNPs). Additionally, the virulence of all mucoid strains was evaluated through serum resistance assay, biofilm-forming assay, and Galleria mellonella survival assay. RESULTS: All mucoid A. baumannii isolates were found to be encapsulated and extremely drug-resistant. Among patients infected with these isolates, 92.3 % had pulmonary infections, and the 30-day mortality rate was 61.5 %. The analysis revealed that not all strains are highly virulent. Whole-genome sequencing (WGS) identified the sequence types as ST136, ST208, ST381, ST195, and ST281, and the capsular types as KL77, KL7, KL33, KL2, and KL3. The ST208 and KL7 isolates exhibited higher virulence and greater biofilm formation, with KL7 isolates also showing higher capsule production. Despite these differences, no significant variations in virulence genes were observed among the mucoid isolates, except for biofilm-associated and quorum-sensing genes. The highly virulent ST208/KL7 strains (AB276, AB313, and AB552) lacked biofilm-associated genes (csuA/BABCDE), indicating these genes do not directly cause differences in biofilm formation. CONCLUSION: The mucoid A. baumannii isolates were extensively drug-resistant, and infections caused by these isolates could lead to higher mortality. However, not all strains had high virulence, with variations likely related to specific sequence types (ST) and K locus (KL) types.

Microbiome-derived antimicrobial peptides show therapeutic activity against the critically important priority pathogen, Acinetobacter baumannii.

Acinetobacter baumannii is designated by the World Health Organisation as a critical priority pathogen. Previously we discovered antimicrobial peptides (AMPs), namely Lynronne-1, -2 and -3, with efficacy against bacterial pathogens, such as Staphylococcus aureus and Pseudomonas aeruginosa. Here we assessed Lynronne-1, -2 and -3 structure by circular dichroism and efficacy against clinical strains of A. baumannii. All Lynronne AMPs demonstrated alpha-helical secondary structures and had antimicrobial activity towards all tested strains of A. baumannii (Minimum Inhibitory Concentrations 2-128 µg/ml), whilst also having anti-biofilm activity. Lynronne-2 and -3 demonstrated additive effects with amoxicillin and erythromycin, and synergy with gentamicin. The AMPs demonstrated little toxicity towards mammalian cell lines or Galleria mellonella. Fluorescence-based assay data demonstrated that Lynronne-1 and -3 had higher membrane-destabilising action against A. baumannii in comparison with Lynronne-2, which was corroborated by transcriptomic analysis. For the first time, we demonstrate the therapeutic activity of Lynronne AMPs against A. baumannii.

Characterization of larval gut microbiota of two endoparasitoid wasps associated with their common host, Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae).

Insect gut microbes play important roles in digestion, metabolism, development, and environmental adaptation. Parasitoid wasps are one of the most important biological control agents in pest control, while the gut microbial species compositions and the associated functions have been poorly investigated. Two endoparasitoid wasps, Cotesia vestalis and Diadromus collaris, parasitize the larval stage and pupal stage of the diamondback moth, Plutella xylostella, respectively. Using whole-genome shotgun metagenomic sequencing, we characterized the gut microbial composition, diversity, and potential functional roles associated with the two parasitoid wasp larvae. The results reveal that Proteobacteria and Firmicutes are the dominant phyla in the gut of C. vestalis and D. collaris larvae, with Rhizobium and Enterococcus being the dominant genera. The putative microbial functions associated with the two parasitoid wasps might play a virtual role in assisting in consuming the host's nutritional composition. The enriched CAZymes family genes are primarily involved in the degradation and synthesis of chitin. Despite the richness of microbial species and communities, the microbes species and the microbial community structure exhibit significant similarity between the two parasitoid wasps and between the parasitoid wasp and the host P. xylostella. Notably, the prevalence of the genus Enterococcus shared among them suggests a possible link of gut microbes between the host and their associated parasitoids. Our study offers insights into the gut microbe-based interactions between the host and parasitoid wasps for the first time, potentially paving the way for the development of an ecologically friendly biocontrol strategy against the pest P. xylostella.IMPORTANCEEndoparasitoid wasps spend the majority of their lifespan within their host and heavily rely on the host's nutrition for survival. There is limited understanding regarding the composition and physiological impacts of gut microbial communities in parasitoid wasps, particularly during the larval stage, which is directly linked to the host. Based on a thorough characterization of the gut microbe and comprehensive comparative analysis, we found the microbial species of the larval parasitoid wasp Cotesia vestalis and the pupal parasitoid wasp Diadromus collaris were similar, sharing 159 genera and 277 species, as were the microbial community structure. Certain of the dominant microbial strains of the two parasitoid wasps were similar to that of their host Plutella xylostella larvae, revealing host insect may affect the microbial community of the parasitoid wasps. The putative microbial functions associated with the parasitoid wasp larvae play an important role in dietary consumption.

Role of Enterococcus mundtii in gut of the tomato leaf miner (Tuta absoluta) to detoxification of Chlorantraniliprole.

Chlorantraniliprole (CAP) is applied worldwide for the control of caterpillars (Lepidoptera). However, with the overuse of CAP, the resistance problem in pest control is becoming increasingly serious. Recent studies have indicated a central role of the gut symbiont in insect pest resistance to pesticides and these may apply to the tomato leaf miner Tuta absoluta, is one of the most destructive insects worldwide. Here, we successfully isolated seven strains of tolerant CAP bacterium from the CAP-resistant T. absoluta gut, of which Enterococcus mundtii E14 showed the highest CAP tolerance, with a minimum inhibitory concentration (MIC) of 1.6 g/L and CAP degradation rate of 42.4%. Through transcriptomics and metabolism analysis, we studied the detoxification process of CAP by the E. mundtii E14, and found that CAP can be degraded by E. mundtii E14 into non-toxic compounds, such as 3,4-dihydroxy-2-(5-hydroxy-3,7-dimethylocta-2,6-dien-1-yl) benzoic acid and 2-pyridylacetic acid. Additionally, 2-pyridylacetic acid was detected both intracellular and extracellular in E. mundtii E14 treated with CAP. Meanwhile, we identified 52 up-regulated genes, including those associated with CAP degradation, such as RS11670 and RS19130. Transcriptome results annotated using KEGG indicated significant enrichment in up-regulated genes related to the glyoxylate cycle, nitrogen metabolism, and biosynthesis of secondary metabolites. Additionally, we observed that reinfection with E. mundtii E14 may effectively enhance resistance of T. absoluta to CAP. The LC50 values of the antibiotic treatment population of T. absoluta reinfection with E. mundtii E14 is 0.6122 mg/L, which was 18.27 folds higher than before reinfection. These findings offer new insights into T. absoluta resistance to CAP and contribute to a better understanding of the relationship between insecticide resistance and gut symbionts of T. absoluta, which may play a pivotal role in pest management.

Structure-Based Discovery of Symmetric Disulfides from Garlic Extract as Pseudomonas aeruginosa Quorum Sensing Inhibitors.

Microorganisms are the most common cause of food spoilage. Pseudomonas aeruginosa is a common foodborne pathogen that causes food spoilage and poses a serious threat to food safety. As a crucial target in antitoxicity strategies, the quorum sensing (QS) system shows promising potential for further development. The garlic extract diallyl disulfide exhibits inhibitory activity against the QS system of P. aeruginosa, with disulfide bonds serving as the active component. However, the biological activity of other symmetric disulfides has not been investigated in this capacity. The study synthesized 39 disulfide bond-containing analogs and evaluated their activity as quorum sensing inhibitors (QSIs). The results showed that p-hydroxyphenyl substitution can replace the allyl groups while maintaining strong biological activity. The virulence factors production was reduced by compound 2i, with the strongest inhibitory effect being observed on elastase production. Synergistic inhibition was observed in the presence of antibiotics like ciprofloxacin and tobramycin. 2i successfully inhibited P. aeruginosa infection in the Galleria mellonella larvae model. Primary mechanism studies using transcriptome, surface plasmon resonance and molecular docking suggested that 2i inhibits the QS system by targeting the LasR protein. Thus, compound 2i could be used in developing QSIs for the control of P. aeruginosa infections.

Paracoccidioides lutzii Infects Galleria mellonella Employing Formamidase as a Virulence Factor.

The formamidase (FMD) enzyme plays an important role in fungal thriving by releasing a secondary nitrogen source as a product of its activity. In Paracoccidioides species, previous studies have demonstrated the upregulation of this enzyme in a wide range of starvation and infective-like conditions. However, Paracoccidioides lutzii formamidase has not yet been defined as a virulence factor. Here, by employing in vivo infections using an fmd-silenced strain in Galleria mellonella larvae model, we demonstrate the influence of formamidase in P. lutzii's immune stimulation and pathogenicity. The formamidase silencing resulted in improper arrangement of the nodules, poor melanogenesis and decreased fungal burden. Thus, we suggest that formamidase may be a piece composing the process of molecular recognition by Galleria immune cells. Furthermore, formamidase silencing doubled the observed survival rate of the larvae, demonstrating its importance in fungal virulence in vivo. Therefore, our findings indicate that formamidase contributes to Galleria's immune incitement and establishes the role of this enzyme as a P. lutzii virulence factor.

Evaluation of Klebsiella pneumoniae pathogenicity through holistic gene content analysis.

Klebsiella pneumoniae is a Gram-negative bacterium that causes both community- and healthcare-associated infections. Although various virulence factors and highly pathogenic phenotypes have been reported, the pathogenicity of K. pneumoniae is still not fully understood. In this study, we utilized whole-genome sequencing data of 168 clinical K. pneumoniae strains to assess pathogenicity. This work was based on the concept that the genetic composition of individual genomes (referred to as holistic gene content) of the strains may contribute to their pathogenicity. Holistic gene content analysis revealed two distinct groups of K. pneumoniae strains ('major group' and 'minor group'). The minor group included strains with known highly pathogenic clones (ST23, ST375, ST65 and ST86). The minor group had higher rates of capsular genotype K1 and presence of nine specific virulence genes (rmpA, iucA, iutA, irp2, fyuA, ybtS, iroN, allS and clbA) compared to the major group. Pathogenicity was assessed using Galleria mellonella larvae. Infection experiments revealed lower survival rates of larvae infected with strains from the minor group, indicating higher virulence. In addition, the minor group had a higher string test positivity rate than the major group. Holistic gene content analysis predicted possession of virulence genes, string test positivity and pathogenicity as observed in the G. mellonella infection model. Moreover, the findings suggested the presence of as yet unrecognized genomic elements that are either involved in the acquisition of virulence genes or associated with pathogenicity.

First report of the association between Wolbachia and Cotesia flavipes (Hymenoptera: Braconidae): effect on life history parameters of the parasitoid.

The symbiosis between microorganisms and host arthropods can cause biological, physiological, and reproductive changes in the host population. The present study aimed to survey facultative symbionts of the genera Wolbachia, Arsenophonus, Cardinium, Rickettsia, and Nosema in Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) and Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) in the laboratory and evaluate the influence of infection on the fitness of these hosts. For this purpose, 16S rDNA primers were used to detect these facultative symbionts in the host species, and the hosts' biological and morphological features were evaluated for changes resulting from the infection caused by these microorganisms. The bacterial symbionts studied herein were not detected in the D. saccharalis samples analysed, but the endosymbiont Wolbachia was detected in C. flavipes and altered the biological and morphological aspects of this parasitoid insect. The results of this study may help to elucidate the role of Wolbachia in maintaining the quality of populations/lineages of C. flavipes.

Trophic Transfer of Metal Oxide Nanoparticles in the Tomato-Helicoverpa armigera Food Chain: Effects on Phyllosphere Microbiota, Insect Oxidative Stress, and Gut Microbiome.

Understanding the trophic transfer and ecological cascade effects of nanofertilizers and nanopesticides in terrestrial food chains is crucial for assessing their nanotoxicity and environmental risks. Herein, the trophic transfer of La2O3 (nLa2O3) and CuO (nCuO) nanoparticles from tomato leaves to Helicoverpa armigera (Lepidoptera: Noctuidae) caterpillars and their subsequent effects on caterpillar growth and intestinal health were investigated. We found that 50 mg/L foliar nLa2O3 and nCuO were transferred from tomato leaves to H. armigera, with particulate trophic transfer factors of 1.47 and 0.99, respectively. While nCuO exposure reduced larval weight gain more (34.7%) than nLa2O3 (11.3%), owing to higher oxidative stress (e.g., MDA and H2O2) and more serious intestinal pathological damage (i.e., crumpled columnar cell and disintegrated goblet cell) by nCuO. Moreover, nCuO exposure led to a more compact antagonism between the phyllosphere and gut microbiomes compared to nLa2O3. Specifically, nCuO exposure resulted in a greater increase in pathogenic bacteria (e.g., Mycobacterium, Bacillus, and Ralstonia) and a more significant decrease in probiotics (e.g., Streptomyces and Arthrobacter) than nLa2O3, ultimately destroying larval intestinal immunity. Altogether, our findings systematically revealed the cascade effect of metal oxide nanomaterials on higher trophic consumers through alteration in the phyllosphere and insect gut microbiome interaction, thus providing insights into nanotoxicity and environmental risk assessment of nanomaterials applied in agroecosystems.