Galleria Mellonella Larvae Research Articles

Characteristics of a Carbapenem-Resistant Acinetobacter baumannii Strain Causing Community-Acquired Pneumonia in a Young Healthy Women.

Multidrug-resistant Acinetobacter baumannii rarely causes community-acquired pneumonia. Here, we report the clinical and genomic characteristics of a multidrug-resistant A. baumannii strain responsible for community-acquired pneumonia in a 31-year-old healthy young women. A. baumannii strain W2LL was recovered from the alveolar lavage fluid sample of a hospitalized patient with pulmonary infection. Growth rate studies were conducted under various conditions, and virulence assessments were performed using Galleria Mellonella larvae. Whole Genome Sequencing (WGS) was carried out using Oxford Nanopore MinIon and Illumina HiSeq. In silico multilocus sequence typing (MLST), plasmid replicons, antimicrobial resistance genes, and virulence genes were determined using the BacWGSTdb webserver. Phylogenetic analysis between strain W2LL and other closely related A. baumannii genomes retrieved from NCBI database was performed. WGS identified strain W2LL as a rare sporadic lineage sequence type (ST) 1431. In addition to the detection of the β-lactamase gene (blaOXA-98) on the chromosome, blaOXA-58 was found on a 92,034 bp plasmid. Antimicrobial susceptibility testing revealed this strain was resistant to cephalosporins and carbapenems, with initial treatment using cefoxitin proving ineffective. Subsequent treatment with piperacillin-sulbactam combined with levofloxacin led to gradual improvement. Compared to A. baumannii ATCC 17978, W2LL exhibited similar growth rates at 37°C and 42°C, as well as in the presence of zinc. However, strain W2LL exhibited higher virulence phenotype compared to ATCC 17978 in G. mellonella model. The closest relative of A. baumannii W2LL was CAM180_1, another isolate recovered from Cambodia, which differed by 191 SNPs. W2LL is a rare ST1431 carbapenem-resistant A. baumannii strain recovered from a patient with no prior hospitalization or typical risk factors. This underscores the growing menace posed by carbapenem-resistant A. baumannii, no longer limited to hospitalized patients, potentially impacting the broader, younger population.

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.

Topical antimicrobial formulations using medicinal plant-derived essential oils targeting methicillin resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) causes a variety of healthcare-associated and community-acquired infections. Due to limited availability of effective antimicrobials for treating MRSA infections, there is a growing need to explore alternative therapeutic approaches. Here, the antimicrobial activities of 19 oils, popularly used for their medicinal properties, were tested against MRSA USA300. Oils obtained from cinnamon, clove, tangerine, and coriander showed the most promising activities, demonstrating bactericidal, anti-adhesive and anti-biofilm activities, and synergistic properties with common antibiotics. Given that clove and cinnamon oils showed the best activities, they were incorporated into topical formulations. Not only did the formulations with oils maintain antimicrobial and anti-adhesive activities, but their anti-biofilm property was potentiated. Tests on Galleria mellonella larvae suggested that the formulation is non-toxic. The formulations proposed here are a great alternative for the decolonisation of surfaces containing MRSA and can help circumventing antimicrobial resistance, a growing threat in the hospital environment.

Utilization of glycoprotein-derived N-acetylglucosamine-L-asparagine during Enterococcus faecalis infection depends on catabolic and transport enzymes of the glycosylasparaginase locus

Enterococcus faecalis is a Gram-positive clinical pathogen causing severe infections. Its survival during infection depends on its ability to utilize host-derived metabolites, such as protein-deglycosylation products. We have identified in E. faecalis OG1RF a locus (ega) involved in the catabolism of the glycoamino acid N-acetylglucosamine-L-asparagine. This locus is separated into two transcription units, genes egaRP and egaGBCD1D2, respectively. RT-qPCR experiments revealed that the expression of the ega locus is regulated by the transcriptional repressor EgaR. Electromobility shift assays evidenced that N-acetylglucosamine-L-asparagine interacts directly with the EgaR protein, which leads to the transcription of the ega genes. Growth studies with egaG, egaB and egaC mutants confirmed that the encoded proteins are necessary for N-acetylglucosamine-L-asparagine catabolism. This glycoamino acid is transported and phosphorylated by a specific phosphotransferase system EIIABC components (OG1RF_10751, EgaB, EgaC) and subsequently hydrolyzed by the glycosylasparaginase EgaG, which generates aspartate and 6-P-N-acetyl-β-d-glucosaminylamine. The latter can be used as a fermentable carbon source by E. faecalis. Moreover, Galleria mellonella larvae had a significantly higher survival rate when infected with ega mutants compared to the wild-type strain, suggesting that the loss of N-acetylglucosamine-L-asparagine utilization affects enterococcal virulence.

Genome sequence of a virulent and hypermucoviscous-like Klebsiella michiganensis clinical isolate

ObjectivesThe hypermucoviscous-like phenotype has been described in Klebsiella pneumoniae species complex (KpSC) and was described as a contributor of increased virulence. This study described the characterization and whole-genome sequencing of an antibiotic susceptible and hypermucoviscous-like Klebsiella michiganensis 9273 clinical isolate.Data descriptionHere, we report the genome sequence of a K. michiganensis clinical isolate obtained from a urinary tract infection exhibiting the hypermucoviscous-like phenotype. The draft genome sequence consisted of 145 contigs and ~ 6.6 Mb genome size. The annotation revealed 6648 coding DNA sequences and 56 tRNA genes. The strain belongs to the sequence type (ST) 50, and the OXY-1 beta-lactam resistance gene, aph(3′)-Ia gene for aminoglycoside resistance and multidrug efflux pumps were identified. The fyuA siderophore receptor of yersiniabactin siderophore was identified. Increased virulence was observed in Galleria mellonella larvae model and increased capsule production was determined by uronic acid quantification. The clinical implications of this phenotype are unknown, but the patient outcome might worsen compared to susceptible- or MDR-classical K. michiganensis isolates.

Tyramine, one quorum sensing inhibitor, reduces pathogenicity and restores tetracycline susceptibility in Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski’s five rules. It also shows high gastrointestinal absorption and the ability to cross the blood–brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

Protective Effect of Procyanidin-Rich Grape Seed Extract against Gram-Negative Virulence Factors.

Biofilm formation and lipopolysaccharide (LPS) are implicated in the pathogenesis of gastrointestinal (GI) diseases caused by Gram-negative bacteria. Grape seeds, wine industry by-products, have antioxidant and antimicrobial activity. In the present study, the protective effect of procyanidin-rich grape seed extract (prGSE), from unfermented pomace of Vitis vinifera L. cv Bellone, on bacterial LPS-induced oxidative stress and epithelial barrier integrity damage has been studied in a model of Caco-2 cells. The prGSE was characterized at the molecular level using HPLC and NMR. The in vitro activity of prGSE against formation of biofilm of Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli was investigated. In vivo, prGSE activity using infected Galleria mellonella larvae has been evaluated. The results show that the prGSE, if administered with LPS, can significantly reduce the LPS-induced permeability alteration. Moreover, the ability of the extract to prevent Reactive Oxygen Species (ROS) production induced by the LPS treatment of Caco-2 cells was demonstrated. prGSE inhibited the biofilm formation of E. coli and S. Typhimurium. In terms of in vivo activity, an increase in survival of infected G. mellonella larvae after treatment with prGSE was demonstrated. In conclusion, grape seed extracts could be used to reduce GI damage caused by bacterial endotoxin and biofilms of Gram-negative bacteria.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

Galleria Mellonella Larvae as an In vitro Model for Testing Microbial Pathogenicity

Abstract The larvae of the insect Galleria mellonella have gained wide acceptance as an in vivo model for the study of virulence in Gram-negative and Gram-positive bacteria and fungi. Importantly, G. mellonella offers an alternative, low-cost, and ethically acceptable method of assessing pathogens. These insects have a number of advantages, including ease of breeding, a rapid developmental cycle, low maintenance costs, and ease of experimental manipulation. Most importantly, G. mellonella larvae have a complete immune system and a nonspecific immune response similar to that of humans, making them a valuable model for studying pathogen-host interactions. Furthermore, using this model, it is possible to test the efficacy of various chemical compounds or natural substances that show antimicrobial activity. In this literature review, we provide a comprehensive overview of recent studies of the use of G. mellonella in virulence analyses and as a platform for testing the efficacy of new drugs.

Within-Host Resistance and Virulence Evolution of a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae ST11 Under Antibiotic Pressure.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused an extremely severe health challenge and public concern. However, the underlying mechanisms of fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we report a hv-CRKP-associated fatal infection and reveal a reduction in virulence due to the acquisition of aminoglycoside resistance. The bacterial identification, antimicrobial susceptibility, hypermucoviscosity, virulence factors, MLST and serotypes were profiled.The clonal homology and plasmid acquisition among hv-CRKP strains were detected by XbaI and S1-PFGE. The virulence potential of the strains was evaluated using Galleria mellonella larvae infection model, serum resistance assay, capsular polysaccharide quantification, and biofilm formation assay. Genomic variations were identified using whole-genome sequencing (WGS). Four K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from an 86-year-old patient with severe pneumonia. Whole-genome sequencing (WGS) showed that all four hv-CRKP strains belonged to the ST11-KL64 clone. PFGE analysis revealed that the four ST11-KL64 hv-CRKP strains could be grouped into the same PFGE type. Under the pressure of antibiotics, the antimicrobial resistance of the strains increased and the virulence potential decreased. Further sequencing, using the Nanopore platform, was performed on three representative isolates (WYKP586, WYKP589, and WYKP594). Genomic analysis showed that the plasmids of these three strains underwent a large number of breaks and recombination events under antibiotic pressure. We found that as aminoglycoside resistance emerged via acquisition of the rmtB gene, the hypermucoviscosity and virulence of the strains decreased because of internal mutations in the rmpA and rmpA2 genes. This study shows that ST11-KL64 hv-CRKP can further evolve to acquire aminoglycoside resistance accompanied by decreased virulence to adapt to antibiotic pressure in the host.

Enteropathogenic Escherichia coli is a predominant pathotype in healthy pigs in Hubei Province of China.

This study aims to investigate the prevalence of intestinal pathogenic Escherichia coli (InPEC) in healthy pig-related samples and evaluate the potential virulence of the InPEC strains. A multiplex PCR method was established to identify different pathotypes of InPEC. A total of 800 rectal swab samples and 296 pork samples were collected from pig farms and slaughterhouses in Hubei province, China. From these samples, a total of 21 InPEC strains were isolated, including 19 enteropathogenic E. coli (EPEC) and 2 shiga toxin-producing E. coli (STEC) strains. By whole-genome sequencing and in silico typing, it was shown that the sequence types and serotypes were diverse among the strains. Antimicrobial susceptibility assays showed that 90.48% of the strains were multi-drug resistant. The virulence of the strains was first evaluated using the Galleria mellonella larvae model, which showed that most of the strains possessed medium to high pathogenicity. A moderately virulent EPEC isolate was further selected to characterize its pathogenicity using a mouse model, which suggested that it could cause significant diarrhea. Bioluminescence imaging (BLI) was then used to investigate the colonization dynamics of this EPEC isolate, which showed that the EPEC strain could colonize the mouse cecum for up to 5 days.

Potential of antimicrobial peptide-fused endolysin LysC02 as therapeutics for infections and disinfectants for food contact surfaces to control Cronobacter sakazakii

Although endolysin (lysin), a muralytic enzyme of bacteriophages, has emerged as a promising antibacterial agent, its use against Gram-negative bacteria is challenging due to the presence of an outer membrane (OM) that acts as a physical barrier. To overcome this limitation, it is crucial to provide lysins with the ability to penetrate or disrupt the OM to access the target peptidoglycan. Here, we fused a lysin LysC02 from phage ΦC02 with nine different amphipathic antimicrobial peptides (AMPs) to enhance its intrinsic bactericidal activity against the notorious opportunistic foodborne Gram-negative pathogen Cronobacter sakazakii. Even without OM permeabilizers, the engineered LysC02 fused at the C-terminus with Cecropin P1, Lactoferricin B, or Thanatin exhibited the most enhanced bactericidal activity against C. sakazakii with ∼3-log reduction within 2 h. The action of LysC02-AMPs, the physical destruction of bacterial cells facilitated by the destabilization of the OM by fused AMPs and the intracellular turgor pressure of the cell, occurred over a wide range of temperatures up to 55 °C and pH from 4 to 10. Not only C. sakazakii but also other Gram-negative pathogens were lysed by LysC02-AMPs. Accordingly, LysC02-AMPs were able to significantly rescue Galleria mellonella larvae infected with C. sakazakii in the in vivo treatment model and significantly reduce C. sakazakii cells in food and food contact surface models. Taken together, LysC02-AMPs could be developed as effective alternative disinfectants and therapeutics to control C. sakazakii.

Screening of two native isolates of entomopathogenic nematodes, Heterorhabditis indica and Heterorhabditis bacteriophora, for temperature and moisture stress tolerance

Entomopathogenic nematodes (EPNs) are obligate parasites infecting a wide range ofinsect species and are oneof promising biocontrol agents with potential in insect pest management. Temperature and soil moisture play an important role for their sustenance in soil and their efficiency varies greatly under different climatic conditions. The present studywas aimed to study the effect oftemperature and soil moisture on survival, infectivity and reproduction of two EPNs viz., Heterorhabditis indica and H. bacteriophora. The IJs of both the EPNs were exposed to different temperatures and soil moisture levels. Results revealed that survival of both EPNs effected at 5ºC. Survival of H. indica decreased from 54% to 29% after exposure to 5ºC for 48 h. None of the EPNs survived at 35ºC and 40ºC. Infectivity and reproduction potential of both the EPNs tested was highest at 30ºC. No decrease in infectivity was observed with increase in exposure time to different temperatures in both the EPN species. Slight decrease in reproductive potential was observed in H. bacteriophora when exposed to 30ºC for 48h. None of the tested EPNs infected Galleria mellonella larva at 5ºC, 35ºC and 40ºC. Both the EPNs tested showed high infectivity at soil moistures more than 10%. Minimum infection was observed at soil moistures below 10%. Our study revealed that temperature range of 20ºC-30ºC and soil moisture of >10% was found optimum for survival, infectivity and reproduction ofthe testedEPNs. Both the EPNs in this study showed similar temperature and moisture requirements.

Emergence of colistin-heteroresistant and carbapenem-resistant hypervirulent Klebsiella pneumoniae

To investigate the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae, and characterize the underlying molecular mechanisms. The population analysis profiles (PAPs) method was used to detect colistin heteroresistance. The time-killing assay was used to examine the effect of colistin on carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro. Galleria mellonella larvae infection model was used to test the potential virulence. qRT-PCR assay was conducted to compare the expression levels of efflux pump genes. Next and third-generation sequencing were conducted to analyse the genomic features. Two colistin-heteroresistant isolates were detected from a multi-center carbapenem-resistant Enterobacterales (CRE) surveillance study in China, which exhibited similar survival rates as the K2 hypervirulent reference strain ATCC 43816 in a G. mellonella larvae model. The two isolates belonged to ST11, harbouring the iucABCD, iutA, iroBCD, and rpmA2 hypervirulent genes and pLVPK-like virulence plasmids. Colistin showed a weak effect on the heteroresistant strains in vitro. The efflux pump genes acrA, acrB, tolC, oqxA, and oqxB were upregulated in this subpopulation compared to the parental strains. This study showed the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae. AcrAB-TolC and OqxAB efflux overexpression were involved in mediating colistin heteroresistance.

In Vivo Production, Development and Storage of Oscheius myriophila (Nematoda: Rhabditida) in Galleria mellonella (Lepidoptera: Pyralidae).

Entomopathogenic nematodes have been used in biological control for some time and are an alternative for the control of insect pests, but during their implementation, situations have arisen that can be improved. These vary with each species and include their production and storage. Oscheius myriophila, an entomopathogenic nematode (EPN), was monitored for its performance when produced in vivo, as well as its development using Galleria mellonella larvae, using the MC5-2014 strain isolated from soil samples in the municipality of Tepalcingo, Morelos, México. For a study with native strains of EPNs, a wide range of tests must be conducted because the required conditions can be very specific. In vivo production was quantified at initial infective juvenile (IJ) inocula of 50, 100 and 500, and we obtained the same production for the three inocula. The life cycle of the EPNs lasted 12 days, and two generations were observed in which adults were found at days 5 and 9. Both evaluations were performed at a temperature of 27 °C in G. mellonella larvae. In addition, the temperatures of 8, 12, 20 and 24 °C were evaluated for their storage, and we observed that the EPNs can be kept for at least 6 months, maintaining a survival rate of 58.67% and a good infective capacity at a temperature of 12 °C, remaining above 60%.

A novel small-molecule compound S-342-3 effectively inhibits the biofilm formation of Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen in both community and hospital settings that often causes persistent and recurrent infections. The continuous emergence of multidrug-resistant strains and the lag in antibiotic development make the treatment of S. aureus more challenging. The biofilm formation of S. aureus is an important reason for persistent infection. In this study, we demonstrated that a small-molecule compound S-342-3 can effectively inhibit the biofilm formation of S. aureus. The crystal violet semi-quantitative assays revealed that at a sub-minimum inhibitory concentration of 4 µg/mL, S-342-3 reduced S. aureus biofilm mass by 57.43%, 52.14%, and 25.49%. Using confocal laser scanning microscopy, we observed that the biofilm was more incompact and less uniform upon the treatment of S-342-3. At a sub-inhibitory concentration (4 µg/mL), the S-342-3 can effectively reduce the production of polysaccharide intercellular adhesin (PIA) and suppress the initial adhesion of cells in the biofilm. Consistently, the results of RT-qPCR revealed that the expression of genes associated with biofilm formation was downregulated by S-342-3 (P < 0.05). However, we found that treatment with S-342-3 resulted in a significant decrease in the expression of global regulatory genes agrA and sarA (P < 0.05), which play a key role in promoting cell surface attachment and PIA production in S. aureus biofilms. Also importantly, we experimentally proved that the S-342-3 was not toxic to A549 alveolar epithelial cells and the Galleria mellonella larvae. Collectively, these results suggest that the S-342-3 may be a promising anti-biofilm drug candidate with excellent biosafety, which can be further explored for its practical application in health care. IMPORTANCE Biofilms are an important virulence factor in Staphylococcus aureus and are characterized by a structured microbial community consisting of bacterial cells and a secreted extracellular polymeric matrix. Inhibition of biofilm formation is an effective measure to control S. aureus infection. Here, we have synthesized a small molecule compound S-342-3, which exhibits potent inhibition of biofilm formation in both MRSA and MSSA. Further investigations revealed that S-342-3 exerts inhibitory effects on biofilm formation by reducing the production of polysaccharide intercellular adhesin and preventing bacterial adhesion. Our study has confirmed that the inhibitory effect of S-342-3 on biofilm is achieved by downregulating the expression of genes responsible for biofilm formation. In addition, S-342-3 is non-toxic to Galleria mellonella larvae and A549 cells. Consequently, this study demonstrates the efficacy of a biologically safe compound S-342-3 in inhibiting biofilm formation in S. aureus, thereby providing a promising antibiofilm agent for further research.

Evaluation of Galleria mellonella immune response as a key step toward plastic degradation

BackgroundPlastic's remarkable durability presents a significant challenge for our planet, leading to widespread environmental damage. However, some organisms, such as Galleria mellonella larvae, have shown a unique capability to consume and degrade plastic, offering potential solutions to plastic pollution. In this study, we investigated the response of G. mellonella larvae to different diets, including artificial diet (AD), polyethylene low density mixed with AD (PELD + AD), and PELD alone. Using various microscopy techniques, we examined the larvae's hemocyte hemogram and mid-gut characteristics to understand their immune response and digestive system when exposed to plastic.ResultsThe results revealed that PELD-only feeding negatively impacted hemocyte immunity, resulting in a significant decrease in total hemocyte counts compared to AD and AD + PELD feeding. Moreover, plastic consumption induced differential hemocyte alterations, affecting specific cell types. The presence of phagosomes in larval hemocytes and mid-gut cells during PELD-only feeding suggested active involvement in plastic breakdown.ConclusionsThese findings highlight the potential of G. mellonella larvae as a model organism to study responses to pollutants, emphasizing the urgent need to address plastic pollution's global threat. Further investigation is warranted to explore larval deformities, weight loss, and appetite changes, potentially influencing mortality rates and enzyme biochemistry. Understanding the impacts of plastic ingestion on G. mellonella larvae is crucial to develop effective strategies for mitigating plastic pollution's ecological implications.

Different Group A Streptococcus pili lead to varying proinflammatory cytokine responses and virulence.

The human pathogen Streptococcus pyogenes, or Group A Streptococcus (GAS), is associated with a variety of diseases ranging from mild skin and soft tissue infections to invasive diseases and immune sequelae such as rheumatic heart disease. We have recently reported that one of the virulence factors of this pathogen, the pilus, has inflammatory properties and strongly stimulates the innate immune system. Here we used a range of nonpathogenic Lactococcus lactis gain-of-function mutants, each expressing one of the major pilus types of GAS, to compare the immune responses generated by various types of fully assembled pili. In vitro assays indicated variability in the inflammatory response induced by different pili, with the fibronectin-binding, collagen-binding, T antigen (FCT)-1-type pilus from GAS serotype M6/T6 inducing significantly stronger cytokine secretion than other pili. Furthermore, we established that the same trend of pili-mediated immune response could be modeled in Galleria mellonella larvae, which possess a similar innate immune system to vertebrates. Counterintuitively, across the panel of pili types examined in this study, we observed a negative correlation between the intensity of the immune response demonstrated in our experiments and the disease severity observed clinically in the GAS strains associated with each pilus type. This observation suggests that pili-mediated inflammation is more likely to promote bacterial clearance instead of causing disruptive damages that intensify pathogenesis. This also indicates that pili may not be the main contributor to the inflammatory symptoms seen in GAS diseases. Rather, the immune-potentiating properties of the pilus components could potentially be exploited as a vaccine adjuvant.

Hyphae of Rhizopus arrhizus and Lichtheimia corymbifera Are More Virulent and Resistant to Antifungal Agents Than Sporangiospores In Vitro and in Galleria mellonella.

Mucorales species cause debilitating, life-threatening sinopulmonary diseases in immunocompromised patients and penetrating wounds in trauma victims. Common antifungal agents against mucormycosis have significant toxicity and are often ineffective. To evaluate treatments against mucormycosis, sporangiospores are typically used for in vitro assays and in pre-clinical animal models of pulmonary infections. However, in clinical cases of wound mucormycosis caused by traumatic inoculation, hyphal elements found in soil are likely the form of the inoculated organism. In this study, Galleria mellonella larvae were infected with either sporangiospores or hyphae of Rhizopus arrhizus and Lichtheimia corymbifera. Hyphal infections resulted in greater and more rapid larval lethality than sporangiospores, with an approximate 10-16-fold decrease in LD50 of hyphae for R. arrhizus (p = 0.03) and L. corymbifera (p = 0.001). Liposomal amphotericin B, 10 mg/kg, was ineffective against hyphal infection, while the same dosage was effective against infections produced by sporangiospores. Furthermore, in vitro, antifungal susceptibility studies show that minimum inhibitory concentrations of several antifungal agents against hyphae were higher when compared to those of sporangiospores. These findings support using hyphal elements of Mucorales species for virulence testing and antifungal drug screening in vitro and in G. mellonella for studies of wound mucormycosis.

Plastic degradation by insect hexamerins: Near-atomic resolution structures of the polyethylene-degrading proteins from the wax worm saliva.

Plastic waste management is a pressing ecological, social, and economic challenge. The saliva of the lepidopteran Galleria mellonella larvae is capable of oxidizing and depolymerizing polyethylene in hours at room temperature. Here, we analyze by cryo-electron microscopy (cryo-EM) G. mellonella's saliva directly from the native source. The three-dimensional reconstructions reveal that the buccal secretion is mainly composed of four hexamerins belonging to the hemocyanin/phenoloxidase family, renamed Demetra, Cibeles, Ceres, and a previously unidentified factor termed Cora. Functional assays show that this factor, as its counterparts Demetra and Ceres, is also able to oxidize and degrade polyethylene. The cryo-EM data and the x-ray analysis from purified fractions show that they self-assemble primarily into three macromolecular complexes with striking structural differences that likely modulate their activity. Overall, these results establish the ground to further explore the hexamerins' functionalities, their role in vivo, and their eventual biotechnological application.