Expression cloning and characterization of a pupal cuticle protein cDNA of Galleria mellonella L

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

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Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella

European Journal of Biochemistry ◽

10.1046/j.1432-1327.2000.01207.x ◽

2000 ◽

Vol 267 (7) ◽

pp. 2046-2053 ◽

Cited By ~ 60

Author(s):

Andreas C. Fröbius ◽

Michael R Kanost ◽

Peter Götz ◽

Andreas Vilcinskas

Keyword(s):

Serine Protease ◽

Protease Inhibitors ◽

Galleria Mellonella ◽

Serine Protease Inhibitors ◽

Isolation And Characterization ◽

Greater Wax Moth ◽

Larval Hemolymph ◽

Wax Moth

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Isolation and partial characterization of the haemolymph lipoproteins of the wax moth, Galleria mellonella

Insect Biochemistry ◽

10.1016/0020-1790(79)90053-2 ◽

1979 ◽

Vol 9 (2) ◽

pp. 211-219 ◽

Cited By ~ 15

Author(s):

Kurian K. Thomas

Keyword(s):

Galleria Mellonella ◽

Partial Characterization ◽

Wax Moth

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Purification and characterization of epidermis-origin hemolymph protein in Galleria mellonella

Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology ◽

10.1016/s1095-6433(99)00164-6 ◽

2000 ◽

Vol 125 (1) ◽

pp. 95-104

Author(s):

Yong-Ho Lee ◽

Haeng-Yeun Lee ◽

Hak R. Kim

Keyword(s):

Galleria Mellonella ◽

Purification And Characterization ◽

Hemolymph Protein

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Characterization of RelA in Acinetobacter baumannii

Journal of Bacteriology ◽

10.1128/jb.00045-20 ◽

2020 ◽

Vol 202 (12) ◽

Cited By ~ 3

Author(s):

María Pérez-Varela ◽

Aimee R. P. Tierney ◽

Ju-Sim Kim ◽

Andrés Vázquez-Torres ◽

Philip Rather

Keyword(s):

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Multidrug Resistant ◽

Cell Physiology ◽

Content Type ◽

Content Model ◽

Transposon Insertion ◽

Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

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Candida guilliermondii Complex Is Characterized by High Antifungal Resistance but Low Mortality in 22 Cases of Candidemia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00099-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 13

Author(s):

Laura Judith Marcos-Zambrano ◽

Mireia Puig-Asensio ◽

Felipe Pérez-García ◽

Pilar Escribano ◽

Carlos Sánchez-Carrillo ◽

...

Keyword(s):

Metabolic Activity ◽

Biofilm Formation ◽

Galleria Mellonella ◽

Sensu Stricto ◽

Candida Guilliermondii ◽

Molecular Techniques ◽

Content Type ◽

Content Model ◽

Crystal Violet Assay

ABSTRACT The objectives of our study were to describe the characteristics of patients with Candida guilliermondii candidemia and to perform an in-depth microbiological characterization of isolates and compare them with those of patients with C. albicans candidemia. We described the risk factors and outcomes of 22 patients with candidemia caused by the C. guilliermondii complex. Incident isolates were identified using molecular techniques, and susceptibility to fluconazole, anidulafungin, and micafungin was studied. Biofilm formation was measured using the crystal violet assay (biomass production) and the XTT reduction assay (metabolic activity), and virulence was studied using the Galleria mellonella model. Biofilm formation was compared with that observed for C. albicans. The main conditions predisposing to infection were malignancy (68%), immunosuppressive therapy (59%), and neutropenia (18%). Clinical presentation of candidemia was less severe in patients infected by the C. guilliermondii complex than in patients infected by C. albicans, and 30-day mortality was lower in C. guilliermondii patients (13.6% versus 33.9%, respectively; P = 0.049). Isolates were identified as C. guilliermondii sensu stricto (n = 17) and Candida fermentati (n = 5). The isolates produced biofilms with low metabolic activity and moderate biomass. The G. mellonella model showed that C. guilliermondii was less virulent than C. albicans (mean of 6 days versus 1 day of survival, respectively; P < 0.001). Patients with candidemia caused by the C. guilliermondii complex had severe and debilitating underlying conditions. Overall, the isolates showed diminished susceptibility to fluconazole and echinocandins, although poor biofilm formation and the low virulence were associated with a favorable outcome.

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