Recombinant Lucihormetica verrucosa Periviscerokinin-2
Description
Sequence and Molecular Properties
LucVe-PVK-2 is a recombinant protein engineered to replicate the native peptide isolated from L. verrucosa. Key structural features include:
| Parameter | Value | Source |
|---|---|---|
| Amino Acid Sequence | GSSGLISMPRV (1–11 aa) | |
| Molecular Weight | 1,103 Da | |
| Host Systems | E. coli, yeast, baculovirus, mammalian cells | |
| Purity | ≥85% (SDS-PAGE validated) |
The sequence aligns with cytoplasmic domain peptides, and its amidated C-terminus (Val-NH₂) is characteristic of bioactive neuropeptides .
Comparative Sequence Analysis
While LucVe-PVK-2 shares homology with periviscerokinin-2 from Periplaneta americana (Gly-Ser-Ser-Ser-Gly-Leu-Ile-Ser-Met-Pro-Arg-Val-NH₂) , it exhibits a truncated N-terminal region, likely due to recombinant expression optimization.
Myotropic Activity
Periviscerokinins are potent visceral muscle stimulants. LucVe-PVK-2 mediates:
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Contractile Activity: Direct stimulation of visceral muscles, critical for processes like digestion .
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Signal Transduction: Acts via G-protein-coupled receptors (GPCRs), triggering intracellular calcium release .
Species-Specific Adaptations
In L. verrucosa, the peptide’s bioactivity may correlate with the cockroach’s specialized digestive physiology, including its omnivorous diet .
Experimental Use Cases
Bioassay Validation
Native periviscerokinins exhibit nanomolar potency in hyperneural muscle assays . Recombinant LucVe-PVK-2 retains this bioactivity when expressed in heterologous systems .
Recombinant Expression Systems
LucVe-PVK-2 is commercially available in multiple formats:
| Host System | Form | Price Range | Purity | Endotoxin |
|---|---|---|---|---|
| E. coli | Lyophilized | $1,225 (0.05 mg) | ≥85% | Low (optional) |
| Yeast | Lyophilized | $1,255 (1 mg) | ≥85% | Low (optional) |
| Baculovirus | Lyophilized | $1,685 (0.5 mg) | ≥85% | Low (optional) |
Storage: -20°C or -80°C for extended use .
References
Product Specs
Target Background
Q&A
What is Phialophora verrucosa and what diseases does it cause?
Phialophora verrucosa is a fungal pathogen that causes several human diseases, most notably chromoblastomycosis. This chronic fungal infection of the skin and subcutaneous tissue is extremely difficult to treat and represents a significant clinical challenge . The infection typically enters through traumatic inoculation, with research showing the fungus can survive and proliferate inside macrophages, contributing to its pathogenicity.
What types of peptidases are secreted by Phialophora verrucosa?
Research has demonstrated that P. verrucosa secretes aspartic-type peptidases capable of cleaving albumin. These enzymes are sensitive to pepstatin A and certain HIV peptidase inhibitors (HIV-PIs), particularly lopinavir, ritonavir, and amprenavir . This represents the first demonstration that this fungus secretes aspartic-type peptidases, an important finding for understanding its virulence mechanisms.
How do peptidases contribute to fungal pathogenicity?
Peptidases serve as crucial virulence factors for fungal pathogens like P. verrucosa. These enzymes facilitate the acquisition of nutrients by degrading host proteins, enable tissue invasion, and can disrupt host defense mechanisms by degrading immune proteins . Research indicates that aspartic peptidases may also be involved in disrupting host cell defense mechanisms, affecting the integrity of important host proteins and other physiological processes essential for pathogen survival.
How do different HIV peptidase inhibitors affect the enzymatic activity of P. verrucosa?
HIV-PIs demonstrate varying inhibitory effects on the aspartic peptidase activity of P. verrucosa. Lopinavir, ritonavir, and amprenavir are the most effective, inhibiting enzymatic activity by approximately 75% . Atazanavir, indinavir, and saquinavir show moderate inhibition (40-50%), while nelfinavir does not significantly inhibit the peptidase activity under the experimental conditions used . These findings suggest biochemical differences between the aspartic peptidases of P. verrucosa and those of other fungal pathogens like Fonsecaea pedrosoi, which show different sensitivity patterns to these inhibitors.
What are the antifungal effects of HIV-PIs against P. verrucosa growth?
Among the HIV-PIs tested, only lopinavir, nelfinavir, and ritonavir demonstrated significant antifungal activity against P. verrucosa, reducing fungal growth by approximately 40%, 55%, and 60% at 400 μM, respectively . Ritonavir showed dose-dependent inhibition with growth reductions of 60%, 45%, and 40% at concentrations of 400, 200, and 100 μM, respectively, with an IC50 of 141.42 μM . This inhibitory profile differs from that observed with F. pedrosoi, where saquinavir and nelfinavir at 100 μM inhibited growth by around 90%, while they had minimal effect on P. verrucosa at this concentration.
What ultrastructural changes occur in P. verrucosa after treatment with HIV-PIs?
Scanning electron microscopy (SEM) revealed that HIV-PI treatment causes dramatic ultrastructural alterations in P. verrucosa cells compared to untreated controls, which maintain typical spherical-to-oval morphology . Treated cells exhibit various morphological changes, including surface invaginations, surface deposits, and cell disruption, which are indicative of cell death . Similar ultrastructural alterations have been observed in other fungi like F. pedrosoi and Candida albicans after HIV-PI treatment, suggesting common mechanisms of cellular damage.
How do HIV-PIs affect the interaction between P. verrucosa and macrophages?
HIV-PIs significantly impair the interaction between P. verrucosa conidia and macrophages. At non-cytotoxic concentrations, ritonavir (25 μM) reduced conidia-macrophage adhesion by approximately 60%, while lopinavir (100 μM) decreased adhesion by about 50% . A combination of lopinavir (50 μM) plus ritonavir (12.5 μM) reduced the adhesion index by approximately 40% . Furthermore, conidia treated with lopinavir (100 μM) and ritonavir (25 μM) showed increased susceptibility to macrophage killing, with reductions in intracellular conidial viability of about 85% and 70%, respectively . Even at subinhibitory concentrations, the combination of these HIV-PIs reduced P. verrucosa viability by approximately 60% during macrophage interaction .
How is the aspartic peptidase activity of P. verrucosa measured experimentally?
Based on the research methodology, P. verrucosa aspartic peptidase activity can be assessed using albumin as a substrate . Typically, fungal cells are grown in appropriate medium, and the peptidase activity is measured in cell-free culture supernatant. The enzymatic reaction occurs in a suitable buffer containing the substrate, with reaction products analyzed to quantify peptidase activity. The enzyme's specificity is determined using inhibitors like pepstatin A (specific for aspartic peptidases) and various HIV-PIs . Inhibitory effects are calculated as a percentage reduction compared to control activity (without inhibitors).
What experimental approaches are used to study the effects of HIV-PIs on fungal-macrophage interactions?
Researchers employ several techniques to study these interactions:
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Adhesion assays: Viable fungal cells are incubated with macrophages (e.g., THP-1 cells) at a specific ratio (5:1 fungi:macrophage) for 1 hour, followed by removal of non-associated fungi. The systems are then incubated with HIV-PIs or control media for 20 hours, fixed with Bouin's solution, and stained with Giemsa . The adhesion index is calculated by multiplying the mean number of attached fungi per macrophage by the percentage of infected macrophages.
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Killing assays: After the adhesion phase and treatment with HIV-PIs, macrophages are lysed with sterile cold water, and the suspensions are plated onto appropriate media to determine the number of viable fungal cells (CFU) . The killing efficiency is assessed by comparing the number of viable fungi in treated versus untreated systems.
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Cytotoxicity assays: The effects of HIV-PIs on macrophage viability are assessed to ensure that observed effects on fungal-macrophage interactions are not due to macrophage toxicity .
How can researchers evaluate the synergistic effects between HIV-PIs and conventional antifungal drugs?
Researchers evaluate potential synergistic effects through combination experiments using subinhibitory concentrations of both HIV-PIs and conventional antifungal agents . Fungal growth is measured in the presence of each agent alone and in combination. A beneficial combinatory effect is indicated when the combination produces significantly greater growth inhibition than either agent alone. In the study with P. verrucosa, ritonavir combined with either ketoconazole or itraconazole showed this synergistic effect, inhibiting fungal growth by 40% and 60% respectively, while subinhibitory concentrations of each agent alone had minimal effects . This approach helps identify promising drug combinations for potential therapeutic applications.
Inhibition of P. verrucosa Aspartic Peptidase Activity by HIV-PIs
| HIV Peptidase Inhibitor | Inhibition Percentage |
|---|---|
| Lopinavir | ~75% |
| Ritonavir | ~75% |
| Amprenavir | ~75% |
| Atazanavir | 40-50% |
| Indinavir | 40-50% |
| Saquinavir | 40-50% |
| Nelfinavir | Not significant |
Data derived from the research article .
Effects of HIV-PIs on P. verrucosa Growth at 400 μM
| HIV Peptidase Inhibitor | Growth Inhibition Percentage |
|---|---|
| Lopinavir | ~40% |
| Nelfinavir | ~55% |
| Ritonavir | ~60% |
| Other HIV-PIs | Not significant |
Data derived from the research article .
Dose-Dependent Inhibition of P. verrucosa Growth by Ritonavir
| Ritonavir Concentration | Growth Inhibition Percentage |
|---|---|
| 400 μM | ~60% |
| 200 μM | ~45% |
| 100 μM | ~40% |
IC50 = 141.42 μM. Data derived from the research article .
Effects of HIV-PIs on P. verrucosa-Macrophage Interaction
| Treatment | Adhesion Inhibition | Intracellular Conidial Viability Reduction |
|---|---|---|
| Lopinavir (100 μM) | ~50% | ~85% |
| Ritonavir (25 μM) | ~60% | ~70% |
| Lopinavir (50 μM) + Ritonavir (12.5 μM) | ~40% | ~60% |
Data derived from the research article .
Synergistic Effects of Ritonavir with Conventional Antifungal Agents
| Combination at Subinhibitory Concentrations | Growth Inhibition Percentage |
|---|---|
| Ritonavir + Ketoconazole | ~40% |
| Ritonavir + Itraconazole | ~60% |
| Ritonavir + Amphotericin B | Not significant |
| Ritonavir + Terbinafine | Not significant |
Data derived from the research article .
What are the molecular mechanisms underlying the antifungal activity of HIV-PIs against P. verrucosa?
While research has established that HIV-PIs inhibit aspartic peptidase activity and fungal growth, the exact molecular mechanisms remain to be fully elucidated. Future studies should investigate the specific molecular targets of HIV-PIs in P. verrucosa and determine whether the antifungal effects result solely from inhibition of aspartic peptidases or if other cellular processes are also affected . Structure-activity relationship studies of HIV-PIs and their interactions with fungal aspartic peptidases would provide valuable insights for developing more specific inhibitors with enhanced antifungal activity.
How might HIV-PIs be optimized for chromoblastomycosis treatment?
Current HIV-PIs were designed primarily to target HIV protease, not fungal aspartic peptidases. Future research should focus on modifying these compounds to enhance their specificity for fungal targets, potentially leading to increased antifungal efficacy at lower concentrations with reduced toxicity . Additionally, pharmacokinetic and pharmacodynamic studies of HIV-PIs in the context of fungal infections would be valuable for optimizing dosing regimens in potential clinical applications against chromoblastomycosis and other fungal diseases.
What is the translational potential of drug repositioning HIV-PIs for fungal infection treatment?
Given the challenges in treating chromoblastomycosis and the promising results with HIV-PIs, drug repositioning represents an attractive approach . HIV-PIs are already approved for clinical use, which could expedite their application in antifungal therapy. Future research should explore the efficacy of HIV-PIs against a broader range of chromoblastomycosis-causing fungi and investigate their effectiveness in animal models of infection . Clinical trials would ultimately be needed to establish the safety and efficacy of HIV-PIs as antifungal agents for human fungal infections that are currently difficult to treat with conventional therapies.
