Recombinant Paracoccidioides brasiliensis Altered inheritance of mitochondria protein 31, mitochondrial (AIM31)

What is Paracoccidioides brasiliensis and what is its clinical significance?

Paracoccidioides brasiliensis is a dimorphic fungal pathogen that causes paracoccidioidomycosis (PCM), one of the most prevalent systemic mycoses in Latin America. It is a soil fungus that undergoes a temperature-dependent dimorphic switch following host inhalation. Approximately 10 million people are infected across South America, with Brazil showing an annual incidence rate of 10-30 infections per million inhabitants and a mortality rate of 1.4 per million per year . PCM primarily presents as a granulomatous pulmonary infection, although disseminated forms affecting the reticuloendothelial system, skin, mucous membranes, and lymph nodes are commonly observed in progressive disease .

What is AIM31 and what is its general function in fungal mitochondria?

AIM31 (Altered Inheritance of Mitochondria protein 31) is a mitochondrial protein involved in maintaining mitochondrial integrity and function. While specific research on P. brasiliensis AIM31 is limited, mitochondrial proteins in pathogenic fungi often play crucial roles in energy metabolism, cellular respiration, adaptation to host environments, and virulence. The protein has been identified in P. brasiliensis genomic analyses and can be produced as a recombinant protein with a histidine tag in E. coli expression systems .

How does P. brasiliensis AIM31 compare to homologous proteins in other pathogenic fungi?

AIM31 appears to be conserved across various fungal species including Neosartorya fumigata (another pathogenic fungus) . While the search results don't provide direct comparative analyses, researchers investigating this protein would typically examine sequence homology, structural similarities, and functional conservation across different fungal species. Understanding these comparisons could provide insights into the evolutionary significance of this protein and its potential as a pan-fungal therapeutic target.

What are the optimal conditions for recombinant expression of P. brasiliensis AIM31?

Based on available data, recombinant full-length P. brasiliensis AIM31 (amino acids 1-144) can be successfully expressed in E. coli with an N-terminal His tag . For optimal expression, researchers should consider the following methodological approaches:

• Vector selection: A bacterial expression vector containing a strong inducible promoter (T7 or tac)

• E. coli strain: BL21(DE3) or Rosetta for enhanced expression of eukaryotic proteins

• Induction conditions: Typically IPTG at 0.5-1mM, at lower temperatures (16-25°C) to enhance proper folding

• Lysis and purification: Native conditions using nickel affinity chromatography leveraging the His-tag

Researchers should optimize these parameters through small-scale expression trials before scaling up production.

What challenges are commonly encountered when working with recombinant mitochondrial proteins from pathogenic fungi?

When working with recombinant mitochondrial proteins like AIM31 from pathogenic fungi, researchers frequently encounter several technical challenges:

• Protein solubility issues due to the hydrophobic nature of many mitochondrial proteins

• Improper folding in bacterial expression systems lacking eukaryotic chaperones

• Lack of post-translational modifications that may be essential for function

• Potential toxicity to the host expression system

• Difficulties in replicating the native mitochondrial environment for functional studies

To address these challenges, researchers may need to explore alternative expression systems, fusion partners to enhance solubility, or refolding protocols for proteins expressed in inclusion bodies.

How does AIM31 contribute to P. brasiliensis pathogenicity and virulence?

While the specific role of AIM31 in P. brasiliensis pathogenicity is not directly addressed in the search results, mitochondrial proteins generally play critical roles in fungal virulence through:

• Energy production required for host colonization and dissemination

• Adaptation to varying nutrient availability within host microenvironments

• Resistance to oxidative stress generated by host immune responses

• Contribution to morphological transitions (particularly relevant for dimorphic fungi like P. brasiliensis)

Research methodologies to investigate these aspects would include gene knockout/knockdown studies, virulence assays in animal models, and transcriptomic analyses comparing expression under different conditions.

What role might AIM31 play in the dimorphic transition of P. brasiliensis?

P. brasiliensis undergoes a temperature-dependent dimorphic switch following host inhalation . Mitochondrial proteins like AIM31 may contribute to this transition through:

• Metabolic reprogramming required for different morphological states

• Energy provision for the cellular remodeling process

• Regulation of cellular responses to temperature changes

Researchers investigating this question would typically employ temperature shift experiments, comparing AIM31 expression and localization between mycelial and yeast forms, potentially utilizing fluorescently tagged AIM31 constructs for visualization.

How does P. brasiliensis interact with host immune receptors, and could AIM31 play a role in this process?

The search results provide insights into how P. brasiliensis components interact with host immunity. Paracoccin (PCN), another P. brasiliensis protein, has been shown to interact with Toll-like receptors (TLRs), specifically TLR2 and TLR4 . This interaction depends on carbohydrate recognition and is affected by mutation of the receptor's N-glycosylation sites .

While not directly established for AIM31, researchers investigating its potential immunomodulatory role should consider:

• Whether AIM31 is exposed to the host immune system during infection

• If AIM31 contains carbohydrate-binding domains similar to paracoccin

• The protein's potential interaction with pattern recognition receptors

• Its ability to stimulate cytokine production by immune cells

Experimental approaches would include receptor binding assays, cytokine profiling of immune cells exposed to recombinant AIM31, and potentially in vivo studies with AIM31-deficient strains.

Could recombinant AIM31 have therapeutic potential against paracoccidioidomycosis, similar to recombinant paracoccin?

The search results describe significant therapeutic effects of recombinant paracoccin (rPCN) in experimental PCM . By analogy, researchers might investigate whether recombinant AIM31 has similar potential through:

• Assessment of rAIM31 administration in murine models of PCM

• Analysis of granuloma formation and fungal clearance in treated vs. untreated mice

• Measurement of cytokine profiles to determine immunomodulatory effects

• Evaluation of different administration regimens (timing, dosage, route)

Based on the rPCN studies, an effective therapeutic protein would ideally promote a balanced Th1 immune response characterized by increased levels of IL-12, IFN-γ, TNF-α, and appropriate levels of IL-10 .

What methodologies are most effective for studying the subcellular localization and trafficking of AIM31 in P. brasiliensis?

For investigating the subcellular localization and trafficking of AIM31 in P. brasiliensis, researchers should consider the following methodological approaches:

• Fluorescent protein tagging: Creating AIM31-GFP fusion constructs for live-cell imaging

• Immunoelectron microscopy: Using gold-labeled antibodies against AIM31 for precise subcellular localization

• Subcellular fractionation: Isolating mitochondria and other organelles followed by Western blotting

• Import assays: In vitro assays using isolated mitochondria to study the import machinery involved in AIM31 localization

Each of these approaches offers distinct advantages and limitations, and combining multiple methods would provide the most comprehensive understanding of AIM31 localization.

How can researchers effectively design gene knockout or knockdown experiments for AIM31 in P. brasiliensis?

Genetic manipulation of P. brasiliensis presents technical challenges due to its complex life cycle and limited genetic tools. For AIM31 functional studies, researchers should consider:

For gene knockout:

• Homologous recombination-based approaches using selectable markers

• CRISPR-Cas9 system adapted for fungal systems

• Careful design of targeting constructs with sufficient homology arms

For gene knockdown:

• RNA interference using siRNA or shRNA constructs

• Antisense oligonucleotides

• Regulatable promoter systems for conditional expression

The experimental design should include appropriate controls and verification of knockout/knockdown efficiency through RT-qPCR, Western blotting, and phenotypic assays.

How do host hormonal factors affect P. brasiliensis infection, and could they influence AIM31 expression or function?

Hormonal factors significantly influence P. brasiliensis infection. Research has demonstrated that 17β-estradiol plays a protective role in females, contributing to their decreased susceptibility to clinical paracoccidioidomycosis . Experimental data shows that:

• After pulmonary infection with conidia, normal male mice showed progressive infection, while normal female mice restricted fungal proliferation

• Castrated male mice reconstituted with 17β-estradiol initially restricted proliferation

• Castrated female mice reconstituted with testosterone were unable to restrict disease progression

Researchers investigating potential links between hormonal status and AIM31 could explore:

• Whether 17β-estradiol affects AIM31 expression levels

• If hormonal factors alter AIM31 subcellular localization or activity

• Whether mitochondrial function in P. brasiliensis is broadly affected by host hormones

What is the role of AIM31 in P. brasiliensis adaptation to different host microenvironments?

Pathogenic fungi must adapt to diverse microenvironments within the host. While specific information on AIM31's role in this adaptation is not provided in the search results, researchers investigating this question should consider:

• Comparative expression analysis of AIM31 under conditions mimicking different host niches (varying pH, nutrient availability, oxygen levels)

• Metabolic profiling of wild-type versus AIM31-deficient strains under different conditions

• Stress response assays examining the susceptibility of AIM31 mutants to various stressors encountered in vivo

• In vivo tracking of infection progression using bioluminescent imaging to identify tissue-specific requirements for AIM31

Such studies would provide insights into whether AIM31 functions as a general housekeeping protein or plays specialized roles in adaptation to specific host environments.

How can structural biology approaches enhance our understanding of AIM31 function in P. brasiliensis?

Structural biology approaches would provide valuable insights into AIM31 function through:

• X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure

• NMR spectroscopy to analyze protein dynamics and interactions

• Molecular docking simulations to identify potential binding partners

• Structure-guided mutagenesis to validate functional domains

Researchers should focus on:

• Identification of conserved structural motifs shared with other fungal AIM31 proteins

• Analysis of potential interaction surfaces for protein-protein interactions

• Investigation of structural changes under different physiological conditions

What bioinformatic approaches can be used to predict functional partners of AIM31 in mitochondrial processes?

To predict functional partners of AIM31, researchers should employ the following bioinformatic approaches:

• Protein-protein interaction network analyses using databases like STRING

• Co-expression analyses across different conditions

• Phylogenetic profiling to identify proteins with similar evolutionary patterns

• Structural prediction of interaction domains

• Analysis of shared regulatory elements in the promoter regions of AIM31 and potential partners

These computational predictions should then be validated through experimental approaches such as co-immunoprecipitation, yeast two-hybrid assays, or proximity labeling techniques.