Recombinant Vibrio vulnificus Purine nucleoside phosphorylase DeoD-type 1 (deoD1)

What is the biochemical function of Vibrio vulnificus Purine nucleoside phosphorylase (deoD1)?

Vibrio vulnificus deoD1 functions as a purine nucleoside phosphorylase that catalyzes the reversible phosphorolysis of purine nucleosides to their corresponding free bases and pentose-1-phosphate. Similar to other characterized PNPs, including the well-studied C. elegans ortholog pnp-1, the enzyme likely converts purine nucleosides like inosine into free bases such as hypoxanthine . The enzyme plays a critical role in the purine salvage pathway, which allows bacteria to recover and reuse nucleosides rather than synthesizing them de novo, providing a metabolic advantage, particularly in nutrient-limited environments.

Metabolomic studies of PNP-deficient organisms reveal characteristic signatures of metabolite accumulation, specifically increased levels of nucleosides (inosine) and decreased levels of free bases (hypoxanthine) . The enzyme likely follows similar kinetics to other characterized bacterial PNPs, with one unit defined as the amount of enzyme that converts 1 μmol of substrate and 1 μmol of phosphate into 1 μmol of product per minute under standard conditions (25°C, pH 7.6) .

How is recombinant V. vulnificus deoD1 typically expressed and purified for research purposes?

While specific protocols for V. vulnificus deoD1 are not detailed in the search results, recombinant expression would typically follow established methods for bacterial PNPs:

• Gene Cloning and Vector Construction: The deoD1 gene would be PCR-amplified from V. vulnificus genomic DNA and cloned into an expression vector (typically pET series) with a suitable affinity tag (His6, GST).

• Expression System: Transformation into an E. coli expression strain (BL21(DE3) or derivatives) is standard practice for bacterial proteins .

• Culture Conditions: Expression typically involves growth at 37°C to mid-log phase followed by induction with IPTG (0.5-1 mM) and further incubation at lower temperatures (16-25°C) to enhance soluble protein yield.

• Purification Process:

  • Affinity chromatography (Ni-NTA for His-tagged proteins)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Buffer Composition: Based on human PNP studies, a suitable buffer would likely be 50 mM Tris-HCl, pH 7.6 .

The purified protein should be assessed for purity via SDS-PAGE and show a specific activity of ≥25 units/mg protein, similar to that reported for human PNP .

What assays are commonly used to measure deoD1 enzymatic activity?

Several complementary methods can be employed to assess deoD1 enzymatic activity:

• Spectrophotometric Assays:

  • The most direct approach involves monitoring the spectral shift that occurs when nucleosides are converted to bases (e.g., inosine to hypoxanthine shows absorbance changes at 290 nm).

  • MESG (2-amino-6-mercapto-7-methylpurine riboside) assay: This substrate releases 2-amino-6-mercapto-7-methylpurine upon phosphorolysis, which can be monitored at 360 nm .

• HPLC-Based Analysis:

  • Reaction products and substrates can be quantified by HPLC, offering high sensitivity and specificity.

  • This method is particularly useful for determining substrate specificity profiles.

• Coupled Enzymatic Assays:

  • PNP activity can be coupled to xanthine oxidase when using inosine/guanosine as substrates, with H2O2 production measured fluorometrically.

• Metabolomic Analysis:

  • LC-MS metabolomics can be used to quantify inosine and hypoxanthine levels, as demonstrated in the C. elegans pnp-1 study .

An example protocol would involve incubating purified enzyme (1-10 μg) with inosine (1 mM) and phosphate buffer (50 mM) at 25°C, pH 7.6, then quantifying reaction products at predetermined timepoints.

What role does deoD1 play in V. vulnificus pathogenesis?

While the specific role of deoD1 in V. vulnificus pathogenesis is not directly addressed in the search results, several lines of evidence suggest potential contributions to virulence:

• Purine Metabolism and Pathogen Survival: Purine salvage pathways are critical for bacterial survival in host environments where purines may be limited. The search results indicate that in C. elegans, mutations in pnp-1 (PNP ortholog) led to enhanced resistance to both intracellular and extracellular pathogens , suggesting that purine metabolism enzymes like deoD1 may be important for pathogen survival.

• Potential Virulence Factor: Although deoD1 is not specifically mentioned among the well-characterized V. vulnificus virulence factors (such as capsule polysaccharide, MARTX toxin, or RtxA1) , other studies have identified purine metabolism genes (like purH) as being important in V. vulnificus pathogenicity .

• Relation to Host Immune Response: PNP activity could potentially modulate host immune responses. In humans, PNP deficiency leads to severe immunodeficiency , suggesting that bacterial PNPs might interact with host purine metabolism in ways that affect immunity.

The search results identify several confirmed virulence factors in V. vulnificus, including:

• Capsule Polysaccharide (CPS) - primary virulence factor

• RtxA1 toxin - plays a significant role in cytotoxicity

• vcgC - virulence-correlated gene

• vvhA - hemolysin gene that plays an additive role in pathogenesis

While deoD1 is not specifically mentioned among these factors, its potential role in bacterial survival during infection warrants further investigation.

How do mutations in deoD1 affect V. vulnificus virulence in animal models?

• Inference from Other Purine Metabolism Genes: Studies have shown that mutations in purH (another purine metabolism gene) altered the lethality and cytotoxic activity of V. vulnificus in mice , suggesting that disruption of purine metabolism pathways, potentially including deoD1, could affect virulence.

• Insights from C. elegans Models: Research on the C. elegans pnp-1 ortholog showed that pnp-1 mutants displayed increased resistance to both intracellular and extracellular pathogens . This suggests that a functional host PNP might somehow benefit pathogens, raising the possibility that bacterial PNPs like deoD1 could contribute to pathogenesis.

• Methodological Approaches: To study deoD1's role in virulence, researchers could:

  • Generate isogenic deoD1 deletion mutants in V. vulnificus

  • Compare wild-type and mutant strains in mouse infection models (both intraperitoneal and oral infection routes)

  • Assess bacterial loads in tissues, cytotoxicity, and mouse survival rates

  • Examine effects on specific virulence mechanisms like biofilm formation or toxin production

It's worth noting that the importance of specific genes in virulence often depends on the infection model. For example, the search results indicate that some V. vulnificus genes (vvn and smcR) were not required for virulence in mice despite their suspected roles in pathogenesis .

Can deoD1 be utilized in gene-directed enzyme prodrug therapy for cancer treatment?

While not specific to V. vulnificus deoD1, the search results provide compelling evidence that bacterial PNPs can be effectively used in gene-directed enzyme prodrug therapy (GDEPT) for cancer treatment:

• Clinical Trial Evidence: A phase I clinical trial (NCT01310179) demonstrated that Escherichia coli PNP could be safely used in combination with fludarabine for treating solid tumors . This approach involves:

  • Intratumoral injection of an adenoviral vector expressing E. coli PNP

  • Systemic administration of fludarabine (a prodrug)

  • Local conversion of fludarabine to fluoroadenine within tumor tissues

• Clinical Outcomes: The trial showed dose-dependent responses with significant tumor regression in higher-dose cohorts (5 of 6 patients in cohorts 3 and 4) without dose-limiting toxicity .

• Applications for V. vulnificus deoD1: Given the broader substrate specificity of bacterial PNPs compared to mammalian enzymes, V. vulnificus deoD1 could potentially be developed for similar applications. Research directions could include:

  • Comparing substrate specificity and catalytic efficiency of deoD1 with E. coli PNP

  • Testing deoD1's ability to convert clinically relevant prodrugs

  • Evaluating immunogenicity of V. vulnificus deoD1 versus other bacterial PNPs

The results from E. coli PNP trials suggest this approach "found that localized generation of fluoroadenine within tumor tissues using E. coli PNP and fludarabine is safe and effective" , providing a foundation for similar applications with V. vulnificus deoD1.

What genetic tools are available for studying deoD1 function in V. vulnificus?

While the search results don't specifically describe genetic tools for deoD1 manipulation, they provide insights into approaches used for genetic analysis of V. vulnificus:

• Genomic Analysis Approaches:

  • Genome-Wide Association Studies (GWAS) have been used to identify genetic variants associated with virulence in V. vulnificus

  • Tools like Pyseer can identify variants associated with clinical phenotypes based on SNPs, insertion/deletion of accessory genes, and k-mers

  • DBGWAS has been used to test associations between k-mers and clinical vs. environmental phenotypes

• Mutation Generation Techniques:

  • Random chromosomal mutagenesis has been conducted to identify virulence factors in V. vulnificus

  • Isogenic mutants have been generated to study specific genes like rtxA1

  • Complementation studies can confirm phenotypes (e.g., "mutation in hlyU resulted in decreased rtxA1 expression" )

• Gene Expression Analysis:

  • RT-PCR has been used to identify toxin mRNA transcripts in vivo

  • Enzyme-linked immunosorbent assay (ELISA) has been employed to detect protein expression

• Typing Systems for Strain Characterization:

  • Random amplified polymorphic DNA PCR

  • 16S rRNA sequence typing

  • Intergenic spacer region typing between 16S and 23S rRNA genes

  • PFGE (Pulse-field gel electrophoresis) patterns have been used to compare banding profiles of different V. vulnificus strains

For specific deoD1 studies, researchers could employ:

• CRISPR-Cas9 for precise gene editing

• Allelic exchange for gene deletion or modification

• Complementation with wild-type or mutant alleles expressed from plasmids

• Reporter gene fusions to study expression patterns

What is known about the regulation of deoD1 expression in V. vulnificus?

While specific information on deoD1 regulation in V. vulnificus is not provided in the search results, insights can be drawn from regulatory patterns of other virulence-associated genes:

• Potential Regulatory Mechanisms:

  • Quorum sensing may affect expression, as seen with the hemolysin vvhA gene which showed "decreased levels of mRNA during swarming and upon loss of the AI-2 quorum sensing system"

  • HlyU protein has been shown to regulate expression of virulence factors like rtxA1 by binding to upstream regions and initiating transcription . Similar transcription factors might regulate deoD1.

  • Iron concentration likely affects expression, as V. vulnificus virulence factors are often regulated by iron availability

• Environmental Influences:

  • Host cell contact might trigger expression changes, as seen with rtxA1 which showed "immediate induction of toxin expression when the bacterium encountered host cells"

  • Temperature fluctuations between marine environments and human hosts could serve as signals for expression regulation

  • Nutrient availability, particularly purine sources, would likely influence expression of purine metabolism genes including deoD1

• Analytical Approaches to Study Regulation:

  • Transcriptional reporter fusions (deoD1 promoter with gfp or lacZ)

  • qRT-PCR to measure expression under different conditions

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factor binding sites

  • RNA-seq to examine global expression patterns in response to environmental changes

How does iron concentration affect deoD1 expression and activity in V. vulnificus?

The search results don't specifically address deoD1 regulation by iron, but they highlight the importance of iron in V. vulnificus virulence:

• Iron and V. vulnificus Pathogenesis:

  • The search results indicate that "V. vulnificus... quickly reached a lethal concentration with enhanced cytotoxicity in the iron-overloaded mice"

  • High serum ferritin levels were identified as "independent and important predictors of survival of the organism in blood"

  • Patients with liver diseases (associated with high serum ferritin) were at elevated risk for V. vulnificus infection

• Potential Mechanisms of Iron Regulation:

  • Iron-responsive transcriptional regulators like Fur (ferric uptake regulator) might control deoD1 expression

  • Iron availability could indirectly affect deoD1 through global regulatory networks

  • Purine metabolism and iron utilization pathways may be coordinately regulated

• Experimental Approaches to Investigate Iron Regulation:

  • Compare deoD1 expression in iron-replete versus iron-limited conditions

  • Assess enzymatic activity with varying iron concentrations

  • Examine potential iron-binding motifs in the deoD1 protein sequence or promoter region

  • Study deoD1 expression in fur mutant backgrounds

Given that iron plays a crucial role in V. vulnificus pathogenesis and many virulence factors are iron-regulated, investigating deoD1's response to iron would be a logical research direction.