Recombinant Vibrio vulnificus Phosphate import ATP-binding protein PstB 2 (pstB2)

What is the role of Phosphate import ATP-binding protein PstB 2 in Vibrio vulnificus pathogenicity?

Phosphate import ATP-binding protein PstB 2 (pstB2) functions as a critical component of the phosphate-specific transport (Pst) system in Vibrio vulnificus, which is essential for bacterial phosphate homeostasis. While not directly characterized as a primary virulence factor like RtxA1 or VvhA (hemolysin), pstB2 plays an important role in bacterial survival within phosphate-limited environments such as human serum.

Research methodologically approaches this by:

• Comparing growth rates of wild-type and pstB2-deficient strains in phosphate-limited media

• Measuring phosphate uptake kinetics in various environmental conditions

• Conducting survival assays in human serum to determine the protein's contribution to pathogenicity

Evidence suggests that phosphate acquisition systems contribute to bacterial fitness during infection, as demonstrated in serum-based survival studies identifying essential genes required for V. vulnificus growth in human serum .

How is recombinant pstB2 typically produced for research applications?

The production of recombinant pstB2 typically employs a systematic expression protocol:

• Gene cloning: The coding sequence for pstB2 is amplified from V. vulnificus genomic DNA (commonly strain CMCP6) using PCR with specific primers containing appropriate restriction sites.

• Expression vector construction: The amplified gene is cloned into a prokaryotic expression vector (commonly pET-based systems) containing:

  • Inducible promoter (typically T7)

  • Affinity tag (6xHis-tag) for purification

  • Appropriate antibiotic resistance marker

• Expression system: The recombinant vector is transformed into E. coli BL21(DE3) or similar expression strains.

• Protein expression:

  • Culture growth at 37°C to mid-log phase (OD600 ≈ 0.6-0.8)

  • Induction with IPTG (typically 0.5-1.0 mM)

  • Post-induction growth at lower temperatures (16-25°C) for 4-16 hours

• Purification:

  • Cell lysis (sonication or pressure-based disruption)

  • Immobilized metal affinity chromatography (IMAC)

  • Size exclusion chromatography for higher purity

The purified protein typically achieves >85% purity as assessed by SDS-PAGE , making it suitable for functional and structural studies.

How can researchers assess the contribution of pstB2 to Vibrio vulnificus survival in human serum?

Methodologically rigorous assessment of pstB2's contribution to serum survival requires multi-faceted approaches:

• Genetic manipulation strategy:

  • Generation of clean deletion mutant (ΔpstB2) using allelic exchange

  • Complementation with wild-type gene on plasmid or chromosomally integrated

  • Construction of point mutants affecting key functional residues

  • Consider potential redundancy by creating double mutants with related genes

• Serum survival assay protocol:

  • Pre-culture bacteria in phosphate-limited media to induce Pst system

  • Standardize inoculum to specific CFU (typically 10³-10⁵ CFU/mL)

  • Expose to human serum (both normal and heat-inactivated) at 37°C

  • Sample at defined timepoints (0, 1, 2, 4, 8h)

  • Determine viable counts on selective media

  • Calculate survival ratio compared to heat-inactivated controls

• Complementary approaches:

  • Transcriptional profiling (RNA-seq) to identify compensatory mechanisms

  • Phosphate uptake kinetics using radiolabeled ³²P

  • Competition assays between wild-type and mutant strains

  • In vivo infection models (mouse) to validate serum survival findings

Sophisticated analysis requires integration of these data sets to distinguish between direct effects of pstB loss versus secondary metabolic consequences. Transposon insertion sequencing (TIS) studies in V. vulnificus have established experimental frameworks for such evaluations, identifying genes essential for serum survival .

What methodological approaches can resolve contradictory findings regarding pstB2 function in different Vibrio species?

Resolving contradictory findings requires systematic methodological approaches:

• Standardized experimental conditions:

  • Use defined minimal media with controlled phosphate concentrations

  • Standardize growth phase for all comparisons

  • Maintain identical temperature, pH, and salinity conditions

  • Employ multiple biological and technical replicates

• Comparative genomic and proteomic analyses:

  • Complete sequence alignment of pstB homologs across Vibrio species

  • Structural modeling to identify species-specific variations

  • Synteny analysis of pst operons to identify organizational differences

  • Phylogenetic analysis to correlate functional differences with evolutionary distance

• Functional complementation experiments:

  • Cross-species gene swap experiments (replacing native pstB with homologs)

  • Domain swap constructs to identify functional regions responsible for differences

  • Heterologous expression systems to test activity under identical cellular backgrounds

• Advanced biochemical characterization:

  • Compare kinetic parameters (Km, Vmax) for ATP hydrolysis

  • Assess phosphate transport efficiency using identical membrane vesicle preparations

  • Determine protein-protein interaction profiles with other Pst components

Research indicates that phosphate transport systems can differ in their regulation and functionality even between closely related bacteria. Genome-wide studies of V. vulnificus have identified distinct ecological adaptations , suggesting species-specific metabolic adaptations that may extend to phosphate acquisition systems.

How might pstB2 interact with virulence regulatory networks in Vibrio vulnificus under phosphate limitation?

Investigation of pstB2's interaction with virulence networks requires integrated methodological approaches:

• Transcriptional profiling strategy:

  • RNA-seq comparing wild-type and ΔpstB2 mutants under varying phosphate conditions

  • ChIP-seq to identify transcription factors responding to pstB2-dependent signals

  • Promoter-reporter fusion assays to validate transcriptional responses

• Regulatory network identification:

  • Two-component system analysis (particularly PhoR-PhoB)

  • Quantification of cyclic-AMP levels and CRP-dependent gene expression

  • Analysis of iron-responsive regulators (Fur) that intersect with phosphate regulation

  • Assessment of quorum sensing systems (LuxO, SmcR) known to regulate virulence

• Virulence factor expression measurement:

  • qRT-PCR quantification of key virulence genes (rtxA1, vvhA)

  • Western blot analysis of toxin production

  • Enzymatic activity assays for secreted virulence factors

  • Immunoblotting for capsular polysaccharide production

Evidence indicates complex regulatory networks in V. vulnificus that integrate multiple environmental signals. For example, CRP activates vvhBA transcription under glucose starvation , while iron can repress vvhA transcription via Fur . The status of phosphate acquisition likely integrates into these networks, with the PhoR-PhoB system potentially serving as a link between phosphate limitation and virulence regulation.

What role might pstB2 play in Vibrio vulnificus biofilm formation and environmental persistence?

A comprehensive research methodology for investigating pstB2's role in biofilm formation includes:

• Biofilm formation assay workflow:

  • Static microtiter plate assays with crystal violet staining

  • Flow cell systems with confocal microscopy for structure analysis

  • Quantification of extracellular polymeric substances (EPS)

  • Comparison between wild-type, ΔpstB2, and complemented strains

  • Testing under varying phosphate concentrations (0.1-2mM)

• Environmental persistence evaluation:

  • Survival in artificial seawater with controlled nutrient levels

  • Microcosm experiments simulating natural environmental conditions

  • Competition assays with environmental isolates

  • Long-term starvation survival assessment

• Molecular basis investigation:

  • Transcriptional profiling of biofilm-associated genes

  • Quantification of second messengers (c-di-GMP, (p)ppGpp)

  • Analysis of extracellular DNA and polysaccharide composition

  • Examination of cell surface hydrophobicity and adhesion properties

Research on V. vulnificus virulence factors suggests links between metabolic systems and biofilm formation. For instance, flagella components (flgK, flgL, flgE) identified in co-adaptation networks have been shown to affect biofilm formation . As phosphate limitation typically triggers biofilm formation in many bacteria, pstB2 likely plays a role in this environmental adaptation process.

How can researchers design an effective knockout strategy for pstB2 that minimizes polar effects on adjacent genes?

An optimal knockout strategy requires careful genetic manipulation approaches:

• In-frame deletion method:

  • Amplify ~1kb flanking regions upstream and downstream of pstB2

  • Fuse fragments with overlap extension PCR to maintain reading frame

  • Clone into suicide vector (e.g., pDM4 with sacB for counter-selection)

  • Perform two-step allelic exchange via homologous recombination

  • Screen for sucrose resistance and antibiotic sensitivity

  • Confirm deletion by PCR and sequencing of the junction region

• Marker-less deletion considerations:

  • Utilize FLP/FRT or Cre/loxP recombination systems

  • Design junction points that maintain translational coupling

  • Preserve ribosome binding sites for downstream genes

  • Avoid disrupting operon regulatory elements

• Validation of non-polar effects:

  • RT-PCR analysis of adjacent gene expression

  • Complementation with pstB2 alone to verify phenotype restoration

  • Transcriptional profiling to assess global effects

  • Growth rate comparison in various media

• Alternative approaches:

  • Site-directed mutagenesis of key functional residues

  • CRISPR/Cas9-based genome editing for precise modifications

  • Conditional knockdown using antisense RNA or inducible promoters

Studies on V. vulnificus virulence genes have successfully employed clean deletion strategies to avoid polar effects. For example, research on capsule biogenesis genes demonstrated the importance of clean deletions when examining closely linked genes such as VV0357 and VV0358 .

What are the most effective experimental designs for comparing wild-type and pstB2-mutant strains in various stress conditions?

Robust experimental designs for stress response comparison require:

• Standardized growth protocol:

  • Define pre-culture conditions (medium, growth phase, temperature)

  • Normalize cell density across all experiments (OD600 ≈ 0.5)

  • Wash cells to remove residual media components

  • Use balanced experimental design with appropriate controls

• Stress exposure matrix:

  Stress Condition	Variables to Test	Measurement Parameters	Timepoints
  Phosphate limitation	0, 0.1, 0.5, 1.0 mM Pi	Growth rate, survival	0, 2, 4, 8, 24h
  Oxidative stress	0.1-5 mM H₂O₂	Survival, enzyme activity	0, 30, 60, 120min
  Serum exposure	10-100% human serum	CFU recovery, gene expression	0, 1, 2, 4h
  pH stress	pH 5.0-9.0	Growth, metabolic activity	0, 2, 4, 8h
  Antimicrobial peptides	1-100 μg/mL	MIC, membrane integrity	0, 2, 4, 8h

• Multiparameter analysis:

  • Viability assessment (plate counts, live/dead staining)

  • Metabolic activity (ATP levels, resazurin reduction)

  • Membrane integrity (propidium iodide uptake)

  • Specific stress responses (ROS levels, pH homeostasis)

• Statistical approach:

  • Minimum of three biological replicates

  • Two-way ANOVA to assess strain × condition interactions

  • Appropriate post-hoc tests with correction for multiple comparisons

  • Effect size calculation to quantify biological significance

This approach allows for systematic identification of conditions where pstB2 contributes to stress resilience, providing insights into its role in V. vulnificus pathogenicity. Similar experimental frameworks have been used in studies identifying virulence-associated genes in V. vulnificus .

What are the most sensitive methods for detecting phosphate transport activity in pstB2-expressing systems?

Detection of phosphate transport requires sensitive methodological approaches:

• Radioisotope-based transport assays:

  • Use ³²P-labeled inorganic phosphate (³²Pi)

  • Prepare inside-out membrane vesicles from expressing cells

  • Initiate transport with ATP addition

  • Sample at short intervals (15, 30, 60, 120 seconds)

  • Rapid filtration through nitrocellulose membranes

  • Scintillation counting for accurate quantification

  • Calculate initial rates from linear phase

• Fluorescent phosphate analog approaches:

  • BCECF-based intracellular pH monitoring (proton-coupled transport)

  • Phosphate analogs with fluorescent properties

  • Real-time continuous monitoring using plate reader

  • Kinetic analysis for Km and Vmax determination

• Indirect coupling assays:

  • ATPase activity measurement (colorimetric phosphate release)

  • Coupled enzyme assays (ATP regeneration systems)

  • Membrane potential sensitive dyes

• Reconstitution system optimization:

  • Proteoliposome preparation with defined lipid composition

  • Co-reconstitution with complete Pst complex components

  • Varying ATP concentrations for kinetic parameter determination

  • Assessment of substrate specificity (Pi, arsenate, phosphite)

• Data analysis considerations:

  • Correction for passive diffusion

  • Controlling for vesicle integrity and size

  • Protein quantification for specific activity calculation

  • Comparison with known phosphate transporters as references

These methodologies provide comprehensive functional assessment, distinguishing between ATP binding, hydrolysis, and actual phosphate translocation activities of pstB2. Similar approaches have been employed for characterizing transport systems in V. vulnificus and related bacteria .

How should researchers interpret differences in pstB2 expression levels across clinical versus environmental Vibrio vulnificus isolates?

Interpretation of expression differences requires rigorous analytical approaches:

• Expression quantification methodology:

  • qRT-PCR with validated reference genes specific for V. vulnificus

  • RNA-seq with appropriate normalization (TPM, RPKM)

  • Western blot using specific antibodies against pstB2

  • Standardize growth conditions and sampling points

• Statistical analysis framework:

  • Minimum sample size calculation based on expected effect size

  • Non-parametric tests for non-normally distributed data

  • Multiple testing correction (Benjamini-Hochberg FDR)

  • Correlation analysis with other genetic markers

• Contextual interpretation guidelines:

  • Relate to phylogenetic grouping of isolates

  • Consider ecological niches and isolation sources

  • Analyze in context of other phosphate metabolism genes

  • Compare with established virulence factor expression patterns

• Validation approaches:

  • Reporter gene fusions to confirm transcriptional differences

  • Complementation experiments across isolate backgrounds

  • Functional assays to correlate expression with phosphate uptake capacity

Research on V. vulnificus isolates has demonstrated significant genetic diversity with clinical implications. Genome-wide studies have identified distinct phylogenetic groups that correlate with virulence potential . Phosphate acquisition mechanisms may follow similar patterns, with analysis requiring consideration of the genetic background and ecological adaptation of each isolate.

What analytical frameworks can distinguish between direct effects of pstB2 mutation and indirect metabolic consequences?

Distinguishing direct from indirect effects requires integrated analytical frameworks:

• Metabolomic profiling strategy:

  • Untargeted LC-MS/MS metabolomics of wild-type vs. mutant

  • Stable isotope labeling to track phosphate incorporation

  • Pathway enrichment analysis to identify affected metabolic modules

  • Temporal sampling to distinguish primary from secondary effects

• Multi-omics integration approach:

  • Paired transcriptomic and proteomic analysis

  • Computational modeling of metabolic networks

  • Flux balance analysis to predict metabolic rerouting

  • Network analysis to identify regulatory hubs

• Genetic interaction mapping:

  • Synthetic genetic array analysis with pstB2 deletion

  • Chemical genetic profiling under varying phosphate conditions

  • Suppressor mutation screening to identify compensatory pathways

• Direct vs. indirect effect criteria:

  Effect Type	Temporal Pattern	Genetic Rescue	Metabolic Signature
  Direct	Immediate	Only by pstB2	Specific to Pi metabolism
  Indirect	Delayed	Multiple genes	Broad metabolic changes
  Compensatory	Adaptive	New equilibrium	Alternative pathway activation

• Statistical approaches:

  • Principal component analysis to separate direct vs. indirect effects

  • Time series analysis to establish causality

  • Bayesian network modeling for probabilistic relationships

This framework provides a systematic approach to deconvolute the complex effects of pstB2 disruption. Similar approaches have been employed in genome-wide epistasis studies (GWES) of V. vulnificus to identify co-adaptation networks and distinguish between direct genetic interactions and indirect effects .

How can structural insights into pstB2 inform the development of targeted inhibitors against Vibrio vulnificus?

Structure-based drug design approaches for pstB2 inhibitors require:

• Structural characterization methodology:

  • X-ray crystallography of pstB2 in multiple conformational states

  • Cryo-EM of complete Pst transport complex

  • Molecular dynamics simulations to identify cryptic binding sites

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Target site identification:

  • ATP binding pocket for competitive inhibitors

  • Interface regions between pstB2 and membrane components

  • Allosteric sites that prevent conformational cycling

  • Species-specific structural features absent in human transporters

• Inhibitor design strategy:

  • Virtual screening of compound libraries against identified pockets

  • Fragment-based approach starting with ATP-competitive scaffolds

  • Structure-activity relationship optimization

  • Incorporation of physico-chemical properties for penetration

• Validation cascade:

  • In vitro ATPase inhibition assays

  • Transport inhibition in membrane vesicles

  • Bacterial growth inhibition under phosphate limitation

  • Toxicity assessment against human cell lines

  • In vivo efficacy in infection models

Research on V. vulnificus has identified several potential therapeutic targets, including the RtxA1 toxin , which has been successfully targeted for vaccine development. Structural insights into essential transport systems like pstB2 could similarly inform therapeutic development, particularly given the increasing antibiotic resistance observed in V. vulnificus .

How might targeting pstB2 function affect Vibrio vulnificus virulence in vivo?

Assessment of pstB2 as a therapeutic target requires systematic in vivo evaluation:

• Animal model selection and design:

  • Iron-overloaded mouse model (mimics human risk factors)

  • Wound infection model with controlled bacterial inoculum

  • Gastrointestinal infection model for food-borne route

  • Use of both wild-type and pstB2-deficient strains with bioluminescent reporters

• Virulence parameter measurement:

  • Bacterial burden in blood and tissues (spleen, liver)

  • Survival curve analysis with Kaplan-Meier statistics

  • Cytokine profiling to assess inflammatory response

  • Histopathological examination of infected tissues

• Inhibitor evaluation protocol:

  • Pharmacokinetic studies to establish dosing regimen

  • Prophylactic vs. therapeutic administration timing

  • Combination with standard antibiotics

  • Different administration routes (oral, intraperitoneal, topical)

• Advanced infection modeling:

  • Humanized mouse models with human serum components

  • Ex vivo human tissue infection models

  • Microfluidic organ-on-chip systems for controlled infection studies

Data from previous virulence studies in V. vulnificus demonstrate that disruption of essential transport systems can significantly attenuate virulence. For example, capsule biosynthesis mutants show dramatically reduced virulence in mouse models , and similar effects might be expected for phosphate transport deficiencies under the phosphate-limited conditions encountered during infection.

What is the relationship between pstB2 genetic variants and clinical outcomes in Vibrio vulnificus infections?

Investigation of pstB2 genetic diversity requires clinical correlation approaches:

• Genetic diversity assessment:

  • Whole genome sequencing of clinical isolates

  • SNP and structural variant identification in pstB2

  • Haplotype analysis and phylogenetic grouping

  • Functional prediction of amino acid substitutions

• Clinical correlation methodology:

  • Retrospective analysis of patient outcomes linked to isolate genetics

  • Case-control studies comparing severe vs. mild infections

  • Standardized clinical data collection with severity scores

  • Multivariate analysis controlling for host factors

• Functional validation:

  • Site-directed mutagenesis to recreate clinical variants

  • Transport activity assays comparing variant proteins

  • Competition assays between variant strains in serum

  • Transcriptional response profiling of variant strains

• Predictive model development:

  • Machine learning approaches integrating genetic and clinical data

  • Risk stratification based on bacterial and host factors

  • Validation in prospective clinical cohorts

Florida Department of Health surveillance data shows significant variability in clinical outcomes of V. vulnificus infections, with mortality rates of approximately 20-25% . Genetic determinants of virulence, including potential variations in essential systems like phosphate transport, may contribute to this clinical heterogeneity.

How does pstB2 expression respond to antibiotic exposure, and could this affect treatment outcomes?

Analysis of pstB2 regulation under antibiotic stress requires:

• Expression analysis methodology:

  • qRT-PCR of pstB2 under sub-inhibitory antibiotic concentrations

  • Reporter fusions (pstB2-promoter:GFP) for real-time monitoring

  • Western blot to confirm protein-level changes

  • Single-cell analysis to detect population heterogeneity

• Antibiotic exposure protocol:

  • Test multiple antibiotic classes (β-lactams, aminoglycosides, tetracyclines)

  • Dose-response assessment (0.1x-1x MIC)

  • Time-course analysis (immediate vs. adaptive responses)

  • Combinatorial antibiotic testing

• Functional consequence evaluation:

  • Phosphate uptake measurement under antibiotic stress

  • Bacterial survival in serum following antibiotic exposure

  • Metabolic activity assessment (ATP levels, respiration)

  • Membrane potential and permeability changes

• Clinical implication assessment:

  • Antibiotic efficacy testing in phosphate-limited conditions

  • Persister cell formation associated with phosphate transport

  • Sequential treatment protocols based on metabolic adaptations

  • Host-relevant microenvironments (varying phosphate availability)

Research indicates that V. vulnificus has begun developing resistance to certain antibiotics , and understanding how essential systems like phosphate transport respond to antibiotic stress could inform improved treatment strategies. Stress responses often engage multiple regulatory networks, and phosphate acquisition systems might be upregulated as part of general stress adaptation mechanisms.

What effect does human serum have on pstB2 expression and activity?

Investigation of serum effects on pstB2 requires systematic methodological approaches:

• Expression analysis in serum:

  • Transcriptional profiling (RNA-seq) comparing growth in serum vs. control media

  • qRT-PCR targeting the complete pst operon

  • Western blotting to confirm protein-level changes

  • Reporter gene constructs for real-time monitoring

• Functional activity assessment:

  • Radiolabeled phosphate uptake assays in serum-exposed bacteria

  • Competition assays with ΔpstB2 mutants in normal vs. heat-inactivated serum

  • Phosphate depletion measurement from growth medium

  • ATP hydrolysis assays with membrane preparations

• Serum factor identification:

  • Fractionation of serum components

  • Testing purified serum proteins (transferrin, albumin)

  • Assessment of complement effects

  • Iron chelation experiments to distinguish from iron-limitation responses

• Physiological relevance determination:

  • Comparison of different host species sera

  • Correlation with phosphate levels in patient samples

  • Ex vivo infection models with human blood

  • Link to disease progression markers