Recombinant Salmonella paratyphi A Probable oxaloacetate decarboxylase gamma chain 1 (oadG1)

What is oadG1 and what is its importance in Salmonella Paratyphi A research?

OadG1 (Probable oxaloacetate decarboxylase gamma chain 1) is a small protein consisting of 79 amino acids with the sequence MNEAVLLGEGFTLMFLGMGFVLSFLFLLIFAIRGMSAVITRFFPEPVAAPAPRAVPVVDDFTRLKPVIAAAIHHHRLNA . It is part of the oxaloacetate decarboxylase complex, which plays a crucial role in the anaerobic energy metabolism of Salmonella. The protein is significant in research as it represents a potential target for antimicrobial development and helps in understanding the metabolic adaptations of Salmonella Paratyphi A during host infection.

How is the Salmonella Paratyphi A strain typically characterized in laboratory settings?

Salmonella Paratyphi A is characterized through both biotyping and serotyping methods. Biotyping involves biochemical tests that show specific patterns: Lactose negative, Catalase positive, Oxidase negative, Indole negative, Methyl Red positive, Vogus Proskaur negative, Citrate negative, Urease negative, Triple Sugar Iron showing K/A glucose acid with gas, Nitrate reductase positive, and Lysine negative .

Serotyping is performed according to the Kauffmann-White scheme, based on somatic O and phase 1/2 flagellar antigens using agglutination tests. Salmonella Paratyphi A is assigned the antigenic formula I 2,12:a:1, distinguishing it from Salmonella Typhi which has the formula I 9,12[Vi]:d:_ .

What are the optimal growth conditions for cultivating Salmonella Paratyphi A for protein extraction?

Research indicates that optimal growth conditions for Salmonella Paratyphi A include:

• Temperature: 37°C

• pH: Maintained at 7.0-7.2 using 2N-NH4OH

• Media: Soyabean casein digest (SCD) media

• Agitation: 125 rpm on rotary shaker

• Supplementation: Addition of 5% glucose (from 40% sterile glucose solution)

• Incubation time: 12-24 hours for complete growth

• Monitoring: Regular purity checks using Gram staining

When scaling up cultures, a stepwise approach is recommended, starting with small volumes (2-50ml) before proceeding to larger cultures (up to 3000ml), as this ensures proper growth curves and higher protein yields .

What genomic approaches can be used to study variation in oadG1 across different Salmonella Paratyphi A strains?

For analyzing oadG1 variation across different strains, researchers should implement whole-genome sequencing followed by comparative genomic analysis. According to recent methodologies, researchers can:

• Employ single nucleotide polymorphism (SNP) analysis to identify variants within the oadG1 gene

• Utilize tools like "Paratype," a genotyping scheme that successfully segregates the global population structure of Salmonella Paratyphi A into three primary, seven secondary, and 18 distinct subclades/genotypes

• Generate maximum-likelihood phylogenetic trees (using tools like RAxML) to visualize evolutionary relationships between different variants

• Correlate specific oadG1 genetic variants with antimicrobial resistance profiles and geographical distribution patterns

This approach has proven valuable in identifying genomic variations between different sampling locations and specific antimicrobial resistance markers that may influence the function of metabolic proteins like oadG1.

How does the oadG1 protein interact with other components of the oxaloacetate decarboxylase complex?

The oadG1 protein (79 amino acids) functions as the gamma subunit of the oxaloacetate decarboxylase complex. While specific interaction data for Salmonella Paratyphi A's oadG1 is limited in the provided research materials, structural biology suggests that:

• The protein likely anchors the complex to the membrane through its hydrophobic regions (GFVLSFLFLLIFAIR sequence)

• It may form a transmembrane channel through which ions are translocated during the decarboxylation reaction

• Interactions with alpha and beta subunits are mediated through specific binding domains

• Post-translational modifications may regulate these interactions

Researchers studying these interactions should consider employing co-immunoprecipitation assays with tagged oadG1 protein, followed by mass spectrometry analysis to identify binding partners within the bacterial proteome.

What are the implications of mutations in the oadG1 gene for Salmonella Paratyphi A virulence and metabolism?

Mutations in oadG1 can significantly impact both virulence and metabolism of Salmonella Paratyphi A. While direct studies on oadG1 mutations are not explicitly covered in the search results, genomic surveillance studies of Salmonella Paratyphi A have identified specific mutations in metabolic genes that correlate with virulence profiles.

Research approaches to study these implications should include:

• CRISPR-Cas9 gene editing to create specific oadG1 mutations

• Comparative growth analysis under varying pH and oxygen conditions

• Mouse infection models to assess virulence changes

• Metabolomic analysis to identify alterations in central carbon metabolism

• Correlation of oadG1 variants with antimicrobial resistance patterns identified through genomic surveillance

For metabolism specifically, mutations could alter anaerobic energy production pathways, potentially affecting survival in the host intestinal environment.

What are the best expression systems for producing recombinant oadG1 protein for functional studies?

The optimal expression system for recombinant oadG1 protein production is E. coli, as demonstrated in successful recombinant protein studies . When designing expression experiments:

• Vector selection: Vectors containing N-terminal His-tag are recommended for purification purposes

• Expression strain: BL21(DE3) or similar strains optimized for membrane protein expression

• Induction conditions: IPTG induction at OD600 0.6-0.8

• Growth temperature: Lowering to 18-25°C after induction may improve proper folding

• Media composition: Consider using SCD media supplemented with glucose as demonstrated effective for Salmonella growth

For purification:

• Employ immobilized metal affinity chromatography (IMAC) using the His-tag

• Consider using mild detergents during lysis and purification to maintain protein structure

• Include glycerol (6% Trehalose) in storage buffer to improve stability

• Maintain pH at 8.0 in Tris/PBS-based buffers

How can researchers effectively design experiments to study oadG1's role in antimicrobial resistance?

When investigating oadG1's potential role in antimicrobial resistance, a multi-faceted experimental approach is recommended:

• Genotype-phenotype correlation:

  • Use the Paratype genotyping tool to classify isolates into genotypes

  • Compare antimicrobial susceptibility profiles across different genotypes

  • Identify specific oadG1 variants associated with resistance

• Gene knockout/complementation studies:

  • Generate oadG1 knockout mutants

  • Complement with wild-type or variant oadG1

  • Test antimicrobial susceptibility under various conditions

• Expression analysis:

  • Measure oadG1 expression levels in response to antimicrobial exposure

  • Compare expression in resistant vs. susceptible strains

• Structural biology:

  • Model potential interactions between oadG1 and antimicrobials

  • Identify binding sites or structural changes affecting drug efflux

• Population genomics:

  • Analyze associations between oadG1 variants and resistance genes in large genomic datasets

  • Track temporal changes in oadG1 sequences as resistance emerges

What methods can be used to assess the immunogenicity of oadG1 for vaccine development purposes?

To evaluate oadG1's potential as a vaccine component, researchers should implement a comprehensive immunological assessment:

• In silico epitope prediction:

  • Identify potential B-cell and T-cell epitopes using prediction algorithms

  • Assess epitope conservation across different Salmonella Paratyphi A genotypes

• Antibody generation:

  • Immunize rabbits with purified recombinant oadG1 following protocols similar to those used for serotyping antisera development:

    • Use 0.25% formalin-killed preparations

    • Follow immunization schedule (0.5ml, 1.0ml, 1.5ml, 2.0ml doses) subcutaneously

    • Test bleed after one week of the last injection

    • Target antibody titers of >1:1000 for evaluation

• Immunological assays:

  • ELISA to measure antibody titers

  • Serum bactericidal assays to assess functional antibody responses

  • T-cell proliferation and cytokine production measurement

• Challenge studies:

  • Mouse models immunized with oadG1 formulations

  • Assessment of protection against Salmonella Paratyphi A challenge

• Cross-protection analysis:

  • Evaluate protection against different genotypes identified through Paratype classification

How should researchers interpret contradictory findings regarding oadG1 function across different Salmonella Paratyphi A isolates?

When facing contradictory findings regarding oadG1 function, researchers should:

What bioinformatic approaches are most effective for analyzing oadG1 sequence conservation and variation across Salmonella strains?

For comprehensive analysis of oadG1 sequence conservation and variation, researchers should employ:

• Sequence alignment and phylogenetic analysis:

  • Multiple sequence alignment using MUSCLE or MAFFT

  • Construct maximum-likelihood phylogenetic trees using RAxML

  • Calculate sequence conservation scores across alignments

• SNP identification and classification:

  • Identify SNPs within oadG1 using genomic analysis pipelines

  • Classify SNPs as synonymous or non-synonymous

  • Calculate dN/dS ratios to assess selective pressure

• Structural impact prediction:

  • Model the effects of amino acid substitutions on protein structure

  • Predict functional impacts using tools like PROVEAN or SIFT

• Population genetics metrics:

  • Calculate nucleotide diversity (π) within and between populations

  • Perform tests for selective sweeps or balancing selection

  • Compare with other genes to identify unusual evolutionary patterns

• Integration with the Paratype framework:

  • Map oadG1 variants to the 18 distinct genotypes identified in global surveillance

  • Analyze temporal and geographic patterns of variation

  • Correlate with antimicrobial resistance profiles

How might genomic surveillance tools like Paratype inform future research on oadG1 and other virulence factors?

Genomic surveillance tools like Paratype represent a significant advancement in Salmonella Paratyphi A research and will impact oadG1 studies in several ways:

• Standardized classification system:

  • Paratype provides a unified framework for classifying isolates into 3 primary clades, 9 secondary clades, and 18 genotypes

  • This enables consistent reporting and comparison of oadG1 studies across different research groups

• Evolutionary context:

  • Researchers can place oadG1 variants within the evolutionary history of Salmonella Paratyphi A

  • This reveals how selection pressures may have shaped protein function over time

• Geographic and temporal tracking:

  • Monitor the spread of specific oadG1 variants globally

  • Track emerging variants that may impact virulence or drug resistance

• Correlation with clinical outcomes:

  • Link specific genotypes to disease severity, treatment response, and antimicrobial resistance

  • Identify high-risk genotypes for targeted intervention

• Vaccine development guidance:

  • Inform the selection of oadG1 variants for inclusion in vaccine formulations

  • Ensure coverage against globally prevalent genotypes

• Integration with other genomic data:

  • Combine oadG1 analysis with mutations in the O2-polysaccharide synthesis locus, a candidate for vaccine development

  • Create comprehensive models of genetic factors influencing pathogenicity

What are the potential applications of recombinant oadG1 protein beyond basic research?

Recombinant oadG1 protein has several potential applications beyond fundamental research:

• Diagnostic development:

  • Creation of antibody-based detection systems for Salmonella Paratyphi A

  • Development of serological assays for paratyphoid fever diagnosis

• Vaccine component:

  • Use as a subunit vaccine antigen alone or in combination with other proteins

  • Carrier protein for polysaccharide conjugate vaccines

• Antibody production:

  • Generation of monoclonal antibodies for research and diagnostic applications

  • Production of therapeutic antibodies for passive immunization

• Structural biology:

  • Crystallization studies to understand membrane protein structure

  • Model system for studying ion transport mechanisms

• Drug target validation:

  • High-throughput screening platform for identifying inhibitors

  • Structure-based drug design targeting the oxaloacetate decarboxylase complex

• Protein-protein interaction studies:

  • Bait protein for identifying binding partners in Salmonella

  • Understanding metabolic complex assembly mechanisms

For these applications, the high-purity recombinant protein (>90% as determined by SDS-PAGE) with proper storage in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 provides an excellent starting material .