Recombinant Yersinia pseudotuberculosis serotype O:1b Glucose-6-phosphate isomerase (pgi), partial

What is Yersinia pseudotuberculosis and its relationship to other Yersinia species?

Yersinia pseudotuberculosis is a gram-negative, facultative anaerobic bacterium belonging to the family Enterobacteriaceae. It shares approximately 73% genetic identity with other pathogenic Yersinia species based on nucleotide sequence analysis and contains similar virulence plasmids (pCD1/pYV) encoding a type three secretion system (T3SS) . Most significantly, Y. pestis is a very recently evolved clone of Y. pseudotuberculosis serotype O:1b, sharing >90% genetic identity based on nucleotide sequence comparison and approximately 75% protein amino acid sequence identity . Y. pseudotuberculosis demonstrates greater genetic stability than Y. pestis, with fewer insertion sequences, and has a broader host range including rodents, dogs, cats, cattle, rabbits, deer, and humans . While taxonomically these organisms could be considered a single species, they remain classified separately due to the extreme virulence of Y. pestis, the causative agent of plague .

What is the significance of serotype O:1b in Yersinia pseudotuberculosis?

Serotype O:1b holds particular significance in Yersinia research for several reasons. Most importantly, Y. pestis evolved directly from Y. pseudotuberculosis serotype O:1b, making it a crucial reference point for understanding pathogen evolution . The O:1b serotype is determined by specific genes in the O-antigen gene cluster that create distinct lipopolysaccharide structures on the bacterial surface, serving as antigenic determinants. The close genetic relationship between Y. pestis and Y. pseudotuberculosis O:1b creates potential difficulties in DNA-based diagnostic methods, requiring specialized techniques for accurate identification . Due to these similarities, O:1b strains are frequently used in comparative genomics and vaccine development studies, offering valuable models for studying pathogenic mechanisms without the extreme biosafety requirements of working with Y. pestis .

What is Glucose-6-phosphate isomerase (pgi) and its role in bacterial metabolism?

Glucose-6-phosphate isomerase (pgi) is a key metabolic enzyme that catalyzes the reversible isomerization between glucose-6-phosphate and fructose-6-phosphate, a critical step in both glycolysis and gluconeogenesis. In bacterial metabolism, pgi serves multiple essential functions:

• Enables carbon flux through glycolysis, facilitating energy production from glucose and other hexose sugars

• Regulates the balance between glycolysis and the pentose phosphate pathway, influencing NADPH production and nucleotide synthesis

• Supports bacterial adaptation to changing environmental conditions by adjusting metabolic pathways based on available carbon sources

• May contribute to virulence through both metabolic functions and potential moonlighting roles in pathogenesis

In Y. pseudotuberculosis, pgi is particularly important for adaptation to different host environments during infection, as the bacterium must transition between external environments and host tissues with varying nutrient availability.

How is recombinant Yersinia pseudotuberculosis serotype O:1b Glucose-6-phosphate isomerase produced in a laboratory setting?

The production of recombinant Y. pseudotuberculosis serotype O:1b Glucose-6-phosphate isomerase typically follows a methodical process that includes gene cloning, expression optimization, and protein purification. The process begins with isolating genomic DNA from Y. pseudotuberculosis serotype O:1b, followed by PCR amplification of the pgi gene using sequence-specific primers. The amplified gene is then cloned into an appropriate expression vector, often from the pET series for E. coli expression, and verified by sequencing to ensure the correct pgi sequence .

For protein expression, the construct is transformed into a suitable E. coli strain, typically BL21(DE3) or derivatives, with expression conditions optimized for temperature, induction parameters, and media composition. Following expression, cells are harvested and lysed, and the recombinant protein is purified through a series of chromatographic steps that may include affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography. Quality control measures include SDS-PAGE, Western blotting, enzymatic activity assays, and mass spectrometry to confirm identity and functionality.

Table 1: Comparison of Expression Systems for Recombinant Y. pseudotuberculosis pgi Production

What are the genetic characteristics of Yersinia pseudotuberculosis serotype O:1b compared to other serotypes?

Y. pseudotuberculosis serotype O:1b possesses distinct genetic characteristics that differentiate it from other serotypes, particularly in the O-antigen gene cluster responsible for lipopolysaccharide biosynthesis. The O-antigen gene cluster contains specific genes encoding enzymes for the synthesis, polymerization, and export of O-antigen components, with sequence variations that can be detected through O-genotyping methods .

Most significantly, Y. pseudotuberculosis serotype O:1b is genetically the closest relative to Y. pestis, with Y. pestis representing a recently evolved clone of this serotype. While Y. pestis contains an O-antigen gene cluster similar to O:1b, it has accumulated mutations that prevent the synthesis of O-antigens . Analysis of these O-antigen gene clusters has identified specific regions that can be used to identify Y. pestis-Y. pseudotuberculosis as a group or Y. pestis alone, with PCR assays targeting these regions demonstrating 100% specificity when tested on collections of Yersinia species and other Enterobacteriaceae .

The different genetic arrangements of the O-antigen gene clusters among the 21 known Y. pseudotuberculosis serotypes have enabled the development of multiplex PCR assays that can replace conventional serotyping methods. These PCR-based approaches can distinguish 14 individual serotypes and two duplex serotypes (O:4a-O:8 and O:12-O:13), with additional PCRs required for serotypes O:7, O:9, and O:10 .

How can recombinant Yersinia pseudotuberculosis serotype O:1b Glucose-6-phosphate isomerase be used in vaccine development?

Recombinant Y. pseudotuberculosis serotype O:1b proteins, including Glucose-6-phosphate isomerase, offer promising applications in vaccine development against Yersinia infections. Y. pseudotuberculosis can serve as a delivery vehicle for vaccine antigens, as demonstrated by recombinant attenuated Y. pseudotuberculosis PB1+ strain (χ10069) with Δ yopK Δ yopJ Δ asd triple mutations engineered to deliver Y. pestis fusion proteins to mice . This approach takes advantage of Y. pseudotuberculosis' natural infection route, where oral administration allows colonization of Peyer's patches and subsequent dissemination to liver, spleen, and other tissues, stimulating both mucosal and systemic immune responses .

Studies have shown that immunization with recombinant attenuated Y. pseudotuberculosis strains induces comprehensive immune responses and protection against multiple Yersinia species. For example, mice immunized with χ10069(pYA5199) exhibited complete protection against lethal oral infections by both Y. enterocolitica WA and Y. pseudotuberculosis PB1+ .

Table 2: Immune Responses Induced by Y. pseudotuberculosis-based Vaccines

The advantages of Y. pseudotuberculosis-based vaccines include greater genetic stability compared to Y. pestis, broader host range, potential for oral administration (providing both social and economic advantages), and ability to induce both mucosal and systemic immunity .

What are the challenges in expressing functional Glucose-6-phosphate isomerase from Yersinia pseudotuberculosis in heterologous systems?

Expressing functional Glucose-6-phosphate isomerase from Y. pseudotuberculosis in heterologous systems presents several technical challenges requiring methodical solutions. Protein folding and solubility issues often arise, as the pgi enzyme requires proper folding to maintain catalytic activity. Expression in E. coli frequently results in inclusion body formation, particularly at high expression levels or elevated temperatures. To address this, researchers can optimize expression conditions by reducing induction temperature (16-25°C), using lower inducer concentrations, or employing specialized E. coli strains that enhance proper folding .

Codon usage bias between Y. pseudotuberculosis and expression hosts can lead to translational pausing, premature termination, or amino acid misincorporation. This can be addressed through codon optimization of the pgi gene for the selected expression host or using strains that supply rare tRNAs. Post-translational modifications may also be required for activity but absent in prokaryotic expression systems, potentially necessitating eukaryotic expression platforms.

Additional challenges include developing specific enzyme activity assays that distinguish between host and recombinant pgi activity, maintaining protein stability during storage and experimental procedures, and effectively separating recombinant pgi from host proteins during purification. Addressing these challenges requires systematic optimization of expression constructs, host systems, and purification strategies to obtain functional Y. pseudotuberculosis pgi suitable for research applications.

How do mutations in the pgi gene affect the virulence of Yersinia pseudotuberculosis serotype O:1b?

Mutations in the pgi gene can significantly impact the virulence of Y. pseudotuberculosis serotype O:1b through multiple mechanisms affecting bacterial metabolism, stress response, and host-pathogen interactions. As pgi catalyzes a key step in glycolysis, mutations can force metabolic rerouting through alternative pathways such as the Entner-Doudoroff pathway or pentose phosphate pathway. This metabolic shift affects energy production efficiency, NADPH generation (influencing oxidative stress resistance), and carbon flux distribution, potentially altering the availability of metabolic intermediates needed for virulence factors.

Pgi mutations can compromise the bacterium's ability to adapt to changing environments during infection, including reduced utilization of specific carbon sources, altered oxidative stress resistance, and compromised acid stress response affecting survival in stomach and phagosome environments. The Type Three Secretion System (T3SS), crucial for Y. pseudotuberculosis virulence, may be indirectly affected through ATP limitation, altered metabolism affecting expression or regulation of T3SS components, or changes in membrane potential impacting functionality.

To investigate these effects, researchers typically construct defined pgi deletion or point mutants in Y. pseudotuberculosis O:1b and perform comparative analyses including in vitro growth assays with various carbon sources, stress resistance tests, tissue culture infection models, and animal infection models. Complementary approaches include transcriptomic and proteomic analyses to identify compensatory changes in gene expression, metabolomic profiling to map redirected metabolic fluxes, and complementation studies using wild-type pgi to confirm phenotype specificity.

What role does Glucose-6-phosphate isomerase play in the pathogenicity of Yersinia pseudotuberculosis?

Glucose-6-phosphate isomerase contributes to Y. pseudotuberculosis pathogenicity through both metabolic functions and potential non-canonical roles. Metabolically, pgi enables efficient glucose utilization, which is critical during different infection stages: in the intestinal lumen where nutrient competition is high, during invasion of host tissues where glucose metabolism powers virulence factor expression, and within host cells where metabolic flexibility contributes to intracellular survival.

Y. pseudotuberculosis encounters various environmental stresses during infection, and pgi activity supports stress resistance through generation of metabolic intermediates feeding into the pentose phosphate pathway (producing NADPH for antioxidant defense), enabling metabolic adaptation to nutrient limitation, and supporting cell envelope maintenance under stress conditions.

Beyond its canonical metabolic role, pgi in some bacteria exhibits moonlighting functions potentially contributing to virulence, including possible cell surface localization where it may participate in adhesion to host cells or extracellular matrix, potential interaction with host immune components, and possible involvement in biofilm formation or host cell signaling.

Metabolic enzymes like pgi can also influence virulence factor expression by affecting levels of metabolic intermediates that serve as signaling molecules, influencing global regulators responsive to metabolic status, and modulating physiological parameters that affect gene expression. Research approaches to investigate these roles include comparative proteomics under infection-relevant conditions, analysis of pgi mutant strains in infection models, protein-protein interaction studies, and transcriptional analysis to determine how pgi activity affects virulence gene expression.

How can structural analysis of recombinant Glucose-6-phosphate isomerase inform drug development against Yersinia infections?

Structural analysis of recombinant Glucose-6-phosphate isomerase from Y. pseudotuberculosis provides critical insights for targeted drug development against Yersinia infections. Detailed structural information enables rational design of inhibitors through identification of the enzyme's active site architecture and catalytic residues, mapping of species-specific structural features distinguishing bacterial pgi from human orthologs, computational screening of virtual compound libraries, and structure-activity relationship studies to optimize lead compounds .

The methodological pipeline typically includes production of high-purity recombinant pgi, crystallization screening, X-ray diffraction data collection and structure determination, with alternative approaches such as cryo-electron microscopy or NMR spectroscopy when crystallization proves challenging. Molecular dynamics simulations complement these experimental approaches by exploring conformational flexibility relevant to inhibitor binding.

Table 3: Comparison of Structural Analysis Methods for Y. pseudotuberculosis pgi

Structural comparison between Y. pseudotuberculosis pgi and human pgi reveals differences in active site architecture that can be exploited for selective inhibition, unique allosteric sites present only in the bacterial enzyme, and surface features potentially involved in pathogen-specific interactions. The advantages of targeting pgi include its essential role in metabolism, structural differences from human orthologs allowing selective targeting, potential broad-spectrum activity against multiple Yersinia species, and a mechanism of action distinct from conventional antibiotics, potentially addressing resistance issues.