Recombinant Listeria monocytogenes serotype 4b Foldase protein prsA 2 (prsA2)

What is PrsA2 and what is its primary function in Listeria monocytogenes?

PrsA2 is one of at least two predicted peptidyl-prolyl cis/trans-isomerases of the Parvulin family encoded within the L. monocytogenes genome (the other being PrsA1, encoded by lmo1444) . It functions as a posttranslocation protein chaperone and/or foldase, assisting in the proper folding of proteins that are secreted across the bacterial membrane into the compartment between the membrane and cell wall . This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH .

How was PrsA2 initially identified in L. monocytogenes?

PrsA2 was identified based on its increased secretion by L. monocytogenes strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression . It was specifically discovered through analysis of bacteria containing prfA mutations that result in the constitutive expression of PrfA-dependent gene products (prfA* mutations) .

How does PrsA2 differ from other chaperone proteins in L. monocytogenes?

PrsA2 is distinct from PrsA1 despite their high degree of amino acid similarity. Research has demonstrated that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis that cannot be complemented by PrsA1 . Experimental evidence indicates no detectable degree of functional overlap between PrsA2 and PrsA1 . While both share homology with the single PrsA protein found in Bacillus subtilis, only PrsA2 appears to be critically required for virulence and optimal secretion of virulence factors .

How can recombinant PrsA2 protein be generated for research purposes?

Based on published research approaches, recombinant PrsA2 protein can be generated by cloning the prsA2 gene into an appropriate expression vector, followed by expression in a suitable bacterial host system. The search results indicate that 500 micrograms of recombinant PrsA2 protein has been used to generate rabbit polyclonal antiserum through a commercial supplier (Cocalico Biologicals) . For researchers seeking to produce recombinant PrsA2, standard protein expression and purification techniques would apply, including:

• PCR amplification of the prsA2 gene from L. monocytogenes serotype 4b

• Cloning into an expression vector with an appropriate tag for purification

• Expression in E. coli or another suitable host

• Protein purification using affinity chromatography

• Validation of protein identity and activity through appropriate assays

What are the methodological approaches to study PrsA2 function in L. monocytogenes?

Multiple experimental approaches have been used to investigate PrsA2 function:

• Gene deletion studies: Creating Δprs2::erm mutant strains to assess the impact on bacterial phenotypes .

• Complementation experiments: Introducing pPL2-prsA2 plasmid to restore PrsA2 function in mutant strains .

• Virulence assays: Measuring bacterial recovery from organs of infected mice to quantify virulence defects .

• Cell culture models: Assessing bacterial invasion, intracellular growth, and cell-to-cell spread in various cell lines .

• Enzymatic activity assays: Measuring the activity of PrsA2-dependent virulence factors such as LLO (using hemolytic activity assays with sheep RBCs) and PC-PLC (using egg yolk agar plates) .

• Protein stability analysis: Western blotting to assess protein degradation patterns .

• Proteomic analyses: Examining the secretome under different conditions to identify PrsA2-dependent proteins .

How can researchers effectively measure the chaperone activity of PrsA2?

To measure the chaperone activity of PrsA2, researchers can employ several approaches:

• Hemolytic activity assays: Measuring LLO-associated hemolytic activity in bacterial supernatants indicates PrsA2 chaperone function. Wild-type strains typically show approximately 60 ± 3.7 units of activity compared to 33 ± 2.5 units for ΔprsA2::erm mutants .

• Phospholipase activity assays: PC-PLC activity can be detected as visible zones of precipitation on appropriate media, with prsA2 mutants exhibiting significantly reduced levels that can be restored with complementation .

• Western blot analysis: Analyzing the stability and degradation patterns of secreted proteins in wild-type versus ΔprsA2 strains. For example, studies have shown that strains lacking prsA2 exhibit different degradation peptide patterns for LLO compared to wild-type bacteria .

• In vitro protein folding assays: While not explicitly mentioned in the search results, standard chaperone activity assays using purified recombinant PrsA2 and substrate proteins could be employed to directly measure folding assistance.

How does PrsA2 contribute to L. monocytogenes virulence?

PrsA2 plays a critical role in L. monocytogenes virulence through several mechanisms:

• Promoting virulence factor activity: PrsA2 enhances the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and the broad-specificity phospholipase C (PC-PLC) .

• Supporting bacterial cell-to-cell spread: Loss of PrsA2 activity severely impairs bacterial cell-to-cell spread in host cells .

• Enhancing bacterial survival in the cytosol: PrsA2 is required for optimal bacterial viability within the host cell cytosol .

• Facilitating invasion: The reduction in bacterial uptake by macrophages observed in prsA2 mutants suggests a role for PrsA2 in proper folding of proteins required for bacterial adhesion and/or invasion .

The magnitude of the prsA2 virulence defect is substantial, with a 5- to 6-log reduction in bacterial recovery from the livers and spleens of infected mice. This defect is comparable to that observed for strains completely lacking hly or prfA, genes encoding well-established essential virulence factors .

What specific virulence factors are affected by PrsA2 activity?

PrsA2 affects the activity and stability of several key virulence factors:

• Listeriolysin O (LLO): Measurement of LLO-associated hemolytic activity in supernatants derived from ΔprsA2::erm strains indicated an approximate twofold decrease in activity compared to wild-type strains (33 ± 2.5 U versus 60 ± 3.7 U) .

• Phospholipase C (PC-PLC): Strains lacking prsA2 exhibited significantly reduced levels of PC-PLC activity . These strains secreted slightly reduced amounts of pro-PC-PLC and approximately three- to fourfold less mature PC-PLC compared to wild-type strains .

• Other proteins: Preliminary analysis of secreted and surface-associated proteins from wild-type and prsA2 mutant strains indicates that a number of proteins appear to be dependent upon PrsA2 activity for secretion and surface association .

How does PrsA2 function change under infection conditions versus laboratory culture?

The function of PrsA2 becomes more critical under conditions relevant to host cell infection:

What are the key functional differences between PrsA2 and PrsA1?

Despite their structural similarities, PrsA2 and PrsA1 exhibit significant functional differences:

• Virulence contribution: PrsA2 is required for L. monocytogenes virulence, while mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth, cell-to-cell spread, and virulence in mice .

• Virulence factor support: PrsA2 is uniquely required for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection, whereas PrsA1's absence does not impact these functions .

• Functional overlap: Despite a high degree of amino acid similarity, no detectable degree of functional overlap has been observed between PrsA2 and PrsA1 .

• Hemolytic activity: Strains lacking prsA1 exhibited levels of hemolytic activity equivalent to wild-type strains (65 ± 5.0 U for ΔprsA1 versus 60 ± 3.7 U for wild-type), while prsA2 mutants showed significantly reduced activity .

• PC-PLC activity: Strains lacking prsA1 exhibited levels of secreted PC-PLC activity equivalent to those of strains containing wild-type prsA1, while prsA2 mutants showed reduced activity .

Can PrsA1 complement the loss of PrsA2 in experimental systems?

Based on the research findings, PrsA1 cannot complement the loss of PrsA2:

• The research explicitly states that "no detectable degree of functional overlap was observed between PrsA2 and PrsA1" .

• Despite their structural similarities, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth, cell-to-cell spread, and virulence in mice, while prsA2 mutants showed severe defects in these areas .

• Experimental evidence indicates that PrsA2 is distinct from PrsA1 in its requirement for L. monocytogenes virulence .

The data suggest that even though both proteins share homology with the single PrsA protein of Bacillus subtilis, they have evolved distinct functions in L. monocytogenes, with PrsA2 specifically adapted for roles in pathogenesis.

What is the impact of PrsA2 on protein degradation patterns of virulence factors?

Research has revealed complex effects of PrsA2 on the stability and degradation patterns of virulence factors:

• LLO degradation patterns: Induction of LLO synthesis in strains lacking prsA2 results in increased amounts of LLO degradation and different degradation peptide patterns compared to bacteria with wild-type prsA2 .

• Structural implications: The altered banding patterns of the LLO degradation products in prsA2 mutant strains may indicate an altered LLO structure that is more sensitive to proteolytic cleavage, consistent with a role for PrsA2 in proper LLO folding .

• Secretion versus degradation balance: Strains with wild-type prsA2 secrete more full-length LLO than strains lacking prsA2, suggesting that PrsA2 helps maintain the balance between secretion and degradation of virulence factors .

• PC-PLC processing: Strains lacking prsA2 secreted slightly reduced amounts of pro-PC-PLC in comparison to strains with wild-type prsA2 and approximately three- to fourfold-less mature PC-PLC, indicating an effect on both secretion and maturation processes .

How does PrsA2 function change under different PrfA activation states?

PrsA2 function shows important differences under various PrfA activation states:

• Enhanced requirement under PrfA activation: Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation .

• Hemolytic activity differences: The presence of prfA(L140F) increased LLO-dependent hemolytic activity four- to fivefold for all strains in comparison to strains carrying the wild-type prfA allele, but strains lacking prsA2 still showed reduced activity (83 ± 10.0 U for the ΔprsA2::erm prfA(L140F) mutant versus 396 ± 29.5 U for prfA(L140F) alone) .

• PC-PLC expression: While the addition of charcoal and phosphorylated glucose has been shown to enhance PC-PLC expression, as does the introduction of the prfA(L140F) allele, strains lacking prsA2 still exhibited reduced PC-PLC activity even under these enhanced expression conditions .

This relationship between PrfA activation and PrsA2 function is particularly significant because PrfA activation normally occurs within the host cell cytosol during infection, highlighting the importance of PrsA2 in the infection process.

What experimental approaches can be used to identify additional proteins dependent on PrsA2 for proper folding?

To identify additional proteins dependent on PrsA2 for proper folding, researchers could employ several advanced approaches:

• Comparative proteomics: Analysis of the secretome and surface proteome of wild-type versus ΔprsA2 strains under various conditions, including PrfA activation. Preliminary analysis has already indicated that a number of proteins appear to be dependent upon PrsA2 activity for secretion and surface association .

• Protein stability assays: Pulse-chase experiments to track the stability and folding kinetics of candidate proteins in wild-type versus ΔprsA2 strains.

• Structural analysis: Techniques such as circular dichroism, fluorescence spectroscopy, or limited proteolysis to assess the folding state of secreted proteins in the presence or absence of PrsA2.

• In vitro folding assays: Reconstitution of folding reactions using purified recombinant PrsA2 and candidate substrate proteins.

• Crosslinking studies: To identify direct interaction partners of PrsA2 during the secretion and folding process.

• Domain swap experiments: Creating chimeric proteins between PrsA1 and PrsA2 to identify domains responsible for substrate specificity and function.

The comprehensive identification of the PrsA2 dependome would provide valuable insights into the scope of this chaperone's influence on L. monocytogenes physiology and pathogenesis.