Recombinant Listeria monocytogenes serotype 4b Serine/threonine phosphatase stp (stp)

What is the functional role of Stp in Listeria monocytogenes virulence?

Stp encodes a functional Mn²⁺-dependent serine-threonine phosphatase similar to PPM eukaryotic phosphatases (Mg²⁺- or Mn²⁺-dependent protein phosphatase) and is required for growth of L. monocytogenes in murine infection models . The phosphatase plays a crucial role in regulating the elongation factor EF-Tu, which is its first identified target. When an stp deletion mutation is introduced, L. monocytogenes exhibits decreased sensitivity to kirromycin, an antibiotic known to inhibit EF-Tu function . This altered antibiotic sensitivity suggests that Stp influences bacterial survival within the infected host through post-translational regulation mechanisms.

Research has demonstrated that signal transduction systems based on reversible phosphorylation are critical for L. monocytogenes to survive and grow in various host and environmental conditions. Among these regulatory elements, Stp stands out as one of only two putative serine-threonine phosphatases identified in the genome sequence .

How does serotype 4b differ from other L. monocytogenes serotypes in clinical significance?

Serotype 4b strains cause the majority of listeriosis clinical cases and outbreaks despite representing only one of 15 serovars within the genus . This clinical predominance occurs because:

• Serotype 4b strains possess specific wall teichoic acid (WTA) glycosylation patterns that contribute to their virulence

• Four major hypervirulent clonal complexes (CCs) - CC1, CC2, CC4, and CC6 - are predominantly found within serotype 4b

• CC4 is significantly overrepresented among human isolates compared to food isolates, suggesting enhanced pathogenicity

Comparative studies have shown that serotype 4b strains account for a significantly higher proportion of clinical isolates (particularly in outbreaks) than would be expected based on their prevalence in food sources, with only 5.1% of clinical isolates carrying premature stop codon mutations compared to 45% of food isolates .

What are the known molecular targets of Stp in L. monocytogenes?

The primary verified target of Stp is the elongation factor EF-Tu . Post-translational phosphorylation of EF-Tu prevents:

• Its binding to amino-acylated transfer RNA

• Its binding to kirromycin (an antibiotic inhibitor of EF-Tu function)

This regulatory mechanism appears critical for bacterial survival during infection. While EF-Tu is the first identified target, the phosphatase likely has multiple substrates given the complexity of phosphorylation-based regulatory networks in bacterial pathogens. Ongoing phosphoproteomic studies are needed to identify additional substrates involved in various aspects of L. monocytogenes physiology and virulence.

What techniques are recommended for expressing and purifying recombinant Stp from L. monocytogenes serotype 4b?

For optimal expression and purification of recombinant Stp, researchers should consider:

Expression Systems:

• E. coli BL21(DE3) with pET-based vectors for high-yield expression

• Baculovirus-insect cell system for eukaryotic post-translational modifications when needed

• Cell-free protein synthesis systems for proteins that may be toxic to host cells

Purification Protocol:

• Transform expression vector containing the stp gene into appropriate host cells

• Induce protein expression (typically with IPTG for E. coli systems)

• Harvest cells and lyse using sonication or pressure-based methods

• Perform initial purification using Ni-NTA affinity chromatography (for His-tagged constructs)

• Apply secondary purification through ion exchange chromatography

• Perform final polishing step using size-exclusion chromatography

• Verify purity using SDS-PAGE and Western blotting

Critical Considerations:

• Include Mn²⁺ in buffers (typically 1-5 mM) to maintain enzyme activity

• Optimize temperature and pH (generally 4°C during purification; pH 7.0-7.5)

• Include protease inhibitors to prevent degradation

• Test activity immediately after purification using para-nitrophenyl phosphate (pNPP) assay

How can researchers generate and verify stp deletion mutants in serotype 4b strains?

To generate stp deletion mutants in L. monocytogenes serotype 4b:

Allelic Exchange Method:

• Construct a deletion vector containing approximately 1 kb of upstream and downstream flanking regions of the stp gene

• Use a temperature-sensitive plasmid (e.g., pKSV7 or pIMK)

• Transform the construct into L. monocytogenes serotype 4b

• Select transformants under permissive conditions

• Promote integration by shifting to non-permissive temperature with antibiotic selection

• Release the integrated plasmid by growth without antibiotic at permissive temperature

• Screen colonies for gene deletion using PCR

Verification Approaches:

• PCR verification with primers flanking the deleted region

• Whole genome sequencing to confirm deletion and absence of secondary mutations

• Southern blot analysis to verify single genomic alterations

• RT-PCR to confirm absence of stp transcript

• Western blot to confirm absence of Stp protein

• Phenotypic verification through kirromycin sensitivity assays

What phenotypic changes occur in L. monocytogenes serotype 4b when stp is deleted?

The deletion of stp in L. monocytogenes results in several observable phenotypes:

In vitro phenotypes:

• Decreased sensitivity to kirromycin (an antibiotic that targets EF-Tu)

• Altered phosphorylation status of EF-Tu

• Potentially altered protein synthesis rates and fidelity

• Changes in stress response mechanisms

In vivo phenotypes:

• Reduced virulence in murine infection models

• Decreased ability to grow and survive within host cells

• Potentially altered interaction with host immune responses

• Changes in tissue tropism or dissemination patterns

These phenotypic changes underscore the importance of Stp in regulating L. monocytogenes virulence and adaptation to the host environment through post-translational modifications of key proteins involved in bacterial physiology.

What methods are most effective for studying Stp phosphatase activity in vitro?

Recommended Methods for Studying Stp Activity:

Substrate-Based Assays:

• pNPP Assay: Measures the release of para-nitrophenol from para-nitrophenyl phosphate

  • Advantages: Simple, colorimetric readout

  • Limitations: Non-specific substrate

• Phosphopeptide-Based Assays:

  • Use synthetic peptides mimicking known Stp substrates like EF-Tu phosphorylation sites

  • Measure dephosphorylation using:

    • Malachite green assay (detects released phosphate)

    • Mass spectrometry (directly detects dephosphorylated peptides)

• In-Gel Phosphatase Assays:

  • For analysis of native complexes and molecular weight determination

  • Incorporate radioactive substrates or fluorescent substrates

Kinetic Analysis:

• Determine Km, Vmax, and kcat values using varying substrate concentrations

• Test enzyme activity under different conditions:

  • pH range (typically 6.0-8.0)

  • Temperature range (25-42°C)

  • Divalent cation concentrations (Mn²⁺, Mg²⁺)

  • Potential inhibitors

Inhibitor Studies:

• Test sensitivity to classical phosphatase inhibitors:

  • Okadaic acid

  • Calyculin A

  • Fluoride

  • Vanadate compounds

How can researchers identify novel Stp substrates in L. monocytogenes serotype 4b?

Identifying novel Stp substrates requires a multi-faceted approach:

Phosphoproteomic Approaches:

• Comparative Phosphoproteomics:

  • Compare phosphorylation profiles between wild-type and Δstp mutants

  • Sample preparation: TCA precipitation followed by tryptic digestion

  • Phosphopeptide enrichment: TiO₂ or IMAC

  • LC-MS/MS analysis with label-free quantification or SILAC

• In vitro Dephosphorylation Assays:

  • Incubate cell lysates with recombinant Stp

  • Identify dephosphorylated proteins by proteomics

Biochemical Approaches:

• Affinity-Based Methods:

  • Use substrate-trapping mutants of Stp (e.g., D→A mutations in catalytic site)

  • Perform pull-down assays followed by mass spectrometry

  • Verify interactions using co-immunoprecipitation

• Yeast Two-Hybrid Screening:

  • Use Stp as bait to identify interacting proteins

  • Confirm physical interactions using other methods

Bioinformatic Analysis:

• Identify proteins with phosphorylation motifs similar to known Stp substrates

• Focus on proteins involved in virulence, stress response, and cell wall functions

• Prioritize candidates from serotype 4b-specific genetic regions

Validation Methods:

• In vitro dephosphorylation assays with purified candidate proteins

• Site-directed mutagenesis of putative phosphorylation sites

• Phenotypic analysis of phosphomimetic and phosphoablative mutants

What experimental models are most appropriate for studying the role of Stp in L. monocytogenes virulence?

In Vitro Cellular Models:

• Macrophage Infection Models:

  • Cell lines: J774, RAW264.7, THP-1

  • Primary mouse bone marrow-derived macrophages

  • Measurements: Bacterial survival, phagosomal escape, cytokine production

• Epithelial Cell Invasion Assays:

  • Cell lines: Caco-2, HepG2, HeLa

  • Measurements: Adhesion, invasion, intracellular growth, cell-to-cell spread

• Tissue Explant Models:

  • Placental tissue for maternal-fetal transmission studies

  • Brain tissue for blood-brain barrier crossing studies

In Vivo Animal Models:

• Mouse Models:

  • Intravenous infection model (systemic listeriosis)

  • Oral infection model (natural route)

  • Galleria mellonella (wax moth larvae) as an alternative model

• Specific Applications:

  • Neutrophil depletion experiments to assess bacterial susceptibility

  • Competitive index assays (wild-type vs. Δstp)

  • In vivo imaging of bioluminescent strains to track dissemination

Model Selection Considerations:

When studying Stp specifically, researchers should consider models that emphasize intracellular growth phases and dissemination, as phosphorylation-based signaling likely plays key roles in adapting to changing host environments during infection.

How does Stp from serotype 4b compare functionally to homologs in other L. monocytogenes serotypes?

While limited comparative data exists, researchers should consider:

• Sequence Comparison Analysis:

  • Perform multiple sequence alignments of Stp from different serotypes

  • Focus on catalytic domains and regulatory regions

  • Identify serotype-specific variations that might affect function

• Enzymatic Activity Comparison:

  • Express and purify Stp from multiple serotypes (1/2a, 1/2b, 4b)

  • Compare substrate specificity and kinetic parameters

  • Test activity under various environmental conditions

• Cross-Complementation Studies:

  • Express serotype 4b Stp in Δstp mutants of other serotypes

  • Assess restoration of virulence phenotypes

  • Identify serotype-specific functional differences

Research indicates that serotype 4b strains generally exhibit enhanced virulence compared to other serotypes, with several hypervirulent clones (CC1, CC2, CC4, and CC6) predominantly found within this serotype . The functional differences in regulatory proteins like Stp may contribute to this enhanced virulence, making comparative studies particularly valuable.

What role might Stp play in L. monocytogenes adaptation to different environmental conditions?

Stp likely plays crucial roles in environmental adaptation through:

• Stress Response Regulation:

  • Temperature fluctuations (growth at refrigeration temperatures)

  • pH changes (survival in acidic food products)

  • Osmotic stress (high salt environments)

• Biofilm Formation:

  • Potential regulation of proteins involved in cell surface adhesion

  • Modulation of extracellular matrix components

  • Influence on bacterial communication systems

• Antimicrobial Resistance:

  • Regulation of cell wall modifications

  • Modulation of efflux pump expression or activity

  • Altered susceptibility patterns to sanitizers and preservatives

Research approaches should include:

• Transcriptomic comparison of wild-type and Δstp strains under various stresses

• Biofilm formation assays under food-relevant conditions

• Testing antimicrobial susceptibility profiles across environmental conditions

How can our understanding of Stp function inform development of novel control strategies against L. monocytogenes serotype 4b?

Targeting Stp or its regulatory pathways could lead to novel control strategies:

• Inhibitor Development:

  • Design small molecule inhibitors specific to Stp active site

  • Target unique structural features not present in host phosphatases

  • Develop peptidomimetic inhibitors based on substrate recognition motifs

• Anti-virulence Approaches:

  • Target downstream effectors of Stp-mediated regulation

  • Develop compounds that disrupt phosphorylation-dependent signaling

  • Consider combination approaches targeting multiple virulence mechanisms

• Biocontrol Strategies:

  • Engineer bacteriophages expressing Stp inhibitors

  • Develop competitive exclusion approaches using attenuated strains

  • Create CRISPR-Cas delivery systems targeting stp gene

These approaches may be particularly effective against serotype 4b strains, which are responsible for the majority of human listeriosis cases despite being less prevalent in food than other serotypes .