Recombinant Leptospira biflexa serovar Patoc Elongation factor Tu (tuf)

What is Elongation Factor Tu (EF-Tu) and what is its primary function in Leptospira?

Recent research has revealed that leptospiral EF-Tu is surface-exposed and performs secondary roles as a cell-surface receptor for host plasma proteins . This moonlighting function allows Leptospira to interact with host factors, contributing to its pathogenic potential through mechanisms distinct from its role in protein synthesis.

How conserved is EF-Tu among different Leptospira species?

Leptospiral EF-Tu is highly conserved across diverse Leptospira species . This conservation reflects not only its essential role in protein synthesis but potentially its importance in pathogenesis. The high degree of sequence conservation makes EF-Tu a potential target for broad-spectrum diagnostic approaches or therapeutics that could be effective against multiple Leptospira species.

The conservation pattern of EF-Tu differs from many other surface-exposed proteins in pathogenic bacteria, which often display significant variation to evade host immune recognition. This unusual conservation suggests that EF-Tu's surface functions may be fundamental to Leptospira biology across species boundaries.

What methods are effective for detecting the surface localization of EF-Tu in Leptospira?

Surface immunofluorescence assay is the primary validated method for demonstrating EF-Tu surface localization in Leptospira . The protocol involves:

• Adhering Leptospira (e.g., 1 × 10^8 L. interrogans) to microscope slides (80 min at 30°C)

• Removing unbound bacteria and fixing with 2% paraformaldehyde

• Blocking with Leptospira Enrichment EMJH medium (90 min at 30°C)

• Incubating with anti-EF-Tu immune sera (1:50 dilution, 60 min at 30°C)

• Detecting with fluorescently labeled secondary antibodies (e.g., Alexa Fluor 488-labeled anti-mouse IgG)

• Visualization using confocal microscopy

This method has successfully demonstrated the unexpected surface localization of EF-Tu in Leptospira species, challenging the traditional view of EF-Tu as strictly cytoplasmic.

How can researchers assess the interaction between Leptospira EF-Tu and host factors?

Multiple complementary approaches can be employed to characterize interactions between Leptospira EF-Tu and host factors:

• Ligand affinity blotting: Purified recombinant EF-Tu is subjected to SDS-PAGE under non-reducing conditions, transferred to nitrocellulose membranes, and incubated with normal human serum (7% dilution in PBS) as a source of host factors. After washing, bound proteins are detected using specific antibodies (e.g., anti-FH antibodies at 1:10000 dilution) .

• Cofactor assay: For functional validation of Factor H binding, researchers can measure Factor I-mediated cleavage of C3b when Factor H is bound to EF-Tu .

• Immunoblot analysis: This approach can detect EF-Tu in different Leptospira fractions using anti-EF-Tu serum (typically at 1:1000 dilution), followed by peroxidase-conjugated secondary antibodies .

These methods have revealed that Leptospira EF-Tu binds to host plasminogen and Factor H, contributing to complement evasion and tissue dissemination.

How does Leptospira EF-Tu contribute to bacterial pathogenesis and immune evasion?

Leptospira EF-Tu contributes to pathogenesis through multiple mechanisms beyond its role in protein synthesis:

• Plasminogen binding and activation: EF-Tu binds host plasminogen, which is then converted to enzymatically active plasmin. This surface-bound plasmin can cleave key host proteins including the central complement component C3b and fibrinogen .

• Complement evasion: By binding Factor H and promoting Factor I-mediated degradation of C3b, EF-Tu helps Leptospira evade complement-mediated killing .

• Tissue dissemination: The plasmin activity associated with EF-Tu-bound plasminogen likely facilitates bacterial spread through host tissues by degrading extracellular matrix components and fibrin clots .

Together, these mechanisms suggest that surface-exposed EF-Tu plays a significant role in Leptospira pathogenesis by promoting both immune evasion and tissue invasion strategies.

How does EF-Tu participate in ribosomal processes and how might this inform its non-canonical functions?

EF-Tu's canonical function involves delivering aminoacyl-tRNA to the ribosome during protein synthesis through a complex mechanism:

• EF-Tu forms a ternary complex with GTP and aminoacyl-tRNA, which engages with the ribosome during the elongation phase of translation .

• After GTP hydrolysis, conformational changes in EF-Tu coordinate the rate-limiting passage of aminoacyl-tRNA through the accommodation corridor toward the peptidyl transferase center .

• Single-molecule fluorescence resonance energy transfer imaging has revealed that EF-Tu dissociates from the ribosome as aminoacyl-tRNA navigates this corridor, but this release can be reversible .

• Intriguingly, new ternary complex formation, accompanied by cycles of GTP hydrolysis, can occur on aminoacyl-tRNA already bound within the ribosome .

These mechanistic insights might inform how EF-Tu's structural flexibility and binding properties enable its moonlighting functions on the bacterial surface, where similar conformational dynamics could facilitate interactions with host proteins.

How do pathogenic and saprophytic Leptospira species differ in their EF-Tu functions?

Research comparing pathogenic Leptospira interrogans serovar Copenhageni (LIC) with saprophytic Leptospira biflexa serovar Patoc (Patoc) provides insights into potential differences in EF-Tu function between these species:

Both pathogenic and saprophytic Leptospira can trigger pro-inflammatory responses in human neutrophils, including upregulation of CD11b expression, adhesion to collagen, and release of IL-8, IL-1β, and IL-6 . These responses involve inflammasome and NFκB pathway activation .

• Pathogenic LIC was observed on the neutrophil surface without being phagocytized

• Saprophytic Patoc generated intracellular reactive oxygen species associated with its uptake

• Only pathogenic LIC selectively increased levels of the AXL receptor protein tyrosine kinase

These observations suggest that while EF-Tu may be surface-exposed in both pathogenic and saprophytic species, its specific interactions with host immune cells could differ, potentially contributing to the different disease-causing potentials of these Leptospira species.

What expression systems are most suitable for producing recombinant Leptospira biflexa serovar Patoc EF-Tu?

While the search results don't provide specific information about expression systems for Leptospira biflexa EF-Tu, successful expression of recombinant leptospiral proteins typically employs the following approaches:

• E. coli expression systems: The BL21(DE3) strain with pET-based vectors is commonly used for recombinant expression of bacterial proteins, including those from Leptospira. The T7 promoter system allows for controlled, high-level expression.

• Purification strategy: A common approach includes:

  • Affinity chromatography using His-tag or GST-tag fusions

  • Ion exchange chromatography as a secondary purification step

  • Size exclusion chromatography for final polishing

  • Endotoxin removal for proteins intended for immunological studies

• Quality control: Validation of recombinant EF-Tu should include:

  • SDS-PAGE and Western blot analysis

  • Mass spectrometry confirmation

  • Circular dichroism to assess secondary structure

  • Functional assays specific to the canonical and non-canonical activities being studied

What challenges exist in studying the dual functions of Leptospira EF-Tu?

Investigating both the canonical translation role and surface-exposed functions of EF-Tu presents several methodological challenges:

What are promising targets for further investigation of Leptospira EF-Tu?

Several key areas warrant further investigation to advance our understanding of Leptospira EF-Tu:

• Structural studies: Determining the three-dimensional structure of Leptospira EF-Tu, particularly in complex with host factors like plasminogen and Factor H, would provide insights into binding mechanisms.

• Surface transport mechanisms: Elucidating how a primarily cytoplasmic protein like EF-Tu reaches the bacterial surface without canonical secretion signals would advance our understanding of bacterial protein export.

• Immunological significance: Investigating whether surface-exposed EF-Tu serves as an antigen during Leptospira infection and its potential as a vaccine candidate.

• EF-Tu inhibitors: Exploring small molecules that could selectively inhibit the surface functions of EF-Tu without disrupting essential translation processes.

• Evolutionary perspective: Comparing EF-Tu sequences and functions across pathogenic and non-pathogenic Leptospira species to understand the evolution of its moonlighting roles.

How might targeted modifications of EF-Tu affect Leptospira virulence?

Understanding the relationship between EF-Tu modifications and virulence could open new therapeutic avenues:

• Domain-specific mutations: Creating Leptospira strains with mutations in EF-Tu domains specifically involved in host interactions while preserving translation functions.

• Expression level modulation: Investigating how altered expression levels of EF-Tu affect both growth and virulence properties.

• Surface localization disruption: Developing approaches to selectively reduce surface exposure of EF-Tu without affecting cytoplasmic pools essential for translation.

• Comparative virulence studies: Testing modified strains in animal models to correlate EF-Tu changes with virulence parameters including bacterial load, dissemination, and host immune responses.