Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni 50S ribosomal protein L36 (rpmJ)

What is the structure and function of the 50S ribosomal protein L36 (rpmJ) in Leptospira interrogans?

L36, encoded by the rpmJ gene, is the smallest protein in the 50S ribosomal subunit of Leptospira interrogans. This protein plays a crucial role in the late steps of 50S ribosomal subunit assembly by facilitating interdomain interactions of 23S rRNA, particularly between helices H92 and H71 . Its incorporation into the ribosome is coordinated with the 2'-O-methylation of U2552 (Um2552) in 23S rRNA, a modification catalyzed by the methyltransferase RlmE .

The functional importance of L36 is highlighted by research showing that its absence in a 45S precursor particle prevents the formation of mature 50S subunits. This indicates that L36 is essential for the completion of ribosome assembly and, consequently, for bacterial protein synthesis. The proper incorporation of L36 ensures correct placement of the central protuberance and other domains during the final stages of ribosome assembly .

Unlike many other bacterial proteins, the role of L36 in Leptospira appears to be highly specialized for ribosomal assembly rather than having dual functions. This contrasts with some other leptospiral proteins that serve multiple roles in cellular functions and pathogenesis.

How does L36 contribute to the assembly of the 50S ribosomal subunit in Leptospira interrogans?

The assembly of the 50S ribosomal subunit in L. interrogans involves a complex, multi-step process requiring the coordinated incorporation of ribosomal proteins and modifications of rRNA. L36 plays a specific role in the late stages of this assembly process.

Research has demonstrated that the formation of the mature 50S subunit from a 45S precursor particle requires both the 2'-O-methylation of U2552 in 23S rRNA (catalyzed by RlmE) and the incorporation of L36 . These two events are mechanistically linked, as the methylation facilitates interdomain interactions in the 23S rRNA, which are then stabilized by L36.

In an experimental setting, researchers have shown that:

• The addition of recombinant RlmE and AdoMet (the methyl donor for the Um2552 modification) to a 45S precursor particle resulted in the formation of mature 50S subunits

• Approximately 25% of the 45S precursor particles were converted to 50S subunits in this reconstitution system

• The reconstituted 50S subunits were capable of associating with 30S subunits to form functional 70S ribosomes

• Analysis of the reconstituted 50S subunits confirmed the presence of Um2552 and the incorporation of L36

This sequential process demonstrates how L36 serves as a critical component in the final stages of ribosome maturation, enabling the transition from precursor to functional ribosomal subunit.

What are the optimal methods for expressing and purifying recombinant L36 from Leptospira interrogans?

While specific protocols for L36 (rpmJ) expression are not detailed in the literature, effective methodologies can be extrapolated from successful approaches used for other Leptospira proteins.

Expression System:

• Escherichia coli BL21 (SI) strain has proven effective as a host expression system for Leptospira proteins

• The rpmJ gene should be PCR-amplified from L. interrogans genomic DNA using primers designed based on published genome sequences

• The gene can be cloned into an expression vector such as pAE, which adds a 6×His tag for purification

Expression Conditions:

• Protein expression can be induced using IPTG or, for the SI strain, by NaCl as described for other Leptospira proteins

• Since L36 is a small, basic protein that interacts with RNA, optimized expression conditions including reduced temperature (16-18°C), shorter induction times, and lower inducer concentrations may prevent aggregation

Purification Strategy:

• Ni-NTA affinity chromatography serves as the primary purification method for His-tagged proteins

• For ribosomal proteins like L36 that interact with RNA, additional purification steps may be necessary:

  • Ion-exchange chromatography to remove bound nucleic acids

  • Size-exclusion chromatography for final polishing

• Expected yields based on similar Leptospira proteins: approximately 10 mg of protein from 1 liter of cultured cells

Quality Assessment:

• SDS-PAGE analysis to confirm molecular weight and purity

• Western blotting with anti-His tag antibodies to verify protein identity

• RNA binding assays to confirm native activity is retained

Since L36 is involved in specific RNA interactions, ensuring proper folding during recombinant expression is critical for functional studies.

What is the relationship between the ribosomal L36 protein and the membrane protein L36 (MPL36) in Leptospira interrogans?

There is a potential source of confusion in the literature between two distinct proteins with similar nomenclature in Leptospira interrogans:

Ribosomal Protein L36 (rpmJ):

• The smallest protein in the 50S ribosomal subunit

• Involved in ribosome assembly and protein synthesis

• Located intracellularly as part of the ribosome

Membrane Protein L36 (MPL36):

• A surface-exposed membrane protein

• Characterized as "a major plasminogen (PLG) receptor in pathogenic Leptospira"

• A rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain

• Functions as an important virulence factor

MPL36 has been extensively characterized as having high plasminogen-binding ability determined by lysine residues in its C-terminal region, with the ability to convert bound-PLG to active plasmin . Research using recombinant MPL36 (rMPL36) confirmed its plasminogen-binding properties, and a mutant of mpl36 showed reduced PLG-binding ability, decreased capacity to adhere and translocate cell monolayers, and attenuated virulence in animal models .

Despite the similar names, these are functionally and structurally distinct proteins:

• Ribosomal L36 is involved in protein synthesis machinery

• MPL36 is involved in host-pathogen interactions and virulence mechanisms

This distinction is important for researchers to avoid confusion when studying either protein.

Current Diagnostic Approaches:

Researchers have identified several immunodominant antigens in Leptospira, including:

• LipL32, LipL41, and LipL48 - major outer membrane proteins

• Lig proteins - particularly their non-identical domains

• GroEL - heat shock protein

• Loa22 - outer membrane protein

• Novel antigens like LIC10215

These proteins have demonstrated value in serological diagnostics. The potential of L36 as a diagnostic marker would depend on several factors:

Key Considerations for L36 as a Diagnostic Target:

• Expression during infection: While L36 is essential for ribosome function, its expression levels during infection compared to in vitro growth are unknown

• Immunogenicity: As an intracellular protein, L36 may not be readily exposed to the host immune system, potentially limiting antibody production

• Conservation: Ribosomal proteins are typically highly conserved, which could limit specificity for pathogenic Leptospira

Potential Diagnostic Applications:

• Recombinant multiepitope approach: If L36 contains immunogenic epitopes, these could be included in a multiepitope construct similar to those described for other Leptospira proteins

• Molecular detection: The rpmJ gene could potentially be targeted in molecular diagnostic assays like PCR, especially if sequence variations exist that allow discrimination between pathogenic and non-pathogenic Leptospira species

A study using protein microarrays comprising 61% of L. interrogans proteome identified 16 antigens that can discriminate between acute cases and healthy individuals . L36 was not specifically mentioned among these discriminatory antigens, suggesting it may not be among the most immunodominant proteins during infection.

How conserved is the L36 protein across different pathogenic Leptospira species?

While specific studies on L36 conservation across Leptospira species are not available, genomic analyses provide insights into the general patterns of conservation within this genus.

Genomic Diversity in Leptospira:

• The genus Leptospira is composed of 2 subclades (S1, S2) of free-living non-pathogenic species and 2 subclades (P1, P2) composed of species with variable pathogenic potential

• The subclade P1 is further divided into P1+ (high-virulence pathogens) and P1- (low-virulence pathogens)

• Genomic comparison among 67 global isolates of L. interrogans identified 1072 SNPs and 258 indels, indicating genetic variation even within a single species

Expected Conservation Patterns for L36:

A comprehensive analysis would require:

• Sequence alignment of rpmJ genes from different Leptospira species

• Comparison of L36 protein sequences to identify conserved domains and variable regions

• Assessment of whether any variations correlate with pathogenicity or host adaptation

Such analysis would be facilitated by whole genome sequences now available for multiple Leptospira species, including L. interrogans, L. weilii, and L. kirschneri , enabling comparative genomic approaches.

The newly available genome sequences of Leptospira weilii strains (FMAS_RT1, FMAS_PD2) and Leptospira kirschneri (FMAS_PN5) provide valuable resources for such comparative analyses .

How does the expression of L36 change during Leptospira infection in mammalian hosts?

The regulation of L36 expression during infection has not been directly studied, but insights can be drawn from broader transcriptomic studies of Leptospira during host interactions.

Transcriptional Responses to Host Immunity:

When Leptospira interacts with host macrophages, it alters the expression of many genes involved in various processes, including:

• Carbohydrate and lipid metabolism

• Energy production

• Signal transduction

• Transcription and translation

• Oxygen tolerance

• Outer membrane proteins

Notably, the expressions of several major outer membrane protein genes (e.g., ompL1, lipL32, lipL41, lipL48, and ompL47) were dramatically down-regulated (10-50 folds) during interaction with macrophages, suggesting an immune evasion strategy .

Potential Regulation of L36:

While L36 is not specifically mentioned in these studies, as a component of the translation machinery, its expression might be regulated as part of the bacterial adaptation to the host environment.

The regulation of ribosomal proteins often responds to growth conditions, and the hostile environment during infection might trigger specific responses. Some possibilities include:

A putative transcription factor (identified as LB333) was found to potentially govern the regulation of outer membrane proteins in Leptospira . Whether this or other transcription factors regulate L36 expression remains to be determined.

What techniques are most effective for studying L36 interactions with other ribosomal components in Leptospira?

Several advanced techniques can be employed to study the interactions of L36 with other ribosomal components in Leptospira:

In vitro Reconstitution Assays:

• Reconstitution of 50S subunits from 45S precursors using purified components

• Use of sucrose density gradient (SDG) analysis to monitor incorporation of L36 into ribosomal precursors

• LC/MS analysis to confirm modifications such as the 2'-O-methylation of U2552 in 23S rRNA

Structural Approaches:

• Cryo-electron microscopy (Cryo-EM) to visualize L36 position within the ribosome

• X-ray crystallography of ribosomal subunits

• Nuclear magnetic resonance (NMR) spectroscopy for studying dynamics of L36-RNA interactions

Crosslinking and Mass Spectrometry:

• Chemical crosslinking to capture interacting partners

• UV-induced crosslinking for RNA-protein interactions

• Analysis of crosslinked complexes by mass spectrometry

Genetic Approaches:

• Construction of L36 variants to identify key residues for function

• Conditional expression systems to study effects of L36 depletion

• Suppressor screens to identify functionally related components

RNA Structure Analysis:

• RNA footprinting to identify protected regions of rRNA

• SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) to analyze RNA structure in the presence and absence of L36

• DMS probing to analyze accessibility of RNA bases

From published research, in vitro reconstitution has been successfully used to study L36 function in ribosome assembly. In this approach, researchers demonstrated that:

• A 45S precursor could be converted to a 50S subunit with the addition of recombinant RlmE (rRNA methyltransferase) and AdoMet (methyl donor)

• About 25% of the 45S precursor was converted to 50S subunits

• The newly formed 50S subunits could associate with 30S subunits to form 70S ribosomes

• LC/MS analysis confirmed Um2552 formation in the reconstituted particles

This experimental system provides a powerful tool for studying the role of L36 in ribosome assembly and could be adapted to investigate specific aspects of L36 function and interactions.

What is the impact of L36 mutations on ribosome function and bacterial fitness in Leptospira interrogans?

While specific studies on L36 mutations in Leptospira are not available, the critical role of L36 in ribosome assembly suggests that mutations would have significant impacts on bacterial physiology.

Predicted Effects of L36 Mutations:

Based on its role in facilitating interdomain interactions in 23S rRNA and promoting the final steps of 50S assembly , mutations in L36 could potentially:

• Disrupt ribosome assembly: Preventing proper formation of 50S subunits

• Alter translation efficiency: Affecting protein synthesis rates and potentially protein folding

• Reduce growth rates: Particularly under stress conditions encountered during infection

• Affect antibiotic susceptibility: Since many antibiotics target the ribosome

Methodological Approaches to Study Mutations:

Several experimental approaches could assess the impact of L36 mutations:

• Site-directed mutagenesis of the rpmJ gene to create specific variants

• In vitro reconstitution assays using mutant L36 proteins to assess effects on ribosome assembly

• Growth analysis of strains expressing mutant L36 under various conditions

• Protein synthesis assays to measure translation efficiency and accuracy

• Animal infection models to assess virulence of L36 mutant strains

The interdependence between L36 incorporation and Um2552 formation in 23S rRNA suggests that L36 mutations might also disrupt this coordination, potentially leading to complex effects on ribosome biogenesis.

Potential Impact on Virulence:

Since efficient protein synthesis is essential for bacterial adaptation and virulence, L36 mutations that compromise ribosome function would likely attenuate pathogenicity. This could manifest as:

• Reduced ability to replicate in host tissues

• Impaired production of virulence factors

• Increased susceptibility to host immune responses

• Decreased stress tolerance

These hypothesized effects would need to be verified through targeted experimentation.

How do environmental conditions affect the expression and function of L36 in Leptospira interrogans?

Leptospira interrogans exists in diverse environments, from soil and water to mammalian hosts, suggesting that adaptation to changing conditions is critical for its survival and virulence.

Environmental Persistence of Leptospira:

Studies have shown that L. interrogans can survive for extended periods in environmental matrices:

• Culturable L. interrogans persisted at low concentrations in soil for at least 16 days

• In spring water, viable bacteria were detected for at least 28 days

These observations suggest that Leptospira has mechanisms to adapt to environmental stress, which may involve regulation of ribosomal components including L36.

Environmental Factors Potentially Affecting L36:

Host vs. Environmental Adaptation:

Transcriptomic studies have shown that when L. interrogans interacts with host macrophages, it alters expression of genes involved in transcription and translation . The transition between environmental survival and host infection may involve specific regulatory changes in ribosomal proteins like L36.

The pathogen's ability to persist in both environments is reflected in genomic adaptations. Comparative genomic analyses of Leptospira species with different lifestyles (free-living vs. pathogenic) could reveal whether differences in ribosomal components, including L36, contribute to this versatility.

Experimental approaches to study these adaptations could include reporter systems using the rpmJ promoter to monitor expression under different environmental conditions, combined with functional assays to assess ribosome assembly and activity.

How does L36 from Leptospira interrogans compare to L36 proteins from other bacterial species?

Ribosomal proteins are generally well-conserved across bacterial species due to their fundamental role in protein synthesis, but species-specific adaptations may exist.

Evolutionary Conservation:

The mechanism involving L36 and Um2552 in ribosome assembly appears to be broadly conserved. Research notes that "RlmE and Um2552 are conserved in other organisms, including human, indicating the functional importance of this process in ribosome biogenesis" .

Comparative Analysis Approaches:

To thoroughly compare L36 across species, researchers could employ:

• Sequence alignment of L36 proteins from diverse bacteria to identify:

  • Core conserved residues essential for function

  • Variable regions that may reflect species-specific adaptations

  • Clustering patterns that correlate with bacterial taxonomy or lifestyle

• Structural comparisons using available ribosome structures to examine:

  • Position of L36 within the ribosome

  • Interactions with rRNA and other proteins

  • Conformational differences

• Functional complementation experiments to test whether:

  • L36 from other species can substitute for Leptospira L36

  • Function is conserved despite sequence differences

Potential Species-Specific Adaptations:

Adaptations in L36 might reflect the unique lifestyle of Leptospira, which transitions between environmental survival and host infection. These could include:

• Modifications affecting protein stability under different temperature conditions

• Alterations in RNA-binding properties

• Differences in integration into partially assembled ribosomes

The coordination between L36 incorporation and specific rRNA modifications (Um2552) suggests a complex and potentially species-specific role in ribosome assembly that merits comparative investigation across bacterial species.

What role does the Leptospira interrogans L36 protein play in antibiotic resistance and susceptibility?

While no studies directly address the role of L36 in antibiotic resistance in Leptospira, its location in the ribosome suggests potential involvement in susceptibility to ribosome-targeting antibiotics.

Ribosome-Targeting Antibiotics:

Many clinically important antibiotics target the bacterial ribosome, including:

• Macrolides (e.g., erythromycin)

• Lincosamides (e.g., clindamycin)

• Aminoglycosides (e.g., gentamicin)

• Tetracyclines (e.g., doxycycline)

Since L36 is involved in the late stages of 50S subunit assembly , alterations in its structure or function could potentially affect the binding of antibiotics that target this subunit.

Potential Mechanisms of L36-Related Resistance:

• Structural alterations: Mutations in L36 could indirectly affect the binding sites of antibiotics by altering ribosome conformation

• Assembly defects: Changes in L36 that affect ribosome assembly might result in heterogeneous ribosome populations with varied antibiotic susceptibility

• Compensatory mechanisms: Altered L36 function might trigger compensatory changes in other ribosomal components that affect antibiotic binding

Research Approaches:

Several experimental strategies could investigate the relationship between L36 and antibiotic susceptibility:

• Minimum inhibitory concentration (MIC) testing of L36 mutant strains against various antibiotics

• Ribosome binding assays to measure antibiotic binding to ribosomes with wild-type versus mutant L36

• Structural studies to determine if L36 mutations affect the conformation of antibiotic binding sites

• Translation assays to assess if L36 variants affect antibiotic-mediated inhibition of protein synthesis

Doxycycline is commonly used to treat leptospirosis, and understanding how ribosomal proteins like L36 might influence susceptibility to this and other antibiotics could have important clinical implications.