Recombinant Haemophilus ducreyi tRNA (guanine-N (1)-)-methyltransferase (trmD)

What is the function of TrmD in Haemophilus ducreyi?

TrmD in H. ducreyi functions as a methyl transferase that catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37 base in tRNA, synthesizing m1G37-tRNA. This methylation is critical for suppressing tRNA frameshifting during protein synthesis on ribosomes. Unlike missense errors, frameshifting errors are almost always lethal because they change the translational reading frame and introduce premature termination codons . This function is particularly important in bacterial survival and pathogenesis, making TrmD essential for H. ducreyi growth and virulence.

How does TrmD differ structurally and functionally from Trm5?

TrmD and Trm5 represent an analogous pair of enzymes that catalyze the same chemical reaction but are fundamentally distinct in several aspects:

These differences make TrmD a highly specific antimicrobial target that would theoretically have minimal cross-reactivity with the human Trm5 enzyme .

What is known about H. ducreyi gene expression under anaerobic conditions?

H. ducreyi resides in the anaerobic environment of an abscess during infection. RNA-seq analysis comparing gene expression under aerobic and anaerobic conditions has revealed distinct transcriptional profiles. During anaerobic growth, H. ducreyi upregulates genes involved in:

• Purine metabolism

• Uptake and use of alternative carbon sources

• Toxin production

• Nitrate reduction

• Glycine metabolism

• Tetrahydrofolate synthesis

Concurrently, genes involved in electron transport, thiamine biosynthesis, DNA recombination, peptidoglycan synthesis, and riboflavin synthesis/modification are downregulated . A substantial component of H. ducreyi gene regulation in vivo overlaps with the organism's response to anaerobiosis in vitro, suggesting that adaptation to anaerobic conditions is a key aspect of the pathogen's successful infection strategy .

What are the optimal conditions for expressing and purifying recombinant H. ducreyi TrmD?

Based on methodologies employed for TrmD studies, the following approach is recommended:

• Cloning: Clone the H. ducreyi trmD gene into an expression vector with a suitable affinity tag (His-tag is commonly used)

• Expression host: Utilize Escherichia coli as an expression system (BL21(DE3) or similar strains)

• Induction conditions:

  • IPTG concentration: 0.5-1.0 mM

  • Temperature: 25-30°C (lower temperatures may improve solubility)

  • Duration: 4-6 hours or overnight

• Purification protocol:

  • Initial capture: Ni-NTA affinity chromatography

  • Buffer composition: 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol

  • Secondary purification: Size exclusion chromatography

  • Final polishing: Ion exchange chromatography if needed

• Storage conditions:

  • Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 50% glycerol

  • Temperature: -80°C for long-term storage

For optimal activity, purified TrmD should be tested with its cofactor requirements, particularly Mg²⁺, which is essential for its catalytic function .

How can the enzymatic activity of recombinant H. ducreyi TrmD be measured?

Several complementary methodologies can be employed to measure TrmD activity:

• Radiometric assay:

  • Substrate: Purified tRNA or synthetic ASL with 9-bp extended stem

  • Methyl donor: [³H]-labeled or [¹⁴C]-labeled S-adenosyl methionine

  • Reaction conditions: 37°C, pH 7.5, with 5-10 mM Mg²⁺

  • Detection: Filter binding assay followed by scintillation counting

• HPLC-based assay:

  • Digest tRNA post-reaction with nucleases

  • Separate modified nucleosides by reverse-phase HPLC

  • Quantify m¹G using UV detection at 254 nm

  • Compare with synthetic m¹G standards

• Mass spectrometry:

  • Analyze tRNA digests by LC-MS/MS

  • Identify and quantify m¹G37 modification

  • High sensitivity for low-abundance modifications

• In vivo frameshifting reporter assay:

  • Construct reporter with frameshifting-prone sequence

  • Express in H. ducreyi or E. coli system with wild-type or mutant TrmD

  • Measure frameshifting frequency through reporter activity

When conducting these assays, it is crucial to include appropriate controls and to consider the asymmetric nature of TrmD catalysis, as one active site in the dimer is catalytically active while the other is inactive .

What is the structural basis for TrmD's unique AdoMet binding and catalytic mechanism?

TrmD employs a unique structural arrangement for AdoMet binding and catalysis:

• Trefoil knot structure:
  The TrmD trefoil knot consists of three β-strands (β3, β4, and β5) at the central β-sheet. This knot forms a complex structure where:

  • β3 is followed by a loop that turns at the back and emerges into β4

  • The end of β4 is followed by another loop that turns into β5

  • β5 makes a circular insertion into the knot by crossing over β3

• AdoMet binding mode:

  • TrmD binds AdoMet in an unusual bent shape

  • This conformation constrains the adenosine and methionine moieties to face each other

  • This differs dramatically from the typical binding in an open space of a dinucleotide fold seen in most methyl transferases including Trm5

• Magnesium requirement:

  • TrmD uniquely requires Mg²⁺ in its catalytic mechanism

  • This requirement distinguishes it from other AdoMet-dependent methyltransferases

  • The Mg²⁺ likely facilitates proper positioning of the G37 substrate

• Asymmetric catalysis:

  • The trefoil knot mediates AdoMet signaling across the dimer interface

  • This confers asymmetry between the two active sites

  • One site is catalytically active while the other remains inactive

  • Mutations disrupting knot stability eliminate this asymmetry but severely compromise catalytic efficiency (by ~1200-fold)

This structural information provides valuable insights for structure-based drug design targeting TrmD.

How does TrmD recognize its tRNA substrate, and what are the minimal requirements for substrate recognition?

TrmD employs a specific recognition mechanism for its tRNA substrate:

Understanding these recognition requirements is essential for designing substrate analogs or competitive inhibitors in research applications.

Why is TrmD considered a high-priority antimicrobial target?

TrmD represents an excellent antimicrobial target for several reasons:

• Essential function:

  • TrmD is essential for bacterial survival

  • The m¹G37 modification prevents lethal frameshifting errors during translation

• Bacterial specificity:

  • TrmD is present only in bacteria

  • Eukaryotes and archaea use the structurally distinct Trm5

  • This provides a wide therapeutic window with minimal off-target effects

• Structural uniqueness:

  • TrmD's trefoil knot and unusual AdoMet binding

  • Distinctive catalytic mechanism requiring Mg²⁺

  • These unique features allow for highly specific inhibitor design

• Conservation across bacterial species:

  • TrmD is broadly conserved among bacterial species

  • Inhibitors could potentially have broad-spectrum activity

  • Conservation exists in both Gram-positive and Gram-negative bacteria

• Pathogen relevance:

  • Present in important pathogens including H. ducreyi

  • H. ducreyi causes chancroid, a genital ulcer disease that facilitates HIV transmission

  • Targeting TrmD could help address diseases with significant public health impacts

The fundamental differences between TrmD and Trm5 suggest that TrmD-specific inhibitors would have minimal interaction with human Trm5, thus limiting potential side effects .

What is the relationship between H. ducreyi infection, TrmD function, and disease progression?

The relationship between H. ducreyi infection, TrmD function, and disease progression can be understood through several key connections:

• H. ducreyi pathogenesis:

  • H. ducreyi causes chancroid, a genital ulcer disease, and cutaneous ulcers in children

  • During infection, H. ducreyi resides in an anaerobic abscess environment

  • Chancroidal ulcers serve as a portal of entry for HIV, establishing an "infectious synergy"

• TrmD function under infection conditions:

  • Gene regulation in H. ducreyi during infection overlaps significantly with its response to anaerobiosis

  • Under anaerobic conditions (similar to in vivo infection), H. ducreyi shows distinct transcriptional profiles

  • These adaptations likely include changes in TrmD expression or activity

• Translational fidelity and virulence:

  • TrmD synthesizes m¹G37-tRNA, preventing frameshifting

  • Proper protein synthesis is essential for virulence factor production

  • Mutations in TrmD that block intramolecular signaling decrease m¹G37-tRNA synthesis, prompting +1-frameshifts and premature termination of protein synthesis

• Survival advantage:

  • Growth under anaerobic conditions fosters the viability of H. ducreyi over time

  • While aerobically grown bacteria show markedly reduced viability by 36h, anaerobically grown bacteria maintain viability for 48h

  • This survival advantage may be linked to translational adaptations involving TrmD

Understanding this relationship provides insights into both basic H. ducreyi biology and potential intervention strategies targeting TrmD.

What approaches can be used to screen for potential inhibitors of H. ducreyi TrmD?

Several complementary approaches can be employed to identify potential inhibitors of H. ducreyi TrmD:

• High-throughput screening (HTS):

  • Biochemical assays:

    • Fluorescence-based AdoMet analog incorporation

    • Scintillation proximity assay with radiolabeled AdoMet

    • FRET-based conformational change detection

  • Throughput: 10,000-100,000 compounds per day

  • Advantages: Direct measure of inhibition

• Structure-based virtual screening:

  • Target the unique trefoil knot AdoMet binding site

  • Focus on compounds that can adopt the bent conformation of AdoMet

  • Molecular docking of compound libraries against crystal structure

  • Follow up with molecular dynamics simulations to assess binding stability

  • Prioritize compounds that interfere with the asymmetric signaling across the dimer

• Fragment-based drug discovery:

  • Screen small molecular fragments for binding to TrmD

  • Methods: NMR, thermal shift assays, X-ray crystallography

  • Optimize hits through fragment linking or growing

  • Advantage: Higher hit rates and chemical diversity

• Whole-cell screening:

  • Test compounds for growth inhibition of H. ducreyi

  • Confirm TrmD as the target through:

    • Overexpression studies

    • Resistant mutant generation and sequencing

    • Enzymatic assays with purified protein

  • Advantage: Identifies compounds with cellular permeability

• Mechanistic inhibitor development:

  • Design inhibitors that target unique aspects:

    • Mg²⁺ coordination interference

    • Disruption of trefoil knot dynamics

    • Prevention of asymmetric catalysis

    • Competitive inhibition of tRNA binding

When screening for TrmD inhibitors, it's critical to include counter-screens against human Trm5 to ensure selectivity and establish a therapeutic window early in the discovery process.

How do mutations in the TrmD trefoil knot affect intramolecular signaling and enzyme function?

Mutations in the TrmD trefoil knot have profound effects on intramolecular signaling and enzyme function:

These findings highlight the critical importance of the trefoil knot structure not merely for AdoMet binding but for the proper orchestration of the entire catalytic cycle through intramolecular signaling.

What is the role of Mg²⁺ in TrmD catalysis and how does it affect inhibitor design?

The requirement for Mg²⁺ in TrmD catalysis is a unique feature with significant implications:

• Catalytic mechanism:

  • TrmD is unusual among AdoMet-dependent methyl transferases in requiring Mg²⁺

  • Mg²⁺ likely plays roles in:

    • Proper positioning of the G37 substrate

    • Stabilization of transition states

    • Facilitation of methyl transfer

• Regulatory significance:

  • The Mg²⁺ dependence is important for regulating Mg²⁺ transport in bacterial pathogens

  • This suggests TrmD may act as a metabolic sensor linking translation fidelity to metal homeostasis

• Inhibitor design implications:

  • Mg²⁺ requirement creates unique opportunities for inhibitor design:

    • Metal chelators with appropriate specificity

    • Compounds that distort the Mg²⁺ binding site

    • Molecules that compete with Mg²⁺ binding

• Structural considerations:

  • Inhibitor screening should be conducted in the presence of Mg²⁺ to capture the physiologically relevant enzyme conformation

  • Crystal structures with and without Mg²⁺ would be valuable for structure-based drug design

  • The Mg²⁺ binding site provides an additional pocket that could be exploited for enhancing inhibitor specificity

This unique requirement for Mg²⁺ provides both mechanistic insights and practical opportunities for developing specific TrmD inhibitors.

How does H. ducreyi TrmD expression and activity change under different environmental conditions?

H. ducreyi adapts to different environmental conditions with distinct gene expression patterns that may affect TrmD function:

• Anaerobic vs. aerobic growth:

  • Anaerobic growth results in distinct transcriptional profiles compared to aerobic growth

  • PCoA plots show clear separation between these conditions

  • Anaerobic growth fosters H. ducreyi viability over extended periods:

    • Aerobically grown bacteria: markedly reduced viability by 36h, absent by 48h

    • Anaerobically grown bacteria: maintain viability for 48h

• Time-dependent changes:

  • Early time points (4h vs. 8h) show few differentially expressed genes (DEGs)

  • By 18h under anaerobic conditions:

    • 18 upregulated DEGs

    • 16 downregulated DEGs

• Metabolic adaptations:

  • Upregulated pathways under anaerobiosis include:

    • Purine metabolism

    • Alternative carbon source utilization

    • Nitrate reduction

    • Glycine metabolism

    • Tetrahydrofolate synthesis

  • Downregulated pathways include:

    • Electron transport

    • Thiamine biosynthesis

    • DNA recombination

    • Peptidoglycan synthesis

    • Riboflavin synthesis/modification

• In vivo relevance:

  • DEGs identified between 4h aerobic and 18h anaerobic growth significantly overlap with those found between inocula and pustules in human infection studies

  • This suggests adaptation to anaerobiosis is a major component of H. ducreyi gene regulation in vivo

While the search results don't specifically mention TrmD regulation under different conditions, the global transcriptional changes observed suggest that translation-related processes are likely affected during adaptation to the anaerobic environment encountered during infection.

How can recombinant H. ducreyi TrmD be used as a tool for studying tRNA modifications?

Recombinant H. ducreyi TrmD offers several research applications for studying tRNA modifications:

• Structural studies of tRNA recognition:

  • Use purified TrmD with various tRNA constructs to define recognition elements

  • Compare D-ASL requirements between H. ducreyi TrmD and other bacterial TrmDs

  • Determine if recognition mechanisms are conserved across bacterial species

• In vitro reconstitution of modification pathways:

  • Use TrmD in sequential modification experiments to study the order of modifications

  • Analyze potential crosstalk between m¹G37 and other modifications

  • Develop complete in vitro systems for studying tRNA maturation

• Translational fidelity assessment:

  • Create in vitro translation systems with and without TrmD-modified tRNAs

  • Measure frameshifting rates with reporter constructs

  • Identify sequence contexts most sensitive to m¹G37 modification

• Evolutionary studies:

  • Compare substrate specificity between H. ducreyi TrmD and other bacterial TrmDs

  • Analyze functional differences from the eukaryotic/archaeal Trm5

  • Understand the evolutionary pressure for maintaining this essential modification

• Development of tRNA-based tools:

  • Engineer specialized tRNAs with controlled modification states

  • Create biosensors based on tRNA modification detection

  • Design synthetic biology applications leveraging TrmD function

These applications would benefit from the understanding that TrmD requires only the D-ASL structure and does not need prior modifications, aminoacylation, or CCA addition to the tRNA substrate .

What are the potential connections between TrmD function and antibiotic resistance in H. ducreyi?

While direct evidence linking TrmD to antibiotic resistance in H. ducreyi is not presented in the search results, several potential connections can be hypothesized based on known mechanisms:

• Translational accuracy and stress responses:

  • TrmD ensures accurate translation by preventing frameshifting

  • Proper protein synthesis is crucial during stress responses, including antibiotic exposure

  • Subtle changes in TrmD activity could affect the translation of stress response proteins

• Adaptation to anaerobic conditions:

  • H. ducreyi shows distinct transcriptional responses to anaerobiosis, which reflects in vivo conditions

  • These adaptations may include changes in membrane permeability and efflux pump expression

  • TrmD-mediated translational control may regulate these resistance mechanisms

• Persister cell formation:

  • Bacterial persistence (a dormant state tolerant to antibiotics) often involves translational pausing

  • TrmD activity could influence persister formation through effects on translation rate

  • This might be particularly relevant given that H. ducreyi maintains viability longer under anaerobic conditions

• Evolutionary considerations:

  • If TrmD inhibitors are developed as antimicrobials, resistance mechanisms would likely emerge

  • Understanding potential resistance pathways proactively would aid drug development

  • This might include TrmD mutations, overexpression, or bypass mechanisms

Future research directions should include examining TrmD expression levels in antibiotic-resistant H. ducreyi isolates and investigating whether modulation of TrmD activity affects susceptibility to different antibiotic classes.

How might understanding H. ducreyi TrmD contribute to novel diagnostic approaches for chancroid?

Understanding H. ducreyi TrmD could contribute to novel diagnostic approaches for chancroid in several ways:

• Serological tests based on TrmD:

  • TrmD is an essential bacterial protein that may elicit antibody responses

  • Similar to the approach with outer membrane proteins described for chancroid diagnostics

  • A recombinant TrmD-based enzyme immunoassay could potentially detect anti-TrmD antibodies in patients

• Molecular detection methods:

  • PCR-based detection of the trmD gene could complement existing molecular diagnostics

  • The high conservation of trmD across bacteria requires careful primer design for specificity

  • Multiplex PCR including trmD and other H. ducreyi-specific genes could improve sensitivity

• Functional diagnostics:

  • Detection of m¹G37-modified tRNAs in clinical samples could indicate active H. ducreyi infection

  • Mass spectrometry approaches could potentially identify this signature

  • This approach would detect metabolically active bacteria rather than just DNA

• TrmD inhibitor-based approaches:

  • If specific TrmD inhibitors are developed, they could be used in selective growth media

  • Such media could improve the currently insensitive culture-based diagnosis

Current diagnostic challenges include:

• Clinical diagnosis of chancroid is inaccurate

• Isolation of H. ducreyi from ulcer specimens is insensitive

• PCR detection of H. ducreyi DNA is more sensitive than culture but not readily available in endemic areas

Given these challenges, novel approaches based on TrmD could potentially improve chancroid diagnostics, especially in resource-limited settings where the disease is endemic.