Recombinant Francisella tularensis subsp. novicida Lipoyl synthase (lipA)

Why is F. novicida preferred as a model organism for studying Francisella metabolic enzymes?

F. novicida serves as a representative biosafety level 2 (BSL-2) model organism for F. tularensis research, offering significant experimental advantages over the highly virulent subspecies tularensis (Type A) strains . While sharing extensive genomic similarity with pathogenic Francisella strains, F. novicida causes disease in mice but is attenuated in humans, making it safer to manipulate in laboratory settings . This biological similarity extends to metabolic pathways, allowing researchers to study enzymes like LipA without requiring BSL-3 containment facilities. Additionally, F. novicida's higher amenability to genetic manipulation facilitates molecular studies of metabolic enzymes that would be challenging in more virulent strains .

How does F. novicida metabolism differ from other Francisella subspecies?

Metabolic differences between F. novicida and highly virulent Francisella subspecies contribute significantly to their distinct pathogenicity profiles. Research has demonstrated that F. novicida exhibits markedly more proinflammatory properties, with differences in TLR signaling, cytokine production, and inflammasome activation that affect bacterial replication in macrophages . A notable example from recent research shows that F. tularensis subspecies tularensis has acquired five amino acid substitutions in RibD, a riboflavin pathway enzyme, that allows it to evade MAIT cell recognition - a feature not present in F. novicida . These metabolic distinctions suggest that studying enzymes like LipA may reveal important adaptations that contribute to virulence differences among Francisella subspecies.

What expression systems are most effective for producing recombinant F. novicida proteins?

For F. novicida proteins, several expression systems have demonstrated efficacy in published research. Complementation experiments frequently utilize the expression plasmid pMP831 containing genes under control of native Francisella promoters, such as the one upstream of FTN_1480 . For recombinant protein production, E. coli-based systems have been successfully employed for F. novicida proteins like LdcF, where structural and functional characterization required substantial protein yields . When expressing iron-sulfur cluster proteins like LipA, specialized E. coli strains with enhanced capacity for proper cofactor incorporation may be necessary. The choice between homologous expression in F. novicida versus heterologous expression in E. coli depends on experimental goals, with the former preserving native post-translational modifications and the latter typically providing higher yields for structural studies.

What methods are most effective for generating lipA deletion mutants in F. novicida?

Creating precise lipA deletion mutants in F. novicida requires a methodical approach similar to that used for other gene deletions in this organism. Based on established protocols from F. novicida research, the following methodology is recommended:

• Design PCR primers to amplify approximately 1kb flanking regions upstream and downstream of the lipA gene

• Create a construct that replaces the lipA coding sequence with an antibiotic resistance cassette (commonly kanamycin)

• Transform the construct into F. novicida using electroporation (typically 2.5kV, 25μF, 600Ω)

• Select transformants on media containing appropriate antibiotics

• Confirm deletion through PCR verification and sequencing

• Complement the mutation by introducing intact lipA on the pMP831 plasmid under a native promoter

This approach has been successfully employed for generating mutations in metabolic genes including lpxF, manB, manC, and kdtA in F. novicida . Proper phenotypic validation should include growth curve analysis in defined media and assessment of metabolic functions dependent on lipoic acid.

How should researchers evaluate intracellular survival of lipA mutants?

Evaluation of intracellular survival for F. novicida lipA mutants should follow a systematic approach:

• Infect murine macrophage cell lines (J774A.1) and primary bone marrow-derived macrophages (BMDMs) at a multiplicity of infection (MOI) of 10-20 bacteria per cell

• Allow for phagocytosis (1-2 hours), then treat with gentamicin (10-50 μg/ml) to kill extracellular bacteria

• Lyse infected cells at defined timepoints (0, 6, 24, 48 hours) and enumerate intracellular bacteria by plating serial dilutions

• Compare growth kinetics of wild-type, ΔlipA mutant, and complemented strains

• Assess macrophage viability using LDH release assays to determine cytotoxicity

• Quantify key inflammatory cytokines (TNF-α, IL-1β, IL-6) to evaluate immunostimulatory properties

This methodology has effectively characterized survival defects in other F. novicida mutants with metabolic deficiencies . For lipA mutants specifically, additional evaluation of host cell metabolic parameters may be informative, as lipoic acid is essential for several metabolic processes that might be affected during infection.

What purification strategy yields optimal activity for recombinant F. novicida LipA?

Purification of enzymatically active recombinant F. novicida LipA requires careful consideration of its iron-sulfur cluster requirements. Based on successful purification of other F. novicida enzymes and iron-sulfur proteins generally, the following protocol is recommended:

• Express LipA in E. coli BL21(DE3) containing pRARE plasmid for rare codon optimization

• Culture at reduced temperature (18-22°C) after induction with 0.1-0.5 mM IPTG

• Include iron supplementation (0.1 mM ferric ammonium citrate) and L-cysteine (0.5 mM) in growth media

• Perform all purification steps under anaerobic conditions or with buffer containing 5 mM DTT to prevent oxidation

• Utilize immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography

• Confirm iron-sulfur cluster incorporation via UV-visible spectroscopy (characteristic absorption at 420 nm)

This approach has been successful for purifying the F. novicida enzyme LdcF in its active form . For LipA specifically, additional consideration should be given to reconstituting the iron-sulfur cluster in vitro if necessary, using established protocols with ferrous ammonium sulfate, L-cysteine, and a sulfur transferase enzyme under anaerobic conditions.

What assays best measure F. novicida LipA enzymatic activity?

Measuring LipA activity requires specialized assays that detect the insertion of sulfur atoms into octanoyl substrates. The following methodological approaches are recommended:

• Direct activity assay: Monitor conversion of octanoyl substrate to lipoylated product using:

  • HPLC analysis with UV detection at 254 nm

  • LC-MS/MS detection of lipoylated products with multiple reaction monitoring

  • Gel-shift assays using native PAGE to detect lipoylation of substrate proteins

• Coupled enzyme assays: Measure activity of lipoic acid-dependent enzymes (pyruvate dehydrogenase, α-ketoglutarate dehydrogenase) after LipA reaction

• Growth complementation: Assess ability of F. novicida LipA to restore growth of E. coli lipA mutants in minimal media

Similar analytical approaches have been employed for characterizing other F. novicida metabolic enzymes like LdcF . When working with LipA specifically, reactions should be performed under strictly anaerobic conditions to preserve iron-sulfur cluster integrity.

How can researchers investigate the structural features of F. novicida LipA?

Structural characterization of F. novicida LipA requires a multi-technique approach:

This approach successfully determined the crystal structure of F. novicida LdcF and enabled comparative analysis with E. coli homologs . For LipA specifically, attention to maintaining anaerobic conditions during purification and crystallization is critical to preserve the integrity of iron-sulfur clusters.

How does LipA contribute to F. novicida stress resistance and pathogenesis?

Investigating LipA's role in stress resistance and pathogenesis requires a comprehensive approach:

• Oxidative stress assays:

  • Challenge wild-type and ΔlipA F. novicida with H₂O₂, paraquat, or SIN-1

  • Measure survival rates at different timepoints

  • Quantify intracellular ROS levels using fluorescent probes

• Proteomic profiling:

  • Perform mass spectrometry-based quantitative proteomics comparing ΔlipA vs. wild-type F. novicida

  • Identify proteins with significantly altered expression levels

  • Focus on DNA repair proteins and oxidative stress response factors

• Metabolomic analysis:

  • Characterize metabolic profiles using LC-MS to identify metabolic pathways affected by lipA deletion

  • Pay particular attention to TCA cycle intermediates and branched-chain amino acid metabolism

Previous research with F. novicida mutants has demonstrated that deletion of metabolic enzymes like LdcF affects expression of approximately 80 proteins, including several DNA repair proteins involved in oxidative stress resistance . Similar approaches would likely reveal how LipA contributes to the metabolic networks supporting F. novicida pathogenesis.

How does F. novicida LipA activity influence host immune responses?

Investigating the immunological consequences of LipA function requires methodical assessment of host immune responses:

• Macrophage infection studies:

  • Compare cytokine profiles (TNF-α, IL-1β, IL-6, IL-8) in macrophages infected with wild-type vs. ΔlipA F. novicida

  • Assess inflammasome activation by measuring caspase-1 activation and IL-1β processing

  • Quantify NF-κB pathway activation using reporter cell lines

• Neutrophil response analysis:

  • Evaluate the effect on neutrophil lifespan, as F. novicida is known to inhibit spontaneous apoptosis

  • Measure PS externalization, nuclear morphology changes, and caspase-3/8/9 processing

  • Determine if ΔlipA mutants differ in their ability to extend neutrophil survival

• MAIT cell activation assays:

  • Test whether metabolites produced in LipA-dependent pathways affect MAIT cell activation

  • Compare TCR-dependent responses between wild-type and ΔlipA F. novicida

  • Evaluate if LipA function affects the bacteria's ability to evade MAIT cell recognition

Research has shown that metabolic enzymes in Francisella can significantly affect immune recognition, as demonstrated by the RibD enzyme's role in MAIT cell evasion . Similar immune evasion mechanisms might be linked to lipoic acid metabolism through LipA activity.

What role does LipA play in F. novicida intracellular adaptation?

Characterizing LipA's contribution to intracellular adaptation requires a multi-faceted approach:

• Transcriptional analysis:

  • Perform RNA-seq comparing gene expression in F. novicida grown in broth versus intracellular environments

  • Specifically monitor lipA expression changes during different stages of infection

  • Identify potential regulatory mechanisms controlling lipA expression

• Metabolic requirement testing:

  • Create conditional lipA mutants using inducible systems

  • Determine if lipA is essential during specific stages of the intracellular lifecycle

  • Investigate whether host-derived lipoic acid can complement bacterial LipA deficiency

• Intracellular trafficking analysis:

  • Track phagosomal escape kinetics of wild-type versus ΔlipA F. novicida

  • Use fluorescence microscopy to monitor co-localization with endosomal/lysosomal markers

  • Determine if LipA affects the bacterium's ability to reach its cytosolic replication niche

Research on F. novicida has shown that metabolic adaptations significantly affect intracellular survival, with mutations in genes like lpxF demonstrating altered host cell interactions . Similar phenotypic analyses would reveal LipA's specific contributions to intracellular adaptation.

How does F. novicida compare to F. tularensis in LipA-dependent metabolic pathways?

Comparative analysis between F. novicida and virulent F. tularensis strains provides insights into the evolution of metabolic pathways:

• Sequence and structural comparison:

  • Align LipA sequences from F. novicida, F. tularensis subsp. tularensis, and F. tularensis subsp. holarctica

  • Identify amino acid substitutions that might affect function, similar to the five substitutions identified in RibD

  • Model structural consequences of any identified variations

• Expression level analysis:

  • Compare lipA transcription and translation between subspecies using qRT-PCR and western blotting

  • Determine if regulatory mechanisms differ between subspecies

  • Assess whether expression changes under various stress conditions differ between strains

• Metabolomic profiling:

  • Compare lipoic acid-dependent metabolite profiles between subspecies

  • Identify metabolic pathways differentially affected by LipA activity across subspecies

  • Correlate metabolic differences with virulence phenotypes

Research has demonstrated that F. tularensis can acquire immune evasion capacity through alteration of metabolic programs during evolution, as exemplified by the RibD enzyme . Similar evolutionary adaptations might be present in the lipoic acid biosynthesis pathway.

What methodologies should be used to evaluate LipA as a potential drug target?

Assessing LipA as a therapeutic target requires systematic evaluation:

• Target validation:

  • Determine essentiality through conditional mutants or CRISPRi approaches

  • Evaluate growth defects in various media and infection models

  • Verify that human cells can synthesize or acquire lipoic acid through pathways independent of bacterial LipA

• High-throughput screening:

  • Develop a miniaturized LipA activity assay suitable for 384-well format

  • Screen compound libraries (10,000-100,000 compounds) at 1-10 μM concentrations

  • Confirm hits using secondary assays that verify on-target activity

• Structure-based drug design:

  • Use crystallographic data to identify unique features of the F. novicida LipA active site

  • Perform virtual screening of compound libraries targeting the SAM binding site or iron-sulfur cluster interface

  • Design compounds that exploit structural differences between bacterial and mammalian lipoic acid synthesis pathways

Similar approaches have identified drug targets in other metabolic pathways of F. novicida, with LdcF recently characterized as a potential target due to its role in oxidative stress resistance .

How should researchers evaluate F. novicida LipA as a vaccine antigen?

Assessment of LipA as a vaccine antigen requires a methodical immunological approach:

• Antigen preparation and formulation:

  • Express and purify recombinant LipA under conditions that maintain native conformation

  • Test multiple adjuvant formulations (alum, MF59, CpG oligonucleotides)

  • Evaluate various delivery platforms (protein subunit, DNA vaccine, viral vectors)

• Immunogenicity testing:

  • Measure antibody responses (IgG, IgA) in serum and mucosal secretions

  • Assess T cell responses through ELISpot assays for IFN-γ, IL-2, and IL-17

  • Determine if immune responses cross-react with LipA from virulent F. tularensis strains

• Protection studies:

  • Challenge immunized mice with lethal doses of F. novicida

  • Evaluate bacterial burdens in organs and survival rates

  • Test cross-protection against F. tularensis LVS or attenuated Type A strains

Previous research has shown that immunization with purified LPS from F. novicida protects against homologous challenge but not against F. tularensis subspecies holarctica, indicating strain-specific protective antigens . Similar evaluation of LipA would determine its potential as a cross-protective antigen.

What approaches can identify host factors that interact with F. novicida LipA?

Identifying host-pathogen protein interactions involving LipA requires multiple complementary techniques:

• Affinity purification-mass spectrometry (AP-MS):

  • Express epitope-tagged LipA in F. novicida

  • Infect host cells and crosslink protein complexes

  • Purify LipA using antibodies against the epitope tag

  • Identify co-purifying host proteins by mass spectrometry

• Proximity-dependent biotin labeling:

  • Create LipA fusions with BioID or APEX2 enzymes

  • Express constructs in F. novicida and infect host cells

  • Activate proximity labeling during infection

  • Purify biotinylated proteins and identify by mass spectrometry

• Yeast two-hybrid screening:

  • Use LipA as bait against human cDNA libraries

  • Validate potential interactions with co-immunoprecipitation

  • Confirm biological relevance through mutational analysis

These approaches have identified important host interactions for other bacterial factors but have not yet been extensively applied to metabolic enzymes like LipA in Francisella. Discovering such interactions could reveal how metabolic enzymes might directly interface with host processes beyond their canonical functions.

How can CRISPR-Cas9 techniques be optimized for F. novicida lipA modification?

Implementing CRISPR-Cas9 genome editing in F. novicida requires specialized approaches:

• Vector system optimization:

  • Use the pCasKm plasmid containing Cas9 under control of a Francisella promoter

  • Design sgRNAs targeting lipA with minimal off-target potential

  • Include homology-directed repair templates with appropriate selectable markers

• Transformation protocol:

  • Prepare electrocompetent F. novicida cells from early log phase cultures (OD₆₀₀ = 0.3-0.5)

  • Transform cells with 500 ng plasmid DNA using 2.5 kV, 25 μF, 600 Ω settings

  • Allow recovery in brain heart infusion (BHI) media for 4 hours before plating on selective media

• Editing verification:

  • Screen transformants by colony PCR

  • Verify modifications by Sanger sequencing

  • Assess potential off-target effects by whole-genome sequencing

This methodology builds upon established genetic manipulation techniques for F. novicida while incorporating more precise CRISPR-based approaches that have been adapted for use in related bacterial species.

What analytical techniques can reveal the impact of lipA disruption on F. novicida metabolism?

Comprehensive metabolic analysis requires integration of multiple analytical platforms:

• Untargeted metabolomics:

  • Extract metabolites from wild-type and ΔlipA F. novicida using methanol/water/chloroform

  • Analyze using HILIC-MS and reverse-phase LC-MS/MS

  • Identify significantly altered metabolites through multivariate statistical analysis

• ¹³C-flux analysis:

  • Culture bacteria with ¹³C-labeled glucose or amino acids

  • Trace carbon flow through central metabolic pathways

  • Quantify differences in metabolic flux distributions between wild-type and mutant strains

• Proteomics integration:

  • Combine metabolomic data with proteomic profiling

  • Identify metabolic enzymes with altered expression in response to lipA deletion

  • Map changes onto metabolic pathway models

Similar multi-omics approaches have revealed how other metabolic enzymes in F. novicida, such as LdcF, affect broader cellular networks involving DNA repair proteins and stress response factors .

How do mutations in F. novicida lipA affect pathogen evolution during host adaptation?

Investigating evolutionary aspects of LipA function requires experimental evolution approaches:

• Serial passage experiments:

  • Culture F. novicida under selective pressures mimicking host environments

  • Sequence lipA and related genes after multiple passages

  • Identify adaptive mutations that arise in metabolic pathways

• Compensatory mutation analysis:

  • Generate lipA point mutants with partially compromised function

  • Select for suppressor mutations that restore fitness

  • Map genetic interactions within metabolic networks

• Comparative genomics:

  • Analyze lipA sequences across clinical and environmental Francisella isolates

  • Identify signatures of selection in metabolic genes

  • Correlate sequence variations with host range or virulence phenotypes

Research has shown that F. tularensis has acquired specific amino acid substitutions in metabolic enzymes like RibD during evolution that contribute to immune evasion . Similar evolutionary studies of lipA would reveal whether lipoic acid metabolism has undergone parallel adaptive changes.