Recombinant Staphylococcus aureus L-lactate dehydrogenase 2 (ldhB)

What is Staphylococcus aureus L-lactate dehydrogenase 2 and how does it function?

S. aureus encodes three lactate biosynthetic enzymes: inducible L-lactate dehydrogenase (Ldh1), a second L-lactate dehydrogenase (Ldh2), and a D-lactate dehydrogenase (Ddh). These enzymes convert pyruvate to lactate while regenerating NAD+ from NADH, helping maintain redox balance during bacterial metabolism. While Ldh1 is unique to S. aureus, Ldh2 is shared among other staphylococcal species and is expressed constitutively even in the absence of stress conditions . The key functional difference is that Ldh2 makes a relatively minor contribution to redox balance during nitric oxide (NO·) stress compared to the more strongly induced Ldh1 .

What expression systems are most effective for producing recombinant S. aureus Ldh2?

For efficient expression of recombinant S. aureus Ldh2, E. coli-based expression systems using pET vectors with T7 promoters have shown good results. The protein should be expressed with an affinity tag (typically His6) to facilitate purification. Optimal expression conditions include:

• Induction with 0.5 mM IPTG at lower temperatures (16-20°C)

• Overnight expression to allow proper protein folding

• Use of E. coli BL21(DE3) or Rosetta strains to overcome potential codon bias issues

• Inclusion of 0.1 mM NADH in purification buffers to stabilize enzyme activity

Following expression, purification via metal affinity chromatography followed by size exclusion chromatography yields high-purity enzyme suitable for biochemical and structural studies.

How does Ldh2 differ from other lactate dehydrogenases in S. aureus?

The three lactate dehydrogenases in S. aureus have distinct characteristics and roles that can be summarized in the following table:

While all three enzymes help maintain redox balance by regenerating NAD+, Ldh1 plays the predominant role during nitric oxide stress, with Ldh2 making a comparatively smaller contribution despite being constitutively expressed .

What is the relationship between Ldh2 and L-lactate-quinone oxidoreductase (Lqo)?

S. aureus possesses an L-lactate-quinone oxidoreductase (Lqo, formerly annotated as Mqo2) that catalyzes the reverse reaction of Ldh2, oxidizing L-lactate back to pyruvate. Unlike lactate dehydrogenases that use NAD+ as an electron acceptor, Lqo transfers electrons directly to the respiratory quinone pool via an FAD cofactor . This enzyme allows S. aureus to reassimilate L-lactate specifically after prolonged NO· exposure, a capability that is important for virulence, particularly in cardiac infections. Mechanistically, while Ldh2 primarily functions in L-lactate production, Lqo enables the bacterium to utilize L-lactate as a carbon source during aerobic growth and NO· stress conditions .

What is the optimal colorimetric assay for measuring Ldh2 activity in research settings?

A highly reliable colorimetric assay for measuring Ldh2 activity employs nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS). This assay is based on the principle that NADH released during the LDH reaction reduces NBT via PMS, resulting in a blue-purple formazan that can be measured spectrophotometrically at ~570 nm .

Optimized Protocol for 96-well Format:

Incubate the reaction at 37°C for 30 minutes in the dark (PMS is light-sensitive) and measure absorbance at 570 nm. This method is suitable for high-throughput screening of potential inhibitors at early stages of drug discovery .

Important Considerations:

• When working with cell lysates, proteins should first be precipitated to remove interfering detergents, then dissolved in a suitable buffer before conducting the assay .

• Include appropriate controls: no-enzyme, no-substrate, and known inhibitors.

How can I design genetic approaches to study Ldh2 function independently from other lactate dehydrogenases?

To isolate and study the specific role of Ldh2, consider the following genetic approach:

• Generate single (Δldh2), double (Δldh1Δldh2), and triple (Δldh1Δldh2Δddh) knockout mutants using CRISPR-Cas9 or allelic replacement methods.

• Create complementation strains by reintroducing ldh2 on a plasmid under its native promoter or an inducible promoter.

• Compare phenotypes across these strains under various conditions:

  • Growth in different carbon sources (glucose, lactate, peptides)

  • Aerobic versus anaerobic conditions

  • Presence or absence of nitric oxide stress

  • Biofilm formation capabilities

• For precise gene editing using CRISPR-Cas9 in S. aureus:

  • Design sgRNAs targeting unique regions of ldh2 with minimal off-target effects

  • Construct a repair template with 500-1000 bp homology arms flanking the desired modification

  • Introduce plasmids sequentially, first into restriction-deficient S. aureus RN4220, then into your strain of interest

  • Confirm edits by PCR, sequencing, and functional assays

This genetic approach will allow you to attribute phenotypes specifically to Ldh2 function rather than compensatory effects from other lactate dehydrogenases.

How does Ldh2 contribute to S. aureus virulence in infection models?

While Ldh1 plays the major role in lactate production during NO· stress, Ldh2 still contributes to S. aureus pathogenesis, particularly in specific infection contexts. Research has shown that:

• Lactate production by S. aureus biofilms (to which Ldh2 contributes) inhibits host immune responses, particularly by inducing IL-10 production in myeloid-derived suppressor cells (MDSCs) .

• S. aureus with mutations in all lactate dehydrogenases (Δddh/Δldh1/Δldh2 triple mutant) shows significantly reduced ability to induce IL-10 in MDSCs and macrophages compared to wild-type bacteria .

• These effects on immunomodulation impact several virulence-associated genes, including those involved in MDSC recruitment and immunosuppression (IFNb1, Nfkbiz, Cxcl1, Cxcl3, Fpr1, and Ptgs2) .

To specifically assess Ldh2's contribution to virulence, experiments comparing wild-type S. aureus with isogenic Δldh2 mutants should be conducted in various infection models, with special attention to conditions where redox balance is challenged.

What is the relationship between Ldh2 and NO· resistance in S. aureus infections?

S. aureus demonstrates remarkable resistance to host-derived nitric oxide (NO·), which is a key component of innate immunity. Research indicates:

• During NO· exposure, S. aureus initially excretes large amounts of L-lactate (primarily via Ldh1, with contributions from Ldh2) to maintain redox balance when respiration is inhibited .

• After prolonged NO· exposure, S. aureus reassimilates L-lactate specifically via Lqo (L-lactate-quinone oxidoreductase) .

• This adaptation is particularly important in cardiac tissue, which naturally contains high levels of L-lactate. Mutants lacking Lqo show attenuated virulence specifically in cardiac infection models, a phenotype that is completely abrogated in mice unable to produce inflammatory NO· (iNOS−/−) .

• The ability of S. aureus to utilize a combination of peptides and L-lactate as carbon sources during NO· stress depends on intact lactate metabolism, including both production (via Ldh enzymes) and reassimilation (via Lqo) .

These findings highlight how the interplay between different components of S. aureus lactate metabolism, including Ldh2, contributes to its ability to survive host immune responses and cause persistent infections.

How can Ldh2 be targeted for potential antimicrobial development?

Targeting Ldh2 as part of S. aureus lactate metabolism offers several potential therapeutic strategies:

• Rational Inhibitor Design:

  • Structural analysis of S. aureus Ldh2 to identify unique pockets not present in human LDH isoforms

  • Virtual screening of compound libraries against these unique sites

  • Structure-activity relationship studies to optimize lead compounds

• High-throughput Screening:

  • The NBT/PMS colorimetric assay provides a robust platform for screening large compound libraries

  • Initial hits should be validated against purified human LDH enzymes to confirm selectivity

  • Promising compounds should be tested against S. aureus growth in vitro and in infection models

• Combined Targeting Approach:

  • Simultaneous inhibition of multiple lactate dehydrogenases (Ldh1, Ldh2, and Ddh) may be necessary for significant antimicrobial effects

  • Combination with inhibitors of Lqo could prevent both lactate production and reassimilation, potentially enhancing efficacy

While direct Ldh2 inhibition alone may have limited antimicrobial effects due to redundancy with Ldh1 and potential compensatory mechanisms, it represents one component of a broader strategy targeting S. aureus metabolism to overcome antimicrobial resistance.

What structural features distinguish S. aureus Ldh2 from human LDH enzymes?

Development of selective inhibitors targeting S. aureus Ldh2 requires understanding key structural differences between bacterial and human LDH enzymes. While comprehensive structural data specifically for S. aureus Ldh2 is still emerging, several general features can guide drug discovery efforts:

• The active site architecture of bacterial LDHs often differs from human isoforms in terms of:

  • Substrate binding pocket dimensions and hydrophobicity

  • Cofactor (NADH) binding region

  • Catalytic residues orientation

• Regions distant from the active site that influence allosteric regulation, oligomerization, or conformational dynamics may offer opportunities for selective targeting.

• The quaternary structure of LDH enzymes (typically tetrameric) presents potential interfaces for disruption that could be specific to the bacterial enzyme.

For definitive structural analysis, X-ray crystallography or cryo-EM studies of S. aureus Ldh2 in various ligand-bound states should be conducted. Molecular dynamics simulations can further identify differences in substrate binding, catalytic mechanism, and conformational changes during catalysis.

How does Ldh2 contribute to S. aureus metabolism in biofilm versus planktonic growth states?

Biofilm formation represents a major virulence determinant for S. aureus, particularly in medical device-associated infections. The role of Ldh2 may differ between planktonic and biofilm growth states:

• In biofilms, S. aureus exists in a heterogeneous environment with gradients of oxygen, nutrients, and pH, creating microniches where different metabolic strategies may be employed .

• Research has shown that lactate production by S. aureus biofilms inhibits phagocyte-based clearance through induction of IL-10, a key anti-inflammatory cytokine . While Ldh1 plays the predominant role, Ldh2 also contributes to this lactate pool.

• The S. aureus ddh/ldh1/ldh2 triple mutant that cannot produce D- or L-lactate shows substantially reduced ability to induce IL-10 in myeloid-derived suppressor cells (MDSCs) compared with wild-type S. aureus biofilm .

To specifically assess Ldh2's contribution to biofilm metabolism, researchers should compare wild-type and Δldh2 biofilms using:

• Transcriptomic and metabolomic analyses to identify altered pathways

• Measurement of oxygen and pH gradients within biofilms using microelectrodes

• Analysis of biofilm matrix composition and architecture

• Assessment of antibiotic tolerance profiles

What is the potential role of Ldh2 in S. aureus adaptation to different host environments?

S. aureus can colonize and infect virtually any human tissue, demonstrating remarkable adaptability to diverse host environments. Ldh2 may contribute to this adaptability in several ways:

• In tissues with high lactate levels (such as cardiac tissue), the interplay between Ldh2 and Lqo may allow S. aureus to utilize host-derived lactate as a carbon source .

• During infection, S. aureus encounters varying levels of oxygen, nutrients, and antimicrobial molecules, necessitating metabolic flexibility. The constitutive expression of Ldh2 ensures a baseline capacity for lactate metabolism across different conditions.

• In specialized niches such as abscesses or within phagocytes, where S. aureus faces extreme stress conditions, redundancy in lactate metabolism (through Ldh1, Ldh2, and Ddh) may provide a survival advantage.

Research exploring the tissue-specific contribution of Ldh2 should employ:

• Infection models in different tissues (skin, bone, heart, lung, kidney)

• Tissue-specific gene expression analysis during infection

• Comparison of wild-type and Δldh2 mutant growth in ex vivo tissue culture models

• In vivo competition assays between wild-type and Δldh2 mutants

How can CRISPR interference (CRISPRi) be applied to study Ldh2 function?

CRISPR interference (CRISPRi) offers advantages over traditional gene knockout approaches for studying Ldh2, particularly for assessing essential functions or for creating tunable repression:

• Design Considerations:

  • Use a catalytically inactive Cas9 (dCas9) expressed under an inducible promoter

  • Design sgRNAs targeting the promoter region or early coding sequence of ldh2

  • For S. aureus, optimize codon usage of dCas9 and ensure efficient sgRNA expression

• Experimental Approach:

  • Create a CRISPRi library with multiple sgRNAs targeting different regions of ldh2

  • Establish a system with tunable repression using inducible promoters

  • Validate knockdown efficiency by RT-qPCR and Western blot

  • Compare phenotypes at different levels of Ldh2 expression

• Applications:

  • Identify minimum Ldh2 levels required for various cellular functions

  • Study epistatic relationships with other metabolic enzymes

  • Perform time-resolved studies by inducing repression at specific stages of growth or infection

CRISPRi provides a complementary approach to gene deletions, allowing for more nuanced analysis of Ldh2 function across different conditions and growth phases.

What advanced analytical techniques can quantify the contribution of Ldh2 to S. aureus metabolism?

Several sophisticated analytical approaches can provide detailed insights into Ldh2's contribution to S. aureus metabolism:

• 13C Metabolic Flux Analysis:

  • Feed cultures 13C-labeled glucose and measure isotope distribution in metabolites

  • Compare flux distributions between wild-type and Δldh2 mutants

  • Quantify the percentage of carbon flow through Ldh2 versus other pathways

• Real-time Metabolite Monitoring:

  • Use biosensors or microelectrodes to measure lactate production in real-time

  • Implement microfluidic systems to control environmental conditions while monitoring metabolism

  • Compare dynamic responses to stressors between wild-type and mutant strains

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from the same samples

  • Use computational modeling to identify regulatory networks involving Ldh2

  • Detect compensatory mechanisms activated in response to Ldh2 deficiency

• Single-cell Analysis:

  • Apply single-cell RNA-seq to detect heterogeneity in ldh2 expression within populations

  • Use fluorescent reporters to monitor ldh2 expression at the single-cell level

  • Identify subpopulations with distinct metabolic states based on Ldh2 activity

These advanced techniques, especially when used in combination, can provide unprecedented insights into the specific contributions of Ldh2 to S. aureus metabolism under different environmental conditions.