Recombinant Bacillus cereus D-alanine--poly (phosphoribitol) ligase subunit 1 (dltA), partial

What is the structural basis for D-alanine selectivity in Bacillus cereus DltA protein?

The DltA protein from Bacillus cereus specifically selects D-alanine through a stereoselective mechanism enhanced by the medium-sized side chain of Cys-269. Crystal structure analysis at 2.0 Å resolution reveals that despite low sequence similarity to other adenylation domains, DltA structurally resembles acetyl-CoA synthetase. The enantiomer selection mechanism appears to be critically dependent on the Cys-269 residue, as demonstrated through mutational studies where the Ala-269 mutant protein shows marked loss of stereoselective properties .

This structural basis for substrate specificity provides important insights for researchers developing DltA inhibitors that could potentially increase gram-positive bacterial susceptibility to antibiotics.

How does the dlt operon function in Bacillus cereus?

The dlt operon in B. cereus consists of dltXABCD genes that collectively enable D-alanylation of teichoic acids in the bacterial cell wall. This process occurs through a well-defined mechanism:

• DltA (D-alanine-D-alanyl carrier protein ligase) activates D-alanine through adenylation

• The activated D-alanine is transferred to DltC (D-alanyl carrier protein)

• DltB is involved in D-alanyl-Dcp secretion

• DltD participates in selection of the Dcp carrier protein for ligation with D-alanine

• The D-alanyl moiety is ultimately incorporated into lipoteichoic acids (LTAs)

This incorporation modifies the net charge of the bacterial cell wall from negative to positive, creating an electrostatic repulsion mechanism against cationic antimicrobial peptides . Notably, D-alanine may be transferred from D-alanylated LTAs to wall-associated teichoic acids through transacylation.

What are the optimal methods for generating dltA knockout mutants in Bacillus cereus?

The most effective methodology for generating dltA knockout mutants in B. cereus involves a double-crossover homologous recombination approach using thermosensitive plasmids. Based on established protocols:

• Amplify DNA fragments flanking the dltA gene using PCR with primers containing appropriate restriction sites

• Insert these fragments on both sides of an antibiotic resistance marker (e.g., aphA3 conferring kanamycin resistance)

• Clone the construct into a thermosensitive plasmid (e.g., pMAD) that can replicate in both E. coli and Bacillus

• Introduce the demethylated plasmid into B. cereus by electroporation

• Select transformants on media containing appropriate antibiotics and X-Gal

• Perform temperature shifts to promote plasmid integration and then excision, resulting in allelic exchange

For instance, successful dltA knockout in B. cereus has been achieved using the pMAD-up-dlt-aphA3Km-down-dlt plasmid with growth at 30°C followed by shifts to 40°C, resulting in the deletion of dltA while inserting the kanamycin resistance marker .

How can researchers effectively express and purify recombinant DltA from Bacillus cereus?

Efficient expression and purification of recombinant B. cereus DltA requires:

• Gene amplification and cloning:

  • Amplify the dltA gene from B. cereus genomic DNA using high-fidelity polymerase

  • Include appropriate restriction sites in primers (e.g., SacI and XmaI)

  • Clone into an expression vector with a compatible promoter

• Expression optimization:

  • Transform into an appropriate E. coli expression strain

  • Induce protein expression (typically using IPTG for T7 promoter systems)

  • Optimize expression conditions: temperature (often lowered to 16-20°C), IPTG concentration, and induction time

• Purification strategy:

  • Lyse cells using sonication or pressure-based methods

  • Perform initial capture using affinity chromatography (His-tag purification is effective)

  • Further purify using ion exchange and size exclusion chromatography

  • Validate protein integrity using SDS-PAGE and functional assays

Researchers have successfully expressed DltA from B. cereus for crystallization studies using these approaches, resulting in properly folded, active protein suitable for structural and biochemical analysis .

How does the dlt operon contribute to Bacillus cereus virulence?

The dlt operon significantly enhances B. cereus virulence through multiple mechanisms:

• Antimicrobial peptide resistance:

  • D-alanylation of teichoic acids modifies the cell surface charge

  • The resulting positive charge repels cationic antimicrobial peptides

  • This increases bacterial survival in host environments rich in AMPs

• Insect pathogenesis:

  • Experiments with Δdlt B. cereus demonstrate that the dlt operon is required for full virulence in lepidopteran models

  • Significant virulence attenuation occurs in both Spodoptera littoralis and Galleria mellonella when dlt is deleted

• Mammalian host interactions:

  • The dlt operon affects interactions with mammalian immune systems

  • D-alanylation impacts recognition by pattern recognition receptors

  • This potentially influences inflammatory responses during infection

Experimental evidence demonstrates that complementation of dlt-deficient mutants with the wild-type dlt operon restores virulence, confirming the direct role of these genes in pathogenicity .

What is the relationship between DltA and inflammasome activation in host cells?

While DltA itself doesn't directly activate inflammasomes, research shows that B. cereus infection triggers NLRP3 inflammasome activation through complex mechanisms that may be indirectly influenced by dlt-mediated cell wall modifications:

• B. cereus primarily activates the NLRP3 inflammasome through its tripartite toxin hemolysin BL (HBL)

• This activation leads to caspase-1 activation, IL-1β and IL-18 release, and pyroptotic cell death

• Cell wall modifications through D-alanylation may affect the bacteria's interaction with host immune cells

In experimental models, B. cereus infection triggered significantly higher NLRP3-dependent inflammasome activation compared to other inflammasome sensors (NLRC4, AIM2), resulting in ASC speck formation and cytokine release. This activation was dependent on potassium efflux through membrane pores formed by the HBL toxin .

The relationship between DltA and inflammasome activation represents an interesting research area, particularly how cell wall modifications might influence host recognition and inflammatory responses.

How can recombinant DltA be employed in developing novel antimicrobial strategies?

Recombinant DltA offers several promising avenues for antimicrobial development:

• Structure-based inhibitor design:

  • The crystal structure of DltA in complex with D-alanine adenylate intermediate provides a template for rational drug design

  • Potential inhibitors can target the D-alanine binding pocket or the ATP-binding domain

  • Computational docking studies can identify compounds that disrupt DltA function

• Combination therapy approaches:

  • DltA inhibitors can potentially sensitize resistant gram-positive bacteria to existing antibiotics

  • Synergistic effects could occur with cationic antimicrobial peptides naturally produced by the host

• Adjuvant development:

  • Targeting DltA could enhance immune clearance by preventing bacterial evasion of host defense peptides

  • This approach might be particularly valuable for immunocompromised patients

• Vaccine development:

  • Recombinant DltA protein could potentially serve as an antigen for vaccine development

  • Antibodies against DltA might interfere with proper cell wall modification

Research indicates that inhibition of DltA would increase bacterial susceptibility to antimicrobial peptides and potentially conventional antibiotics, representing a promising strategy for combating multi-drug resistant gram-positive pathogens .

What insights can be gained from comparative analysis of DltA across the Bacillus cereus group?

Comparative analysis of DltA across the B. cereus group (including B. cereus, B. anthracis, B. thuringiensis, and related species) reveals important evolutionary and functional insights:

This comparative analysis provides insights into bacterial evolution, host adaptation, and potential species-specific targeting strategies for antimicrobial development.

What in vitro and in vivo models are most appropriate for studying DltA function?

In vitro models:

• Biochemical assays:

  • ATP-pyrophosphate exchange assays to measure adenylation activity

  • D-alanine incorporation assays using purified LTAs

  • Fluorescent or radioactive labeling for tracking D-alanine transfer

• Cell wall analysis techniques:

  • Nuclear magnetic resonance (NMR) spectroscopy for detecting D-alanine modifications

  • Mass spectrometry for quantitative analysis of teichoic acid composition

  • Zeta potential measurements to assess cell surface charge

• Antimicrobial susceptibility testing:

  • Minimum inhibitory concentration (MIC) determination for various antimicrobial peptides

  • Time-kill assays to assess kinetics of bacterial killing

  • Fluorescent microscopy with labeled AMPs to visualize binding patterns

In vivo models:

• Insect models:

  • Galleria mellonella (greater wax moth) provides a well-established system for analyzing B. cereus virulence

  • Spodoptera littoralis offers another lepidopteran model with demonstrated sensitivity to dlt-dependent virulence

• Mammalian infection models:

  • Mouse systemic infection models can assess bacterial dissemination and survival

  • Local infection models (skin, eye) to study tissue-specific effects

• Organoid and tissue culture systems:

  • Intestinal epithelial models to study interaction with gut barriers

  • Macrophage infection models to assess phagocytosis and intracellular survival

  • 3D printed bladder cancer-on-a-chip models have been used for related research on recombinant B. cereus applications

The integration of these models provides comprehensive insights into DltA function across different biological contexts.

How do the biological effects of recombinant DltA differ between in vitro and in vivo systems?

Research shows several important differences in recombinant DltA effects between in vitro and in vivo systems:

For example, in studies of recombinant BCG expressing dltA (rBCG-dltA), in vitro assays showed dose-dependent decreases in cancer cell proliferation, while in vivo models demonstrated more complex patterns of efficacy that varied with administration timing and route .

How can high-throughput screening approaches be optimized for identifying DltA inhibitors?

Optimizing high-throughput screening (HTS) for DltA inhibitors requires specialized approaches tailored to this enzymatic system:

• Assay development considerations:

  • Primary assays should target D-alanine adenylation, which can be monitored through ATP consumption or pyrophosphate release

  • Coupling enzymes (e.g., pyruvate kinase/lactate dehydrogenase) can link ATP hydrolysis to measurable NADH oxidation

  • Fluorescence polarization assays using labeled D-alanine can detect competitive binding

• Compound library selection:

  • Focus on compound classes that typically interact with ATP-binding pockets

  • Include natural products with known antimicrobial properties

  • Diversity-oriented libraries to capture novel scaffolds

• Screening cascade optimization:

  • Primary screens should balance throughput with sensitivity

  • Secondary confirmation assays must validate hits through orthogonal methods

  • Counter-screens against related adenylation domains to assess selectivity

  • Bacterial survival assays to confirm biological relevance

• Data analysis considerations:

  • Machine learning algorithms can help identify structure-activity relationships

  • Time-resolved data collection captures kinetic inhibition patterns

  • Cluster analysis identifies chemically diverse hits with similar mechanisms

The crystal structure of DltA from B. cereus provides valuable information for structure-based virtual screening approaches that can complement traditional HTS, potentially increasing the efficiency of inhibitor discovery .

What are the challenges in expressing and purifying functional recombinant DltA for structural studies?

Researchers face several specific challenges when expressing and purifying recombinant DltA for structural studies:

• Protein solubility issues:

  • DltA has hydrophobic regions that can promote aggregation

  • Expression optimization often requires testing multiple fusion tags (His, GST, MBP)

  • Solubility enhancers like SUMO tags may be necessary

• Maintaining enzymatic activity:

  • DltA requires proper folding to maintain adenylation activity

  • Lower temperature expression (16-20°C) often preserves function better than standard conditions

  • Careful buffer optimization is needed to maintain stability during purification

• Crystallization challenges:

  • DltA may adopt multiple conformations, complicating crystal formation

  • Co-crystallization with substrates or substrate analogs can stabilize the protein

  • The presence of D-alanine adenylate intermediate has been crucial for successful crystallization

• Protein heterogeneity:

  • Post-translational modifications may occur in heterologous expression systems

  • Proteolytic degradation during purification can produce heterogeneous samples

  • Size-exclusion chromatography as a final purification step is essential to obtain monodisperse protein

• Scale-up considerations:

  • Obtaining sufficient quantities for structural studies requires optimized large-scale expression

  • Consistency between batches is critical for reproducible crystallization

Successful structural studies of DltA from B. cereus at 2.0 Å resolution demonstrate that these challenges can be overcome with careful optimization of expression, purification, and crystallization conditions .

How might genetic manipulation of dltA be used to develop attenuated bacterial strains for research or biotechnological applications?

Genetic manipulation of dltA offers promising strategies for developing attenuated bacterial strains with various applications:

• Vaccine development platforms:

  • Controlled attenuation through dltA modification can create strains with reduced virulence but maintained immunogenicity

  • Such strains could serve as live attenuated vaccines or antigen delivery vehicles

  • The specific immune response can be modulated by the degree of dltA expression

• Biotechnology applications:

  • Engineered B. cereus with modified dltA could serve as production platforms for heterologous proteins

  • Altered cell surface properties affect protein secretion and anchoring efficiency

  • These modifications might enhance the display of recombinant proteins on bacterial surfaces

• Research tools:

  • dltA-modified strains serve as valuable tools for studying host-pathogen interactions

  • They allow investigation of the specific role of D-alanylated teichoic acids in various biological processes

  • Reporter gene fusions with dltA provide insights into gene regulation during infection

• Therapeutic applications:

  • Recombinant BCG strains expressing dltA (rBCG-dltA) show promise in bladder cancer treatment

  • At optimal dosages (30 MOI), rBCG-dltA demonstrates superior anti-tumor activity compared to conventional BCG

  • The system produces elevated levels of immunomodulatory cytokines like TNF-α and IL-6

Future research should focus on fine-tuning these genetic manipulations to achieve the optimal balance between attenuation and functionality for specific applications.

What are the emerging areas of research regarding interactions between DltA and the host immune system?

Several cutting-edge research areas are exploring the complex interactions between DltA and host immunity:

• Inflammasome modulation:

  • Recent studies have begun to explore how cell wall modifications through DltA activity might indirectly influence inflammasome activation

  • The NLRP3 inflammasome appears particularly responsive to B. cereus infection

  • Investigation of potential cross-talk between D-alanylated teichoic acids and pattern recognition receptors is ongoing

• Trained immunity effects:

  • Emerging evidence suggests bacterial cell wall components can induce epigenetic reprogramming in innate immune cells

  • How D-alanylated versus non-D-alanylated teichoic acids might differentially affect trained immunity represents an exciting research frontier

• Microbiome interactions:

  • The role of DltA in competitive fitness within polymicrobial communities is being explored

  • How D-alanylation affects colonization resistance against pathogens represents an important ecological dimension

• Tissue-specific immune responses:

  • Different tissue environments (gut, respiratory tract, skin) may respond differently to DltA-modified bacteria

  • Tissue-resident immune cells show specialized responses to bacterial cell wall components

• Immunomodulatory applications:

  • The potential to harness DltA or its inhibitors as immunomodulatory agents is being investigated

  • In cancer immunotherapy contexts, manipulating DltA activity might enhance anti-tumor immune responses