Recombinant Neisseria meningitidis serogroup B Acyl carrier protein (acpP)

What is Neisseria meningitidis and what disease does it cause?

Neisseria meningitidis is a Gram-negative bacterium that causes meningococcal disease, including meningitis. The pathogen can be carried in the throat of some individuals without causing illness, yet these carriers can transmit the bacterium to others who may develop severe disease. In the United States, N. meningitidis causes meningitis in approximately 25% of people who contract the illness annually . The bacterium is capable of causing both sporadic cases and outbreaks of meningitis, with outbreaks being more common outside the United States, particularly in sub-Saharan Africa .

What is the Adhesin Complex Protein (ACP) of Neisseria meningitidis?

The Adhesin Complex Protein (ACP) is a 13-kDa outer membrane protein encoded by the acp gene in Neisseria meningitidis. It has been identified in several proteomic studies of meningococcal outer membrane and outer membrane vesicles . This protein functions primarily as an adhesin, facilitating the attachment of meningococci to human host cells, and has also been shown to be upregulated under iron-depleted conditions . ACP has garnered significant research interest due to its highly conserved nature across meningococcal strains and its potential as a vaccine candidate .

How conserved is the ACP sequence across different meningococcal strains?

Analysis of ACP amino acid sequences from 13 meningococcal strains isolated from patients and colonized individuals, along with 178 strains in the Bacterial Isolate Genome Sequence (BIGS) database, revealed only three distinct sequence types (I, II, and III) with high similarity (>98%) . This high level of conservation is significant for vaccine development, as it suggests that a vaccine targeting ACP would potentially be effective against a broad range of meningococcal strains. Additionally, the protein is expressed at similar levels across different meningococcal strains regardless of the sequence type (I, II, or III) .

What methodologies are used to produce recombinant ACP for research studies?

The production of recombinant ACP (rACP) typically involves:

• Gene cloning: The acp gene from N. meningitidis serogroup B (such as strain MC58) is amplified and cloned into an expression vector.

• Expression in E. coli: The recombinant protein is expressed in E. coli under controlled conditions.

• Purification: The expressed protein is purified using appropriate chromatographic techniques.

• Formulation: For immunization studies, the purified rACP may be incorporated into different formulations, including:

  • Detergent micelles

  • Liposomes (with or without adjuvants)

  • Saline solution alone

Research has shown that the formulation used can significantly impact the immunogenicity of rACP. For example, studies have demonstrated that incorporation of monophosphoryl lipid A (MPLA) as an adjuvant can actually reduce bactericidal titers, possibly by interfering with the native conformation of the protein .

How does recombinant ACP induce immunity against Neisseria meningitidis?

Immunization studies with type I recombinant ACP have demonstrated the induction of high levels of serum bactericidal activity (SBA) against both homologous and heterologous strains. Specifically:

• Immunization with type I rACP in various formulations (detergent micelles, liposomes, or saline solution) induced high SBA titers (1/512) against the homologous strain MC58 .

• The antibodies generated were also effective against strains expressing heterologous sequence types II and III, with SBA titers ranging from 1/128 to 1/512 .

This cross-strain bactericidal activity is particularly valuable for vaccine development, as it suggests that an ACP-based vaccine could provide broad protection against diverse meningococcal strains. The mechanism likely involves antibody-mediated complement activation leading to bacterial lysis or opsonophagocytosis.

How does ACP mediate meningococcal interactions with human cells?

ACP functions as an adhesin facilitating meningococcal attachment to various human cell types. This has been demonstrated through multiple experimental approaches:

• Knockout studies: Deletion of the acp gene (MC58 ΔACP) resulted in significant reduction in bacterial association with human cells in vitro :

  • Approximately 75% reduction in adhesion to Chang and Hep2 epithelial cells

  • 30-50% reduction in adhesion to human umbilical vein endothelial cells (HUVECs) and meningioma cells

• Complementation studies: Restoration of the acp gene in the knockout strain restored bacterial association to levels similar to the wild-type strain .

• Direct binding studies: Purified rACP protein was shown to bind directly to human cell surfaces.

• Inhibition studies: Anti-rACP sera inhibited the adherence of wild-type bacteria to human cells .

These findings collectively establish ACP as an important adhesin mediating meningococcal interactions with human host cells.

What are the differences in ACP function between capsulated and non-capsulated meningococci?

The function of ACP differs significantly between capsulated (Cap+) and non-capsulated (Cap-) meningococci:

This differential function may be related to capsule interference with surface exposure or accessibility of ACP to host cell receptors.

How does ACP compare to other meningococcal adhesins as a vaccine candidate?

ACP offers several advantages as a vaccine candidate compared to other meningococcal adhesins:

These properties suggest that ACP merits serious consideration for inclusion in next-generation serogroup B meningococcal vaccines . The high conservation, universal presence, and ability to induce cross-protective immunity make it particularly attractive as a vaccine antigen.

What experimental approaches can be used to study ACP-mediated adhesion and invasion?

Several established methodologies can be employed to study ACP-mediated adhesion and invasion:

• Generation of knockout strains:

  • Create an acp gene deletion mutant (e.g., MC58 ΔACP)

  • Create complemented strains by reintroducing the acp gene

• Cell association assays:

  • Incubate wild-type, knockout, and complemented strains with various human cell types

  • Quantify associated bacteria after washing to remove unbound bacteria

  • Compare association levels between strains to determine the contribution of ACP

• Invasion assays:

  • Perform gentamicin protection assays to kill extracellular bacteria

  • Lyse cells and quantify internalized bacteria by plating

  • Compare invasion levels between wild-type and knockout strains

• Direct binding assays:

  • Study binding of purified recombinant ACP to human cells

  • Use labeled rACP to visualize binding patterns

  • Perform competition assays with unlabeled protein

• Inhibition studies:

  • Use anti-rACP sera to block adhesion/invasion

  • Quantify the reduction in bacterial association/invasion

These approaches can be applied to various human cell types, including epithelial cells (e.g., Chang, Hep2), endothelial cells (HUVECs), and meningioma cells, to comprehensively characterize ACP function .

How can researchers evaluate the immunogenicity and protective efficacy of recombinant ACP?

A systematic approach to evaluating rACP as a vaccine candidate should include:

• Immunization studies:

  • Test different formulations (micelles, liposomes, adjuvants)

  • Establish immunization schedules (primary doses, boosters)

  • Compare different routes of administration

• Antibody response assessment:

  • Measure antibody titers using ELISA

  • Characterize antibody isotypes and subclasses

  • Assess avidity maturation over time

• Functional assays:

  • Serum bactericidal activity (SBA) assays against homologous and heterologous strains

  • Opsonophagocytic killing assays

  • Adhesion inhibition assays

• Cross-protection analysis:

  • Test activity against strains expressing different ACP sequence types

  • Evaluate protection against diverse clinical isolates

• In vivo protection studies:

  • Challenge immunized animals with virulent meningococci

  • Assess bacterial clearance and survival

Research has shown that when evaluating different formulations, it's important to consider potential effects on protein conformation. For example, the addition of monophosphoryl lipid A (MPLA) to liposomes containing rACP abolished serum bactericidal activity, possibly by altering protein folding and native-like conformation .

What strategies can be employed to optimize recombinant ACP production for research applications?

Optimizing recombinant ACP production requires attention to several factors:

• Expression system selection:

  • E. coli-based systems have been successfully used

  • Consider codon optimization for the expression host

  • Evaluate different promoter systems for optimal expression

• Protein solubility enhancement:

  • Test different fusion tags (His-tag, GST, etc.)

  • Optimize induction conditions (temperature, inducer concentration)

  • Consider solubility-enhancing co-expression partners (chaperones)

• Purification strategy development:

  • Implement multi-step chromatography procedures

  • Optimize buffer conditions to maintain native conformation

  • Develop protocols to remove endotoxin contamination

• Quality assessment:

  • Verify protein integrity by mass spectrometry

  • Confirm proper folding using circular dichroism

  • Assess functional activity through binding assays

• Stability optimization:

  • Identify optimal storage conditions

  • Evaluate freeze-thaw stability

  • Test different formulation buffers and excipients

Maintaining the native-like conformation of ACP is particularly important, as research has shown that alterations in protein folding can significantly impact the functional properties and immunogenicity of the protein .

What are the key unresolved questions regarding ACP structure and function?

Several important questions about ACP remain to be fully addressed:

• Structural characterization:

  • What is the three-dimensional structure of ACP?

  • Which domains are critical for adhesion function?

  • How does ACP integrate into the bacterial outer membrane?

• Host receptor identification:

  • What are the specific host cell receptors for ACP?

  • Do these receptors vary across different human cell types?

  • How does receptor binding trigger downstream events?

• Regulatory mechanisms:

  • Beyond iron regulation, what other factors control ACP expression?

  • How is ACP expression coordinated with other virulence factors?

  • What role does phase variation play in ACP expression?

• Interaction with other bacterial components:

  • Does ACP function independently or as part of a larger adhesin complex?

  • How does the capsule modulate ACP function?

  • Are there synergistic interactions with other adhesins?

Addressing these questions will provide deeper insights into the molecular mechanisms of meningococcal pathogenesis and potentially identify new approaches for intervention.

How might research on ACP contribute to new therapeutic strategies beyond vaccines?

Research on ACP could lead to several novel therapeutic approaches:

• Anti-adhesive therapies:

  • Development of small molecule inhibitors of ACP-host cell interactions

  • Design of peptide mimetics that compete with ACP for receptor binding

  • Creation of recombinant soluble receptors as decoys

• Targeted drug delivery:

  • Utilization of ACP as a targeting moiety for delivering antimicrobials to meningococci

  • Development of ACP-based nanoparticles for specific targeting of colonized tissues

• Diagnostic applications:

  • Development of ACP-based detection systems for rapid identification of meningococci

  • Creation of ACP antibody-based assays for serological testing

• Host-directed therapeutics:

  • Identification of host pathways activated by ACP binding

  • Development of modulators of host response to prevent excessive inflammation

These approaches could complement vaccination strategies and provide alternative options for prevention and treatment of meningococcal infections, particularly in individuals who cannot be effectively vaccinated or in outbreak situations requiring rapid intervention.