Recombinant Yersinia pseudotuberculosis serotype IB Acyl carrier protein (acpP)

What is the functional role of acpP in Y. pseudotuberculosis metabolism?

The acpP protein in Y. pseudotuberculosis functions as the primary acyl carrier protein involved in fatty acid synthesis (FAS) and serves as an essential carrier for acyl intermediates in metabolic pathways. Similar to other bacterial systems, acpP is initially synthesized as an inactive apo-form that requires conversion to the active holo-form through the addition of a 4′-phosphopantetheine (Ppant) group by a phosphopantetheinyl transferase (PPTase) . This conversion is critical for functionality.

Research suggests that Y. pseudotuberculosis acpP likely shares functional properties with other bacterial ACPs, primarily facilitating the shuttling of growing acyl chains between enzymes in the fatty acid synthesis pathway. Studies in related bacteria have demonstrated that among multiple ACP homologs, only the primary AcpP (AcpP1) functions in essential fatty acid synthesis pathways . The acpP protein participates in both primary metabolism (fatty acid synthesis) and secondary pathways like lipid A biosynthesis, which is crucial for bacterial membrane integrity and host-pathogen interactions.

How is acpP gene expression regulated in Y. pseudotuberculosis?

Temperature is a critical environmental cue for Y. pseudotuberculosis, regulating the expression of numerous genes including those involved in lipid metabolism . While specific data on acpP regulation in Y. pseudotuberculosis isn't fully characterized, research on related Yersinia species provides valuable insights.

In Y. enterocolitica, the expression of several lipid A acyltransferases that utilize acyl-ACPs as substrates exhibits temperature-dependent regulation. Expression levels of msbB and lpxP are higher at 21°C than at 37°C, whereas htrB expression increases at 37°C . This temperature-dependent regulation affects lipid A acylation patterns, with hexa-acylated lipid A predominating at 21°C and tetra-acylated lipid A at 37°C . Since these acyltransferases use acyl-ACP as substrates, it's reasonable to infer that acpP expression or activity is similarly regulated in response to temperature shifts encountered during host infection.

Additionally, Y. pseudotuberculosis demonstrates significant transcriptional remodeling when growing in human plasma compared to laboratory media, suggesting that acpP and related metabolic genes likely respond to host-derived environmental signals .

What structural characteristics define the acpP protein in Y. pseudotuberculosis?

The acpP protein in Y. pseudotuberculosis likely contains the highly conserved DSL (Asp-Ser-Leu) motif characteristic of bacterial ACPs, with the serine residue serving as the attachment site for the essential 4′-phosphopantetheine prosthetic group . Based on structural studies of ACPs from other bacteria, the Y. pseudotuberculosis acpP protein is predicted to adopt a four α-helix bundle structure .

The functional states of acpP include:

• Apo-acpP: The initial translation product lacking the prosthetic group

• Holo-acpP: The active form carrying the 4′-phosphopantetheine prosthetic group

• Acyl-acpP: Holo-acpP carrying various acyl intermediates

The 4′-phosphopantetheine prosthetic group provides the critical thiol group that forms thioester bonds with acyl chains, creating a flexible "swinging arm" that allows acpP to interact with multiple enzymes in the fatty acid synthesis pathway. This structural flexibility enables acpP to sequentially interact with different enzymes while protecting reactive intermediates from the aqueous environment.

What expression systems are most effective for producing functional recombinant Y. pseudotuberculosis acpP?

Based on successful expression of other Y. pseudotuberculosis proteins, Escherichia coli expression systems are highly suitable for recombinant Y. pseudotuberculosis acpP production . For optimal results, consider the following experimental design factors:

Expression vector selection:

• pTac85 has been successfully used for ACP expression in other bacterial systems

• pET-based vectors with T7 promoters provide high expression levels

• N-terminal His-tag facilitates purification without interfering with C-terminal protein interactions

Host strain considerations:

• BL21(DE3) and derivatives provide high expression levels

• Rosetta strains supply rare codons that may be present in Y. pseudotuberculosis genes

• ACP phosphopantetheinyl transferase (PPTase) co-expression strains ensure production of holo-acpP

Expression conditions optimization:

Co-expression with a compatible phosphopantetheinyl transferase is critical for obtaining active holo-acpP rather than inactive apo-acpP. The E. coli AcpS or Bacillus subtilis Sfp are commonly used PPTases for this purpose .

What methodological approaches can verify the functional activity of recombinant Y. pseudotuberculosis acpP?

Multiple complementary methods should be employed to verify the functional activity of recombinant Y. pseudotuberculosis acpP:

Genetic complementation assays:
Test whether recombinant acpP can restore growth in E. coli acpP mutant strains like CY1877, which requires arabinose-induced expression of E. coli acpP for viability . Functional complementation would indicate that the recombinant protein can participate in bacterial fatty acid synthesis.

In vitro acylation assays:
Utilize Vibrio harveyi acyl-ACP synthetase (AasS) to test if the recombinant holo-acpP can be acylated with fatty acids such as hexanoic acid or dodecanoic acid . Successful acylation detected by altered migration patterns on conformationally-sensitive urea-PAGE confirms functionality.

Mass spectrometry analysis:

• Verify conversion from apo-acpP to holo-acpP by detecting the mass increase (~340 Da) corresponding to the phosphopantetheine group

• Confirm acylation by identifying mass additions corresponding to specific acyl chains

• Perform top-down proteomics to characterize specific modifications

Protein-protein interaction studies:
Test interactions with purified enzymes from fatty acid synthesis pathway (FabB, FabF, FabH, FabI) or lipid A biosynthesis (LpxA, LpxD) using techniques like surface plasmon resonance or pull-down assays.

Research in Ralstonia solanacearum demonstrated that AcpP1 complemented an E. coli acpP mutant while other ACP homologs did not, confirming its role in fatty acid synthesis . Similar approaches can be applied to verify Y. pseudotuberculosis acpP functionality.

What purification strategy provides the highest yield of functional recombinant acpP?

An optimized purification strategy for recombinant Y. pseudotuberculosis acpP should include multiple steps to ensure purity and functionality:

1. Affinity chromatography:

• If using His-tagged acpP, Ni-NTA affinity chromatography is highly effective

• Recommended buffer: Tris/PBS at pH 8.0 with 6% Trehalose

• Imidazole gradient elution (20-250 mM) to minimize non-specific binding

• Consider on-column refolding if the protein forms inclusion bodies

2. Ion exchange chromatography:

• ACPs are typically acidic proteins (pI ~4.0-5.0), making anion exchange chromatography (Q Sepharose) effective

• Buffer conditions: 20 mM Tris-HCl pH 7.5-8.0, with gradual NaCl increase (0-500 mM)

• This step separates charged variants and removes nucleic acid contamination

3. Size exclusion chromatography:

• Final polishing step to remove aggregates and ensure homogeneity

• Recommended column: Superdex 75 or equivalent for small proteins

• Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl

4. Separation of apo and holo forms:

• Urea-PAGE can be used preparatively to separate apo-acpP from holo-acpP

• Reverse-phase HPLC can also separate these forms based on hydrophobicity differences

Stabilization and storage:
After purification, store the protein in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, with 5-50% glycerol for long-term storage at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation.

Yield optimization:
Typical yields of 10-20 mg of purified protein per liter of bacterial culture can be expected, with >90% purity achievable following this protocol .

How can Y. pseudotuberculosis acpP contribute to vaccine development research?

Y. pseudotuberculosis has emerged as a promising vaccine vector platform, particularly for plague vaccines targeting Y. pestis infection . While acpP itself may not be a direct antigen target, its role in lipid metabolism offers several research applications for vaccine development:

Attenuated strain engineering:
Modifying acpP-dependent pathways can create attenuated Y. pseudotuberculosis strains with altered membrane properties and reduced virulence while maintaining immunogenicity. Research has demonstrated that recombinant attenuated Y. pseudotuberculosis strains with mutations in virulence factors (ΔyopK ΔyopJ Δasd) can effectively deliver Y. pestis antigens .

Lipid adjuvant development:
ACPs participate in the synthesis of lipid A, which has potent immunostimulatory properties. Engineering acpP-dependent pathways could produce modified lipid structures with tailored adjuvant properties. The temperature-dependent regulation of lipid A acylation in Yersinia could be exploited to create temperature-responsive adjuvants.

Type III secretion system optimization:
Y. pseudotuberculosis strains engineered to express the Y. pestis YopE-LcrV fusion protein via the type III secretion system (T3SS) induced significant protection (80% survival) against intranasal challenge with Y. pestis . Understanding how acpP-dependent lipid metabolism affects T3SS assembly and function could optimize heterologous antigen delivery.

Outer membrane vesicle (OMV) vaccines:
Recent research has explored Y. pseudotuberculosis outer membrane vesicles as immunogenic self-adjuvanting vesicles for vaccine development . The lipid composition of these OMVs, influenced by acpP activity, directly impacts their immunostimulatory properties and stability.

Studies by Sun et al. demonstrated that oral immunization with a recombinant Y. pseudotuberculosis strain expressing Y. pestis antigens provided 90% protection against intranasal challenge with Y. pestis , highlighting the potential of this system for vaccine development.

How does acpP contribute to Y. pseudotuberculosis virulence and host adaptation?

The acpP protein plays multiple crucial roles in Y. pseudotuberculosis virulence and host adaptation:

Lipid A modification and immune evasion:
In Yersinia species, lipid A acylation patterns change with temperature and significantly affect virulence . At 37°C (mammalian host temperature), Y. pestis synthesizes tetra-acyl lipid A lacking secondary acylation, whereas at 21°C, lipid A is mainly hexa-acylated . These modifications, which involve acyltransferases using acyl-ACPs as substrates, directly implicate acpP in virulence-associated lipid modifications that alter recognition by host immune receptors like TLR4.

Membrane adaptation to host environments:
When Y. pseudotuberculosis grows in human plasma, it undergoes major transcriptional regulatory events and key metabolic reorientations . The acpP-dependent fatty acid synthesis pathway likely contributes to membrane remodeling that helps the bacterium adapt to the host environment.

Virulence factor regulation:
Research in Y. enterocolitica demonstrated that lipid A acylation status affects the expression of multiple virulence factors:

• Flagellar master regulatory operon flhDC (affecting motility)

• Phospholipase yplA (involved in virulence)

• Invasin inv (mediating host cell invasion)

Similar regulatory mechanisms likely exist in Y. pseudotuberculosis, connecting acpP-dependent lipid metabolism to virulence factor expression.

Temperature-dependent virulence regulation:
Temperature is a critical cue regulating Y. pseudotuberculosis virulence genes. Growth in human plasma upregulates the yadA adhesin gene and most transcriptional units of the pYV-encoded type III secretion apparatus, while strongly repressing the pH6 antigen locus . The acpP-dependent lipid composition changes at different temperatures may contribute to these regulatory effects.

Research by Rebeil et al. showed that mutations in lipid A acyltransferases in Y. enterocolitica led to reduced motility, decreased invasion of HeLa cells, and attenuation in a mouse infection model , highlighting the importance of acpP-dependent acylation in virulence.

What methodological approaches can elucidate acpP interaction networks in Y. pseudotuberculosis?

Understanding acpP-protein interactions is essential for fully characterizing its biological functions. Several complementary methodological approaches can identify and characterize the acpP interaction network:

Bacterial two-hybrid screening:

• Create a fusion library of Y. pseudotuberculosis proteins for screening against acpP bait

• Identify positive interactions through reporter gene activation

• Validate interactions with individual protein pairs

• Advantage: Can detect interactions in a bacterial cellular environment

Co-immunoprecipitation and pull-down assays:

• Express His-tagged acpP for pull-down experiments with bacterial lysates

• Identify binding partners by mass spectrometry

• Validate interactions with reciprocal pull-downs

• Quantify binding affinities using varying stringency conditions

Crosslinking mass spectrometry:

• Utilize chemical crosslinkers that capture transient protein-protein interactions

• Identify crosslinked peptides by tandem mass spectrometry

• Map specific interaction interfaces at amino acid resolution

• Advantage: Can detect weak or transient interactions

Surface plasmon resonance (SPR):

• Immobilize purified acpP on a sensor chip

• Measure real-time binding kinetics with potential interaction partners

• Determine association and dissociation rates

• Evaluate effects of mutations on binding efficiency

Based on research in related systems, expected interaction partners include:

• Fatty acid synthesis enzymes (FabB, FabF, FabH, FabI)

• Lipid A biosynthesis enzymes (LpxA, LpxD)

• Acyltransferases (MsbB, HtrB, LpxP)

• Enzymes involved in phospholipid synthesis

• Regulatory proteins that modulate ACP activity

Research in Ralstonia solanacearum demonstrated that only AcpP1 functioned in fatty acid synthesis and interacted with the corresponding enzymes, while other ACP homologs had distinct interaction networks related to secondary metabolism .

What evolutionary insights can be gained from comparing Y. pseudotuberculosis acpP with homologs in other Yersinia species?

Comparative analysis of acpP across Yersinia species provides valuable insights into bacterial evolution and adaptation:

Sequence conservation patterns:
Y. pseudotuberculosis is the evolutionary ancestor of Y. pestis, sharing 96-100% genetic identity across most genes . This high conservation suggests that acpP, as an essential gene, is likely highly conserved at the sequence level among Yersinia species. Analyzing subtle sequence variations could reveal regions under selective pressure versus those with functional constraints.

Genomic context analysis:
Examining the genetic organization surrounding acpP in different Yersinia species can reveal evolutionary events like gene rearrangements, acquisitions, or losses. For instance, Y. pestis lost the opgGH operon during its evolution from Y. pseudotuberculosis . This operon encodes glucosyltransferases that use ACP as a cofactor, suggesting shifts in ACP-dependent metabolic networks during evolution.

Functional divergence assessment:
While sequence conservation may be high, functional roles could diverge based on species-specific adaptations. For example, Y. pestis must cycle between mammalian hosts (37°C) and flea vectors (25°C), potentially requiring unique acpP-dependent lipid adaptations compared to the primarily enteric Y. pseudotuberculosis.

Evolutionary selection pressures:
Analyzing synonymous versus non-synonymous substitution rates in acpP across Yersinia species can identify regions under positive or negative selection, potentially correlating with host adaptation or niche specialization.

Comparative genomic studies between Y. pestis and Y. pseudotuberculosis have identified species-specific regions and gene loss/acquisition events that contributed to the emergence of plague . Similar approaches focused specifically on acpP and related lipid metabolism genes could reveal how metabolic adaptations contributed to pathogen evolution.

How do acpP-dependent lipid modifications differ between Yersinia species and what are their functional implications?

AcpP-dependent lipid modifications show significant variation between Yersinia species, with important implications for pathogenesis and host adaptation:

Temperature-dependent lipid A modifications:
Studies in Y. enterocolitica and Y. pestis demonstrate that lipid A acylation patterns change with temperature :

• At 37°C, Y. pestis synthesizes tetra-acyl lipid A lacking any secondary acylation

• At 21°C, Y. pestis lipid A is mainly hexa-acylated, resembling E. coli lipid A

• Y. enterocolitica similarly produces less acylated lipid A at 37°C than at 21°C

These modifications involve acyltransferases (MsbB, HtrB, LpxP) that use acyl-ACPs as substrates, directly implicating acpP in these temperature-responsive changes.

Functional consequences of species-specific modifications:

• Immune recognition: Differences in lipid A structure dramatically affect recognition by host TLR4 receptors, with Y. pestis tetra-acylated lipid A (37°C) being less stimulatory than hexa-acylated forms, promoting immune evasion

• Membrane properties: Species-specific acpP-dependent lipid modifications alter membrane fluidity, permeability, and resistance to antimicrobial peptides

• Virulence factor expression: In Y. enterocolitica, lipid A acylation affects expression of motility genes (flhDC), phospholipase (yplA), and invasin (inv)

Methodological approaches to study these differences:

• Lipidomic analysis of different Yersinia species grown under various conditions

• Construction of chimeric strains expressing acpP or acyltransferases from different Yersinia species

• In vitro reconstitution of lipid A synthesis with purified components from different species

Research by Rebeil et al. demonstrated that in Y. enterocolitica, an htrB mutant affecting lipid A acylation was attenuated for virulence , highlighting how species-specific differences in acpP-dependent lipid modifications could contribute to the distinct pathogenic strategies of Yersinia species.

How does the role of acpP in Y. pseudotuberculosis compare with acpP function in model organisms like E. coli?

Comparative analysis of acpP function between Y. pseudotuberculosis and model organisms reveals both conserved and divergent aspects:

Conserved features:

• Essential role in fatty acid synthesis: In both Y. pseudotuberculosis and E. coli, acpP serves as the primary acyl carrier in the type II fatty acid synthesis pathway, which is essential for bacterial viability

• Structural conservation: Both proteins likely share the characteristic four-helix bundle structure with the conserved DSL motif at the attachment site for the 4′-phosphopantetheine prosthetic group

• Functional complementation: Research with other bacterial ACPs suggests that Y. pseudotuberculosis acpP could likely complement E. coli acpP mutants, similar to how Ralstonia solanacearum AcpP1 partially restored growth in an E. coli acpP mutant

Species-specific differences:

• Temperature-dependent regulation: Yersinia species show distinctive temperature-dependent lipid modifications that may involve unique regulatory mechanisms controlling acpP function or its downstream pathways

• Interaction partners: Y. pseudotuberculosis likely possesses species-specific enzymes that interact with acpP, particularly those involved in virulence-associated lipid modifications

• Metabolic integration: The integration of acpP into metabolic networks may differ between species based on their ecological niches and lifestyle adaptations

Experimental approaches for comparative studies:

• Heterologous expression of Y. pseudotuberculosis acpP in E. coli with phenotypic analysis

• Construction of chimeric acpP proteins to identify functionally important regions

• Interspecies protein-protein interaction studies to identify conserved versus species-specific binding partners

• Comparative transcriptomic and proteomic analyses to understand regulatory differences

Research in Ralstonia solanacearum showed that multiple ACP homologs exist with specialized functions, where only AcpP1 functions in fatty acid synthesis while others participate in secondary metabolism . Similar functional specialization might exist in the acpP-dependent pathways of Y. pseudotuberculosis compared to model organisms.

What experimental strategies can overcome challenges in expressing active recombinant Y. pseudotuberculosis acpP?

Expressing functionally active Y. pseudotuberculosis acpP presents several technical challenges. Here are methodological solutions to common issues:

Challenge 1: Insufficient conversion of apo-acpP to holo-acpP

Challenge 2: Poor solubility or stability

Challenge 3: Low expression levels

Research with other bacterial ACPs demonstrated that expression at moderate temperatures (25-30°C) in E. coli with careful optimization of induction parameters often yields active protein . Successful expression of other Y. pseudotuberculosis proteins in E. coli suggests that similar approaches would be effective for acpP.

What analytical methods can distinguish between different forms of acpP and verify their functional status?

Distinguishing between different forms of acpP (apo, holo, and acylated) is critical for functional studies. Multiple complementary analytical approaches should be employed:

Electrophoretic methods:

• Urea-PAGE (Conformationally-sensitive gel electrophoresis):

  • The apo, holo, and acylated forms of ACPs show distinct migration patterns in urea-containing gels

  • Typically, holo-ACP migrates faster than apo-ACP

  • Acylated ACPs show additional mobility shifts depending on the acyl chain length

  • Running standards of known forms alongside samples enables identification

Mass spectrometry-based approaches:

• MALDI-TOF or ESI-MS for intact protein analysis:

  • Apo to holo conversion: mass increase of +339 Da (Ppant group)

  • Acylation: additional mass increase corresponding to specific acyl chains

  • High resolution allows detection of multiple acylated species

• Top-down proteomics:

  • Fragmenting intact protein in mass spectrometer

  • Locates specific modifications on the protein sequence

  • Distinguishes between different acylation sites

Functional biochemical assays:

• Acylation assays with acyl-ACP synthetase:

  • Only holo-acpP can be acylated by AasS

  • Monitor acylation by gel shift or mass increase

  • Different acyl chain lengths produce distinct migration patterns

• Enzyme activity assays:

  • Test function in reconstituted fatty acid synthesis reactions

  • Only active forms support acyl transfer reactions

Biophysical characterization:

• Circular dichroism: Detects conformational differences between forms

• Fluorescence techniques: Monitor binding to fluorescent probes or partners

• NMR analysis: Provides detailed structural information about different states

Research by Cao et al. demonstrated that holo-AcpP1 and holo-AcpP3 showed altered gel migration patterns after incubation with fatty acids, ATP, and V. harveyi AasS, confirming successful acylation . This demonstrates the utility of conformationally-sensitive gel electrophoresis for distinguishing between different functional states of acpP.

What approaches enable study of acpP function without compromising bacterial viability?

Studying acpP function is challenging because it is essential for bacterial growth. Several methodological approaches can overcome these limitations:

Genetic approaches:

• Conditional expression systems:

  • Temperature-sensitive promoters

  • Inducible/repressible systems (tetracycline, arabinose)

  • Degradation tag systems (e.g., SsrA tags with regulated proteases)

  • CRISPR interference (CRISPRi) for tunable repression

• Point mutations and domain-specific alterations:

  • Engineer mutations that affect specific functions while maintaining essential activities

  • Create temperature-sensitive acpP variants for conditional studies

  • Construct chimeric proteins to analyze domain-specific functions

Chemical biology approaches:

• Small molecule modulators:

  • Sub-inhibitory concentrations of ACP inhibitors

  • Competitive inhibitors of specific ACP interactions

  • Activity-based probes for selective labeling of acpP forms

• Metabolic engineering strategies:

  • Supplement with fatty acids to bypass essentiality

  • Engineer alternate metabolic routes to reduce dependency on acpP

  • Express heterologous fatty acid synthases with different carrier proteins

Systems biology approaches:

• Multi-omics integration:

  • Transcriptomics after perturbation of acpP levels

  • Lipidomics to identify changes in lipid profiles

  • Network analysis to identify compensatory pathways

• In situ imaging and tracking:

  • Fluorescent protein fusions for localization studies

  • FRET-based sensors for monitoring acpP interactions

  • Time-resolved analyses of acpP-dependent processes

Research on Y. enterocolitica demonstrated that mutations affecting lipid A acylation (which involves acyl-ACPs) resulted in specific phenotypes related to motility, invasion, and virulence while maintaining viability . Similar approaches in Y. pseudotuberculosis could provide valuable insights into acpP-dependent processes without completely inactivating this essential function.