SURA E.Coli

What is SurA and what is its primary function in E. coli?

SurA is a periplasmic prolyl isomerase/chaperone that plays a critical role in outer membrane protein (OMP) biogenesis and pilus assembly in Escherichia coli. It functions primarily to facilitate the correct folding and assembly of OMPs as they transit from the inner membrane to the outer membrane. SurA accomplishes this by binding to unfolded outer membrane proteins (uOMPs) and preventing their aggregation during transport across the periplasm. As the most important member of the periplasmic chaperone network, SurA maintains direct genetic interaction with the β-barrel assembly machinery (BAM) complex, which is essential for proper OMP insertion into the outer membrane .

What is the structural organization of SurA protein?

The crystal structure of SurA reveals an asymmetric dumbbell-shaped protein consisting of four segments. The N-terminal, C-terminal, and first peptidyl-prolyl isomerase (PPIase) segments form a core structural module that includes an extended crevice implicated in membrane protein folding. The second PPIase segment forms a satellite domain tethered approximately 30 Å from this core module. This unique architectural arrangement enables SurA to bind uOMPs in a groove formed between the core and P1 domains, causing a dramatic expansion of the uOMP that has significant biological implications for protein delivery to the BAM complex .

How does SurA contribute to E. coli pathogenesis?

SurA plays a crucial role in the pathogenic cascade of uropathogenic E. coli (UPEC), particularly during urinary tract infections (UTIs). Studies demonstrate that SurA is required within bladder epithelial cells for UPEC to undergo morphological changes underlying the maturation of intracellular bacterial communities (IBCs). When surA is genetically disrupted in UPEC strains, the bacteria are unable to persist in the urinary tract despite initial invasion abilities. The formation of IBCs is severely compromised, with intracellular collections containing fewer bacteria, looser organization, and lacking the normal transition to a densely packed, coccoid morphology. This indicates that SurA is essential for the completion of the UPEC pathogenic cascade in the urinary tract .

Which outer membrane proteins (OMPs) are most dependent on SurA for proper assembly?

Research using label-free differential proteomics approaches based on 2D-LC-MS/MS has identified several β-barrel proteins that are significantly affected by the loss of SurA. Among the 23 β-barrel proteins identified in one study, 8 were negatively affected in SurA-deficient strains. These include FadL, LptD, FhuA, OmpX, FecA, and the major OMPs OmpA, OmpF, and LamB. Notably, LptD and FhuA are considered true SurA substrates as their decreased abundance in SurA-deficient cells cannot be attributed to lower mRNA levels, suggesting a direct role for SurA in their assembly. Additionally, these experiments revealed that while several proteins are affected by SurA deficiency, the decreased outer membrane density observed in surA mutants is primarily due to reduction in the major OMPs (OmpA, OmpF, and LamB), which constitute a large portion of the bulk mass of OMPs .

What phenotypes are associated with SurA deficiency in E. coli?

E. coli strains lacking SurA exhibit multiple defects indicative of outer membrane perturbations. These include:

• Hypersensitivity to detergents and hydrophobic antibiotics

• Decreased outer membrane density compared to wild-type strains

• Lower levels of several outer membrane proteins, particularly OmpA, LamB, OmpF, and OmpC

• Impaired ability to form mature intracellular bacterial communities during infection

• Reduced persistence in the urinary tract in infection models

• Compromised outer membrane integrity affecting bacterial survival under stress conditions

These phenotypes collectively demonstrate SurA's essential role in maintaining outer membrane architecture and function, which directly impacts bacterial viability and pathogenicity .

How does SurA interact with the β-barrel assembly machinery (BAM) complex?

SurA interacts with the BAM complex through a functional partnership that facilitates the final stages of OMP assembly. Current models suggest that SurA delivers unfolded OMPs to the BAM complex by binding to the POTRA domains of BamA, the central component of the BAM complex. The unique binding mechanism of SurA, which causes expansion of bound uOMPs, potentially primes these substrates for efficient handoff to the BAM complex. Experimental evidence from crosslinking, mass spectrometry, and solution scattering studies supports the existence of multiple binding modes between SurA and its substrates, allowing for versatile interactions that facilitate the escort function across the periplasm to the BAM complex .

What experimental approaches are most effective for studying SurA-substrate interactions?

Several complementary techniques have proven effective for characterizing SurA-substrate interactions:

These approaches have revealed that SurA utilizes at least three distinct binding modes to interact with uOMPs and that multiple SurA molecules can simultaneously bind a single uOMP substrate .

How can researchers effectively generate and validate conditional SurA depletion systems?

For studying essential proteins like SurA, conditional depletion systems offer superior control compared to knockout strains. An effective methodology includes:

• Design of Complementation Plasmid: Create a plasmid (e.g., pDH15) containing surA under control of an inducible promoter (arabinose-inducible pBAD promoter has been successfully employed).

• Construction of Depletion Strain: Introduce the surA::kan allele into the desired E. coli strain, then complement with the inducible surA plasmid.

• Validation Strategy:

  • Confirm depletion kinetics using Western blot analysis over time after removing inducer

  • Verify phenotypic changes (membrane permeability, OMP levels) correlate with depletion timing

  • Establish dose-response relationship between inducer concentration and SurA levels

  • Include controls with wild-type surA expression to account for plasmid effects

• Experimental Application: This system enables time-resolved studies of SurA function by allowing bacterial growth with normal SurA levels followed by controlled depletion at specific experimental timepoints, such as after invasion into host cells .

What are the current models explaining SurA's mechanism for expanding unfolded outer membrane proteins?

Based on integrative structural biology approaches, researchers have proposed several models for how SurA expands uOMPs:

• Cryptic Groove Model: SurA binds uOMPs in a groove formed between the core and P1 domains, which becomes accessible through conformational changes in SurA. This binding event leads to a dramatic expansion of the rest of the uOMP structure.

• Multi-Site Engagement Model: SurA utilizes three distinct binding modes to interact with different regions of uOMPs simultaneously, preventing collapse into compact states and maintaining extended conformations.

• Iterative Binding Mechanism: Multiple SurA molecules bind and release segments of uOMP polypeptides sequentially, preventing inappropriate interactions and maintaining expanded states conducive to proper folding.

These models are supported by experimental data from crosslinking, mass spectrometry, and solution scattering techniques, which collectively demonstrate that SurA binding results in significantly more extended uOMP conformations compared to the compact states observed with other chaperones. This expansion may represent a key mechanism by which SurA prepares uOMPs for efficient delivery to and engagement with the BAM complex .

What controls should be included when studying SurA-dependent phenotypes?

When investigating SurA-dependent phenotypes, researchers should incorporate the following controls:

• Genetic Complementation: Include a plasmid-based wild-type surA complementation to confirm phenotypes are specifically due to SurA deficiency.

• Alternative Chaperone Mutants: Compare surA mutant phenotypes with those of other periplasmic chaperones (Skp, DegP) to distinguish SurA-specific effects from general chaperone deficiency.

• Double Mutant Analysis: Examine synthetic phenotypes in strains lacking both SurA and secondary chaperones to identify functional redundancy.

• Substrate Overexpression: Test whether overexpression of specific SurA-dependent OMPs can suppress or exacerbate phenotypes.

• Dose-Dependent Response: For conditional depletion systems, establish correlation between SurA protein levels and phenotype severity.

• Background Strain Considerations: Verify phenotypes in multiple E. coli genetic backgrounds to ensure observations aren't strain-specific.

• Internal Standardization: When quantifying protein levels by immunoblotting, use internal standards (such as the 55 kDa protein recognized by LptD antiserum) to normalize between samples .

How should researchers design experiments to differentiate direct and indirect effects of SurA on OMP biogenesis?

Differentiating direct from indirect effects requires multi-faceted experimental approaches:

• Transcriptional Analysis: Combine proteomic data with mRNA level measurements (RT-qPCR or RNA-seq) to identify proteins whose decreased abundance cannot be explained by transcriptional changes, as demonstrated for LptD and FhuA.

• Pulse-Chase Experiments: Use radioactive or fluorescent labeling to track newly synthesized OMPs in real-time, comparing maturation rates in wild-type versus SurA-deficient strains.

• In Vitro Binding Assays: Purify SurA and candidate substrate proteins to measure direct binding affinities and kinetics, establishing which OMPs are bona fide SurA substrates.

• Targeted Crosslinking: Employ site-specific crosslinking approaches to capture direct SurA-OMP interactions in vivo, particularly under conditions where other chaperones are present.

• Sequential Depletion Experiments: Use conditional expression systems to control the timing of SurA depletion relative to synthetic processes, helping distinguish primary (direct) from secondary effects.

This multi-method approach has successfully identified LptD as a direct SurA substrate, as its decreased abundance in SurA-deficient cells cannot be attributed to lower mRNA levels, suggesting specific dependence on SurA for proper assembly .

What methodological approaches are recommended for analyzing SurA's role in pathogenic E. coli strains?

To effectively study SurA's role in pathogenic E. coli strains, researchers should consider these methodological approaches:

• In Vitro Infection Models:

  • Bladder epithelial cell infection assays to distinguish binding from invasion phenotypes

  • Time-course analysis of intracellular bacterial community (IBC) formation

  • Quantitative assessment of bacterial morphology changes during infection

• In Vivo Models:

  • Murine cystitis models to assess urinary tract persistence

  • Controlled complementation using inducible promoters (e.g., arabinose-inducible)

  • Competition assays between wild-type and surA mutant strains to measure fitness differences

• Conditional Genetic Systems:

  • Deploying arabinose-inducible surA expression in surA knockout strains

  • Utilizing the absence of inducer in host environments to achieve post-invasion depletion

  • Carefully timing inducer removal to separate invasion from IBC maturation stages

• Microscopic Analysis:

  • High-resolution imaging to characterize morphological changes in IBCs

  • Quantification of bacterial numbers, organization patterns, and coccoid morphology transitions

  • Immunofluorescence labeling to track SurA levels within intracellular bacteria

These approaches have revealed that SurA is specifically required for the morphological changes underlying IBC maturation, distinguishing its role in pathogenesis from its general OMP biogenesis function .

How should researchers interpret apparent contradictions in SurA substrate specificity across different studies?

When confronting contradictory findings regarding SurA substrate specificity, researchers should consider:

• Methodological Differences: Compare experimental techniques used across studies, as different approaches (genetic knockouts vs. conditional depletion; in vivo vs. in vitro analysis) may yield varying results. For example, proteomics methods like 2D-LC-MS/MS provide different insights than targeted immunoblotting.

• Strain Variability: Assess whether different E. coli strains were used, as genetic background can significantly influence results. Laboratory strains (K-12 derivatives) may show different dependencies than pathogenic isolates.

• Growth Conditions: Evaluate whether bacteria were grown under comparable conditions, as temperature, media composition, and growth phase affect OMP expression and SurA dependency.

• Direct vs. Indirect Effects: Distinguish between primary (direct SurA substrates) and secondary effects (downstream consequences of membrane perturbation). This requires integrating transcriptomics with proteomics data, as demonstrated in studies identifying LptD and FhuA as true SurA substrates.

• Functional Redundancy: Consider compensation by other chaperones (Skp, DegP) which may mask SurA dependency in certain experimental setups or for specific substrates.

A systematic comparison table categorizing findings by methodology, strain, and conditions can help identify patterns explaining apparent contradictions .

What quantitative approaches provide the most reliable assessment of outer membrane protein levels in SurA studies?

For reliable quantitative assessment of OMP levels in SurA studies, researchers should consider these approaches:

The most reliable approach involves combining multiple methods. For example, initial screening using LC-MS/MS proteomics (as demonstrated in studies identifying 64 OM proteins including 23 β-barrel proteins) followed by targeted verification of key findings using quantitative immunoblotting with appropriate internal standards (such as the 55 kDa protein used as internal standard for LptD quantification) .

How can researchers distinguish between SurA's chaperone and PPIase activities when analyzing experimental data?

Distinguishing between SurA's chaperone and peptidyl-prolyl isomerase (PPIase) activities requires specialized experimental design:

• Domain-specific Mutants: Utilize SurA variants with mutations that specifically disrupt either:

  • PPIase active sites in P1 and/or P2 domains

  • Substrate binding regions in the core module

  • Interdomain interfaces that affect conformational dynamics

• Functional Complementation Analysis: Compare the ability of different SurA domain constructs to complement surA deletion phenotypes:

  • Core module only (without PPIase domains)

  • PPIase-deficient variants (with inactivating mutations)

  • Individual domain deletions (ΔP1 or ΔP2)

• In Vitro Activity Assays:

  • Measure prolyl isomerization using model peptide substrates

  • Assess chaperone activity through prevention of protein aggregation

  • Compare activities using full-length SurA versus domain-specific variants

• Structure-Function Correlation: Relate experimental observations to the crystal structure of SurA, which reveals that the core module (formed by N-terminal, C-terminal, and first PPIase segments) contains an extended crevice implicated in chaperone function, while the second PPIase domain forms a satellite linked by a flexible tether.

Studies using these approaches have demonstrated that the chaperone function resides primarily in the core module, while the PPIase activities, though present, are dispensable for the primary function of SurA in OMP biogenesis .

What are the most promising approaches for targeting SurA as an antibiotic development strategy?

Given SurA's critical role in OMP biogenesis and bacterial pathogenesis, several promising approaches for antibiotic development include:

• Small Molecule Inhibitors: Design compounds that:

  • Bind to the substrate-binding groove between the core and P1 domains

  • Disrupt SurA-BAM complex interactions

  • Lock SurA in conformations incompatible with chaperone function

• Peptide-based Inhibitors: Develop peptides that mimic natural substrates but bind irreversibly or with higher affinity, competing with uOMPs for SurA binding.

• Allosteric Modulators: Target regions that regulate conformational changes in SurA, preventing the protein from cycling between states needed for chaperone function.

• Combination Therapies: Design inhibitors that simultaneously target SurA and secondary chaperones (Skp, DegP) to overcome redundancy in the periplasmic chaperone network.

• Structure-guided Drug Design: Utilize the crystallographic structure of SurA (PDB: 1M5Y) and models of SurA-uOMP complexes to identify druggable pockets and design targeted inhibitors.

These approaches are particularly promising because SurA has been identified as the most important member of the periplasmic chaperone network due to its genetic interaction with the essential BAM complex. Furthermore, SurA's role in pathogenesis, as demonstrated in uropathogenic E. coli models, suggests that inhibitors could potentially reduce virulence without directly affecting bacterial viability, potentially reducing selection pressure for resistance .

What unresolved questions remain regarding SurA's binding mechanism to unfolded outer membrane proteins?

Despite significant advances in understanding SurA function, several important questions remain unresolved:

• Binding Specificity Determinants: What sequence or structural features in uOMPs determine preferential binding by SurA? Current data suggest SurA recognizes aromatic-rich motifs, but precise specificity rules remain elusive.

• Multiple Binding Modes: How do the three distinct binding modes identified for SurA-uOMP interactions function in concert during chaperone activity? The relationship between these modes and folding progression requires further investigation.

• Conformational Dynamics: What are the rates and energy barriers for conformational changes in SurA during substrate binding and release cycles? Real-time observations of these dynamics would enhance mechanistic understanding.

• Quantitative Binding Parameters: What are the binding affinities, on/off rates, and thermodynamic profiles for different classes of SurA substrates? How do these parameters correlate with in vivo dependence on SurA?

• Cooperative Binding: How does binding of multiple SurA molecules to a single uOMP affect folding outcomes? The spatial arrangement and potential cooperativity between multiple bound SurA molecules remains poorly understood.

• Substrate Handoff: What is the molecular mechanism by which SurA transfers expanded uOMPs to the BAM complex? The structural details of this critical handoff event have not been fully elucidated.

Addressing these questions will require advanced biophysical techniques including single-molecule fluorescence, advanced nuclear magnetic resonance methods, and time-resolved structural biology approaches .

How might systems biology approaches enhance our understanding of SurA's role in the broader context of bacterial envelope homeostasis?

Systems biology approaches offer powerful frameworks for understanding SurA's role in bacterial envelope homeostasis:

• Network Analysis: Mapping protein-protein interaction networks connecting SurA to other components of envelope biogenesis machinery would reveal functional relationships and potential compensatory mechanisms.

• Multi-omics Integration: Combining transcriptomics, proteomics, metabolomics, and phenotypic data from SurA-deficient strains could identify:

  • Regulatory networks responding to SurA deficiency

  • Metabolic adaptations to membrane stress

  • Secondary effects cascading from primary OMP assembly defects

• Mathematical Modeling: Developing quantitative models of OMP biogenesis incorporating:

  • Rates of protein synthesis, transport, and assembly

  • Chaperone availability and substrate competition dynamics

  • Feedback mechanisms governing envelope homeostasis

• Synthetic Biology Approaches: Engineering strains with modified or expanded chaperone networks to:

  • Test predictions about network robustness

  • Identify minimal requirements for functional OMP assembly

  • Develop tunable systems for controlling outer membrane composition

• Comparative Genomics: Analyzing SurA conservation and co-evolution with substrate proteins across diverse bacteria could reveal specialized adaptations and functional constraints.

These approaches would help place SurA's function in broader context, potentially revealing unexpected connections to other cellular processes and identifying new targets for antimicrobial development .

What are the key takeaways from current research on SurA in E. coli?

Current research on SurA in E. coli has established several fundamental concepts with significant implications for bacterial physiology, pathogenesis, and antibiotic development:

• SurA functions as the primary chaperone in the periplasmic space, playing a critical role in the biogenesis of outer membrane proteins by preventing their aggregation during transport across the periplasm.

• The unique structural organization of SurA, featuring a core module with an extended crevice and a satellite PPIase domain, enables it to bind unfolded OMPs in a distinctive manner that causes dramatic expansion of the substrate proteins.

• SurA exhibits substrate specificity, with certain OMPs (particularly LptD and FhuA) showing high dependence on SurA for proper assembly independent of transcriptional effects.

• In pathogenic E. coli strains, SurA is required for the maturation of intracellular bacterial communities during urinary tract infections, linking OMP biogenesis directly to virulence mechanisms.

• SurA has been identified as a promising target for antibiotic development due to its essential role in maintaining outer membrane integrity and its contribution to pathogenesis.

These findings collectively highlight SurA as a critical component of bacterial envelope biogenesis with direct implications for bacterial survival, stress resistance, and pathogenicity .

How has our understanding of SurA function evolved over the past decade?

The past decade has witnessed significant evolution in our understanding of SurA function:

• From General Chaperone to Specific Mediator: Earlier views of SurA as a general periplasmic chaperone have evolved toward recognition of its specific role in OMP biogenesis, with evidence for preferential handling of certain substrate proteins.

• Structural Insights: Crystallographic studies revealed SurA's unique architecture, while newer integrative structural biology approaches have elucidated how SurA engages uOMPs through multiple binding modes, causing significant expansion of substrates.

• Pathogenesis Connection: Research has established direct links between SurA function and bacterial pathogenesis, particularly in uropathogenic E. coli, demonstrating roles beyond basic envelope maintenance.

• Substrate Specificity: Advanced proteomics approaches have refined our understanding of which OMPs truly depend on SurA, distinguishing direct from indirect effects through multi-omics integration.

• BAM Complex Relationship: The functional partnership between SurA and the BAM complex has been clarified, revealing how SurA prepares uOMPs for efficient transfer to this essential assembly machinery.

• Therapeutic Potential: Recognition of SurA as a promising antibiotic target has emerged, based on its essential role in maintaining outer membrane integrity and pathogenesis.

This evolution reflects a shift from descriptive to mechanistic understanding, enabled by advances in structural biology, proteomics, and infection models .

What methodological advances are needed to address current gaps in SurA research?

Addressing current gaps in SurA research will require several methodological advances:

• Real-time Single-molecule Techniques: Development of methods to visualize SurA-substrate interactions in real-time at single-molecule resolution would provide unprecedented insights into binding dynamics and conformational changes during the chaperoning process.

• In-cell Structural Biology: Adaptation of structural biology techniques (cryo-electron tomography, in-cell NMR) to study SurA-substrate complexes in their native cellular environment would bridge the gap between in vitro and in vivo observations.

• Quantitative Imaging of OMP Biogenesis: Advanced fluorescence microscopy approaches to track the spatiotemporal dynamics of OMP biogenesis in living cells would reveal how SurA coordinates with other cellular machinery.

• Targeted Protein Degradation Tools: Development of methods for rapid, specific degradation of SurA protein would enable precise temporal control for studying immediate versus long-term consequences of SurA deficiency.

• High-throughput Substrate Profiling: Creation of comprehensive libraries of potential SurA substrates paired with quantitative binding assays would enable systematic mapping of specificity determinants.

• Improved Animal Models: Development of refined in vivo models that allow visualization and manipulation of SurA activity during actual infection processes would strengthen the connection between molecular mechanisms and disease outcomes.