Secreted zinc-dependent alkaline metalloendopeptidase (serralysin / aeruginolysin) of the peptidase M10B (RTX-metalloprotease) family. The mature enzyme is a two-domain protein comprising an N-terminal catalytic metallopeptidase domain bearing a single catalytic zinc ion (coordinated by an HEXXH-type motif) and a C-terminal parallel beta-roll domain built from hemolysin-type (RTX) calcium-binding repeats; calcium binding drives folding and activation and stabilises the fold. It lacks a cleavable Sec signal peptide and is exported across both membranes by a dedicated type I secretion system (the AprDEF transporter), acting in the extracellular environment. Catalytically it preferentially cleaves peptide bonds N-terminal to hydrophobic residues and degrades a broad, immune-biased set of substrates. In the host it cleaves monomeric flagellin, complement components (notably C2, plus C1q, C1s, C3, C5a), cytokines, and the epithelial sodium channel, contributing to innate-immune evasion and modulation of host epithelial physiology.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0004222
metalloendopeptidase activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: Core molecular function. AprA is a zinc-dependent metalloendopeptidase (serralysin, EC 3.4.24.40) of the peptidase M10B family, with a catalytic zinc ion coordinated by an HEXXH-type motif (His185, His189, His195; active-site Glu186) and an endopeptidase mechanism cleaving internal peptide bonds.
Reason: Strongly supported by the solved crystal structures, the assigned EC number, MEROPS family classification (M10.056), and the catalytic zinc-binding active site. This is the central, defining molecular function of the protein.
Supporting Evidence:
UniProtKB:Q03023
RecName: Full=Serralysin; EC=3.4.24.40. Active site His185/His189/His195 coordinate catalytic Zn(2+); Belongs to the peptidase M10B family.
|
|
GO:0005509
calcium ion binding
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: Structural calcium binding. The C-terminal beta-roll is formed by hemolysin-type (RTX) calcium-binding repeats and the protein binds 8 Ca(2+) ions per subunit; the calcium-loaded beta-roll is required for folding, activation, and type I secretion.
Reason: Directly demonstrated by X-ray crystallography (calcium-binding parallel beta-roll motif) and recorded as a UniProt cofactor (binds 8 Ca(2+) per subunit). This is a genuine, structurally essential function that contributes to rather than constitutes the core catalytic function.
Supporting Evidence:
UniProtKB:Q03023
COFACTOR Name=Ca(2+); Note=Binds 8 Ca(2+) ions per subunit. Hemolysin-type calcium-binding repeats form a parallel beta-roll.
|
|
GO:0005576
extracellular region
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: Correct localization. AprA is a secreted protein exported by a dedicated type I secretion system (AprDEF) and acts extracellularly on host and microbial substrates.
Reason: UniProt records the subcellular location as "Secreted", and all characterised activities (flagellin/complement/ENaC cleavage) occur extracellularly. The more precise child term GO:0005615 (extracellular space) would also be appropriate, but extracellular region is correct.
Supporting Evidence:
UniProtKB:Q03023
SUBCELLULAR LOCATION: Secreted.
|
|
GO:0006508
proteolysis
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: Core biological process. AprA is a proteolytic enzyme; proteolysis is the process directly carried out by its metalloendopeptidase activity on host and microbial protein substrates.
Reason: Proteolysis is the fundamental process executed by this protease and is well supported by biochemical characterisation of its catalytic activity and substrate specificity.
Supporting Evidence:
UniProtKB:Q03023
CATALYTIC ACTIVITY: Preferential cleavage of bonds with hydrophobic residues in P1'.; EC=3.4.24.40
|
|
GO:0008233
peptidase activity
|
IEA
GO_REF:0000117 |
ACCEPT |
Summary: Correct but uninformatively broad. Peptidase activity is a high-level parent of the specific metalloendopeptidase activity (GO:0004222) already annotated for this enzyme.
Reason: The annotation is not wrong, but GO:0004222 (metalloendopeptidase activity) already captures this function at the appropriate level of specificity. Retained as a correct broad parent; it is not the core (most informative) molecular-function term.
Supporting Evidence:
UniProtKB:Q03023
RecName: Full=Serralysin; EC=3.4.24.40; Belongs to the peptidase M10B family.
|
|
GO:0008237
metallopeptidase activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: Correct but broad. Metallopeptidase activity is a parent of the specific metalloendopeptidase activity (GO:0004222) already annotated.
Reason: AprA is genuinely a zinc metallopeptidase, so the term is accurate, but it is subsumed by the more informative GO:0004222. Retained as a correct broad parent rather than the core molecular-function term.
Supporting Evidence:
UniProtKB:Q03023
Binds 1 zinc ion per subunit; catalytic Zn(2+) coordinated by His185/His189/His195; peptidase M10B (serralysin) family.
|
|
GO:0008270
zinc ion binding
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: Catalytic zinc binding. AprA binds one catalytic Zn(2+) ion in its active site, coordinated by the HEXXH-derived histidines (His185, His189, His195).
Reason: Directly supported by crystallography and UniProt cofactor/binding-site annotations; the catalytic zinc is essential for the metalloendopeptidase mechanism.
Supporting Evidence:
UniProtKB:Q03023
COFACTOR Name=Zn(2+); Note=Binds 1 zinc ion per subunit. BINDING 185/189/195 ligand=Zn(2+) ligand_note=catalytic.
|
|
GO:0031012
extracellular matrix
|
IEA
GO_REF:0000002 |
REMOVE |
Summary: Incorrect localization from an over-propagated electronic inference. This InterPro-to-GO mapping (peptidase M10 / matrixin signature) places AprA in the extracellular matrix, but AprA is a freely secreted bacterial protease, not a structural component of or resident in a host extracellular matrix.
Reason: Extracellular matrix (GO:0031012) is part_of extracellular region, so this is a more specific localization claim than GO:0005576, not a looser one, and the specific claim is unsupported. The annotation derives from InterPro IPR001818 (peptidase M10), a signature shared with eukaryotic matrix metalloproteinases (matrixins/MMPs) that genuinely reside in and act on the ECM; for a bacterial secreted serralysin this is an over-propagated IEA. AprA is secreted via the type I secretion system (AprDEF) and acts on soluble host substrates (complement C2/C1q/C1s, cytokines, monomeric flagellin); there is no evidence it localizes to or is a component of the extracellular matrix. Its correct localization is already captured by GO:0005576 (extracellular region). This is the demonstrably-wrong, argue-against-on-biological-grounds case for which REMOVE (rather than MARK_AS_OVER_ANNOTATED) is appropriate.
Supporting Evidence:
UniProtKB:Q03023
SUBCELLULAR LOCATION: Secreted.
|
|
GO:0141141
symbiont-mediated evasion of recognition by host pattern recognition receptor
|
EXP
PMID:22131330 Pseudomonas aeruginosa alkaline protease blocks complement a... |
ACCEPT |
Summary: Well-supported virulence function. AprA enables P. aeruginosa to evade host pattern-recognition-receptor detection. Its best-characterised mechanism is degradation of free monomeric flagellin, the ligand for the PRRs TLR5 (mammals) and FLS2 (plants), while flagellar (polymeric) flagellin and the receptors themselves are spared, preserving motility.
Reason: The term is genuinely correct for AprA and experimentally established. The most apt primary reference is the flagellin/TLR5/FLS2 study (PMID:21901099), which directly demonstrates evasion of PRR recognition; the cited complement paper (PMID:22131330) also bears on innate-immune evasion (lectin-pathway initiators MBL/ficolins are soluble PRRs). Added the flagellin reference as supporting evidence.
Supporting Evidence:
PMID:21901099
monomeric flagellin, while polymeric flagellin (involved in bacterial motility)
PMID:21901099
and TLR5 itself resist degradation.
PMID:21901099
This was due to decreased activation of the receptor FLS2 and
|
|
GO:0001869
negative regulation of complement activation, lectin pathway
|
IDA
PMID:22131330 Pseudomonas aeruginosa alkaline protease blocks complement a... |
ACCEPT |
Summary: AprA blocks lectin-pathway complement activation. It degrades complement C1s and C2 and, by cleaving C2, prevents formation of the C4b2a C3 convertase and C3b deposition via the lectin (and classical) pathway, while the alternative pathway is unaffected.
Reason: Directly demonstrated experimentally (IDA). Serum degradation and repletion assays show the inhibitory mechanism is cleavage of C2, blocking lectin-pathway C3b deposition. This is a specific, downstream (non-core) virulence consequence of the protease activity.
Supporting Evidence:
PMID:22131330
deposition via the classical and lectin pathways, whereas the alternative
PMID:22131330
of action for complement inhibition is cleavage of C2. In summary, we showed
|
|
GO:0008233
peptidase activity
|
IDA
PMID:22859302 Activation of the epithelial sodium channel (ENaC) by the al... |
ACCEPT |
Summary: Experimental confirmation of proteolytic activity. Purified AprA (AP) was shown to be folded and proteolytically active and to activate ENaC by cleavage.
Reason: Direct experimental evidence (IDA) of peptidase activity for purified AprA. The activity is more specifically a zinc metalloendopeptidase activity (GO:0004222), but the experimental peptidase annotation is correct as recorded.
Supporting Evidence:
PMID:22859302
further characterize this AP-induced ENaC activation, AP was purified, and its
PMID:22859302
folding, activity, and ability to activate ENaC were assessed.
|
|
GO:0010765
positive regulation of sodium ion transport
|
IDA
PMID:22859302 Activation of the epithelial sodium channel (ENaC) by the al... |
ACCEPT |
Summary: AprA proteolytically activates the epithelial sodium channel (ENaC). Apically applied AprA increased basal ENaC current consistent with sustained channel activation in human bronchial epithelia (CF and non-CF), with activation mapped to the channel's gamma-subunit.
Reason: Directly demonstrated experimentally (IDA) by short-circuit current measurements on polarized epithelial monolayers. A specific, downstream (non-core) effect of the protease on host ion transport, relevant to airway pathophysiology in cystic fibrosis.
Supporting Evidence:
PMID:22859302
trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To
PMID:22859302
ENaC in polarized monolayers indicated that AP activated ENaC in immortalized
|
|
GO:0045959
negative regulation of complement activation, classical pathway
|
IDA
PMID:22131330 Pseudomonas aeruginosa alkaline protease blocks complement a... |
ACCEPT |
Summary: AprA blocks classical-pathway complement activation. By degrading C1s and cleaving C2 it prevents C3b deposition via the classical (and lectin) pathway, inhibiting opsonization, C5a formation, and complement-dependent neutrophil phagocytosis/killing.
Reason: Directly demonstrated experimentally (IDA); the C2-cleavage mechanism blocks classical-pathway complement activation while sparing the alternative pathway. A specific, downstream (non-core) virulence consequence of the protease activity.
Supporting Evidence:
PMID:22131330
both human C1s and C2. However, repletion assays demonstrated that the mechanism
PMID:22131330
complement activation via cleavage of C2.
file:PSEAE/aprA/aprA-deep-research-falcon.md
AprA was identified as the first bacterial protease demonstrated to cleave C2
|
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
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Gene: aprA (PA1249) | UniProt: Q03023 | Organism: Pseudomonas aeruginosa PAO1
The aprA gene (ordered locus name PA1249) of Pseudomonas aeruginosa strain PAO1 encodes the alkaline protease (AP), also known as aeruginolysin, a secreted zinc-dependent metalloproteinase classified as a serralysin (EC 3.4.24.40). The mature protein is approximately 50 kDa and belongs to the peptidase M10B family within the metzincin superfamily of metalloproteases (laarman2012pseudomonasaeruginosaalkaline pages 2-3, butterworth2014modulationofthe pages 1-2). AprA is recognized as a key extracellular virulence factor of P. aeruginosa that functions primarily as an immune-modulating protease rather than a conventional tissue-degrading enzyme (krumina2026molecularmechanismsunderlying pages 8-10).
AprA is a two-domain metalloprotease precursor protein with the following architecture:
N-terminal catalytic domain: Contains the canonical HEXXHXXGXXH zinc coordination motif that is the hallmark of the metzincin superfamily. The active-site zinc ion (Zn²⁺) is essential for catalytic activity and is coordinated by three histidine residues within this motif (butterworth2014modulationofthe pages 1-2). This domain shares evolutionary similarity with mammalian matrix metalloproteinases (MMP2 and MMP9) (butterworth2014modulationofthe pages 4-5).
C-terminal calcium-binding RTX domain: Contains multiple RTX (Repeats-in-ToXin) nonapeptide repeats that are rich in aspartate and glycine residues. These repeats form a parallel β-roll motif upon binding calcium ions. The RTX domain is intrinsically disordered in the absence of Ca²⁺, and calcium binding at millimolar concentrations (apparent affinity of 50–60 mM) induces proper folding of the entire protein and is essential for activation of the N-terminal protease domain (butterworth2014modulationofthe pages 4-5, butterworth2014modulationofthe pages 1-2, butterworth2014modulationofthe pages 10-10). This calcium-dependent folding mechanism is a key regulatory feature of serralysin-type proteases and may also facilitate secretion, as the unfolded protein can be threaded through the type I secretion channel.
The structural and functional features of AprA are summarized below:
| Domain/Motif | Location | Function | Key Features |
|---|---|---|---|
| N-terminal catalytic domain | N-terminal region of mature AprA | Catalyzes proteolytic cleavage of host and microbial substrates as a Zn²⁺-dependent metalloprotease | Contains the conserved HEXXHXXGXXH zinc-binding motif characteristic of serralysin-type metalloproteases; the active-site Zn²⁺ is essential for catalysis; responsible for cleavage of substrates such as complement proteins and ENaC (butterworth2014modulationofthe pages 1-2) |
| C-terminal calcium-binding RTX domain | C-terminal region | Promotes folding, stabilization, and activation of the protease after Ca²⁺ binding | Contains RTX-associated Asp/Gly-rich nonapeptide repeats that form a Ca²⁺-bound parallel β-roll; intrinsically disordered or poorly folded without Ca²⁺; Ca²⁺ binding induces proper folding and enables full protease activity (butterworth2014modulationofthe pages 4-5, butterworth2014modulationofthe pages 1-2, butterworth2014modulationofthe pages 10-10) |
| Metalloprotease superfamily classification | Whole protein / family-level annotation | Places AprA within the serralysin branch of secreted bacterial metalloproteases | AprA is a serralysin-family alkaline metalloprotease belonging to peptidase family M10B within the metzincin-like metalloprotease superfamily; this classification is consistent with the Zn²⁺ catalytic motif and Ca²⁺-regulated RTX architecture (krumina2026molecularmechanismsunderlying pages 8-10, butterworth2014modulationofthe pages 1-2) |
| AprI inhibitor interaction | Functional interaction with AprA catalytic machinery | Protects the bacterium from uncontrolled AprA proteolysis and can block AprA activity extracellularly in vitro | AprI is the cognate alkaline protease inhibitor; it inhibits AprA near-stoichiometrically and blocks AprA-dependent ENaC activation; inhibitor action is linked to interaction with the Zn²⁺-containing protease active site, consistent with structural studies of the AprA-AprI system (butterworth2014modulationofthe pages 4-5, butterworth2014modulationofthe pages 7-9, butterworth2014modulationofthe pages 10-10) |
Table: This table summarizes the major structural and functional features of Pseudomonas aeruginosa AprA, including its catalytic and RTX calcium-binding regions, family classification, and interaction with the cognate inhibitor AprI. It is useful for connecting domain organization to secretion, activation, catalysis, and regulation.
AprA catalyzes the proteolytic cleavage of peptide bonds in a Zn²⁺-dependent manner, with Ca²⁺ serving as an essential cofactor for structural integrity and enzymatic activation. Without Ca²⁺, the enzyme exhibits minimal activity (butterworth2014modulationofthe pages 4-5). The enzyme retains sustained catalytic activity in the extracellular environment, maintaining 63% activity after 24 hours at 37°C (butterworth2014modulationofthe pages 4-5).
AprA has a broad but selective substrate specificity, with a marked preference for host immune proteins rather than generic structural extracellular matrix components. The primary substrates and their biological consequences are summarized in the following table:
| Substrate | Pathway/System Affected | Biological Consequence | Key Reference |
|---|---|---|---|
| Complement C2 | Classical and lectin complement pathways | AprA cleaves C2, preventing formation of the C4b2a C3 convertase, reducing C3b opsonization, C5a generation, neutrophil recruitment, phagocytosis, and complement-mediated killing (laarman2012pseudomonasaeruginosaalkaline pages 7-8, gonzalezalsina2023pseudomonasaeruginosaand pages 10-12) | (laarman2012pseudomonasaeruginosaalkaline pages 7-8, gonzalezalsina2023pseudomonasaeruginosaand pages 10-12) |
| C1q | Classical complement pathway | Degradation of C1q impairs complement recognition/activation and contributes to immune evasion (laarman2012pseudomonasaeruginosaalkaline pages 2-3, gonzalezalsina2023pseudomonasaeruginosaand pages 10-12) | (laarman2012pseudomonasaeruginosaalkaline pages 2-3, gonzalezalsina2023pseudomonasaeruginosaand pages 10-12) |
| C1s | Classical complement pathway | Cleavage of C1s contributes to suppression of classical pathway activation, although C2 cleavage appears to be the dominant inhibitory mechanism (laarman2012pseudomonasaeruginosaalkaline pages 7-8, laarman2012pseudomonasaeruginosaalkaline pages 1-2, laarman2012pseudomonasaeruginosaalkaline pages 2-3) | (laarman2012pseudomonasaeruginosaalkaline pages 7-8, laarman2012pseudomonasaeruginosaalkaline pages 1-2, laarman2012pseudomonasaeruginosaalkaline pages 2-3) |
| C3 | Central complement cascade | At higher concentrations/longer incubation, AprA can cleave C3, further reducing opsonization and downstream complement effector functions (laarman2012pseudomonasaeruginosaalkaline pages 7-8, laarman2012pseudomonasaeruginosaalkaline pages 2-3) | (laarman2012pseudomonasaeruginosaalkaline pages 7-8, laarman2012pseudomonasaeruginosaalkaline pages 2-3) |
| IFN-γ | Host cytokine signaling / innate immunity | Degradation of IFN-γ dampens host inflammatory and antimicrobial signaling, aiding immune evasion (laarman2012pseudomonasaeruginosaalkaline pages 2-3) | (laarman2012pseudomonasaeruginosaalkaline pages 2-3) |
| TNF-α | Host cytokine signaling / inflammation | Degradation of TNF-α reduces inflammatory signaling and host defense activation (laarman2012pseudomonasaeruginosaalkaline pages 2-3, akter2026structuralandfunctional pages 2-5) | (laarman2012pseudomonasaeruginosaalkaline pages 2-3, akter2026structuralandfunctional pages 2-5) |
| Flagellin | TLR5-mediated innate immune recognition | Cleavage of monomeric flagellin prevents TLR5 activation, reducing host detection of P. aeruginosa (laarman2012pseudomonasaeruginosaalkaline pages 2-3, keim2024polymicrobialinteractionsbetween pages 30-34) | (laarman2012pseudomonasaeruginosaalkaline pages 2-3, keim2024polymicrobialinteractionsbetween pages 30-34) |
| ENaC | Airway epithelial ion transport | Proteolytic cleavage activates epithelial sodium channel activity, potentially altering airway surface physiology; activation is slower than trypsin and can be blocked by AprI (butterworth2014modulationofthe pages 4-5, butterworth2014modulationofthe pages 7-9) | (butterworth2014modulationofthe pages 4-5, butterworth2014modulationofthe pages 7-9) |
| Fibrin | Tissue/extracellular host substrate in corneal infection | Fibrin degradation contributes to tissue damage and virulence in infected corneal tissue (akter2026structuralandfunctional pages 2-5) | (akter2026structuralandfunctional pages 2-5) |
| S. aureus SasG | Polymicrobial aggregation / biofilm formation | AprA cleaves SasG, removing/exposing domains that promote S. aureus aggregation, enhancing polymicrobial biofilm formation, persistence, and antibiotic tolerance in chronic wounds (keim2024polymicrobialinteractionsbetween pages 8-10, keim2024polymicrobialinteractionsbetween pages 10-13, keim2024polymicrobialinteractionsbetween pages 13-14) | (keim2024polymicrobialinteractionsbetween pages 8-10, keim2024polymicrobialinteractionsbetween pages 10-13, keim2024polymicrobialinteractionsbetween pages 13-14) |
Table: This table summarizes experimentally supported and review-supported AprA substrates, the host or microbial systems they affect, and the resulting biological consequences. It is useful for linking AprA's biochemical specificity to its roles in immune evasion, tissue damage, and polymicrobial biofilm biology.
The best-characterized enzymatic activity of AprA is its cleavage of complement component C2. AprA was identified as the first bacterial protease demonstrated to cleave C2 (laarman2012pseudomonasaeruginosaalkaline pages 7-8). At physiologically relevant concentrations (200 nM, within the range secreted by P. aeruginosa), AprA specifically cleaves C2 into C2a and C2b fragments. The resulting fluid-phase C2a cannot bind to surface-deposited C4b, preventing formation of the active C3 convertase (C4b2a) required for classical and lectin pathway complement activation (laarman2012pseudomonasaeruginosaalkaline pages 7-8). AprA cleaves C2 at sites similar to the natural cleavage sites of the complement proteases C1s and MASP, suggesting it targets accessible, structurally exposed regions (laarman2012pseudomonasaeruginosaalkaline pages 7-8). At higher concentrations and longer incubation times, AprA can also cleave C3 and C4 (laarman2012pseudomonasaeruginosaalkaline pages 7-8). Importantly, AprA does not inhibit the alternative complement pathway, as C2 is not involved in that route (laarman2012pseudomonasaeruginosaalkaline pages 7-8, laarman2012pseudomonasaeruginosaalkaline pages 1-2). The IC₅₀ for AprA-mediated inhibition of complement-dependent phagocytosis is approximately 100 nM (laarman2012pseudomonasaeruginosaalkaline pages 6-6). AprA also degrades C1q, C1s, and C5a, further suppressing complement recognition and downstream effector functions (laarman2012pseudomonasaeruginosaalkaline pages 2-3, gonzalezalsina2023pseudomonasaeruginosaand pages 10-12).
Beyond complement proteins, AprA degrades cytokines IFN-γ and TNF-α, dampening host inflammatory and antimicrobial signaling (laarman2012pseudomonasaeruginosaalkaline pages 2-3). AprA cleaves monomeric bacterial flagellin, preventing Toll-like receptor 5 (TLR5) activation and thus reducing innate immune detection of P. aeruginosa (laarman2012pseudomonasaeruginosaalkaline pages 2-3). AprA also disrupts neutrophil extracellular traps (NETs), a key antimicrobial defense mechanism (krumina2026molecularmechanismsunderlying pages 8-10). Additionally, AprA proteolytically activates the epithelial sodium channel (ENaC) on the apical surface of airway epithelial cells, leading to increased sodium absorption; this activation is slower than trypsin-mediated activation (approximately 5-fold slower kinetics) and can be blocked by the cognate inhibitor AprI (butterworth2014modulationofthe pages 4-5, butterworth2014modulationofthe pages 7-9). In corneal tissue, AprA degrades fibrin and contributes to tissue necrosis (akter2026structuralandfunctional pages 2-5).
AprA is secreted into the extracellular milieu via the Type I Secretion System (T1SS), a one-step secretion apparatus that directly translocates substrates across both inner and outer membranes without a periplasmic intermediate (akter2026structuralandfunctional pages 2-5, krumina2026molecularmechanismsunderlying pages 8-10, sousa2021genomicandmetabolic pages 5-7). The T1SS responsible for AprA secretion is encoded by the aprDEF gene cluster, organized in a dedicated operon located immediately upstream of the aprA structural gene (krumina2026molecularmechanismsunderlying pages 8-10). This system comprises three essential components: AprD, an ABC transporter that provides energy for translocation from the inner membrane; AprE, a membrane fusion protein (MFP) that bridges the periplasm; and AprF, an outer membrane factor (OMF) (krumina2026molecularmechanismsunderlying pages 8-10, sousa2021genomicandmetabolic pages 5-7). The protein is secreted with its propeptide intact (krumina2026molecularmechanismsunderlying pages 22-23). Once released, AprA functions exclusively in the extracellular environment, where it encounters and degrades host immune molecules at sites of infection (laarman2012pseudomonasaeruginosaalkaline pages 2-3, akter2026structuralandfunctional pages 2-5).
A periplasmic inhibitor, AprI, is co-expressed with AprA and serves to protect the bacterium from unregulated protease activity during secretion. AprI inhibits AprA at near-stoichiometric concentrations and can block AprA-mediated ENaC activation in a dose-dependent manner (butterworth2014modulationofthe pages 4-5, butterworth2014modulationofthe pages 7-9).
Expression of aprA is tightly regulated by multiple signaling systems:
Quorum sensing: AprA expression is controlled by the LasR-LasI (Las) quorum sensing system, with additional input from the RhlI-RhlR (Rhl) system. The LasR transcriptional activator directly regulates the apr gene cluster, linking AprA production to bacterial population density (krumina2026molecularmechanismsunderlying pages 8-10, gonzalezalsina2023pseudomonasaeruginosaand pages 10-12). The aprA gene was found to be part of the approximately 61% of LasR-regulated genes that were dysregulated in stringent response mutants (pletzer2020thestringentstress pages 12-13).
Stringent stress response: AprA is a downstream effector of the ppGpp-mediated stringent stress response. Under stringent response activation (e.g., serine hydroxamate induction), aprA is upregulated 4.1-fold, whereas in ppGpp-deficient mutants (relA spoT double mutant), aprA is downregulated 9.6-fold (pletzer2020thestringentstress pages 8-9, pletzer2020thestringentstress pages 7-8). This regulation may reflect a bacterial strategy to produce extracellular proteases for nutrient acquisition during amino acid starvation (pletzer2020thestringentstress pages 7-8).
Two-component regulatory systems: AprA is also subject to regulation by two-component signal transduction systems, including indirect control through hierarchical regulators such as AlgR and GacA (pletzer2020thestringentstress pages 9-11).
AprA is a critical immune evasion factor. By degrading C2, C1q, C1s, C3, and C5a, AprA potently inhibits complement-mediated opsonization, phagocytosis, and neutrophil killing of P. aeruginosa (laarman2012pseudomonasaeruginosaalkaline pages 1-2, laarman2012pseudomonasaeruginosaalkaline pages 7-8, gonzalezalsina2023pseudomonasaeruginosaand pages 10-12). This complement resistance operates as part of a redundant system in P. aeruginosa, alongside elastase (LasB) and Protease IV, which also degrade complement components (laarman2012pseudomonasaeruginosaalkaline pages 7-8). The cytokine-degrading and flagellin-cleaving activities further suppress innate immune activation (laarman2012pseudomonasaeruginosaalkaline pages 2-3).
In a murine cutaneous infection model, deletion of aprA significantly enhanced mouse survival to 77% compared with only 33% survival in wild-type PAO1-infected mice after 3 days (P = 0.025), despite no differences in initial lesion sizes or bacterial counts (pletzer2020thestringentstress pages 8-9). This directly demonstrates that AprA contributes to P. aeruginosa virulence through its proteolytic degradation of host defense molecules. AprA has also been identified as the downstream effector responsible for the reduced cytotoxicity observed in stringent response mutants (pletzer2020thestringentstress pages 1-2).
Recent research has revealed a novel role for AprA in mediating polymicrobial interactions. In chronic wound co-infections of P. aeruginosa and Staphylococcus aureus, AprA cleaves the S. aureus surface protein SasG by removing its A domain and exposing the B domain, enabling homodimeric interactions between adjacent S. aureus cells (keim2024polymicrobialinteractionsbetween pages 8-10). This SasG processing induces MRSA aggregation and promotes polymicrobial biofilm formation. Heterologous expression of AprA alone in E. coli is sufficient to induce SasG-dependent aggregation at levels comparable to wild-type P. aeruginosa (keim2024polymicrobialinteractionsbetween pages 8-10, keim2024polymicrobialinteractionsbetween pages 10-13). AprA works synergistically with LasB elastase for maximal SasG processing, and the resulting aggregates display increased tolerance to vancomycin and ciprofloxacin (keim2024polymicrobialinteractionsbetween pages 13-14). AprA expression is upregulated in vivo in clinical wound specimens (keim2024polymicrobialinteractionsbetween pages 8-10). These findings indicate that AprA promotes polymicrobial coexistence, delayed wound healing, and increased antimicrobial resistance in chronic wound infections (keim2024polymicrobialinteractionsbetween pages 13-14, keim2024polymicrobialinteractionsbetween pages 14-17).
AprA also contributes to pyocyanin production, an important virulence factor in P. aeruginosa (krumina2026molecularmechanismsunderlying pages 8-10, krumina2026molecularmechanismsunderlying pages 22-23). In corneal infections, AprA causes tissue necrosis, degrades fibrin, and increases bacterial attachment to corneal epithelium by exposing host cell receptors (akter2026structuralandfunctional pages 2-5).
The apr gene is highly conserved among clinical P. aeruginosa isolates. In a study of Mexican clinical isolates from hospital- and community-acquired infections, the alkaline protease gene (apr) was detected in 99.1% of strains, underscoring its near-ubiquitous presence in pathogenic P. aeruginosa populations (gonzalezalsina2023pseudomonasaeruginosaand pages 10-12). AprA has been highlighted as a promising target for anti-virulence therapeutic development, particularly given that aprA deletion enhances host survival in infection models without affecting bacterial growth, suggesting that targeting AprA could reduce virulence without imposing strong selective pressure for resistance (pletzer2020thestringentstress pages 8-9, pletzer2020thestringentstress pages 12-13).
AprA (alkaline protease/aeruginolysin) of P. aeruginosa PAO1 is a secreted, calcium-activated, zinc-dependent metalloprotease of the serralysin (M10B) family. It is exported via the AprDEF type I secretion system and functions exclusively in the extracellular environment. Its primary enzymatic activity is the proteolytic cleavage of host immune proteins—most notably complement component C2, which blocks classical and lectin pathway activation. AprA also degrades complement factors C1q, C1s, C3, and C5a, cytokines IFN-γ and TNF-α, and monomeric flagellin, collectively enabling immune evasion. Expression is regulated by the Las/Rhl quorum sensing hierarchy and the ppGpp-mediated stringent stress response. Recent work has expanded its functional repertoire to include promotion of polymicrobial biofilm formation through processing of the S. aureus surface protein SasG. Deletion of aprA significantly reduces virulence in murine infection models, establishing it as a key contributor to P. aeruginosa pathogenesis and a potential anti-virulence therapeutic target.
References
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(butterworth2014modulationofthe pages 1-2): Michael B. Butterworth, Liang Zhang, Xiaoning Liu, Robert M. Shanks, and Patrick H. Thibodeau. Modulation of the epithelial sodium channel (enac) by bacterial metalloproteases and protease inhibitors. PLoS ONE, 9:e100313, Jun 2014. URL: https://doi.org/10.1371/journal.pone.0100313, doi:10.1371/journal.pone.0100313. This article has 36 citations and is from a peer-reviewed journal.
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(butterworth2014modulationofthe pages 4-5): Michael B. Butterworth, Liang Zhang, Xiaoning Liu, Robert M. Shanks, and Patrick H. Thibodeau. Modulation of the epithelial sodium channel (enac) by bacterial metalloproteases and protease inhibitors. PLoS ONE, 9:e100313, Jun 2014. URL: https://doi.org/10.1371/journal.pone.0100313, doi:10.1371/journal.pone.0100313. This article has 36 citations and is from a peer-reviewed journal.
(butterworth2014modulationofthe pages 10-10): Michael B. Butterworth, Liang Zhang, Xiaoning Liu, Robert M. Shanks, and Patrick H. Thibodeau. Modulation of the epithelial sodium channel (enac) by bacterial metalloproteases and protease inhibitors. PLoS ONE, 9:e100313, Jun 2014. URL: https://doi.org/10.1371/journal.pone.0100313, doi:10.1371/journal.pone.0100313. This article has 36 citations and is from a peer-reviewed journal.
(butterworth2014modulationofthe pages 7-9): Michael B. Butterworth, Liang Zhang, Xiaoning Liu, Robert M. Shanks, and Patrick H. Thibodeau. Modulation of the epithelial sodium channel (enac) by bacterial metalloproteases and protease inhibitors. PLoS ONE, 9:e100313, Jun 2014. URL: https://doi.org/10.1371/journal.pone.0100313, doi:10.1371/journal.pone.0100313. This article has 36 citations and is from a peer-reviewed journal.
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(gonzalezalsina2023pseudomonasaeruginosaand pages 10-12): Alex González-Alsina, Margalida Mateu-Borrás, Antonio Doménech-Sánchez, and Sebastián Albertí. Pseudomonas aeruginosa and the complement system: a review of the evasion strategies. Microorganisms, 11:664, Mar 2023. URL: https://doi.org/10.3390/microorganisms11030664, doi:10.3390/microorganisms11030664. This article has 31 citations.
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(keim2024polymicrobialinteractionsbetween pages 10-13): Klara Keim, Mohini Bhattacharya, H. A. Crosby, C. Jenul, Krista B. Mills, Michael J. Schurr, and A. Horswill. Polymicrobial interactions between staphylococcus aureus and pseudomonas aeruginosa promote biofilm formation and persistence in chronic wound infections. bioRxiv, Nov 2024. URL: https://doi.org/10.1101/2024.11.04.621402, doi:10.1101/2024.11.04.621402. This article has 18 citations.
(keim2024polymicrobialinteractionsbetween pages 13-14): Klara Keim, Mohini Bhattacharya, H. A. Crosby, C. Jenul, Krista B. Mills, Michael J. Schurr, and A. Horswill. Polymicrobial interactions between staphylococcus aureus and pseudomonas aeruginosa promote biofilm formation and persistence in chronic wound infections. bioRxiv, Nov 2024. URL: https://doi.org/10.1101/2024.11.04.621402, doi:10.1101/2024.11.04.621402. This article has 18 citations.
(laarman2012pseudomonasaeruginosaalkaline pages 6-6): A. Laarman, B. Bardoel, M. Ruyken, Job Fernie, F. Milder, J. V. van Strijp, and S. Rooijakkers. Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways. The Journal of Immunology, 188:386-393, Jan 2012. URL: https://doi.org/10.4049/jimmunol.1102162, doi:10.4049/jimmunol.1102162. This article has 160 citations.
(sousa2021genomicandmetabolic pages 5-7): Telma de Sousa, Michel Hébraud, Maria L. N. Enes Dapkevicius, Luís Maltez, José Eduardo Pereira, Rosa Capita, Carlos Alonso-Calleja, Gilberto Igrejas, and Patricia Poeta. Genomic and metabolic characteristics of the pathogenicity in pseudomonas aeruginosa. International Journal of Molecular Sciences, 22:12892, Nov 2021. URL: https://doi.org/10.3390/ijms222312892, doi:10.3390/ijms222312892. This article has 225 citations.
(krumina2026molecularmechanismsunderlying pages 22-23): Angelika Krūmiņa, Aigars Reinis, Agneta Jeske, Indra Zeltiņa, and Ludmila Vīksna. Molecular mechanisms underlying the pathogenicity of pseudomonas aeruginosa. Medicina, 62:462, Feb 2026. URL: https://doi.org/10.3390/medicina62030462, doi:10.3390/medicina62030462. This article has 1 citations.
(pletzer2020thestringentstress pages 12-13): Daniel Pletzer, Travis M. Blimkie, Heidi Wolfmeier, Yicong Li, Arjun Baghela, Amy H. Y. Lee, Reza Falsafi, and Robert E. W. Hancock. The stringent stress response controls proteases and global regulators under optimal growth conditions in pseudomonas aeruginosa. Aug 2020. URL: https://doi.org/10.1128/msystems.00495-20, doi:10.1128/msystems.00495-20. This article has 39 citations and is from a peer-reviewed journal.
(pletzer2020thestringentstress pages 8-9): Daniel Pletzer, Travis M. Blimkie, Heidi Wolfmeier, Yicong Li, Arjun Baghela, Amy H. Y. Lee, Reza Falsafi, and Robert E. W. Hancock. The stringent stress response controls proteases and global regulators under optimal growth conditions in pseudomonas aeruginosa. Aug 2020. URL: https://doi.org/10.1128/msystems.00495-20, doi:10.1128/msystems.00495-20. This article has 39 citations and is from a peer-reviewed journal.
(pletzer2020thestringentstress pages 7-8): Daniel Pletzer, Travis M. Blimkie, Heidi Wolfmeier, Yicong Li, Arjun Baghela, Amy H. Y. Lee, Reza Falsafi, and Robert E. W. Hancock. The stringent stress response controls proteases and global regulators under optimal growth conditions in pseudomonas aeruginosa. Aug 2020. URL: https://doi.org/10.1128/msystems.00495-20, doi:10.1128/msystems.00495-20. This article has 39 citations and is from a peer-reviewed journal.
(pletzer2020thestringentstress pages 9-11): Daniel Pletzer, Travis M. Blimkie, Heidi Wolfmeier, Yicong Li, Arjun Baghela, Amy H. Y. Lee, Reza Falsafi, and Robert E. W. Hancock. The stringent stress response controls proteases and global regulators under optimal growth conditions in pseudomonas aeruginosa. Aug 2020. URL: https://doi.org/10.1128/msystems.00495-20, doi:10.1128/msystems.00495-20. This article has 39 citations and is from a peer-reviewed journal.
(pletzer2020thestringentstress pages 1-2): Daniel Pletzer, Travis M. Blimkie, Heidi Wolfmeier, Yicong Li, Arjun Baghela, Amy H. Y. Lee, Reza Falsafi, and Robert E. W. Hancock. The stringent stress response controls proteases and global regulators under optimal growth conditions in pseudomonas aeruginosa. Aug 2020. URL: https://doi.org/10.1128/msystems.00495-20, doi:10.1128/msystems.00495-20. This article has 39 citations and is from a peer-reviewed journal.
(keim2024polymicrobialinteractionsbetween pages 14-17): Klara Keim, Mohini Bhattacharya, H. A. Crosby, C. Jenul, Krista B. Mills, Michael J. Schurr, and A. Horswill. Polymicrobial interactions between staphylococcus aureus and pseudomonas aeruginosa promote biofilm formation and persistence in chronic wound infections. bioRxiv, Nov 2024. URL: https://doi.org/10.1101/2024.11.04.621402, doi:10.1101/2024.11.04.621402. This article has 18 citations.
UniProt: Q03023 (APRA_PSEAE); EC 3.4.24.40; MEROPS M10.056; peptidase M10B
(serralysin) family. 479 aa precursor (propeptide 1–9, mature chain 10–479).
AprA is a secreted, zinc-dependent alkaline metalloendopeptidase of the
serralysin (RTX-type metalloprotease, peptidase M10B) family. It is a major
extracellular virulence factor of P. aeruginosa. Structurally it is a
two-domain protein: an N-terminal catalytic metallopeptidase domain (HEXXH zinc
motif) and a C-terminal β-roll domain formed by hemolysin-type (RTX)
calcium-binding repeats that bind multiple Ca²⁺ ions. It has no cleavable
N-terminal signal peptide and is exported by a dedicated type I secretion system
(the AprDEF ABC-transporter / RTX exporter encoded in the same operon), with a
C-terminal secretion signal. It is co-secreted with, and held in check by, the
periplasmic/secreted inhibitor AprI.
Catalytically it preferentially cleaves peptide bonds N-terminal to (in P1' of)
hydrophobic residues. Biologically it acts on a broad range of host substrates,
and its best-characterized roles are in innate-immune evasion and modulation of
host epithelial physiology.
aprA mutants give >100-fold enhanced TLR5 activation. PMID:21901099Core function:
- Molecular function: zinc metalloendopeptidase activity (GO:0004222), with
catalytic Zn²⁺ binding (GO:0008270) and structural Ca²⁺ binding (GO:0005509).
- Cellular component: secreted / extracellular region (GO:0005576;
GO:0005615 extracellular space is the more precise term).
- Biological process: proteolysis (GO:0006508), executed on host substrates
to drive virulence (immune evasion; host ion-transport modulation = non-core,
pleiotropic downstream consequences).
Likely over-/under-annotations to flag:
- GO:0008233 (peptidase) and GO:0008237 (metallopeptidase) IEA are correct but are
high-level parents of the more informative GO:0004222 metalloendopeptidase
activity → general/redundant.
- GO:0031012 "extracellular matrix" (located_in, IEA from InterPro) is a likely
over-annotation — AprA is freely secreted into the extracellular space, not a
structural ECM component. Prefer GO:0005576 / GO:0005615.
Broader experimentally/review-supported substrate range and roles (not all yet GO-annotated):
- Complement: primary substrate is C2 (cleaved into C2a/C2b, ~200 nM, blocks
C4b2a convertase); also degrades C1q, C1s, C3, C5a. "AprA was identified as
the first bacterial protease demonstrated to cleave C2."
- Cytokines: degrades IFN-γ and TNF-α, dampening inflammatory signaling.
- Disrupts neutrophil extracellular traps (NETs).
- Corneal infection: degrades fibrin, causes tissue necrosis, increases
bacterial attachment to corneal epithelium.
- Polymicrobial biofilm: cleaves S. aureus surface protein SasG (removes
A domain, exposes B domain → MRSA aggregation, biofilm, antibiotic tolerance)
[keim2024, bioRxiv preprint — treat as provisional].
- Virulence in vivo: aprA deletion raised murine survival to 77% vs 33% for
WT PAO1 (cutaneous infection), with no change in bacterial counts [pletzer2020].
- Secretion: type I secretion system encoded by the aprDEF operon (AprD ABC
transporter, AprE membrane-fusion protein, AprF outer-membrane factor); secreted
with propeptide intact; folding/activation is Ca²⁺-dependent (RTX β-roll).
- Regulation: Las/Rhl quorum sensing and the ppGpp stringent response.
- Conservation: apr gene present in ~99% of clinical P. aeruginosa isolates;
proposed anti-virulence drug target.
Note: the falcon report mis-attributes the flagellin/TLR5 finding to Laarman 2012;
the correct primary source is Bardoel 2011 (PMID:21901099).
id: Q03023
gene_symbol: aprA
aliases: [APRA, PA1249, AP, aeruginolysin, "alkaline metalloproteinase"]
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:208964
label: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM
14847 / LMG 12228 / 1C / PRS 101 / PAO1)
description: Secreted zinc-dependent alkaline metalloendopeptidase (serralysin / aeruginolysin)
of the peptidase M10B (RTX-metalloprotease) family. The mature enzyme is a two-domain
protein comprising an N-terminal catalytic metallopeptidase domain bearing a single
catalytic zinc ion (coordinated by an HEXXH-type motif) and a C-terminal parallel
beta-roll domain built from hemolysin-type (RTX) calcium-binding repeats; calcium
binding drives folding and activation and stabilises the fold. It lacks a cleavable
Sec signal peptide and is exported across both membranes by a dedicated type I secretion
system (the AprDEF transporter), acting in the extracellular environment. Catalytically
it preferentially cleaves peptide bonds N-terminal to hydrophobic residues and degrades
a broad, immune-biased set of substrates. In the host it cleaves monomeric flagellin,
complement components (notably C2, plus C1q, C1s, C3, C5a), cytokines, and the epithelial
sodium channel, contributing to innate-immune evasion and modulation of host epithelial
physiology.
existing_annotations:
- term:
id: GO:0004222
label: metalloendopeptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: Core molecular function. AprA is a zinc-dependent metalloendopeptidase
(serralysin, EC 3.4.24.40) of the peptidase M10B family, with a catalytic zinc
ion coordinated by an HEXXH-type motif (His185, His189, His195; active-site
Glu186) and an endopeptidase mechanism cleaving internal peptide bonds.
action: ACCEPT
reason: Strongly supported by the solved crystal structures, the assigned EC number,
MEROPS family classification (M10.056), and the catalytic zinc-binding active
site. This is the central, defining molecular function of the protein.
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: 'RecName: Full=Serralysin; EC=3.4.24.40. Active site His185/His189/His195
coordinate catalytic Zn(2+); Belongs to the peptidase M10B family.'
- term:
id: GO:0005509
label: calcium ion binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: Structural calcium binding. The C-terminal beta-roll is formed by hemolysin-type
(RTX) calcium-binding repeats and the protein binds 8 Ca(2+) ions per subunit;
the calcium-loaded beta-roll is required for folding, activation, and type I
secretion.
action: ACCEPT
reason: Directly demonstrated by X-ray crystallography (calcium-binding parallel
beta-roll motif) and recorded as a UniProt cofactor (binds 8 Ca(2+) per subunit).
This is a genuine, structurally essential function that contributes to rather
than constitutes the core catalytic function.
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: 'COFACTOR Name=Ca(2+); Note=Binds 8 Ca(2+) ions per subunit.
Hemolysin-type calcium-binding repeats form a parallel beta-roll.'
- term:
id: GO:0005576
label: extracellular region
evidence_type: IEA
original_reference_id: GO_REF:0000120
qualifier: located_in
review:
summary: Correct localization. AprA is a secreted protein exported by a dedicated
type I secretion system (AprDEF) and acts extracellularly on host and microbial
substrates.
action: ACCEPT
reason: UniProt records the subcellular location as "Secreted", and all characterised
activities (flagellin/complement/ENaC cleavage) occur extracellularly. The more
precise child term GO:0005615 (extracellular space) would also be appropriate,
but extracellular region is correct.
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: 'SUBCELLULAR LOCATION: Secreted.'
- term:
id: GO:0006508
label: proteolysis
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: involved_in
review:
summary: Core biological process. AprA is a proteolytic enzyme; proteolysis is
the process directly carried out by its metalloendopeptidase activity on host
and microbial protein substrates.
action: ACCEPT
reason: Proteolysis is the fundamental process executed by this protease and is
well supported by biochemical characterisation of its catalytic activity and
substrate specificity.
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: "CATALYTIC ACTIVITY: Preferential cleavage of bonds with hydrophobic\
\ residues in P1'.; EC=3.4.24.40"
- term:
id: GO:0008233
label: peptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000117
qualifier: enables
review:
summary: Correct but uninformatively broad. Peptidase activity is a high-level
parent of the specific metalloendopeptidase activity (GO:0004222) already annotated
for this enzyme.
action: ACCEPT
reason: The annotation is not wrong, but GO:0004222 (metalloendopeptidase activity)
already captures this function at the appropriate level of specificity. Retained
as a correct broad parent; it is not the core (most informative) molecular-function
term.
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: 'RecName: Full=Serralysin; EC=3.4.24.40; Belongs to the peptidase
M10B family.'
- term:
id: GO:0008237
label: metallopeptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: Correct but broad. Metallopeptidase activity is a parent of the specific
metalloendopeptidase activity (GO:0004222) already annotated.
action: ACCEPT
reason: AprA is genuinely a zinc metallopeptidase, so the term is accurate, but
it is subsumed by the more informative GO:0004222. Retained as a correct broad
parent rather than the core molecular-function term.
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: Binds 1 zinc ion per subunit; catalytic Zn(2+) coordinated by
His185/His189/His195; peptidase M10B (serralysin) family.
- term:
id: GO:0008270
label: zinc ion binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: Catalytic zinc binding. AprA binds one catalytic Zn(2+) ion in its active
site, coordinated by the HEXXH-derived histidines (His185, His189, His195).
action: ACCEPT
reason: Directly supported by crystallography and UniProt cofactor/binding-site
annotations; the catalytic zinc is essential for the metalloendopeptidase mechanism.
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: 'COFACTOR Name=Zn(2+); Note=Binds 1 zinc ion per subunit. BINDING
185/189/195 ligand=Zn(2+) ligand_note=catalytic.'
- term:
id: GO:0031012
label: extracellular matrix
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: located_in
review:
summary: Incorrect localization from an over-propagated electronic inference.
This InterPro-to-GO mapping (peptidase M10 / matrixin signature) places AprA
in the extracellular matrix, but AprA is a freely secreted bacterial protease,
not a structural component of or resident in a host extracellular matrix.
action: REMOVE
reason: 'Extracellular matrix (GO:0031012) is part_of extracellular region, so
this is a more specific localization claim than GO:0005576, not a looser one,
and the specific claim is unsupported. The annotation derives from InterPro
IPR001818 (peptidase M10), a signature shared with eukaryotic matrix metalloproteinases
(matrixins/MMPs) that genuinely reside in and act on the ECM; for a bacterial
secreted serralysin this is an over-propagated IEA. AprA is secreted via the
type I secretion system (AprDEF) and acts on soluble host substrates (complement
C2/C1q/C1s, cytokines, monomeric flagellin); there is no evidence it localizes
to or is a component of the extracellular matrix. Its correct localization is
already captured by GO:0005576 (extracellular region). This is the demonstrably-wrong,
argue-against-on-biological-grounds case for which REMOVE (rather than
MARK_AS_OVER_ANNOTATED) is appropriate.'
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: 'SUBCELLULAR LOCATION: Secreted.'
- term:
id: GO:0141141
label: symbiont-mediated evasion of recognition by host pattern recognition receptor
evidence_type: EXP
original_reference_id: PMID:22131330
qualifier: involved_in
review:
summary: Well-supported virulence function. AprA enables P. aeruginosa to evade
host pattern-recognition-receptor detection. Its best-characterised mechanism
is degradation of free monomeric flagellin, the ligand for the PRRs TLR5 (mammals)
and FLS2 (plants), while flagellar (polymeric) flagellin and the receptors themselves
are spared, preserving motility.
action: ACCEPT
reason: The term is genuinely correct for AprA and experimentally established.
The most apt primary reference is the flagellin/TLR5/FLS2 study (PMID:21901099),
which directly demonstrates evasion of PRR recognition; the cited complement
paper (PMID:22131330) also bears on innate-immune evasion (lectin-pathway initiators
MBL/ficolins are soluble PRRs). Added the flagellin reference as supporting evidence.
additional_reference_ids:
- PMID:21901099
supported_by:
- reference_id: PMID:21901099
supporting_text: monomeric flagellin, while polymeric flagellin (involved in
bacterial motility)
- reference_id: PMID:21901099
supporting_text: and TLR5 itself resist degradation.
- reference_id: PMID:21901099
supporting_text: This was due to decreased activation of the receptor FLS2 and
- term:
id: GO:0001869
label: negative regulation of complement activation, lectin pathway
evidence_type: IDA
original_reference_id: PMID:22131330
qualifier: involved_in
review:
summary: AprA blocks lectin-pathway complement activation. It degrades complement
C1s and C2 and, by cleaving C2, prevents formation of the C4b2a C3 convertase
and C3b deposition via the lectin (and classical) pathway, while the alternative
pathway is unaffected.
action: ACCEPT
reason: Directly demonstrated experimentally (IDA). Serum degradation and repletion
assays show the inhibitory mechanism is cleavage of C2, blocking lectin-pathway
C3b deposition. This is a specific, downstream (non-core) virulence consequence
of the protease activity.
supported_by:
- reference_id: PMID:22131330
supporting_text: deposition via the classical and lectin pathways, whereas the
alternative
- reference_id: PMID:22131330
supporting_text: of action for complement inhibition is cleavage of C2. In summary,
we showed
- term:
id: GO:0008233
label: peptidase activity
evidence_type: IDA
original_reference_id: PMID:22859302
qualifier: enables
review:
summary: Experimental confirmation of proteolytic activity. Purified AprA (AP)
was shown to be folded and proteolytically active and to activate ENaC by cleavage.
action: ACCEPT
reason: Direct experimental evidence (IDA) of peptidase activity for purified AprA.
The activity is more specifically a zinc metalloendopeptidase activity (GO:0004222),
but the experimental peptidase annotation is correct as recorded.
supported_by:
- reference_id: PMID:22859302
supporting_text: further characterize this AP-induced ENaC activation, AP was
purified, and its
- reference_id: PMID:22859302
supporting_text: folding, activity, and ability to activate ENaC were assessed.
- term:
id: GO:0010765
label: positive regulation of sodium ion transport
evidence_type: IDA
original_reference_id: PMID:22859302
qualifier: involved_in
review:
summary: AprA proteolytically activates the epithelial sodium channel (ENaC).
Apically applied AprA increased basal ENaC current consistent with sustained
channel activation in human bronchial epithelia (CF and non-CF), with activation
mapped to the channel's gamma-subunit.
action: ACCEPT
reason: Directly demonstrated experimentally (IDA) by short-circuit current measurements
on polarized epithelial monolayers. A specific, downstream (non-core) effect
of the protease on host ion transport, relevant to airway pathophysiology in
cystic fibrosis.
supported_by:
- reference_id: PMID:22859302
supporting_text: trypsin-inducible ENaC current, consistent with sustained activation
of ENaC. To
- reference_id: PMID:22859302
supporting_text: ENaC in polarized monolayers indicated that AP activated ENaC
in immortalized
- term:
id: GO:0045959
label: negative regulation of complement activation, classical pathway
evidence_type: IDA
original_reference_id: PMID:22131330
qualifier: involved_in
review:
summary: AprA blocks classical-pathway complement activation. By degrading C1s
and cleaving C2 it prevents C3b deposition via the classical (and lectin) pathway,
inhibiting opsonization, C5a formation, and complement-dependent neutrophil
phagocytosis/killing.
action: ACCEPT
reason: Directly demonstrated experimentally (IDA); the C2-cleavage mechanism
blocks classical-pathway complement activation while sparing the alternative
pathway. A specific, downstream (non-core) virulence consequence of the protease
activity.
supported_by:
- reference_id: PMID:22131330
supporting_text: both human C1s and C2. However, repletion assays demonstrated
that the mechanism
- reference_id: PMID:22131330
supporting_text: complement activation via cleavage of C2.
- reference_id: file:PSEAE/aprA/aprA-deep-research-falcon.md
supporting_text: AprA was identified as the first bacterial protease demonstrated
to cleave C2
core_functions:
- description: Secreted zinc metalloendopeptidase that degrades extracellular (host
and microbial) protein substrates
molecular_function:
id: GO:0004222
label: metalloendopeptidase activity
directly_involved_in:
- id: GO:0006508
label: proteolysis
locations:
- id: GO:0005576
label: extracellular region
supported_by:
- reference_id: UniProtKB:Q03023
supporting_text: 'RecName: Full=Serralysin; EC=3.4.24.40; catalytic Zn(2+) coordinated
by His185/His189/His195; SUBCELLULAR LOCATION: Secreted.'
- reference_id: PMID:22859302
supporting_text: folding, activity, and ability to activate ENaC were assessed.
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO
terms
findings: []
- id: GO_REF:0000117
title: Electronic Gene Ontology annotations created by ARBA machine learning models
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: PMID:22131330
title: Pseudomonas aeruginosa alkaline protease blocks complement activation via
the classical and lectin pathways.
findings:
- statement: >-
AprA degrades human C1s and C2; the mechanism of complement inhibition is
cleavage of C2, blocking C3b deposition via the classical and lectin pathways
while sparing the alternative pathway.
supporting_text: of action for complement inhibition is cleavage of C2. In summary,
we showed
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: PubMed-verified abstract; directly supports the classical- and lectin-pathway
complement-inhibition annotations (GO:0045959, GO:0001869) via C2 cleavage.
- id: PMID:22859302
title: Activation of the epithelial sodium channel (ENaC) by the alkaline protease
from Pseudomonas aeruginosa.
findings:
- statement: >-
Purified AprA proteolytically activates ENaC (mapped to the gamma-subunit),
increasing basal sodium current in primary human bronchial epithelia from CF
and non-CF patients.
supporting_text: ENaC in polarized monolayers indicated that AP activated ENaC
in immortalized
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: PubMed-verified abstract; supports the peptidase-activity (IDA) and
positive-regulation-of-sodium-ion-transport (GO:0010765) annotations.
- id: PMID:21901099
title: Pseudomonas evades immune recognition of flagellin in both mammals and plants.
findings:
- statement: >-
AprA degrades free monomeric flagellin (the TLR5/FLS2 ligand) but not polymeric
flagellar flagellin or the receptors, allowing the bacterium to evade
pattern-recognition-receptor detection while retaining motility.
supporting_text: monomeric flagellin, while polymeric flagellin (involved in
bacterial motility)
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: Full text (PMC) reviewed; the most apt primary reference for GO:0141141
(symbiont-mediated evasion of recognition by host PRR), establishing degradation
of monomeric flagellin to evade TLR5/FLS2.
- id: file:PSEAE/aprA/aprA-deep-research-falcon.md
title: Deep research report (falcon/Edison) for APRA
findings:
- statement: >-
AprA is a calcium-activated, zinc-dependent serralysin (M10B) secreted by the
AprDEF type I secretion system, with an immune-biased substrate range (complement
C2/C1q/C1s/C3/C5a, cytokines IFN-gamma/TNF-alpha, monomeric flagellin, ENaC,
fibrin); aprA deletion attenuates virulence in a murine infection model.