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).
Summary
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.
Structure / cofactors (from UniProt Q03023 + crystallography)
- Catalytic Zn²⁺: coordinated by His185, His189, His195; catalytic Glu186
(active site His-Glu-X-X-His … His "zincin/metzincin" motif). [UniProt Q03023
FT ACT_SITE 186; BINDING 185/189/195 Zn] [PMID:8253063 EMBO J 1993, crystal
structure: "two-domain protein with a calcium binding parallel beta roll motif"]
- Binds 8 Ca²⁺ per subunit and 1 Zn²⁺ per subunit. [UniProt Q03023 COFACTOR]
- C-terminal β-roll formed by hemolysin-type (RTX) Ca-binding repeats (FT REPEAT
341–354, 355–367, 368–385; PROSITE PS00330 HEMOLYSIN_CALCIUM). The Ca²⁺-loaded
β-roll is also the structural element required for type I secretion/folding.
- Cognate inhibitor complex solved: AprA + AprI ("inhibition by a zinc-NH2
coordinative bond"). PMID:11445573
- Subcellular location: Secreted. [UniProt Q03023]
Catalytic activity / specificity
- EC 3.4.24.40; "Preferential cleavage of bonds with hydrophobic residues in P1'."
[UniProt Q03023 CATALYTIC ACTIVITY; ECO:0000269|PubMed:4199986]
- Specificity established with synthetic peptides. [PMID:4199986 Morihara 1973]
Biological roles / host substrates (virulence)
1. Innate-immune evasion via degradation of monomeric flagellin (TLR5/FLS2)
- AprA degrades free monomeric flagellin (the TLR5/FLS2 ligand) but not
polymeric flagellin in the flagellum, so the bacterium evades pattern-recognition
receptor detection while keeping motility.
[PMID:21901099 Bardoel 2011, "AprA effectively degrades the TLR5 ligand
monomeric flagellin, while polymeric flagellin (involved in bacterial motility)
and TLR5 itself resist degradation"]
["by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition
by the innate immune systems of both mammals and plants"]
aprA mutants give >100-fold enhanced TLR5 activation. PMID:21901099- This is the strongest experimental basis for the GO term
GO:0141141 "symbiont-mediated evasion of recognition by host pattern recognition
receptor". (Note: the GOA EXP annotation for GO:0141141 currently cites the
complement paper PMID:22131330; the flagellin paper PMID:21901099 is the more
apt primary reference. Complement lectin-pathway initiators (MBL/ficolins) are
also soluble PRRs, so the term is still defensible for the gene.)
2. Inhibition of complement (classical + lectin pathways) via C2 cleavage
- AprA degrades human C1s and C2; complement inhibition mechanism is cleavage of
C2; blocks C3b deposition via the classical and lectin (not alternative)
pathways; blocks neutrophil phagocytosis/killing and C5a formation.
[PMID:22131330 Laarman 2012, "AprA specifically blocked C3b deposition via the
classical and lectin pathways, whereas the alternative pathway was not affected";
"the mechanism of action for complement inhibition is cleavage of C2"]
- Basis for GO:0045959 (neg. reg. complement, classical) and GO:0001869 (neg. reg.
complement, lectin), both IDA.
3. Activation of the epithelial sodium channel (ENaC)
- Purified AP proteolytically activates ENaC by cleaving the γ-subunit,
increasing basal Na⁺ current in human bronchial epithelia (CF and non-CF);
proposed to reduce airway-surface-liquid volume / mucociliary clearance in CF.
[PMID:22859302 Butterworth 2012, "This activation was mapped to the γ-subunit of
ENaC"; "an increase in basal ENaC current and a loss of trypsin-inducible ENaC
current, consistent with sustained activation of ENaC"]
- Basis for GO:0010765 (positive regulation of sodium ion transport, IDA) and a
second experimental confirmation of peptidase activity (GO:0008233, IDA).
GO annotation review orientation
Core 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.
Additional findings (from falcon/Edison deep research, APRA-deep-research-falcon.md)
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).
References
- UniProt Q03023 (APRA_PSEAE)
- PMID:4199986 — substrate specificity (synthetic peptides), EC 3.4.24.40
- PMID:8253063 — crystal structure, two-domain + Ca β-roll
- PMID:11445573 — AprA–AprI inhibitor complex structure
- PMID:21901099 — flagellin/TLR5/FLS2 immune evasion (Bardoel 2011)
- PMID:22131330 — complement classical/lectin inhibition via C2 cleavage (Laarman 2012)
- PMID:22859302 — ENaC γ-subunit activation (Butterworth 2012)