(A) Intestinal neutrophil response to conventional (CV), germ free (GF), and mono-associations of wild-type Aeromonas (Aer ΔT2C), and an isogenic mutant of the type II secretion system (ΔT2). ΔT2 …
Aeromonas secretes a protein that regulates intestinal neutrophil response to a general model of inflammation.
(A) veronii strain Hm21 isolates ΔT2, the type two secretion mutant, and ΔT2C, the compliment isolate with a wild-type phenotype, colonize the fish to similar levels. Each dot represents one fish. n …
Colonization of A. veronii strain Hm21 type 2 secretion mutant.
(A) The 2.3 Å structure of AimA displays two calycin domains (PDB ID 6B7L). (B) The amino terminal domain (orange) of AimA is connected by a short linker to the carboxy terminal domain (teal) and …
(A) Structural overlay of N-term domain of AimA with top structural homology hit from PDBeFold search (Supplemental Table 2), Streptomyces avindinii streptavidin (PDB ID 1KL3). (B) Structural …
Shown are Rfree (black line, solid squares), Rwork, (gray line, solid triangles), and Rfree-Rwork (dotted line, open circles) calculated at 2.9 Å for paired refinements in which the model was first …
(A) Feeding zebrafish soy saponin induces increased intestinal neutrophil response, and treating those fish with 100 ng/mL purified AimA prevents the increased intestinal neutrophil response. …
AimA contains regions structurally conserved with the lipocalin family and reduces soysaponin induced inflammation.
(A) Shown is a ribbon diagram of the structure of human LCN2 (PDB ID 1L6M) with ferric enterobactin bound. Enterobactin is shown in pink with oxygen in red, nitrogen in blue, and iron as a yellow …
AimA does not bind enterobactin.
(A) Overlay of C-term and N-term domains of AimA using PDBeFold. (B) Conventionally raised (CV) fish fed soysaponin (SS) and treated with either purified full-length AimA or purified N-term (NT) or …
Both domains of AimA retain neutrophil modulating function.
(A) Structure-based sequence alignment of N-term and C-term domains of AimA. Black text indicates regions that don’t align in the structures, colored text are regions that do. Blue arrows, …
(A) Structural overlay (PDBeFold) of AimA (blue) and the model of AimB (purple) generated by I-TASSER using AimA as a threading structure. (B) Colonization level of wild-type ZF Aeromonas and ZF Aer …
AimA reduces neutrophil influx and promotes colonization of Aeromonas.
(A) Clustal Omega alignment of six AimA homologues with AimA. The predicted secretion signal, the linker between the N- and C-term, and two possible lipocalin SCRs are indicated on the alignment. (B)…
Homologues to AimA are found across the Aeromonas genus.
Comparison of WT ZF Aeromonas (ZOR001) and ZF Aer ΔAimA colonizing the zebrafish intestine for 24 hr (4–5 dpf), 48 hr (4–6 dpf), or 72 hr (4–7 dpf). *p<0.05, Students T test. N > 14, from at least …
ZF Aer DAimA suffers the greatest colonization defect 7 dpf.
(A) The per capita effect of wild-type Aeromonas, ΔAimA, ΔAimB, and ΔAΔB. Each dot represents the average neutrophil response from a flask of 15 fish divided by the average colonization level from a …
The increased neutrophil response to Aeromonas ΔAΔB causes decreased survival rate.
Strain | Characteristics | Ref. or source | Manuscript abbreviation |
---|---|---|---|
Hm21S | Parent strain, SmR | Graf, 1999 | Aer |
HE-1095 | Hm21S::interrupted exeM mTn5 KmR SmR | Maltz and Graf, 2011 | Aer ΔT2 |
HEC-1344 | HE-1095::Tn7 containing TpR exeMN + promoter region | Maltz and Graf, 2011 | Aer ΔT2C |
ASRC7 | Hm21S aimA::cmR | This study | Aer ΔaimA |
ASRD5 | Hm21S ΔaimB | This study | Aer ΔaimB |
ASRD4 | Hm21S aimA::cmR; ΔaimB | This study | Aer ΔAΔB |
ZOR0001 | Zebrafish Aeromonas isolate | Stephens | ZF Aer |
ASRC9 | ZOR0001 aimA::cmR | This study | ZF Aer ΔaimA |
Data collection | Iodide | Native | ||||
---|---|---|---|---|---|---|
space group | P622 | P622 | ||||
unit cell a, b, c (Å) | 161.4 | 161.4 | 66.6 | 160.5 | 160.5 | 66.2 |
alpha, beta, gamma (degrees) | 90 | 90 | 120 | 90 | 90 | 120 |
resolution (Å) | 50.0–2.7 (2.75–2.70) | 41.1–2.30 | ||||
completeness (%) | 100 (100) | 100 (100) | ||||
no. unique reflections | 14772 (711) | 22818 (3244) | ||||
multiplicity | 37.7 (38.1) | 70.2 (71.6) | ||||
I/sigma> | 44.2 (3.5) | 16.1 (1.4) | ||||
CC1/2 | 1 | 0.861 | 1.0 (0.9) | |||
CC1/2 anomolous | 1 | 0.873 | ||||
R merge (%) | 19.6 (486) | 19.1 (574) | ||||
Refinement | ||||||
R work (%) | 17.2 | |||||
R free (%) | 20.4 | |||||
no. of molecules in the asymetric unit | 1 | |||||
no. protein residues | 290 | |||||
no. of waters | 69 | |||||
rmsd for lengths (Å) | 0.008 | |||||
rmsd for angles (deg) | 1.1 | |||||
Ramachandran plot (%) | ||||||
preferred | 96.2 | |||||
allowed | 3.4 | |||||
outliers | 0.4 | |||||
Avg. B factor (Å^2) | ||||||
mainchain* | 81 | |||||
waters | 75 | |||||
new PDB entry | 6B7L |
* Several loop regions display high mobility but have been modeled due to a visible chain path in the electron density, resulting in an increase in the average observed B-factors of the main chain
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Escherichia coli) | BL21 DE3 | New England Biolabs | C2527 | |
Strain, strain background (E. coli) | K12 strain (MG1655) | doi: 10.1128/JB.188.3.928–933.2006 | Dr. Matthew Mulvey, Univ. of Utah | |
Strain, strain background (Aeromonas veronii) | HM21S; Aer; Aeromonas Hm21; Hm21 | doi:10.1128/AEM.01621–10 | Parent strain, SmR | |
Strain, strain background (A. veronii) | HE-1095; Aer DT2 | doi:10.1128/AEM.01621–10 | Hm21S::interrupted exeM mTn5 KmR SmR | |
Strain, strain background (A. veronii) | HEC-1344; Aer DT2C | doi:10.1128/AEM.01621–10 | HE-1095::Tn7 containing TpR exeMN + promoter region | |
Strain, strain background (A. veronii) | ASRC7; Aer DaimA | this study | Hm21S aimA::cmR | |
Strain, strain background (A. veronii) | ASRD5; Aer DaimB | this study | Hm21S DaimB | |
Strain, strain background (A. veronii) | ASRD4; Aer DADB | this study | Hm21S aimA::cmR; DaimB | |
Strain, strain background (A. veronii) | ZOR0001; ZF Aer | doi:10.1038/ismej.2015.140 | Zebrafish Aeromonas isolate | |
Strain, strain background (A. veronii) | ZOR0001; ZF Aer DaimA | this study | ZOR0001 aimA::cmR | |
Genetic reagent (Danio rerio) | AB x Tu strain; wild type zebrafish | UO Zebrafish facility | ||
Genetic reagent (Danio rerio) | myd88-/- | PMID: 28973938 | ||
Genetic reagent (Danio rerio) | Tg(BACmpx:GFP) i114; mpx:GFP | doi:10.1182/blood -2006-05-024075 | ||
Recombinant DNA reagent | pET21B | Genscript | ||
Chemical compound, drug | LPS, E. Coli O111:B4 | Sigma | L2630 | |
Peptide, recombinant protein | Lipocalin 2, LCN2, Siderocalin | Biolegend | 588002 | |
Peptide, recombinant protein | AimA | this study | NCBI hypothetical protein WP_021230730.1 | see Materials and Methods |
Peptide, recombinant protein | AimB | this study | NCBI hypothetical protein WP_021230165.1 | see Materials and Methods |
Peptide, recombinant protein | mLCN2 | this study | NCBI gene NM_008491.1 | see Materials and Methods |
Mass spectrophotometry results comparing cell-free supernatant from the complement of the type II secretion mutant A. veronii strain Hm21 (WT, ∆T2C) compared to the type II secretion mutant (mut, ∆T2).
Mass-spectrophotometry was performed on the CFS from ∆T2C and ∆T2 determined which proteins were enriched in the ΔT2C compared to the ΔT2 strain. This table lists the top 22 proteins that were enriched by greater than 10 counts in the ΔT2C CFS.
Structural homology hits to the N and C terminal domains of AimA.
Structural homology searches performed against the N and C terminal domains of AimA separately revealed that both domains have similarity to proteins in the calycin superfamily, primarily avidins and lipocalins.