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Quorum sensing in aeromonas salmonicida subsp. achromogenes and the effect of the autoinducer synthase asai on bacterial virulence

Contents lists available at Veterinary Microbiology Quorum sensing in Aeromonas salmonicida subsp. achromogenes andthe effect of the autoinducer synthase AsaI on bacterial virulence Johanna Schwenteit ,, Lone Gram , Kristian F. Nielsen , Olafur H. Fridjonsson ,Uwe T. Bornscheuer Michael Givskov , Bjarnheidur K. Gudmundsdottir a Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, IS-112 Reykjavı´k, Icelandb National Food Institute, Technical University of Denmark, Søltofts Plads bldg 221, DK-2800 Kgs. Lyngby, Denmarkc DTU Sytems Biology, Technical University of Denmark, Søltofts Plads bldg 221, DK-2800 Kgs. Lyngby, Denmarkd Prokaria, Matis ohf, Vı´nlandsleið 12, 113 Reykjavı´k, Icelande Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix Hausdorff-Str. 4, 17487 Greifswald, Germanyf Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark The Gram-negative fish pathogenic bacterium Aeromonas salmonicida possesses the LuxIR- Received 5 July 2010 type quorum sensing (QS) system, termed AsaIR. In this study the role of QS in A.
Received in revised form 15 July 2010 salmonicida subsp. achromogenes virulence and pigment production was investigated. Five Accepted 19 July 2010 wild-type Asa strains induced the N-acyl-homoserinelactone (AHL) monitor bacteria.
HPLC–HR-MS analysis identified only one type of AHL, N-butanoyl-L-homoserine lactone (C4-HSL). A knock out mutant of AsaI, constructed by allelic exchange, did not produce a Aeromonas salmonicida subsp. achromogenes detectable QS signal and its virulence in fish was significantly impaired, as LD50 of the AsaI- deficient mutant was 20-fold higher than that of the isogenic wt strain and the mean day to death of the mutant was significantly prolonged. Furthermore, the expression of two virulence factors (a toxic protease, AsaP1, and a cytotoxic factor) and a brown pigment Quorum sensing inhibitorsArctic charr were reduced in the mutant. AsaP1 production was inhibited by synthetic QS inhibitors (N-(propylsulfanylacetyl)-L-homoserine lactone; N-(pentylsulfanylacetyl)-L-homoserine lac-tone; and N-(heptylsulfanylacetyl)-L-homoserine lactone) at concentrations that did notaffect bacterial growth.
It is a new finding that the AHL synthase of Aeromonas affects virulence in fish and QS has not previously been associated with A. salmonicida infections in fish. Furthermore,AsaP1 production has not previously been shown to be QS regulated. The simplicity of theA. salmonicida subsp. achromogenes LuxIR-type QS system and the observation thatsynthetic QSI can inhibit an important virulence factor, AsaP1, without affecting bacterialgrowth, makes A. salmonicida subsp. achromogenes an interesting target organism to studythe effects of QS in disease development and QSI in disease control.
ß 2010 Elsevier B.V. All rights reserved.
Quorum sensing (QS) is a cell–cell communication system that enables bacteria to synchronize gene expres- * Corresponding author. Tel.: +354 5855100; fax: +354 5673979.
sion with population density. LuxIR-type QS via N-acyl- E-mail addresses: (J. Schwenteit), homoserinelactones (AHLs) autoinducers are used by (L. Gram), (K.F. Nielsen), many Gram-negative proteobacteria for intraspecies QS.
(O.H. Fridjonsson), LuxI is the autoinducer synthase and LuxR is an AHL- (U.T. Bornscheuer), (M. Givskov), (B.K. Gudmundsdottir).
dependent transcriptional regulator. Pathogenic bacteria 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved.
J. Schwenteit et al. / Veterinary Microbiology 147 (2011) 389–397 Table 1Bacterial strains and plasmids used in this study.
Strain or plasmid Source or reference StrainsA. salmonicida subsp. achromogenes Isolate from diseased Atlantic salmon (Salmo salar) DasaP1::kan derived from 265-87 DasaI::kan derived from 265-87 From diseased Atlantic cod (Gadus morhua) From diseased Arctic charr (Salvelinus alpinus) From diseased Atlantic halibut (Hippoglossus hippoglossus) From diseased Brown trout (Salmo trutta) F- mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15DlacX74recA1 araD139 D(ara-leu)7697 galU galK rpsL (StrR)endA1 nupG thi pro hsdR hsdM+ recA [RP4 2-Tc::Mu-Km::Tn7 (TprSmr)Tra+] Mobilizing donor for conjugation C. violaceum, CV026 AHL-Monitorbacteria, AHL receptor CviR A. tumefaciens,pZLR4 AHL-Monitorbacteria, AHL receptor TraR Cloning vector for TOPO TA cloning1 Mobilizable suicide vector, SucS (Mob+sacRB+Tetr) KmR determinant from Tn903 NCMB, National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
often possess one or more QS pathways ( relation to bacterial phenotypes and virulence in Arctic charr (Salvelinus alpinus L.).
There are data showing that luxRI homologs are universally present in the genus Aeromonas ( 2. Materials and methods ). Aeromonas salmonicida is a fish pathogenthat causes furunculosis and related diseases worldwide 2.1. Bacterial strains, plasmids and growth conditions The species is divided intofive subspecies: subsp. salmonicida, subsp. achromogenes, All bacterial strains and plasmids used in mutant subsp. masoucida, subsp. smithia and subsp. pectinolytica.
construction, AHL detection and growth and challenge ) A. salmonicida subsp.
experiments are described in . Growth medium was salmonicida causes typical furunculosis of salmonid fish, supplemented with 1.5% (w/v) agar (Difco) when appro- while the A. salmonicida subsp. achromogenes strains cause priate. Bacterial concentrations were determined by atypical furunculosis of various fish species plating and counting of colony forming units (CFU)/ml.
Aeromonas hydrophila is a All cultures and samples were stored at 80 8C until used.
related species that has a broad host range and infects both The A. salmonicida subsp. achromogenes strains used in aquatic and terrestrial animals ). The this study were isolated from the head–kidney of diseased LuxIR-type like QS systems in A. salmonicida and A.
fish by cultivation on blood agar. Authenticity and purity of hydrophila are termed AsaIR and AhyIR, respectively ( the isolates were established by examination of Gram- ). N-butanoyl-L-homoserine lactone (C4-HSL) is stained smears and by whole cell granular pattern the main AHL molecule produced by both bacteria, but A.
agglutination tests, using the MONO-As kit (BIONOR AS, salmonicida subsp. salmonicida also produce three addi- Skien, Norway). The strains used have been characterized by tional AHLs, i.e. N-decanoyl-L-homoserine lactone (C10- various typing methods and found to group with type HSL), N-hexanoyl-L-homoserine lactone (C6-HSL) and N-3- strains for A. salmonicida subsp. achromogenes. The typing methods include standard bacteriological tests, profile of secreted enzymes and AFLP-fingerprinting A group of A. salmonicida subsp. achromogenes strains, including type strains, secrete an aspzincin metallopro- A. salmonicida subsp. achromogenes, strains Keldur265- tease, AsaP1, which is highly lethal toxic for both fish and 87, F47-97, F19-99 and F106-02, were isolated from fish suffering from atypical furunculosis in our laboratory (The strains were routinely cultured on 5% horse An AsaP1-deficient mutant has decreased virulence, blood agar (blood agar base, Oxoid) (BA). Culture was both in Atlantic salmon and Atlantic cod performed at 16 8C. Liquid cultures were grown in brain ). These strains also secrete a cytotoxic factor(s) heart infusion (BHI, BD) broth with agitation (200 rpm) inoculated to an initial cell density of 103–104 CFU/ml from ) and a brown water-diffusible static overnight cultures.
Growth kinetics of the A. salmonicida subsp. achromo- The aim of this research was to investigate the QS genes strains was done in three parallel broth cultures and system of A. salmonicida subsp. achromogenes and its all samples were then analysed in triplicates. Samples J. Schwenteit et al. / Veterinary Microbiology 147 (2011) 389–397 (2 ml) were taken at different time points. Cell density was determined according to a standard curve obtained, using monitored by determining CFU/ml.
known concentrations of synthetic C4-HSL (Fluka serial Extracellular products (ECP) were isolated by collecting #9945; Sigma).
supernatants from bacterial cultures following centrifuga-tion (10,000  g for 15 min at 4 8C) and sterile filtering 2.3. Characterization of AHLs with HPLC–HR-MS analysis (Whatman, 0.45 mm), using either liquid cultures (BHIbroth) at different time of growth (B-ECP) or samples from Three millilitre of C-ECP of strain Keldur-265-87 were 72 h cultures on cellophane (TOPIPLAST) covered BHI agar acidified with 0.5% formic acid until dissolved. The extracts plates washed off the plate with 0.5 ml of phosphate- were then evaporated to dryness under N2-flow, redis- buffered saline (PBS) (C-ECP) ( solved in 1 ml acidified ethyl acetate, transferred to a glass ). C-ECP was produced in order to obtain more vial and stored at 80 8C. Extracts were analysed by HPLC– concentrated ECP samples.
HR-MS, as previously described ).
Protein concentration of ECP was measured using a Shortly, 300 ml ethyl acetate extract was evaporated using Bradford protein assay kit (Coomassie Plus, Pierce) and an N2-flow and redissolved in 100 ml acetonitrile water bovine serum albumin (Sigma) for plotting a standard containing 1% formic acid and filtered. Two microlitre were curve. Absorbance was measured at 590 nm and each injected and separated on a Luna C18 II column (Phenom- sample measured in triplicate.
enex), using an acetonitrile water gradient system and E. coli strains were grown in LB media, using 0.5% NaCl detected by positive electrospray (ESI+)-HPLC–HR-MS on a (LB5) at 37 8C.
LCT orthogonal Time of Flight mass spectrometer (LCT- When required for the selection and maintenance of recombinant plasmids, antibiotics (Sigma) were added tothe culture media at the following final concentrations: 2.4. Construction of a DasaI mutant of A. salmonicida subsp.
ampicillin (100 mg/ml); tetracycline (25 mg/ml); cepha- lothin (200 mg/ml); gentamicin (20 mg/ml); and kanamy-cin (50 mg/ml for Aeromonas and 20 mg/ml for monitor All plasmids used are listed in Degenerate primers, listed in were designed for PCR Agrobacterium tumefaciens, strain NT1 with pZLR4 ( amplification of a sequence from asaI, the gene that and Chromobacterium violaceum, strain CV026 encodes AsaI of A. salmonicida subsp. salmonicida (GenBank (grown at 25 8C, were used to U65741), and the sequence of the kan gene on the plasmid monitor production of AHLs. A. tumefaciens was grown in pSSVI186- The asaI gene and its AB medium supplemented with flanking regions were isolated using the PstI-f1 and PstI-r1 2.5 mg/l thiamine (Sigma), 0.5% (w/w) casamino acids primers. The isolated fragment was cloned into pCR1II- (Sigma), 0.5% (w/w) glucose (Sigma) and 20 mg/ml TOPO1, sequenced and used for further construction of the gentamicin. C. violaceum was grown in LB5 supplemented mutant and for initial sequencing of the asaI gene of A.
with 20 mg/ml kanamycin.
salmonicida subsp. achromogenes, using asaI-wt-f/asaI-wt-rand PstI-f1/PstI-r1 primers.
2.2. Detection of AHLs activity produced by A. salmonicida The DasaI mutant of A. salmonicida subsp. achromo- subsp. achromogenes genes,strain Keldur265-87-3 was constructed by splicing ofoverlapping extensions (soe) PCR ), and AHLs in B-ECPs and C-ECPs were detected using A.
following allelic exchange. Thus, the two flanking sites of tumefaciens (5) and C. violaceum (5, 17) in a well-diffusion the asaI gene were amplified using PstI-f2/5-soe-r and 3- assay (22). The plates were incubated for 48 h at 25 8C and soe-f/PstI-r2 primers, respectively. The soe-primers have a the diameter of pigment production by the monitor strains 20 bp overhang complementary to the kan-gene. The soe- measured. The C4-HSL concentration of samples was kan-f and soe-kan-r primers, used to amplify the kan gene Table 2Oligonucleotide primers and their application in this study.
Isolation of asaI with flanking sites out of the A. salmonicida supsp. achromogenes genome Amplification of the 50- and -30 flanking site of asaI as template for soe-PCR Amplification of the kan J. Schwenteit et al. / Veterinary Microbiology 147 (2011) 389–397 from the plasmid pSSVI186-IN, have a 20 bp overhang prepared in 96 wells flat bottom microtiter plates as complementary to the 50-flanking site and the 30-flanking previously described After site of the asaI gene, respectively. The three resulting DNA culturing for 2 days, cells were washed twice with serum fragments were used as templates for the final soe-PCR, free medium and incubated with C-ECP diluted 1:20 (v/v) using PstI-f2/PstI-r2 primers, resulting in an asaI::kan in the medium at 16 8C for 48 h. Cytotoxicity was construct. The construct was TOPO1 cloned and the determined by observing cell culture microscopically colonies screened by PCR using the kan-f/kan-r primers.
and by percent cell mortality by measuring the release Colonies giving a PCR product of the expected size (a 472 nt of lactate dehydrogenase in EPC cell culture supernatant fragment of a 624 bp gene) were picked and the correct via a cytotoxicity detection kit (LDH, Roche, Basel, construct confirmed by sequencing.
Switzerland). The assay was performed in triplicate The construct was cloned into the suicide vector according to the manufacturer's instructions.
pSUP202sac containing the sacB gene. Expression of thisgene is lethal for Gram-negative bacteria and can be induced 2.8. Experimental fish and challenge experiments by sucrose ). The vector was thentransformed into E. coli S17.1 and conjugated into strain Arctic charr (38  6 g SD) free of infections, according to Keldur265-87 by mating Nine ml of standard routine diagnostic procedures performed at the Keldur265-87 cell culture in the end log-phase and 1 ml of E.
Icelandic Fish-Disease Reference Laboratory, were used for coli S17.1 were pelleted and both pellets resuspended in challenge. Fish were kept in 70 l tanks supplied with 50 ml BHI and spotted on a BHI agar and mating performed continuously running fresh water at 9  1 8C. Oxygen con- at room temperature for 24 h. The cells were then solved in centration, temperature and mortality in each tank were 500 ml BHI and selection of transconjugants performed on monitored daily throughout the experiments. Prior to treat- BHI agar supplemented with cephalothin and kanamycin ment, the fish were anaesthetized with tricaine methane (50 mg/ml). Selection of double-crossover DasaI negative sulfonate (50 mg/l) and marked with Alcian blue dye. A.
mutants was performed by cultivation on BHI agar salmonicida subsp. achromogenes, strain Keldur265-87, and its supplemented with cephalothin, kanamycin (50 mg/ml) isogenic DasaI mutant, Keldur265-87-3, were passaged three and sucrose (15%, w/v) at 16 8C for 7 days. Cephalothin was times in Arctic charr before their use in challenge experiments.
used to select against E. coli and for Aeromonas. The Challenges were performed by i.p. injection of 103–108 CFU/ successful exchange of the asaI gene with the asaI::kan fish from liquid cultures. Control fish received PBS only. Ten construct was selected by sucrose and kanamycin. The fish were in each injection group. The head–kidney from all resulting colonies were screened by PCR using the kan-f/ dead and surviving fish were sampled, inoculated onto blood kan-r primers and deletion of asaI was confirmed by agar and incubated for seven days at 16 8C. A. salmonicida subsp. achromogenes isolated from dead fish was identifiedserologically using MONO-As kit (BIONOR AS) and the DasaI 2.5. DNA sequencing and sequence analysis mutant and wt strains were recognised by cultivation on BHIagar with or without kanamycin, respectively, and confirmed DNA sequencing was performed at Matis, ohf using an by PCR, using the kan-f/kan-r primers.
ABI 3730 genetic analyser (Applied Biosystems). Sequence Fish experiments were approved and performed editing and alignment was performed using Sequencher according to the Icelandic Animal Research Authority 4.8 (Gene Codes Corporation).
(approval no. YDL03080041/023BE).
2.6. Detection of pigment and protease production 2.9. Complementation of AHL production of the DasaImutant Pigment and protease concentrations were measured in B-ECPs obtained from three separate growth kinetics Strain Keldur265-87, and its isogenic DasaI mutant, experiments. Pigment production of A. salmonicida subsp.
Keldur265-87-3, were cultivated for 72 h in broth. The achromogenes strains Keldur265-87, and Keldur265-87-3 DasaI mutant was cultivated with the addition of 0.1 mM was monitored spectrophotometically (A405) ( or 1 mM of C4-HSL to the culture medium before using BHI as a control.
inoculation and also in media without AHL supplementa- Caseinolytic activity of strains Keldur265-87 and tion. Three independent cultures were obtained from each Keldur265-87-2 was detected by a spectrophotometric of the four experimental groups. Caseinolytic activity and (A450) azocasein assay ), pigment production was monitored in the B-ECP of the using the AsaP1-deficient mutant strain, Keldur265-87-2, respective cultures, as described above.
and PBS as controls.
AsaP1 protease was detected by Western blotting using 2.10. The effect of QS inhibitors (QSI) on growth and AsaP1 polyclonal mouse a-AsaP1 antibodies (prepared at our laboratory) as a probe ( The QSI compounds N-(propylsulfanylacetyl)-L-homo- 2.7. Cytotoxic effect serine lactone (ProS-AHL), N-(pentylsulfanylacetyl)-L-homoserine lactone (PenS-AHL) and N-(heptylsulfanyla- Confluent cell monolayers of a cultured Epithelioma cetyl)-L-homoserine lactone (HepS-AHL) were used dis- papulosum cyprini (EPC) cell line (passage 251) were solved in dimethylsulfoxide (DMSO) J. Schwenteit et al. / Veterinary Microbiology 147 (2011) 389–397 A. salmonicida subsp. achromogenes, strains Keldur265- ABO91724). Furthermore, a sequence (frame +2, bases 87(wt) and Keldur265-87-2 (AsaP1-deficient), were grown 1394-1664) encoded 90 aa that were 98% identical to the in liquid cultures, as described above, with a starting cell N-terminal end of the 301 aa long oriC replication inhibitor density of 1.5  103 CFU/ml. The wt strain was grown with protein of strain 449 (GenBank ABO91722).
or without addition of 10 mM ProS-AHL, PenS-AHL andHepS-AHL and 0.1% DMSO (solvent used as control). The 3.3. AsaI-deficient mutant does not produce AHL signals and AsaP1-deficient mutant strain was grown without QSI produces less extracellular proteins supplementation. Samples were taken at regular intervalsfor determination of cell density and caseinolytic activity.
An AsaI-deficient mutant, Keldur265-87-3, was suc- cessfully constructed by soe-PCR and allelic exchange.
2.11. Calculations and statistical analysis Sequencing of the asaIR locus of the mutant verified thatthe whole asaI gene was replaced by the kanamycin Three independent experiments were performed where resistant gene and that the kanamycin resistant gene was applicable and the samples then analysed in triplicates.
in frame with the upstream reverse asaR gene (frame -3). A Students's t test was used to analyse differences between total number of 70 colonies were screened by PCR, using groups. Fisher's exact test was used to analyse the the kan-f/kan-r primers and 5 positive clones tested for significance of differences in mortality between groups.
AHL production by the monitor strain CV026, before The LD50 was calculated according to the method of sequencing. None of the isolates induced the monitor Mean day to death (MDD) were calculated strain, indicating that they did not produce the AHL signal using the following formula: MDD = molecule produced by its isogenic wt strain. One isolate m = number of mortalities; d = days post challenge was selected and used for further analyses. The growth of The threshold level for significance was 0.05.
the AsaI-deficient mutant did not differ from that of the wtstrain in liquid cultures with initial cell density of 104 CFU/ 2.12. Nucleotide sequence accession number ml A). The growth of the mutant was, however,delayed compared to the wt strain when the inoculum was The nucleotide sequence of the asaI gene of strain 103 CFU/ml (data not shown). Furthermore, the growth Keldur265-87 and its flanking regions has been deposited rate of the wt and the mutant strains on cellophane in the GenBank nucleotide sequence database under covered agar plates was not different (p = 0.23780) ( accession GU811648.2 ). Knock out of the asaI gene resulted in a complete lack ofAHL production and the mutant secretedsignificantly less proteins than the wt strain at all time points tested in liquid cultures (data not shown) and also 3.1. Detection of AHL in A. salmonicida subsp. achromogenes on cellophane covered agar plates ().
Extracts from C-ECPs of all five strains induced zones 3.4. AsaP1 production is QS regulated with similar size in both AHL monitors with the mostpronounced reaction in C. violaceum (Supplementary data The caseinolytic activity as detected spectrophotome- Table 1). N-butanoyl homoserine Lactone (C4-HSL) was the trically B) was paralleled by production of AsaP1 as only AHL molecule detected in extracts of strain Keldur265- detected by Western blotting AsaP1 production 87 by HPLC–HR-MS (Supplementary data Fig. 1). The was detectable in the B-ECP of the wt strain after 42 h calculated C4-HSL concentration of B-ECP harvested from cultivation (4.5  108 CFU  7.2  107 SD), but after 62 h a 100 h broth culture with a cell density of 8.2  108 (5.4  108 CFU  5.3  107 SD) in the B-ECP of the DasaI (0.2  108 SD) CFU/ml of strain Keldur265-87 was 0.260 mM.
mutant. After 204 h cultivation 5-fold higher caseinolyticactivity/108 CFU was detected in the B-ECP of the wt strain 3.2. The amplified DNA fragment of strain Keldur265-87 compared to its isogenic AsaI-deficient mutant. Complemen- encodes the AsaI AHL synthase and partial sequences of the tation of the culture medium of the AsaI-deficient mutant at transcriptional regulator AsaR and replication inhibitor IciA the start of incubation with 0.1 or 1 mM of C4-HSL resulted inincreased caseinase production (The induction of The sequenced DNA fragment amplified by primers caseinolytic activity was positively associated with the C4- PstI-f1 and PstI-r1 was 1665 bp long (GU811648.2). The HSL concentration. This indicates that AsaP1 is QS regulated.
asaI ORF of A. salmonicida subsp. achromogenes (frame +1bases 511-1134) was 624 bp with 99% identity to that of 3.5. Pigment production of A. salmonicida subsp.
the asaI ORF of A. salmonicida subsp. salmonicida, A449 achromogenes is QS regulated (GenBank U65741). The only base differences found weresilent nucleotide substitutions. The AsaI peptides of A.
As shown in B, the wt strain Keldur265-87 produced salmonicida have 94% aa identity to those of the AhyI of A.
a pigment (dark brown) from about 72 h cultivation hydrophila ). The sequence also (5.5  108 CFU/ml), but pigment production of the AsaI- included 448 bases (frame -3 bases 1-448) encoding 149 deficient mutant (Keldur265-87-3), was detected after aa from the N-terminal end of the 260 aa long transcrip- 109 h (5.5  108 CFU/ml) and it produced significantly less tional activator AsaR with 97% aa identity to AsaR of pigment during cultivation (p < 0.0001). After 204 h culti- vation the cell density of the wt strain and the mutant was

J. Schwenteit et al. / Veterinary Microbiology 147 (2011) 389–397 Fig. 2. Spectrophotometrically measured caseinolytic activity/108 CFU (A)and brown pigment/108 CFU (B) in B-ECPs after 240 h growth of A.
salmonicida subsp. achromogenes, Keldur265-87 (solid columns), and itsisogenic AsaI-deficient mutant, Keldur265-87-3 (open columns) cultures.
Initial cell densities of the cultures were 2–4  104 CFU/ml. Response ofthe AsaI-deficient mutant to C4-HSL (added to give 0, 0.1, or 1 mM) in theculture medium was monitored. The error bars represent 1 standarddeviation (n = 3).
as the release of LDH in EPC cell culture supernatant isshown in . Cell mortality induced by the AsaI-deficient mutant was not different from that of thenegative control (p = 0.1680), but significantly less than Fig. 1. Comparison of A. salmonicida subsp. achromogenes, Keldur265-87 that of the wt strain (p = 0.0004).
(solid bars), and its isogenic DasaI mutant, Keldur265-87-3 (open bars).
Bacterial growth in BHI at 16 8C, measured in log10 CFU/ml (solid lines),and AHL production measured as a zone induced by bacterial 3.7. The QS mutant has impaired virulence properties extracellular products in a well-diffusion assay with the monitorbacterium C. violaceum (dashed lines) (A). Production of caseinase Virulence of strains Keldur265-87 and Keldur265-87-3 (solid lines) and a brown pigment (dashed lines) during growth measured in Arctic charr was compared in i.p. challenges. No spectrophotometrically, A450 and A405, respectively (B). Immunostainingof AsaP1 in the ECP with anti-AsaP1 antibodies during growth (C). The mortality occurred when the mutant was injected in error bars represent 1 standard deviation (n = 3).
concentrations 104 CFU/fish, but 104 CFU/fish of the wtstrain caused 71% mortality and 103 CFU/fish induced 8% 2.2  108 and 2.8  108 CFU/ml, respectively, but the mortality ().
difference in A405 of the two respective cultures was 11- The calculated LD50 of the wt strain was 20-fold higher fold. Complementation of the culture medium of the AsaI- than that of the AsaI-deficient mutant, or 5  103 CFU/fish deficient mutant at the time of incubation with 0.1 or 1 mM and 1  105 CFU/fish, respectively.
of C4-HSL resulted in increased pigment production. The Calculated MDDs were always higher when the fish induction of pigment production was positively associated were injected with the mutant compared to the wt strain with the C4-HSL concentration (B). This indicates that pigment production is QS regulated.
3.8. QSIs inhibit protease (AsaP1) production 3.6. Cytotoxic effect of the AsaI-deficient mutant is impaired The bacterial growth was neither affected by any of the C-ECP of the wt strain and the AsaI-deficient mutant three QSIs in the concentrations used, nor the DMSO caused ruptures in the cell monolayer of the EPC cells after solvent Caseinolytic activity was not detected in 48 h incubation, but a difference was not observed any sample from the AsaP1-deficient mutant (Keldur265- between the two strains. Percent cell mortality measure 87-2), indicating that the bacterium does not secrete

J. Schwenteit et al. / Veterinary Microbiology 147 (2011) 389–397 Table 3Growth on cellophane covered agar plates (CFU/ml); protein concentration in supernatants obtained from cell cultures (C-ECP); and percent cell death inEpithelioma papulosum cyprini (EPC) cells treated for 48 h with C-ECPs of A. salmonicida subsp. achromogenes, Keldur265-87, or its isogenic AsaI-deficientmutant, Keldur265-87-3. All values are given in means (SD) (n = 3). An asterisk (*) signifies a significant difference of the AsaI-deficient mutant compared to itsisogenic wt strain (p < 0.05).
mg protein/108 cells Keldur265-87 (wt) 1.8  109 (6.3  108) Keldur265-87-3 (DasaI) 1.3 109 (1.1  108) another caseinase at the experimental conditions. A Accumulated percent mortality (mortality %) and mean day of death caseinase was produced from late exponential phase by (MDD) of Arctic charr following i.p. injection of A. salmonicida subsp.
the wt strain (Keldur265-87). All three QSI compounds achromogenes strain Keldur265-87 and its isogenic AsaI-deficient mutantKeldur265-87-3 (n = 10).
inhibited production of AsaP1 significantly (p < 0.001), butHepS-AHL was most effective, resulting in 1.7-fold reduction of AsaP1 production. Addition of DMSO did not affect caseinase production A. salmonicida comprises the LuxIR-type QS system, termed AsaIR, where AsaI is the autoinducer synthase and AsaR the transcriptional regulator. The results of this studyshow that QS of A. salmonicida subsp. achromogenes hasregulatory effects on virulence and pigment production, aspigment production was significantly reduced and thepathogenicity of an AsaI knock out mutant was impaired in Fig. 3. The effect of the QSI compounds ProS-AHL, PenS-AHL and HepS-AHL and the solvent DMSO on growth of A. salmonicida subsp. achromogenes, strainKeldur265-87, and its AsaP1-deficient mutant, Keldur265-87-2, in BHI at 16 8C (A) and on caseinolytic activity of the respective ECP's, monitored by an azo-casein assay (B). The error bars represent 1 standard deviation (n = 3).
J. Schwenteit et al. / Veterinary Microbiology 147 (2011) 389–397 Arctic charr. The LuxIR-like QS system of the bacterium growth phase of the wt strain, but in the early stationary was found to be simple, as only one type of acylated phase of the AsaI-deficient mutant. After 204 h cultivation homoserine lactones (C4-HSL) was produced. Further- in an experiment, where cell density of both strains was more, QSIs, which compete with autoinducers for binding comparable during growth, the wt strain produced 5-fold to AsaR, were found to inhibit the expression of an more of the AsaP1 protease than the AsaI-deficient mutant.
important virulence factor, AsaP1.
Addition of 0.1 or 1 mM C4-HSL to the culture medium of The main AHL produced by Aeromonas bacteria, C4-HSL, the AsaI-deficient mutant resulted in increased AsaP1 was the only AHL synthesized by strain Keldur265-87, as expression that was dose dependent and the higher C4- detected by HPLC–HR-MS analyses. All five A. salmonicida HSL dose completely restored proteolytic activity. Com- subsp. achromogenes strains, including the type strain plementation of the phenotype instead of the genotype (NCIMB 1110), tested in the present study induced zones was chosen, as a replicating plasmid for A. salmonicida was of similar size in a reaction with the two monitor bacteria, C.
not found, leaving in trans complementation complicated.
violaceum and A. tumefaciens, indicating a similar AHL In a previous study QS regulation of a metalloprotease production, but AHL characterization by HPLC–HR-MS of A. hydrophila, strain AH-1N, was reported. Strain AH-1N analysis was only performed on strain Keldur265-87. In a is a spontaneous mutant of strain AH-1 that lacks the S- previous study ) five A. salmonicida subsp.
layer and the O-11 antigen Strain AH-1 achromogenes isolates, originating from diseased fish in secretes a metalloprotease, EprA1, which is 91% identical Scotland, Iceland, Denmark and Norway, were all found to to AsaP1 of A. salmonicida subsp. achromogenes be QS negative. Furtheremore, one type strain of A.
There are no available reports on the effect of salmonicida subsp. achromogenes (NCIMB 1110) produced the AhyI deletion on A. hydrophila pathogenicity or on C6-HSL in addition to C4-HSL, but a type strain of the virulence function of the EprA1 protease, but deletion of subspecies from a different culture collection with the same AhyR transcriptional regulator of A. hydrophila has been origin (ATCC 19261) did not induce either of the AHL found to affect bacterial pathogenesis and protease monitor bacteria (). This indicates that the bacterium may loose the ability to produce AHL and also Cultivation of the wt strain (Keldur265-87) with 3 that strains classified as A. salmonicida subsp. achromogenes different synthesised sulfide AHL analogues (ProS-AHL, are heterogenic with respect to AHL production.
PenS-AHL and HepS-AHL), which bind the QS pathway by The deduced aa sequence of the AsaI peptide of A.
competing with AHL molecules for specific binding to the salmonicida subsp. achromogenes was identical to that of LuxR-type signal receptor ), resulted in the previously described AsaI peptide of A. salmonicida significantly reduced expression of the AsaP1 protease. In subsp. salmonicida this study nt bases encoding 149 aa (57% of the whole A knock out mutant of AsaI was established by protein) from the N-terminal end of the asaR gene of strain replacing the asaI gene with a kanamycin resistant marker.
Keldur265-87 were sequenced and found to have high To minimize any polar effects the resistant gene was set in similarity to the asaR sequence of A. salmonicida subsp.
frame with the upstream asaR gene. Autoinducer produc- salmonicida, 449. This indicates that strain Keldur265-87 tion was completely knocked out in the mutant. Previously possesses a functional AsaR signal receptor and is a further it has been shown that a knock out of both the ahyI and support of the statement that AsaP1 is QS regulated.
ahyR genes of A. hydrophila, strain SSU, resulted in total lack HepS-AHL was found to be the most effective compound of AHL production ), but it is a new of the three tested in inhibiting AsaP1 expression. This is in finding that knock out of asaI alone has this effect.
agreement with , who found HepS-AHL The AsaI mutation had a significant effect on the to be the most potent QSI of the three that were tested in this pathogenicity of the A. salmonicida subsp. achromogenes study. The QSI were not found to affect bacterial growth, bacterium. The lowest bacterial doses injected in challenge which makes it unlikely to pose a selective pressure for the experiments were 104 CFU/ml (i.e. 103 CFU/fish) at which development of resistant mutants, if the compound will be level growth of the wt and mutant in vitro were identical.
used as an antimicrobial agent.
The results therefore suggest that the decreased patho- Pigment production of the AsaI-deficient strain was genicity of the AsaI-deficient mutant is due to difference in significantly impaired and was not spectrophotometrically availability of virulence factors rather than impaired detected until after 109 h cultivation (late stationary growth of the mutant. The production of total extracellular phase). On the other hand pigment production of the wt proteins of the mutant was also impaired. QS regulation of strain was spectrophotometrically detected after 70 h A. salmonicida subsp. achromogenes virulence was also cultivation (early stationary phase). The addition of C4- supported by results showing that the mutation lead to HSL to the culture medium of the mutant resulted in reduced expression of a lethal toxic metalloprotease, complementation of pigment production, showing that it AsaP1, which is an important virulence factor ( is QS regulated. The name of the A. salmonicida subsp.
), reduced secretion of a cytotoxic factor(s) and achromogenes, meaning lacking colouring, may therefore reduced pigment production. Many virulence factors have originate from an AsaI-deficient strain. QS regulation of A.
cytotoxic effects and pigments, especially melanin-like salmonicida pigment production has already been sug- pigments, have been associated with virulence in several gested by , who observed that atypical A.
bacteria ().
salmonicida strains lacking AHL signals are not producing a The findings of this study show that the AsaP1 exotoxin brown pigment, which is characteristic for typical A.
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