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Antigens of Aeromonas salmonicida subsp. salmonicida specifically induced in vivo in Oncorhynchus mykiss

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Simon Menanteau-Ledouble at Norce Research
Simon Menanteau-Ledouble
Mansour El-Matbouli at University of Veterinary Medicine, Vienna
Mansour El-Matbouli
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Short communication
Antigens of Aeromonas salmonicida subsp. salmonicida
specifically induced in vivo in Oncorhynchus mykiss
S Menanteau-Ledouble and M El-Matbouli
Clinical Division of Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of
Veterinary Medicine, Vienna, Austria
Keywords: Antigenic profile, AopO, In vivo-
induced antigen technology, lactoylglutathione
lyase, LamB, Virulence.
Furunculosis is a major fish disease, associated with
high mortality rates, and its effect is particularly sig-
nificant in farmed salmonids (Hiney & Olivier
1999). Efficacious vaccines, generally injected
alongside an oil adjuvant, are available. However,
certain side effects have been reported (Midtlyng,
Reitan & Speilberg 1996; Anderson et al. 1997)
and protection can lapse in stressed fish and in
non-salmonid species (Gudmundsdottir &
Bj€ornsdottir 2007).
In vivo-induced antigen technology (IVIAT)
uses antibodies raised by individuals exposed to
the pathogen of interest. These antibodies are then
adsorbed against an in vitro culture of both the
pathogen and, to avoid cross-reaction, the organ-
ism in which the genomic library will be expressed
(Handfield et al. 2000). This removes antibodies
binding antigens expressed under culture condi-
tions. The remaining antibodies, recognizing anti-
gens specifically expressed during growth within
the host, are used to probe a genomic library
expressing random segments from the genome of
the pathogen of interest. Reactive clones are
sequenced and identified based on homology,
identifying genes overexpressed in vivo (Rollins
et al. 2005). Antigens associated with the infec-
tious phenotype are likely to act as virulence fac-
tors and constitute interesting targets for vaccine
development.
Consequently, we have previously applied
IVIAT to Aeromonas salmonicida subsp. salmoni-
cida (clinical isolate A14390) infecting rainbow
trout weighing an average of 92 grams
(Menanteau-Ledouble et al. 2014a; Menanteau-
ledouble et al. 2014b) in a study approved by the
university institutional ethics committee and the
national authority according to §26 of the Aus-
trian Law for Animal Experiments (Tierversuchs-
gesetz 2012–TVG 2012-91 under the No. GZ
68.205/140-II/3b/2012). Sera were harvested at
multiple time points to sample a wide array of the
immune response and screening identified four
antigens: UDP-3-O-acyl-N-acetylglucosamine deac-
etylase (involved in cell wall synthesis), RNA
polymerase sigma factor RpoD (a regulator of
gene expression) as well as TonB (that provides
energy for transport across the cell membrane)
and a hypothetical protein (Menanteau-ledouble
et al. 2014b).
In this study, we report on further screening of
this library using the same pool of adsorbed sera.
A significant difference between both studies is
that more time points were included in the RT-
qPCR to confirm that the genes discovered were
overexpressed throughout the course of the infec-
tion: This time, six time points were included: 1,
6, 12 and 48 h as well as a 1 and 2 weeks post-
infection. Mean fold changes in gene expression
were calculated between in vitro cultures and the
Correspondence M El-Matbouli, Clinical Division of Fish
Medicine, Department for Farm Animals and Veterinary Public
Health, University of Veterinary Medicine, Veterinarplatz 1,
Vienna 1210, Austria
(e-mail: Mansour.El-Matbouli@vetmeduni.ac.at)
1
Ó2015 The Authors.
Journal of Fish Diseases
Published by John Wiley
& Sons Ltd.
This is an open access
article under the terms
of the Creative
Commons Attribution
License, which permits
use, distribution and
reproduction in any
medium, provided the
original work is properly
cited.
Journal of Fish Diseases 2015 doi:10.1111/jfd.12430
various infected fish tissues according to the
2
DDC
t method (Livak & Schmittgen 2001).
Four more proteins were detected during this
renewed screening and were identified based on
sequence homology (Table 1): AopO; lactoylglu-
tathione lyase; a LamB-like maltoporin; and a
hypothetical conserved protein. Each sequence dis-
played a very high level of homology with genes
from the A. salmonicida subsp. salmonicida A449
genome (Reith et al. 2008). These sequences were
then further analysed in silico.
When RT-qPCRs were performed, they con-
firmed that all four genes were more highly
expressed in vivo: the average expression ratio of the
four genes was 8.94E
+04
(4.69E
+04
) between bacte-
ria in infected tissue samples and bacterial cultures.
The first protein identified was AopO (Genbank
identification number: DQ386862). A homologue
to the effector protein YopO of Yersinia ruckeri
(Dacanay et al. 2006), AopO is secreted through
the type III secretion system (T3SS) (Vanden
Bergh et al. 2013a Vanden Bergh et al. 2013b), a
virulence mechanism that is considered particularly
important in A. salmonicida (Burr et al. 2003).
Previously, it had been shown that mutants defi-
cient in the expression of three effector proteins of
the T3SS (AopO, AopH and AexT) displayed a
significantly reduced intracellular survival at 24
HPI in adherent head kidney macrophages (Fast
et al. 2009). However, inactivation of aopO alone
only had a moderate effect when the fish were
infected by immersion and none during injection
challenge (Dacanay et al. 2006). Despite being
identified in our genomic library, aopO is carried
on a motile genetic element (Stuber et al. 2003).
However, the plasmid carrying aopO is large,
approximately 140 kb, and such large plasmids are
difficult to separate from bacterial chromosomes
and are often found in genomic preparations.
The change in the transcription of aopO was
7.04E
+04
3.43E
+04
in average between infected
organs and in vitro cultures (Fig. 1), as calculated by
the 2
DDC
t method. Notably, this gene was found
not to be significantly overtranscribed in the kidney
at 48 HPI (mean fold change of 1.36 6.28E
01
).
This was the exception as, otherwise, all investigated
genes were found to have significantly higher expres-
sion levels in all three organs at every time point
compared to the cultures.
Table 1 List of the genes identified in this study
Sequence
identified
GenBank
accession
number
Percentage
of identity (%)
Query
cover (%)
aopO DQ386862 99 93
Lactoylglutathione
lyase
CP000644
Region:
1286580 to
1286990
100 94
Hypothetical protein CP000644
Region:
1072365 to
1073015
99 97
LamB CP000644
Region:
2545944 to
2547230
99 91
Figure 1 Relative gene expression of aopO calculated between the bacteria in the spleens, livers and kidneys of rainbow trout
in vitro. 16S rRNA expression was used for normalization, and relative gene expression changes were determined. Each value repre-
sents the mean of triplicates.
2
Journal of Fish Diseases 2015 S. Menanteau-Ledouble & M. El-Matbouli IVIAT in Aeromonas salmonicida
Ó2015 The Authors.
Journal of Fish Diseases
Published by John Wiley
& Sons Ltd.
Indeed, the gene coding for lactoylglutathione
lyase (CP000644 region: 1286580 to 1286990,
belonging to the cluster of orthologous groups
(COG) 3324) appeared significantly overexpressed
in all three organs sampled and at all time points
with an average value of 2.65E
+05
1.74E
+05
(Fig. 2). Lactoylglutathione lyase is known to be
involved in cellular detoxification and resistance to
oxidative stress (Alsop & Vijayan 2009), for exam-
ple in Lactococcus lactis (Li et al. 2003). In Strepto-
coccus mutans, this molecule is overexpressed under
acidic condition and is involved in the bacterial
resistance to low pH (Korithoski, Levesque &
Cvitkovitch 2007). It has also been shown to play
an important role in the intracellular invasiveness
of Salmonella as well as in the translocation of
effectors encoded on the Salmonella pathogenicity
island 2, and mutants deficient in this enzyme dis-
play a reduced invasiveness into epithelial cells
(Chakraborty et al. 2014).
The third protein identified was a conserved
hypothetical protein (CP000644 region: 1072365
to 1073015) that has yet to be characterized. The
mean fold change in the expression of the gene
encoding for this protein was 2.76E
+03
8.16E
+02
(Fig. 3). PSORTb found that the protein was
likely located within the cytoplasm (localization
score of 8.96), while Pfam described two
endonuclease domains, although their scores were
low: 57.2 and 32.1. Indeed, ProtFun found the
likelihood of this protein to play an enzymatic
role to be low: 0.353.
Finally, we identified a LamB-like maltoporin
(CP000644 region: 2545944 to 2547230),
belonging to the COG 4580. Unfortunately,
because of the low numbers of bacteria at the later
time points, only very low copy numbers could be
detected for this gene at 1 and 2 weeks post-infec-
tions and the results from the expression ratio
analysis appeared as outliers compared to that of
the other genes or time points. It was therefore
decided to exclude these values from the analysis.
After exclusion of these two later time points, the
mean fold change of lamB was 5.98
+03
1.12E
+03
Figure 2 Mean fold change in the expression of the gene coding for lactoylglutathione lyase over the course of this study.
Figure 3 Mean fold change in the expression of the gene coding for the conserved hypothetical protein over the course of this study.
3
Journal of Fish Diseases 2015 S. Menanteau-Ledouble & M. El-Matbouli IVIAT in Aeromonas salmonicida
Ó2015 The Authors.
Journal of Fish Diseases
Published by John Wiley
& Sons Ltd.
(Fig. 4). LamB has been well studied as a specific
diffusion channel for the uptake of maltodextrins
(Ranquin & Van Gelder 2004). Interestingly, a
similar LamB homologue termed Omp48 has
been implicated in the binding of Aeromonas vero-
nii to the extracellular matrix as well as to HeLa
epithelial cells (Vazquez-Juarez et al. 2004). More-
over, recombinant vaccines that targeted this
molecule were found to be protective against
infection with Aeromonas hydrophila (Khushira-
mani et al. 2012) as well as to offer cross-protec-
tion against Edwardsiella tarda and a number of
Vibrio species (Khushiramani et al. 2012; Lun
et al. 2014).
Two inserts were also sequenced that were
identical to the ones previously identified
(Menanteau-Ledouble et al. 2014a; Menanteau-
ledouble et al. 2014b). The first was identical to
the UDP-3-O-acyl-N-acetylglucosamine and likely
originated from the same original insertion event.
The sequence of the other insert (a hypothetical
protein) originated from a different segment on the
same gene, suggesting that the same gene had been
identified independently twice.
Eight genes were identified through both
screenings. This relatively low number is expected
for IVIAT: this technique only identifies strongly
differentially expressed proteins and only if they
are sufficiently immunogenic to generate a detect-
able antibody response.
In the future, it would be interesting to further
investigate the significance of these genes in the dis-
ease process, for example by inhibiting their
expression using defined deletion mutants (Vipond
et al. 1998) before testing these mutants in an
infection challenge. Similarly, the hypothetical pro-
tein could be characterized and the vaccine poten-
tial of LamB could be investigated.
Acknowledgements
The authors recognize the assistance of Bernhard
Eckel and Dr. Andrea Dressler. This study was
funded by the Austrian Science Fund (FWF)
project no. P 23850-B17.
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Ó2015 The Authors.
Journal of Fish Diseases
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Received: 20 July 2015
Revision received: 2 October 2015
Accepted: 3 October 2015
5
Journal of Fish Diseases 2015 S. Menanteau-Ledouble & M. El-Matbouli IVIAT in Aeromonas salmonicida
Ó2015 The Authors.
Journal of Fish Diseases
Published by John Wiley
& Sons Ltd.
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Full-text available
  • Aug 2020
Gram-negative bacteria have developed several nanomachine channels known as type II, III, IV and VI secretion systems that enable export of effector proteins/toxins from the cytosol across the outer membrane to target host cells. Protein secretion systems are critical to bacterial virulence and interactions with other organisms. Aeromonas utilize various secretion machines e.g. two-step T2SS, a Sec-dependent system as well as one-step, Sec-independent T3SS and T6SS systems to transport effector proteins/toxins and virulence factors. Type III secretion system (T3SS) is considered the dominant virulence system in Aeromonas. The activity of bacterial T3SS effector proteins most often leads to disorders in signalling pathways and reorganization of the cell cytoskeleton. There are also scientific reports on the pathogenicity mechanism associated with host cell apopotosis/pyroptosis resulting from secretion of a cytotoxic enterotoxin, i.e. the Act protein, by the T2SS secretion system and an effector protein Hcp by the T6SS system. Type IV secretion system (T4SS) is the system which translocate protein substrates, protein-DNA complexes and DNA into eukaryotic or bacterial target cells. In this paper, the contribution of virulence determinants involved in the pathogenicity potential of Aeromonas is discussed. Considering that the variable expression of virulence factors has a decisive impact on the differences observed in the virulence of particular species of microorganisms, it is important to assess the correlation between bacterial pathogenicity and their virulence-associated genes.
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... Aeromonas are well-known fish pathogens ). The species with the highest clinical significance is Aeromonas salmonicida, the causative agent of furunculosis, a major disease with high mortality rates that causes great economic losses in aquaculture, particularly affecting salmonids (Beaz-Hidalgo, Alperi, Bujan, Romalde & Figueras, 2010;Khor, Puah, Tan, Puthucheary & Chua, 2015;Menanteau-Ledouble & El-Matbouli, 2016). Other species, such as A hydrophila, Aeromonas bestiarum, A. veronii and Aeromonas sobria have also been associated with diseases in fish Kozinska, 2007;Orozova, Barker, Austin & Austin, 2009;Rasmussen-Ivey et al., 2016;Yi et al., 2013). ...
Limited performance of MALDI-TOF for identification of fish Aeromonas isolates at species level
Article
  • Aug 2018
The aim of this study was to evaluate the usefulness of the MALDI‐TOF MS to identify 151 isolates of Aeromonas obtained mostly from diseased fish. MALDI‐TOF MS correctly identified all isolates to the genus level but important differences in the percentage of isolates correctly identified depending on the species were observed. Considering exclusively the first identification option, Aeromonas bestiarum, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii and Aeromonas sobria were the best identified with results >95%. However, considering the first and second identification options, the only species that showed values >90% was A. hydrophila. Overall, when the database was supplemented with 14 new spectra, the number of accurate identifications increased (41% vs. 55%) and the number of inconclusive identifications decreased (45% vs. 29%), but great differences in the success of species‐level identifications were found. Species‐distinctive mass peaks were identified only for A. hydrophila and A. bestiarum (5003 and 7360 m/z in 95.5% and 94.1% of their isolates, respectively). This work demonstrates the utility of MALDI‐TOF MS for Aeromonas identification to the genus level, but there is no consistency for the accurate identification of some of the most prevalent species implicated in fish disease.
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... Regarding aquatic species, IVIAT has been used to investigate Edwardsiella tarda [86], Vibrio anguillarum [87] as well as A. salmonicida subsp. salmonicida [88,89] and, more recently, Photobacterium damselae subsp. piscicida [90]. ...
A new age in AquaMedicine: Unconventional approach in studying aquatic diseases
Article
Full-text available
  • Jun 2018
  • BMC Vet Res
Background: Marine and aquaculture industries are important sectors of the food production and global trade. Unfortunately, the fish food industry is challenged with a plethora of infectious pathogens. The freshwater and marine fish communities are rapidly incorporating novel and most up to date techniques for detection, characterization and treatment strategies. Rapid detection of infectious diseases is important in preventing large disease outbreaks. Main text: One hundred forty-six articles including reviews papers were analyzed and their conclusions evaluated in the present paper. This allowed us to describe the most recent development research regarding the control of diseases in the aquatic environment as well as promising avenues that may result in beneficial developments. For the characterization of diseases, traditional sequencing and histological based methods have been augmented with transcriptional and proteomic studies. Recent studies have demonstrated that transcriptional based approaches using qPCR are often synergistic to expression based studies that rely on proteomic-based techniques to better understand pathogen-host interactions. Preventative therapies that rely on prophylactics such as vaccination with protein antigens or attenuated viruses are not always feasible and therefore, the development of therapies based on small nucleotide based medicine is on the horizon. Of those, RNAi or CRISPR/Cas- based therapies show great promise in combating various types of diseases caused by viral and parasitic agents that effect aquatic and fish medicine. Conclusions: In our modern times, when the marine industry has become so vital for feed and economic stability, even the most extreme alternative treatment strategies such as the use of small molecules or even the use of disease to control invasive species populations should be considered.
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A review on the beneficial effect of thymol on health and production of fish
Article
Full-text available
  • Aug 2020
The thymol molecule is a food additive, used to boost the growth, production and feed efficiency via improving the digestive secretion and activating the structure and function of digestive system. The major application of thymol essential oil is used in animal rations, particularly as a natural feed additive to decrease hazardous compounds in a wide range of animal species. Thymol essential oil plays an important role in improving the nutrient bioavailability, reproductive and productive performance, general health and immunity of livestock besides reducing problems of many animal diseases such as cancer and some other diseases. These roles may be attributed to the ability of thymol as anticancer, antimicrobial, antiviral, antioxidant, immunomodulators, anti‐inflammatory and antispasmodic factors through suppressing free radicals and harmful constituents from interacting with cellular biological components and its capability to manipulate the balance of the intestinal microbiota, increased digestion, metabolism and absorption of nutrients. This review elucidates the chemical structure and natural sources, physical proprieties and practical applications of thymol in fish nutrition.
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The role of oregano herb and its derivatives as immunomodulators in fish
Article
  • Jun 2020
The motivation behind this article is to give point‐by‐point data about the beneficial applications of oregano feed supplement in fish diets as immunomodulators, antioxidant, antiviral, antifungal and antiparasitic. Use of this plant as feed additive plays an important role in the fish diet when compared to other synthetic feed additives. Oregano is rich in phytochemical compounds including carvacrol and thymol in addition to other phenolic compounds with antioxidant and immune‐enhancing activities. Origanum vulgare extract improved the immunological responses and enhanced non‐specific immunity. Also, non‐specific immunity and the lysosomal activity were significantly increased in rainbow trout fed diet enriched with 3.0 mL essential oil of Origanum onites L kg⁻¹ diet for 60 days. Furthermore, non‐specific immune stimulant, antioxidant and nitric oxide activities were improved due to O. vulgare oil supplementation. In some recent studies, Origanum heracleoticum L essential oil as a growth enhancer increased the antioxidant status. In rainbow trout, the hepatic levels of antioxidant enzymes and the total antioxidant capacity increased by feeding diet enriched with 6 and 10 g kg⁻¹ diet of O. vulgare extract. Therefore, the addition of oregano and/or derivatives as a dietary supplement in fish diet may promote growth and enhance the immunity and health of fish and this will be useful for nutritionists, physiologists and veterinarians.
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Use of in vivo induced antigen technology to identify genes from Aeromonas salmonicida subsp. salmonicida that are specifically expressed during infection of the rainbow trout Oncorhynchus mykiss
Article
Full-text available
  • Dec 2014
  • BMC Vet Res
Background Aeromonas salmonicida is a major fish pathogen associated with mass mortalities in salmonid fish. In the present study, we applied In Vivo Induced Antigen Technology (IVIAT), a technique that relies on antibodies adsorbed against in vitro cultures of the pathogen, to a clinical isolate of A. salmonicida subsp. salmonicida.ResultsThe results from IVIAT allowed identification of four proteins that were upregulated in the fish samples: A UDP-3-O-acyl-N-acetylglucosamine deacetylase, an RNA polymerase sigma factor D as well as TonB and a hypothetical protein. Subsequent investigations were performed using real-time PCR and cDNA synthesised from infected spleen, liver and anterior kidneys. These confirmed that the transcription level of each of these genes was significantly upregulated during the infection process compared to bacteria in vitro.Conclusions The present studied identified four genes that were upregulated during the infectious process and are likely to play a role in the virulence of A. salmonicida. Because these are antigenic they might constitute potential targets for the development of new vaccine as well as therapeutic agents.
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Salmonella methylglyoxal detoxification by STM3117 encoded Lactoylglutathione lyase affects virulence in Coordination with SPI-2 and Phagosomal acidification.
Article
Full-text available
  • Jun 2014
Intracellular pathogens like Salmonella Typhimurium manipulate their host cells through interplay of various virulence factors. A multitude of such virulence factors are encoded on the genome of S. Typhimurium and are usually organised in pathogenicity islands. The virulence associated genomic stretch of STM3117-3120 is shown to have structural features of pathogenicity islands and is exclusively present in non-typhoidal serovars of Salmonella. It encodes metabolic enzymes predicted to be involved in methylglyoxal metabolism. STM3117 encoded lactoylglutathoine lyase significantly impacts the proliferation of intracellular Salmonella. The deletion mutant of STM3117 (Δlgl) fails to grow in epithelial cells but hyper-replicates in macrophages. This difference in proliferation outcome was the consequence of failure to detoxify methylglyoxal by Δlgl, which also reflected in the form of oxidative DNA damage and up-regulation of kefB in the mutant. Within macrophages, the toxicity of methylglyoxal adducts elicits the potassium efflux channel (KefB) in the mutant which subsequently modulates the acidification of mutant containing vacuoles (MCVs). The perturbation in the pH of MCV milieu and bacterial cytosol enhances the SPI-2 translocation in Δlgl, increasing its net growth within macrophages. In epithelial cells, however, the maturation dynamics of Δlgl containing vacuoles were affected as these non-phagocytic cells do not maintain acidic vacuoles like macrophages. Remarkably, ectopic expression of TLR2 and 4 on epithelial cells partially restored the survival of Δlgl. This study identified a novel metabolic enzyme in S. Typhimurium whose activity during intracellular infection within a given host cell type differentially affects the virulence of the bacteria.
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Protein expression and transcription profiles of three strains of Aeromonas salmonicida ssp. salmonicida under normal and iron-limited culture conditions
Article
Full-text available
  • May 2014
  • PROTEOME SCI
Background Aeromonas salmonicida is an important fish pathogen that produces a wide and varied array of virulence factors. Here we used iron deprivation by addition of the chelator 2’2-dipyridyl to induce the expression of several such virulence factors in three isolates of Aeromonas salmonicida (one avirulent and two virulent). By using SDS-PAGE followed by mass spectrometry, we identified proteins that appeared differentially expressed under these conditions. The differential transcription of the identified gene products were subsequently measured by reverse transcription quantitative real-time PCR (RT-qPCR). Results Our initial screening using SDS-PAGE identified five proteins that appeared differentially expressed in virulent and avirulent isolates or, within the same isolates, between bacteria cultivated under iron-rich or iron-deprived conditions. The transcription of the genes coding for these proteins were subsequently quantified by RT-qPCR. Results of this analysis demonstrated that the gene coding for alkyl hydroperoxide reductase (AhpC), a protein involved in oxidative stress response, was transcribed at a higher rate in the virulent strain as compared to the avirulent strain. Additionally, it was observed that addition of an iron chelator to the culture medium lead to a reduction of the transcription levels of the regulatory histone-like nucleoid structuring protein (H-NS). This was consistent in all three isolates. On the other hand, the transcription levels of the virulence array protein (VapA) and the protein ATP-synthetase F (ATPF) displayed only limited changes, despite being the dominant component of a protein fraction that displayed changes during the preliminary SDS-PAGE screening. This was true regardless of the culture conditions and of the isolates considered. Finally, transcription of the enzyme enolase was upregulated in the iron-deprived broths in all isolates. Conclusions We identified several genes differentially expressed under culture conditions known to lead to the overexpression of virulence factors. In addition, we identified alkyl hydroperoxide as being overexpressed in the virulent isolates compared to the avirulent isolates. The results from this study will contribute to enhance our understanding of the virulence of A. salmonicida and may suggest new directions for further research.
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The Aeromonas salmonicida subsp. salmonicida exoproteome: Global analysis, moonlighting proteins and putative antigens for vaccination against furunculosis
Article
Full-text available
  • Oct 2013
  • PROTEOME SCI
Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Despite the identification of several virulence factors the pathogenesis is still poorly understood. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic DeltaascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth. Among the different experimental conditions we obtained semi-quantitative values for a total of 2136 A. salmonicida proteins. Proteins of specific A. salmonicida species were proportionally less detected than proteins common to the Aeromonas genus or those shared with other Aeromonas species, suggesting that in vitro growth did not induce the expression of these genes. Four detected proteins which are unidentified in the genome of reference strains of A. salmonicida were homologous to components of the conjugative T4SS of A. hydrophila pRA1 plasmid. Polypeptides of three proteins which are specific to the 01-B526 strain were also discovered. In supernatants (SNs), the number of detected proteins was higher in SP (326 for wt vs 329 for mutant) than in GP (275 for wt vs 263 for mutant). In pellets, the number of identified proteins (a total of 1536) was approximately the same between GP and SP. Numerous highly conserved cytoplasmic proteins were present in A. salmonicida SNs (mainly EF-Tu, EF-G, EF-P, EF-Ts, TypA, AlaS, ribosomal proteins, HtpG, DnaK, peptidyl-prolyl cis-trans isomerases, GAPDH, Enolase, FbaA, TpiA, Pgk, TktA, AckA, AcnB, Mdh, AhpC, Tpx, SodB and PNPase), and several evidences support the theory that their extracellular localization was not the result of cell lysis. According to the Cluster of Orthologous Groups classification, 29% of excreted proteins in A. salmonicida SNs were currently poorly characterized. In this part of our work we elucidated the whole in vitro exoproteome of hypervirulent A. salmonicida subsp. salmonicida and showed the secretion of several highly conserved cytoplasmic proteins with putative moonlighting functions and roles in virulence. All together, our results offer new information about the pathogenesis of furunculosis and point out potential candidates for vaccine development.
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The Aeromonas salmonicida subsp. salmonicida exoproteome: Determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors
Article
Full-text available
  • Sep 2013
  • PROTEOME SCI
Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic DeltaascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the DeltaascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the DeltaascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent DeltaascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.
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Association of Type III secretion genes with virulence of Aeromonas salmonicida subsp. salmonicida.
Article
  • Jan 2003
  • DIS AQUAT ORGAN
Aeromonas salmonicida subsp. salmonicida possesses a number of potential virulence factors, including a recently identified plasmid-encoded Type III secretion system. A number of field isolates of A. salmonicida subsp. salmonicida were examined for the presence of Type III secretion genes. Using in vitro experiments, it was found that field isolates containing such genes are cytotoxic to fish cell lines, whereas those that lack these genes are not. Using a rainbow trout in vivo model, the virulence of a wild type A. salmonicida subsp. salmonicida strain (Strain JF2267), which possesses Type III secretion genes, was compared to that of a laboratory derivative of the same strain that has lost these genes. While Strain JF2267 was virulent towards rainbow trout, its derivative was not. The A. salmonicida subsp. salmonicida Type Strain ATCC 33658T, which also lacks Type III secretion genes, was also found to be avirulent by this challenge model. The findings from both the in vitro and in vivo experiments suggest that the presence of Type III secretion genes is associated with the virulence of this important fish pathogen.
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Adjuvants and Immunostimulants for Potentiating Protection Against Furunculosis in Fish
Chapter
  • Dec 1997
Effective bacterins against furunculosis disease in salmonids are now commercially available. The adjuvants are the substances having potentiating effects on the specific immune response and are used with vaccines. When these adjuvants are used without vaccine, these are termed as “immunostimulants.” The addition of adjuvant heightens the humoral antibody titres and enhances protection against furunculosis, that is, increases the effectiveness of bacterins used against furunculosis. Complete Freund's adjuvant (CFA) was used as the important potentiating component for the first time. Beta-glucans, muramyl dipeptide, streptococcal extracts, light-oils, and levamisole induce marked elevations of the non-specific defence mechanisms, as well as the specific immune response of fish. The immunostimulants affect the nonspecific defense mechanisms. The CFA when injected without a bacterin has the capability to induce significant protection against furunculosis. Nonspecific defense mechanisms, including phagocytic cell adherence, oxidative radical release, and engulfment, are among the most important early cellular factors in protection.
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Experimental studies on the efficacy and side-effects of intraperitoneal vaccination of Atlantic salmon (Salmo salar L.) against furunculosis
Article
  • Jul 1996
Efficacy and side-effects of anti-furunculosis vaccination were studied in an experimental trial. One unadjuvanted and eight adjuvanted bacterins were administered by intraperitoneal injection to Atlantic salmon (Salmo salarL.) presmolts held in fresh water. Adjuvant systems represented were mineral oil, aluminium salts, glucan and levamisole. Protection, plasma antibodies, and side-effects were determined six weeks, three months, and six months after vaccination. After six weeks, four vaccination groups were significantly protected compared to unvaccinated fish. Only two groups, being vaccinated with a trivalent and a monovalent mineral oil adjuvanted vaccine, respectively, were protected after three and six months. Fish from these two groups were increasingly protected with time. These two groups also displayed significant intra-abdominal lesions. Plasma antibody levels measured against A-layer protein and sonicated cells ofAeromonas salmonicidadiffered greatly between groups, though all vaccinated groups mounted a significant response in at least one of the samplings. High antibody levels increasing with time were found in the two long-term protected groups. At all sampling times, a positive association between antibody levels and survival was found. In conclusion, only use of mineral oil adjuvanted vaccines induced durable protective immunity against virulent waterborne furunculosis challenge.
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