The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease
outbreaks affecting several fish species including flatfish Solea senegalensis (Kaup). Phdp is able to avoid host defences
by invasion and intracellular survival in non-phagocytic cells, mainly epithelial cells. Virulence factors reported in Phdp
include restricting complement-mediated activity, apoptosis of phagocytes caused by exotoxins secretion, iron acquisition
mechanisms such as siderophores that enable the pathogen to obtain iron from transferrin and ability to bind haemin and
antioxidant enzymatic activities capable to counteract superoxide radicals (Do Vale et al., 2005; Andreoni and Magnano,
2014). Commonly, genes expressed during pathogen infection are important for pathogenicity. In vivo-induced antigen
technology (IVIAT) (Handfield et al., 2000) has been used to identify in vivo-induced genes using pooled sera from fish
that have experienced photobacteriosis.
Materials and methods
Sera were obtained from surviving S. senegalensis specimens after sublethal infection with Phdp (Lg41/01) and subsequently
pooled and adsorbed against in vitro grown Phdp Lg41/01 and Escherichia coli BL21 (DE3) cells and lysates according to
Handfield et al. (2000). The efficiency of sera adsorption was evaluated based on the immunoreactivity after each adsorption
step with whole and lysed Phdp cells grown in vitro. A genomic expression library of Phdp Lg41/01 was generated in E.
coli BL21 (DE3) using pET-30 expression system (Novagen, San Diego, CA, USA). The expression library was probed
with adsorbed and non-absorbed sera using immunoblot technique. Reactive clones of in vivo-induced and in vitro antigens
were obtained, purified and their inserted DNA sequenced (Macrogen Europe, Amsterdam, The Netherlands). Nucleotide
sequences were compared against the NCBI protein database using BLASTx.
A progressive reduction in sera immunoreactivity against in vitro grown Phdp cells was detected after the adsorption
rounds, especially after the first adsorption step. Thus, following adsorption steps substantially removed antibodies against
in vitro expressed antigens and resulted in relative enrichment in antibodies recognizing in vivo expressed antigens. The
library from Phdp Lg14/01 constructed in E. coli BL21 (DE3) consisted of approximately 6500 recombinants.
A total of 117 clones were selected for their reactivity with pooled adsorbed and non-adsorbed sera from convalescent
S. senegalensis specimens after a first round of screening. In a second screening, 14 out of 117 candidate clones showed
positive reaction, among which two clones were clearly positive and two gave weak reaction against adsorbed sera. Predicted
proteins codified by inserted sequences have intracellular and membrane cell location and are involved in virulence,
synthesis of intermediary products, energy metabolism and gene replication. Inosine-5’-monophosphate dehydrogenase
(IMPDH) and alkyl hydroperoxide reductase (AhpC) have been identified as in vivo induced antigens expressed during
S. senegalensis infection with Phdp. Iron/manganese superoxide dismutase (Fe/Mn-SOD) and alanyl-tRNA synthetase
(AlaRS) proteins have also been identified, though with weak signal.
Discussion and conclusion
Identification of immunogenic bacterial proteins during Phdp infection is essential for understanding bacterial pathogenesis
and development of effective vaccines. AhpC peroxidase activity has a protective role by reducing hydrogen peroxide,
peroxynitrite and organic hydroperoxides. Immunization with AhpC conferred protection against Helicobacter pylori
infection (O’Riordan et al., 2012). IMPDH catalyzes the conversion of products essential in de novo synthesis of guanine
nucleotides. Adequate levels of purine nucleotides are critical for cell proliferation, nucleic acid replication, cell signaling
and as a biochemical energy source. This gene is an important therapeutic target against bacterial diseases (Shu and Nair,2008). In conclusion, different genes expressed during Phdp infection in S. senegalensis have been identified. Among them,
IMPDH and AhpC have been identified as in vivo induced antigens expressed during S. senegalensis infection with Phdp.
Thus, they are likely to play a role in the virulence of Phdp. The antigenic character of these proteins makes them potential
targets for the development of new vaccines.
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