Analysis of the Flavobacterium psychrophilum outer-membrane subproteome and identification of new antigenic targets for vaccine by immunomics

Abstract

Flavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture. INTRODUCTIONFlavobacterium psychrophilum is a yellow-pigmented, Gram-negative, gliding bacterium that predominantly affects salmonid fish (Borg, 1960), such as coho salmon (Oncorhynchus kisutch) or rainbow trout (Oncorhynchus mykiss), and occasionally other fish species, such as ayu (Plecoglossus altivelis) (Iida & Mizokami, 1996). This bacterium is therefore responsible for considerable economic losses in fish aquaculture. Infections with F. psychrophilum have several clinical manifestations, the most significant of which include mortality associated with haemorrhagic septicaemia and spleen hypertrophy in juvenile fish, referred to as rainbow trout fry syndrome, and in adults, septicaemia preceded by extensive necrotic lesions, called cold-water disease (CWD) (Bernardet & Bowman, 2006). However, the actual mechanism of pathogenesis is not well understood, although virulence has been suspected to be related to the ability of F. psychrophilum to produce exotoxins (Dalsgaard, 1993), extracellular metalloproteases (Fpp1-2; Secades et al., 2001Secades et al., , 2003 or enzymes involved in the degradation of products such as chondroitin sulfate, collagen and fibrinogen (Bertolini et al., 1994). Clearly, the flavobacterial outer membrane (OM) is important when we consider interactions of bacteria with host cells and tissues in the context of pathogenesis and immunity to infection. Several surface components of F. psychrophilum have been implicated in flavobacterial pathogenesis and identified as possible vaccine and diagnostic candidate macromolecules; they include lipopolysaccharide O antigen (MacLean et al., 2001) and surface-exposed antigens [e.g. 20 kDa antigen and OmpA (Merle et al., 2003;Dumetz et al., 2007)], some of which may be good candidates for an F. psychrophil...

Different Phenotypes of Mature Biofilm in Flavobacterium psychrophilum Share a Potential for Virulence That Differs from Planktonic State

“…It is likely that the active protein product of the FP0546 gene and some other upregulated adhesins (Table 1) are WGA-binding glycoproteins with N -acetyl sugar modifications. Indeed, some glycoproteins and OmpA-family proteins may participate in modulation of the adherence and pathogenicity in F. psychrophilum (Dumetz et al, 2007, 2008) and other fish-pathogenic bacteria such as F. columnare (Laanto et al, 2014). In addition, two putative adhesins were encoded by non-DEGs (FP0595 and FP183; Figures 4C,D), suggesting that these proteins may have a similar adhesion function in biofilm and planktonic states.…”

“…For instance, it has been proposed that gene fmo , which encodes a pore-forming toxin known as flavomodulin (downregulated in the biofilm state, Table 1), is product of a horizontal gene transfer event from a Gram-positive bacterium (Dumetz et al, 2008). Some virulent strains of F. psychrophilum (Castillo et al, 2016) possess genomic islands that carry genes associated with toxins, drug resistance, and putative virulence factors such as virulence-associated protein E (VapE).…”

PROTEOMICS in aquaculture: Applications and trends

“…Several envelop proteins were identified with protein VP088 further characterized with the aim to better understand the molecular mechanisms of iridoviral pathogenesis and disease prevention in marine aquaculture industry. Dumetz in his 2008 paper [39] reveals a number of novel candidate proteins for developing vaccines against flavobacteriosis infection in aquaculture, using 2-DE and LC-MS/MS techniques. In bivalves, the majority of studies on its interaction molecular mechanisms with bacteria are mainly on mRNA and recombinant protein levels.…”

“…The effect of probiotics administration in diets has also been reported in rainbow trout and cod [35,36] with interesting results at the level of immune-stimulation. In vitro [37] and in vivo infections proteomic studies on cultured fish like gilthead seabream [38], rainbow trout [39] and Atlantic salmon [40,41] are also commonly addressed since infection and concomitant death is one of the main causes of heavy economic losses in marine fish cultures. These works lead not only to a better understanding of the molecular mechanisms of pathogenesis as well as provide novel protein candidates for vaccines development.…”

“…Here most are on health aspects, with focus on viral diseases [41,[110][111][112][113][114][115][116][117][118], bacterial diseases [38,110,[119][120][121][122][123][124][125][126][127][128] and vaccine development [39,[129][130][131][132][133][134][135][136][137][138] but also parasites [139][140][141], hepatic tumours [142] and skeletal deformities in fish [34] have been studied. Besides these, immunoproteomic studies also encompass results on immunostimulants [35], antimicrobial peptides [143][144][145] and composition of mucosal secretions [146,147], along with other topics related to a better understanding of mechanistic aspects of immune responses to certain stimuli [40,[148]…”