Purification and characterization of a membrane glycoprotein from the fish pathogen<i>Flavobacterium psychrophilum</i>

Merle, Christine; Faure, D.; Urdaci, María C.; Hénaff, Michel Le

doi:10.1046/j.1365-2672.2003.01946.x

Cited by 27 publications

(39 citation statements)

References 33 publications

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“…This suggests that the presence of the ~35 kDa OmpA (P60) protein is not an artifact due to sample preparation, but there may be processing or degradation occurring in in vitro culture. The OmpA (P60) protein of F. psychrophilum has been extensively characterized and suggested to play a potential role in protective immunity (Merle et al 2003, Dumetz et al 2007. In these studies, rabbit polyclonal serum was prepared against purified OmpA (P60), and Western blot analysis of crude F. psychrophilum extracts revealed a single band in 1-dimensional electrophoresis (Dumetz et al 2007).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, recent efforts have been aimed at identifying specific bacterial antigens to target for vaccine development. Such research has resulted in the identification of numerous proteins or specific fractions of F. psychrophilum (Rahman et al 2002, Merle et al 2003, LaFrentz et al 2004, Massias et al 2004, Crump et al 2005, Dumetz et al 2007, 2008, LaFrentz 2007, Sudheesh et al 2007), some of which have been shown to be protective (Rahman et al 2002, LaFrentz et al 2004, Dumetz et al 2006, Crump et al 2007). These studies demonstrate the potential for identifying vaccine candidates; however, the work was performed using in vitro cultured bacteria, and our knowledge of differential gene expression by F. psychrophilum grown in vivo is lacking.…”

Section: Introductionmentioning

confidence: 99%

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Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media

LaFrentz

LaPatra²,

Call³

et al. 2009

Dis. Aquat. Org.

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Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease, but the pathogenic mechanisms of this important fish pathogen are not fully understood. Identifying bacterial genes of F. psychrophilum differentially expressed in vivo may lead to a better understanding of pathogenesis and provide targets for vaccine development. Therefore, the present study used a proteomic approach to identify and quantify proteins of F. psychrophilum following growth in vivo and under iron-limited growth conditions. As determined by 2D polyacrylamide gel electrophoresis (2D-PAGE), numerous proteins exhibited different spot intensities following culture of the bacterium in vivo, and of these, 20 were selected and identified by liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) analysis and Mascot searches of the F. psychrophilum genome. Eighteen proteins exhibited increased spot intensities in vivo, and these included: several chaperone and stress proteins, gliding motility protein GldN, outer membrane protein OmpH, 2 probable outer membrane proteins (OmpA family), probable aminopeptidase precursor, probable lipoprotein precursor, 3-oxoacyl-[acyl-carrier-protein]-reductase, and several proteins with unknown function. Two proteins exhibited decreased spot intensities in vivo and were identified as ferritin FtnA and outer membrane protein OmpA (P60). Culture of F. psychrophilum in iron-limited media resulted in similar protein spot intensity changes for 6 of the 20 proteins identified following growth in vivo. Results from the present study suggest a role of upregulated proteins in the pathogenesis of F. psychrophilum and these may represent potential vaccine candidate antigens.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media

LaFrentz

LaPatra²,

Call³

et al. 2009

Dis. Aquat. Org.

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“…28), P60 (ref. 29) and FspA (ref. 30), as well as two proteins similar to those that induce protective immunity against the poultry pathogen Ornithobacterium rhinotracheale 31 .…”

Section: Cell Surface Proteinsmentioning

confidence: 99%

Complete genome sequence of the fish pathogen Flavobacterium psychrophilum

et al. 2007

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We report here the complete genome sequence of the virulent strain JIP02/86 (ATCC 49511) of Flavobacterium psychrophilum, a widely distributed pathogen of wild and cultured salmonid fish. The genome consists of a 2,861,988-base pair (bp) circular chromosome with 2,432 predicted protein-coding genes. Among these predicted proteins, stress response mediators, gliding motility proteins, adhesins and many putative secreted proteases are probably involved in colonization, invasion and destruction of the host tissues. The genome sequence provides the basis for explaining the relationships of the pathogen to the host and opens new perspectives for the development of more efficient disease control strategies. It also allows for a better understanding of the physiology and evolution of a significant representative of the family Flavobacteriaceae, whose members are associated with an interesting diversity of lifestyles and habitats.

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“…The OMP content was estimated using our standard procedures (Merle et al, 2003). The OM preparation was finally adjusted to 10 mg protein ml 21 and stored at 280 uC.…”

Section: Methodsmentioning

confidence: 99%

“…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. psychrophilum subunit vaccine, such as the surface-localized Flavobacterium-specific protein (FspA; Crump et al, 2005). Indeed, a protective immune response has been shown experimentally in rainbow trout by P18 (Massias et al, 2004), a surface-exposed protein belonging to the OmpH family .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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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...

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Purification and characterization of a membrane glycoprotein from the fish pathogenFlavobacterium psychrophilum

Cited by 27 publications

References 33 publications

Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media

Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media

Complete genome sequence of the fish pathogen Flavobacterium psychrophilum

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

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