Iron Uptake Mechanisms in the Fish Pathogen Tenacibaculum maritimum

Abstract

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding. Tenacibaculum maritimum (formerly Flexibacter maritimus)(48) is the etiological agent of an ulcerative disease known as marine flexibacteriosis or tenacibaculosis, which affects a large number of marine fish in the world and thus has considerable economic significance to aquaculture producers. The infection has diverse clinical manifestations depending on the species and age of fish, the most significant symptom being the presence of gross lesions on the body surface (11,16).Although the phenotypic, antigenic, and molecular characteristics of T. maritimum have been examined by several authors (for a review, see reference 49), the actual factors determining the virulence of this pathogen have not yet been elucidated. Some synergistic interactions of the toxins contained in extracellular products and a hemolysin might be involved in T. maritimum infections (8). Moreover, pathological properties of the bacterium, such as a strong adherence to the skin mucus of different fish species and the capacity to resist its bactericidal activity (31), have been pointed out as possible virulence factors.It is well known that the ability to take up iron during infection is an essential factor in the pathogenicity of several bacteria, being necessary for the pathogen multiplication. However, the levels of free iron in the biological fluids within the animal host is often v...

Tenacibaculosis infection in marine fish caused by Tenacibaculum maritimum: a review

“…The homogeneity of Tenacibaculum maritimum facilitates the use of the miniaturized systems API ZYM and API 50CH for its identification (Bernardet & Grimont 1989, Pazos et al 1993, Bernardet et al 1994, Chen et al 1995, Ostland et al 1999, Avendaño-Herrera et al 2004b, 2005c. In fact, within the API ZYM gallery, the majority of isolates display a characteristic profile (positive results in the first 11 enzymatic reactions), whereas within the API 50CH not all T. maritimum strains were able to produce acid from any carbohydrate substrate.…”

“…Avendaño-Herrera et al (2005c) have demonstrated that this bacterium, regardless of its serotype, grows and multiplies in the presence of the chelating agent ethilenediamine-di-(o-hydroxyphenyl acetic acid) (EDDHA) using at least 2 different systems of iron acquisition. One mechanism involves the production of an unidentified siderophore, which is neither a phenolate nor a hydroxamate type, as demonstrated by the chromoazurol S (CAS) agar (Fig.…”

“…(Hikida et al 1979, Kimura & Kusuda 1983, Pépin & Emery 1993, Handlinger et al 1997) and saprophytic organisms, mainly ciliated protozoans (McVicar & White 1979, Devesa et al 1989) such as Trichodina and Uronema spp. (Chen et al 1995, Handlinger et al 1997), makes visualization difficult and increases the possibility of misdiagnosis.Because isolation of the bacterium from diseased fish is not always successful, the definitive diagnosis must be supported by the isolation of colonies of Tenacibaculum maritimum on appropriate specific media, followed by the determination of at least a limited number of morphological and biochemical characteristics, or by the use of specific molecular DNA-based methods.The homogeneity of Tenacibaculum maritimum facilitates the use of the miniaturized systems API ZYM and API 50CH for its identification (Bernardet & Grimont 1989, Pazos et al 1993, Bernardet et al 1994, Chen et al 1995, Ostland et al 1999, Avendaño-Herrera et al 2004b, 2005c. In fact, within the API ZYM gallery, the majority of isolates display a characteristic profile (positive results in the first 11 enzymatic reactions), whereas within the API 50CH not all T. maritimum strains were able to produce acid from any carbohydrate substrate.…”

Fur-Regulated Iron Uptake System of Edwardsiella ictaluri and Its Influence on Pathogenesis and Immunogenicity in the Catfish Host

“…Antibiotics and reagents were from Sigma (St. Louis, MO). LB broth (tryptone, 10 g; yeast extract 5 g; NaCl, 10 g; glucose, 1 g; double-distilled water [ddH 2 O], 1 liter) (5), Bacto brain heart infusion (BHI) broth, chromoazurol S (CAS) broth (58), and Trypticase soy broth (TSB) were used routinely. When required, media were supplemented with 1.5% agar, 5% sucrose, colistin sulfate (Col) (12.5 g/ml), ampicillin (Amp) (100 g/ml), chloramphenicol (Cm) (25 g/ml), kanamycin (Km) (50 g/ml), FeSO 4 (150 M) (Sigma), or 2,2=-dipyridyl (150 M) (Sigma).…”

“…These siderophore mechanisms of iron acquisition have been linked to the virulence of different fish bacterial pathogens, such as Vibrio anguillarum (Listonella anguillarum) (38,73), Aeromonas salmonicida (24,27), Photobacterium damselae subsp. piscicida (13), Edwardsiella tarda (25), and Tenacibaculum maritimum (2). Direct iron uptake systems from host proteins rely on the interaction between specific microbial receptors and host transferrin or heme-containing compounds (31,41) and often involve bacterial hemolytic or proteolytic activity (10).…”