The type IX secretion system is required for virulence ...

AEM Accepted Manuscript Posted Online 22 September 2017 Appl. Environ. Microbiol. doi:10.1128/AEM.01769-17 Copyright © 2017 American Society for Microbiology. All Rights Reserved.

9/15/17 Revised manuscript AEM01769-17

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The type IX secretion system is required for virulence of the fish pathogen Flavobacterium

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columnare.

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Nan Lia, b, Yongtao Zhub, Benjamin R. LaFrentzc, Jason P. Evenhuisd, David W. Hunnicutte,

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Rachel A. Conradb, Paul Barbierb, Connor W. Gullstrande, Jack E. Roetse, Jonathan L. Powerse,

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Surashree S. Kulkarnib, Devon H. Erbesb, Julio C. Garcíac, Pin Niea, and Mark J. McBrideb.

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State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese

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Academy of Sciences, Wuhan, Hubei Province, Chinaa;

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Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USAb;

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United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Aquatic

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Animal Health Research Unit, Auburn, AL, USAc;

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USDA-ARS, National Center for Cool and Cold Water Aquaculture, Kearneysville, WV, USAd;

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Department of Biology, St. Norbert College, De Pere, WI, USAe

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N. L. and Y. Z. contributed equally to this article.

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Address correspondence to Mark J. McBride, [email protected], or Pin Nie, [email protected].

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Downloaded from http://aem.asm.org/ on September 27, 2017 by UNIV OF WISCONSIN

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Corresponding author:

Mark J. McBride Telephone: (414) 229-5844

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Fax: (414) 229-3926

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[email protected]


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Co-corresponding author: Pin Nie

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Telephone: 86-27-68780736

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Fax: 86-27-68780123

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[email protected]

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Running Title: Type IX secretion system and F. columnare virulence

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Abstract:

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Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in

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wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease

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are not known. Many members of the phylum Bacteroidetes use type IX secretion systems

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(T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F.

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columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core

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component of the T9SS, was deleted in wild type strains of F. columnare. The F. columnare

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gldN mutants were deficient in secretion of several extracellular proteins and lacked gliding

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motility. The gldN mutants exhibited reduced virulence in zebrafish, channel catfish, and

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rainbow trout, and complementation restored virulence. PorV is required for secretion of a subset

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of proteins targeted to the T9SS. A F. columnare porV mutant retained gliding motility but

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exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild

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type and complemented strains caused rapid mortality but spent media from gldN and porV

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mutants did not, suggesting that soluble toxins are secreted by the T9SS.

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Importance:

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Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture.

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Little is known regarding the virulence factors produced by F. columnare and control measures

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are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX

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protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are

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common in members of the phylum Bacteroidetes and likely contribute to virulence of other

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animal and human pathogens.

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Introduction Flavobacterium columnare is a common fish pathogen that causes columnaris disease (1-3). F. columnare causes epidemics in wild and cultured fish and is a major problem in freshwater

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aquaculture worldwide, resulting in significant mortality. Many species of freshwater fish are

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susceptible to columnaris disease (4, 5). The virulence mechanisms of F. columnare are

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incompletely understood and current control strategies are inadequate. F. columnare isolates are

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genetically diverse and have been assigned to multiple ‘genomovars’ (6, 7). F. columnare

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genomovars I and II exhibit significant differences including host ranges (8-10). Described

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outbreaks in salmonid aquaculture systems have almost invariably been associated with

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genomovar I strains (8, 11-14) whereas epidemics in catfish and other warm water fish have

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involved members of diverse genomovars (6, 7, 9, 10, 15).

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Secreted enzymes such as proteases and chondroitin sulfate lyases have been suggested as

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possible F. columnare virulence factors (3). Many virulence factors of pathogenic bacteria are

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either secreted proteins or the secretion systems themselves (16, 17). Gram-negative bacteria use

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at least nine secretion systems, named type I secretion system to type IX secretion system (T1SS-

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T9SS), to transport proteins across their outer membranes (16). The T9SS (previously called the

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Por secretion system) was originally described in the human periodontal pathogen

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Porphyromonas gingivalis and in the environmental bacterium Flavobacterium johnsoniae (18).

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T9SSs are common in, but confined to, the phylum Bacteroidetes (19, 20). In F. johnsoniae they

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are involved in secretion of cell-surface components of the gliding motility apparatus and thus

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the T9SS is required for gliding motility (18, 21, 22). The F. johnsoniae T9SS is also involved in

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secretion of the soluble extracellular chitinase ChiA and in the secretion of many other proteins

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(23, 24). The P. gingivalis T9SS secretes gingipain proteases and cell-surface adhesins that are

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thought to function as virulence factors in periodontitis (18, 25). Genetic analyses suggest that GldK, GldL, GldM, GldN, SprA, SprE, SprT, PorU, and PorV

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(Fig. 1) are T9SS components (18, 21, 24, 26, 27). Proteins secreted by T9SSs have N-terminal

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signal peptides that facilitate export across the cytoplasmic membrane by the Sec system. They

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also typically have conserved C-terminal domains (CTDs) that target them for secretion across

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the outer membrane by the T9SS (24, 28-34). The CTDs are often removed by the protease PorU

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during or after secretion (28). Some secreted proteins remain attached to the cell surface whereas

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others are released in soluble form (23, 24, 34).

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Techniques to genetically manipulate F. columnare were recently developed (35, 36)

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allowing exploration of the roles of individual genes in columnaris disease. We used these tools

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to generate T9SS mutants. Analysis of these mutants demonstrated that the T9SS is required for

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virulence and that secreted toxins have a role in pathogenesis.

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Results

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Numerous proteins are predicted to be secreted by the F. columnare T9SS.

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Bioinformatic analyses revealed that the genomes of F. columnare strain IA-S-4 (genomovar I)

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and F. columnare strain C#2 (genomovar II) had orthologs of the F. johnsoniae T9SS genes,

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gldK, gldL, gldM, gldN, sprA, sprE, sprT, porU, and porV (Table S1). The proteins encoded by

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these genes are thought to be components of the T9SS (19, 21, 24). Proteins secreted by T9SSs

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can often be identified by their conserved C-terminal domains that target them for secretion (24,

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28-34, 37). Most of these CTDs belong to either the TIGRFAM protein domain family

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TIGR04183 (referred to as type A CTDs) or to the TIGR04131 family (referred to as type B

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CTDs) (37). F. columnare strains IA-S-4 and C#2 had 39 and 43 genes encoding such proteins

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respectively (Table S2). Included in this list are predicted proteases, chondroitin sulfate lyases,

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and adhesins, that might have roles in virulence.

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F. columnare gldN mutants exhibit defects in gliding motility and protein secretion.

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The T9SS gene gldN was deleted in F. columnare strains IA-S-4 and C#2 (Fig. 2). F. johnsoniae

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gldN mutants are defective in gliding motility. This is at least partially explained by failure of the

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mutants to secrete motility adhesins such as SprB to the cell surface (18, 21). The F. columnare

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gldN mutants were also defective in gliding motility. They formed nonspreading colonies on agar

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(Fig. 3), and individual cells exhibited no movement on glass in tunnel slides (Fig. 4, and Movies

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S1 and S2). Complementation with pLN5 and pLN8 which carry wild type F. columnare IA-S-4

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gldN and F. columnare C#2 gldN respectively restored gliding motility in each case. The genome

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analyses described above and in Table S2 suggested that proteases and chondroitin sulfate lyases

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are likely secreted by the F. columnare T9SS. The F. columnare gldN mutants were defective in

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digestion of proteins and chondroitin (Fig. 5) supporting the suggestion that enzymes involved in

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digestion of these polymers are secreted by the T9SS.

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Cell-free culture fluid of wild type mutant and complemented strains of F. columnare

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C#2 were examined by SDS-PAGE to determine the effect on secreted proteins. Several

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prominent bands from the culture fluids of wild type and complemented strains were absent in

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the culture fluid from the gldN mutant (Fig. 6). Proteins present in the culture fluid of wild type

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and complemented cells but absent or greatly reduced in the gldN mutant were identified by

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LC/MS analyses (Table 1). Soluble proteins secreted by the T9SS included potential virulence

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factors such as the chondroitin sulfate lyases CslA (AX766_RS05135) and CslB

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(AX766_RS01510), the predicted peptidases AX766_RS05330 and AX766_RS13405, and the

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predicted thiol-activated cytolysins AX766_RS03975 and AX766_RS13970. F. columnare C#2

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CslA and CslB exhibited 96.6% and 98.1% amino acid identity with F. columnare strain G4

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CslA and CslB respectively (35). The identification of chondroitin sulfate lyases and peptidases

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in the cell-free culture fluids of wild type cells that were absent or greatly reduced in cell-free

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culture fluids from the gldN mutant is consistent with the enzyme activity results shown in Fig.

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5. Four of the seven secreted proteins identified had recognizable T9SS CTDs that are predicted

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to target these proteins to the secretion system (Table 1). The other three proteins may have

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novel targeting sequences, or may be released by a process that only indirectly involves the

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T9SS. Perhaps significantly, each of these three proteins that lacked obvious T9SS CTDs were

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predicted lipoproteins (Table 1).

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To directly assess protein secretion by wild type and gldN mutant cells we introduced a

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plasmid expressing the foreign protein mCherry carrying an N-terminal signal peptide to allow

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export across the cytoplasmic membrane by the Sec system, and carrying the CTD of F.

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johnsoniae ChiA to target the protein for secretion across the outer membrane by the T9SS. The

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CTD of F. johnsoniae ChiA functioned in F. columnare strain C#2, as demonstrated by the

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accumulation of mCherry in the spent culture fluid of wild type cells but not of the gldN mutant

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(Fig. S2).

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Cells of a F. columnare porV mutant are defective in protein secretion but retain

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gliding motility. F. johnsoniae PorV is needed for secretion of a subset of proteins targeted to

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the T9SS but it is not needed for secretion of the gliding motility adhesin SprB (24). F.

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johnsoniae porV mutants thus retain the ability to glide. In order to separate F. columnare

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protein secretion from motility a porV deletion mutant of wild type strain C#2 was constructed

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and examined. The mutant was defective in digestion of extracellular protein and chondroitin

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(Fig. 5) suggesting a protein secretion defect. Analysis of secreted proteins by SDS-PAGE

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demonstrated that several soluble proteins failed to be secreted in the porV mutant (Fig. 6). porV

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mutants retained the ability to glide (Fig. 3, 4, and Movie S3), similar to F. johnsoniae porV

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mutants. The F. columnare porV mutant spread less well on agar and produced smaller colonies

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than did wild type cells, but motility of individual porV mutant and wild type cells on glass was

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equivalent. The F. columnare porV mutant allowed us to partially separate motility from

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secretion, and thus examine the roles of each on virulence.

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F. columnare T9SS mutants are defective in virulence toward zebrafish, rainbow

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trout and channel catfish. Wild type F. columnare C#2, the gldN mutant, and the gldN

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mutant complemented with pLN8 which carries wild type gldN, were examined for ability to kill

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zebrafish (Fig. 7 and Fig. S3). The growth rates in culture of wild type, mutant and

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complemented strains were similar (Fig. S4). The gldN mutant exhibited decreased virulence

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compared to the wild type and complemented strains, suggesting that the T9SS has an important

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role in F. columnare virulence. The porV mutant was also examined to begin to separate the

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roles of secretion and motility on virulence. The porV mutant was less virulent than were the

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wild type and complemented strains (Fig. 7 and Fig. S3). This suggests that lack of secretion

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rather than lack of motility may account for much of the reduced virulence of T9SS mutants. For

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all of the zebrafish infection experiments described above, greater than 50% of mortalities

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exhibited clinical signs of columnaris disease including external lesions, tail rot, and/or damaged

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gills. In contrast, none of the uninfected control fish, and none of the fish infected with gldN or

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porV mutants, exhibited any of these signs or exhibited mortalities. Results similar to those

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described above for F. columnare strain C#2 were obtained when virulence of wild type F.

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columnare strain IA-S-4 (genomovar I), the gldN mutant of IA-S-4, and the complemented

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mutant were examined for virulence against zebrafish (data not shown). Wild type F. columnare, gldN mutants, and complemented mutants were also examined

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for ability to kill rainbow trout and channel catfish. In each case the gldN mutants exhibited

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decreased virulence compared to the wild type and complemented strains (Fig. 8 and Fig. S5).

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For the rainbow trout challenges, clinical signs of columnaris disease including external lesions,

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fin rot, and/or necrotic gills were observed in greater than 50% of mortalities, and such signs

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were observed on all channel catfish mortalities. None of the uninfected control fish, and none of

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the fish infected with gldN mutants, exhibited these signs.

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During the zebrafish, rainbow trout, and channel catfish challenges described above, re-

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isolation of F. columnare was attempted from ~ 20-30% of dead/moribund fish by inoculating

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fish tissue onto agar plates, culturing, and examining for yellow, rhizoid, adherent colonies. F.

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columnare was re-isolated from all challenge mortalities, suggesting that the morbidity and

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mortality observed were due to F. columnare infections. For isolates from rainbow trout, 16S

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rRNA genes were amplified and sequenced and each was identical to the sequence from the

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challenge strain, F. columnare strain IA-S-4. Finally, in each study, negative control fish that

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were not exposed to F. columnare experienced no mortalities.

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Wild type F. columnare but not T9SS mutants secreted materials that were toxic to

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fish. The requirement of the T9SS for virulence suggested the possibility that soluble secreted

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proteins may be important virulence factors. Filtered, cell-free spent media from cultures of wild

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type, mutant, and complemented cells were examined for ability to kill zebrafish. Cell-free spent

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media from exponential cultures of wild type and complemented cells caused rapid mortality,

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whereas spent media from the gldN and porV mutant cells did not (Fig. 9). The toxicity of the

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spent medium from wild type cells of F. columnare strain C#2 was eliminated by heating at 60oC

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for 60 min and by treatment with trypsin (Fig. 10), suggesting that the toxins may be proteins.

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Incubation of cell-free spent culture fluid at 37oC for 12 h without trypsin resulted in apparent

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decreased toxicity (compare untreated trials from Fig. 10 panels A and B). This may be the result

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of digestion of the toxins by secreted F. columnare proteases, although other explanations are

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also possible.

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Discussion

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F. columnare is an important pathogen of freshwater fish and is especially problematic in

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the high-density conditions often employed in aquaculture systems. Columnaris disease causes

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substantial economic losses and has received considerable attention. Previous studies identified

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possible virulence factors, including proteases and chondroitin sulfate lyases (38-40). However,

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definitive proof for the involvement or lack of involvement of these enzymes in virulence was

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lacking. Techniques for gene transfer (36) and gene deletion (35) were recently developed for F.

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columnare, and allow experiments to test the roles of individual genes and proteins in virulence.

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Genomic analysis identified the components of the F. columnare T9SS. As identified by

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genome analyses, T9SSs are common throughout most of the phylum Bacteroidetes and are

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found in all members of the genus Flavobacterium whose genomes have currently been

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sequenced and analyzed (20, 37). Further, recent comparative analyses identified all of the T9SS

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genes in the genomes of each of nine F. columnare strains examined (41, 42). For all members of

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the phylum Bacteroidetes that have the T9SS genes, gldK, gldL, gldM, and gldN are invariably

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located together in this order as an operon (20, 21). Most of the other T9SS genes are not

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clustered together and are not located near the gldKLMN operon. Phylogenetic analyses of T9SS

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genes and of 16s rRNA genes suggested that the T9SS genes are part of the core genome of most

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genera within the phylum Bacteroidetes (20, 21). Our genome analyses also identified dozens of

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proteins that have CTDs predicted to target them for secretion by the T9SS (Table S2). This is

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likely to be an underestimate of the actual number of T9SS-secreted proteins because F.

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columnare proteins with similarity to T9SS CTDs that were slightly below the trusted

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TIGRFAM cutoffs were also identified, and because some proteins known to be secreted by

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T9SSs exhibit no obvious sequence similarity to these conserved CTD sequences (23, 24, 37).

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Included in the lists of predicted secreted proteins were chondroitin sulfate lyases, proteases, and

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adhesins, any of which could contribute to virulence.

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Since secreted proteins are known virulence factors in other bacteria (16, 17) we targeted

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the T9SS for analysis. Deletion of gldN and porV, which encode components of the T9SS,

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resulted in decreased secretion of extracellular enzymes. These results are similar to those

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observed for F. johnsoniae and P. gingivalis and support the roles of GldN and PorV in protein

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secretion (18, 24, 26, 43, 44). The F. columnare T9SS mutants exhibited decreased levels of

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extracellular proteases, chondroitin sulfate lyases, and proteins of unknown function. The

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mutants also exhibited reduced virulence for zebrafish, rainbow trout, and channel catfish. In

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addition, whereas the cell-free culture fluid from wild type cells was toxic for zebrafish, the

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culture fluids from gldN and porV mutants were not. The identity of the secreted toxin(s) are

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not known, but they were destroyed by elevated temperature and by protease treatment,

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suggesting that they are proteins. The toxins may be one or more of the seven proteins that were

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identified in the cell-free culture fluid of wild type cells. This possibility can now be addressed

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by deletion of the genes encoding these proteins, either singly or in combination.

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In addition to soluble secreted proteins such as those mentioned above, many proteins secreted by T9SSs are found on the cell surface (34). These cell-surface enzymes and adhesins

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may also be important for virulence, and future studies are needed to address this possibility.

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One cell-surface protein secreted by the T9SS whose function is known is F. johnsoniae SprB,

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which is involved in gliding motility. This adhesin is propelled rapidly along the cell surface (45,

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46). The action of the gliding motor on SprB proteins that are attached to the substratum results

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in gliding motility. F. columnare has an SprB ortholog that is thought to be involved in gliding

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(42). Not surprisingly, deletion of the core F. columnare T9SS gene gldN resulted not only in

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secretion defects but also in loss of motility. This motility defect may be explained by the

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inability to secrete SprB to the cell surface. In F. johnsoniae, the T9SS protein PorV is less

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essential for secretion than is GldN. F. johnsoniae PorV is required for secretion of many

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proteins that are targeted to the T9SS, but it is not required for secretion of SprB and related

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proteins (24). For this reason, porV mutants of F. johnsoniae exhibit gliding motility. In this

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study, we demonstrated that an F. columnare porV mutant behaved similarly. It was defective

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for secretion and virulence, but retained gliding motility. This result partially separates the

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potential roles of motility and secretion in virulence. Motility may play a role in virulence, but

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even in the presence of a functional motility system, a secretion defective porV mutant exhibited

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reduced virulence.

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Secretion is important for virulence of F. columnare, but the identities of the most

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important secreted virulence factors are not yet known. Recently, both secreted chondroitin

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sulfate lyases of F. columnare strain G4 were examined for their roles in virulence. Cells lacking

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both chondroitin sulfate lyases retained virulence when fish were challenged by intraperitoneal

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injection but exhibited decreased competitiveness during co-infection by immersion (35).

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Chondroitin sulfate is found in connective tissues, and the chondroitin sulfate lyases may allow

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the bacteria to penetrate fish tissues during infection. Similar genetic experiments to those

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described in this study should allow identification of the most critical secreted proteins involved

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in columnaris disease.

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The results presented here demonstrate that the T9SS is involved in F. columnare

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virulence. They also suggest strategies to control this pathogen. Secreted proteins appear to be

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important in the disease process. Deletion of the genes encoding one or more of these proteins

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may result in attenuated strains that fail to cause disease, but that may still interact with fish and

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generate a protective immune response. Such strains could potentially function as effective live

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attenuated vaccines. T9SSs are common in members of the phylum Bacteroidetes (20). Some of

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these bacteria are animal and human pathogens, and their T9SSs may play important roles in

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their ability to cause disease. Continued studies may result in a better understanding of the

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virulence mechanisms employed by these pathogens, and suggest strategies to control them.

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Materials and Methods Bacterial strains, plasmids, and growth conditions. F. columnare genomovar I strain

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IA-S-4 and genomovar II strain C#2 (47, 48), were the wild-type strains used in this study. Strain

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IA-S-4 was isolated in Iowa (USA) in 2011 from the skin of a walleye with columnaris disease

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(B. LaFrentz, unpublished). F. columnare strains were grown at 25 to 30 °C in Shieh medium

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(49), Modified Shieh medium (50), tryptone yeast extract salts (TYES) medium (51), or in 10%

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CYE, which consisted of CYE medium (52) that had been diluted ten-fold with distilled water.

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For solid media, 15 g agar was added per l. Escherichia coli strains were grown in lysogeny

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broth (LB) at 37°C (53). Strains and plasmids used in this study are listed in Table 2 and primers

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are listed in Table 3. Antibiotics were used at the following concentrations when needed:

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ampicillin, 100 μg/ml; tobramycin, 1 μg/ml; and tetracycline, 10 μg/ml unless indicated

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otherwise.

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Growth of F. columnare in liquid media. F. columnare IA-S-4 and C#2 strains were

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streaked from -80°C freezer tubes onto TYES and Shieh agar respectively and incubated 48 h at

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30°C. All strains were restreaked on fresh agar and incubated 48 h at 30°C, and were then used

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to inoculate broth cultures. F. columnare IA-S-4 and C#2 were grown overnight at 28°C with

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shaking at 200 rpm in 20 ml TYES and Shieh liquid media respectively (starter cultures) with 1

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µg/ml of tetracycline included for the complemented strains. 2 ml of F. columnare IA-S-4 or

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C#2 starter cultures (normalized to OD600=0.6) were introduced into 48 ml of TYES or Shieh

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liquid medium respectively in 250-ml side-arm flasks and incubated at 28°C with shaking.

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Turbidity was monitored using a Klett-Summerson photoelectric colorimeter (Klett Mfg. Co.,

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NY). Growth experiments were performed in triplicate.

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Conjugative transfer of plasmids into F. columnare strains. Plasmids were transferred

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from E. coli S17-1 λ pir into F. columnare strains by conjugation. E. coli and F. columnare

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strains were incubated overnight with shaking in LB at 37°C or in Shieh broth at 30°C,

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respectively. 500 μl overnight culture were inoculated in fresh LB and Shieh broth and shaken at

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37°C and 30°C, respectively, until the OD600 value reached 0.4. The cells were collected by

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centrifugation at 4,200 × g for 5 min. The pellets of F. columnare were washed twice with 1 ml

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Shieh medium and suspended in 50 μl Shieh medium. The suspensions of E. coli and F.

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columnare were mixed and spotted on mixed cellulose ester filter membranes, pore size 0.45 m

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(EMD Millipore, Billerica, MA) which had been placed on Shieh agar. Conjugation was allowed

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to proceed at 30°C for 24 h. The cells were removed from the filter membrane with a scraper and

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suspended in 1 ml Shieh medium. Aliquots were spread on Shieh agar containing 1 μg/ml

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tobramycin and 10 μg/ml tetracycline and incubated at 30°C for 48-72 h. Construction of gldN and porV deletion mutants. Gene deletions in F. columnare were

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constructed essentially as previously described (35). To delete gldN from F. columnare strain

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C#2, a 2,024-bp product spanning gldM and including the first 144 bp of gldN was amplified by

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PCR using Phusion DNA polymerase (New England Biolabs, Ipswich, MA) and primers 1618

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(introducing a BamHI site) and 1619 (introducing a SalI site). The product was digested with

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BamHI and SalI and ligated into pMS75, which had been digested with the same enzymes, to

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generate pLN6. A 1,723-bp product spanning AX766_RS08575 (encoding an FAD-dependent

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oxidoreductase) and the final 30 bp of gldN was amplified with primers 1620 (introducing a SalI

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site) and 1621 (introducing an SphI site). The product was digested with SalI and SphI and fused

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to the upstream region of gldN by ligation with pLN6 which had been digested with the same

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enzymes, to generate the deletion construct pLN7. Plasmid pLN7 was transferred by conjugation

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into F. columnare C#2, and colonies that had the plasmid integrated into the chromosome by

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recombination were obtained by selecting for tetracycline resistance. Colonies were streaked for

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isolation, and were then inoculated into 3 ml Shieh medium without antibiotics to allow loss of

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the integrated plasmid. The gldN deletion mutant (gldNC#2) was obtained by selecting for

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sucrose resistance, and was confirmed by PCR using primers 1682 and 1683, which flank the

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gldN coding sequence. A gldN deletion mutant of F. columnare strain IA-S-4 (gldNIA-S-4) was

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obtained in the same way, except that primers 1893, 1894, 1895, and 1896 were used to construct

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deletion plasmid pLN31.

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To delete F. columnare C#2 porV a 1,967-bp product downstream of porV was amplified using primers 1722 (introducing a BamHI site) and 1723 (introducing a SalI site). The amplified

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product was digested with BamHI and SalI and cloned into pMS75, generating pLN9. A 2,046-

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bp product upstream of porV was amplified by PCR using primers 1724 (introducing a SalI site)

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and 1725 (introducing a SphI site). The amplified product was digested with SalI and SphI and

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cloned into pLN9, generating pLN10. pLN10 was introduced into F. columnare C#2 by

335

conjugation and the deletion mutant (porVC#2) was obtained as described above.

336

Complementation of deletion mutants. Plasmids carrying gldN or porV were constructed

337

using shuttle vector pCP23. To construct a plasmid containing F. columnare C#2 gldN, primers

338

1682 (introducing a BamHI site) and 1683 (introducing an SphI site) were used to amplify a

339

1,369-bp product spanning gldN from F. columnare C#2 genomic DNA. To construct a plasmid

340

containing F. columnare IA-S-4 gldN, primers 1648 (introducing a BamHI site) and 1649

341

(introducing an SphI site) were used to amplify a 1,152-bp product spanning gldN from F.

342

columnare IA-S-4 genomic DNA. To construct a plasmid containing F. columnare C#2 porV,

343

primers 1747 (introducing a BamHI site) and 1748 (introducing an SphI site) were used to

344

amplify a 1,542-bp product spanning porV from F. columnare C#2 genomic DNA. The products

345

were digested with BamHI and SphI and ligated into pCP23, which had been digested with the

346

same enzymes, to generate pLN8, pLN5, and pLN11. The plasmids were transferred to

347

appropriate F. columnare mutants by conjugation.

348

Analysis of colony spreading and cell motility. F. columnare IA-S-4 and C#2 colonies

349

were grown for 2 d at 30oC on TYES and Shieh agar respectively. Colonies of wild type, mutant,

350

and complemented strains were examined using a Photometrics Cool-SNAPcf2 camera mounted

351

on an Olympus IMT-2 phase-contrast microscope. Gliding of individual cells was also examined

352

microscopically. F. columnare IA-S-4 and C#2 were grown to early stationary phase (14 h) with

353

shaking at 28oC in TYES and Shieh liquid media respectively. Tunnel slides were constructed

16


331

using double stick tape, glass microscope slides, and glass cover slips, as previously described

355

(22). Ten microliters of cultures were introduced into the tunnel slides, incubated for 5 min, and

356

observed for motility using an Olympus BH2 phase-contrast microscope at 25oC. Images were

357

recorded with a Photometrics CoolSNAPcf2 camera and analyzed using MetaMorph software

358

(Molecular Devices, Downingtown, PA). Rainbow traces of cell movements were made using

359

ImageJ version 1.45s (http://rsb.info.nih.gov/ij/) and macro Color FootPrint (45).

360

Measurement of protease activity. Azocasein protease assays were performed to

361

quantitate proteolytic activity. F. columnare strains were grown in triplicate 5 ml volumes of

362

TYES broth for 24 h at 28°C with shaking at 175 rpm. The cultures were centrifuged at 19,980 ×

363

g for 10 min. Residual cells were removed from the supernatants by passage through 0.45 µM

364

HT Tuffryn® syringe filters (PALL Life Sciences, Ann Arbor, MI) and the cell-free culture fluids

365

were stored at 4°C for 24 h prior to the azocasein assay. The open tubes containing the bacterial

366

pellets were placed in an 80°C heat block for 3 h, and the dry weights of the cell pellets were

367

determined.

368

Proteolytic activity for each strain was quantified as previously described (40) with some

369

modifications. A 2% azocasein (Sigma) solution was prepared in 0.05 M Tris-HCl (pH 7.4).

370

Cell-free supernatant (50 µl) was mixed with 50 µl of the azocasein substrate and incubated at

371

28°C for 2 h. Duplicate assays were performed for each supernatant sample and negative

372

controls were included consisting of 50 µl of sterile TYES broth. Following incubation, 130 µl

373

of 10% trichloroacetic acid was added to each sample, mixed, and held at room temperature for

374

10 min. The samples were then centrifuged at 19,980 × g for 20 min to remove precipitated

375

azocasein. 100 µl of the soluble fraction of each sample was removed, added to a flat bottom 96-

376

well plate, and 200 µl of 1 M NaOH was added to each well and mixed. The OD450 was

17


354

determined using an iMark™ microplate reader (Bio-Rad). For each supernatant sample, the raw

378

OD450 values obtained from the duplicate assays were averaged and the mean negative control

379

OD450 was subtracted from these values. The adjusted OD450 values were converted to units of

380

proteolytic activity by dividing by 0.001 (40). The units of proteolytic activity were multiplied

381

by 100 to obtain the total proteolytic activity for each 5-ml culture, and divided by the dry weight

382

of the cells to obtain the proteolytic activity per mg of dry cells. For each strain, the proteolytic

383

activities determined for triplicate cultures were averaged.

384

Measurement of chondroitin sulfate lyase activity. Chondroitin sulfate lyase activity

385

was measured as previously described with modifications (35, 54). Briefly, strains derived from

386

F. columnare IA-S-4 and C#2 were grown with shaking at 200 rpm at 28°C to mid-exponential

387

phase in TYES and Shieh broth, respectively. The cultures were normalized to the same

388

concentration (Klett of 55 measured with a Klett colorimeter, which is equivalent to OD600 of

389

0.4), centrifuged at 16,873 × g for 10 min at 4°C, and the supernatants were harvested. 20 µl of

390

supernatants and 150 µl of 0.2 mg/ml chondroitin sulfate A (Sigma-Aldrich) in 20 mM Tris

391

buffer (pH 7.0) were added to wells in a 96-well flat-bottom microplate and incubated at 30°C

392

for 30 min. 20 µl of TYES or Shieh broth were used as no-chondroitin sulfate lyase controls. 30

393

µl of 0.5% BSA in 0.45 M acetate buffer (pH 4.0) was added to each well and opaque white

394

color developed, which was directly proportional to the amount of undigested chondroitin sulfate

395

A present. The optical density of each well was measured at 405 nm with a microplate reader

396

(Infinite 200 PRO, Tecan Group Ltd., Männedorf, Switzerland). Percentage of chondroitin

397

sulfate degradation (%) = (ControlOD405 - SampleOD405)/ControlOD405 × 100.

398 399

Detection of recombinant mCherry secretion. Cells of F. columnare strain C#2 or of its T9SS mutant gldN were grown overnight in Shieh broth (5 μg/ml tetracycline) at 30°C with

18


377

shaking. Cells carried either pSSK54 which expresses mCherry with the N-terminal signal

401

peptide of F. johnsoniae ChiA (SP-mCherry), or pSSK52, which expresses SP-mCherry fused to

402

the 105-amino acid CTD of ChiA. Cells were pelleted by centrifugation at 22,000 × g for 15 min

403

at 4°C, and the culture supernatant (spent medium) was separated. The spent medium was

404

ultracentrifuged at 352,900 × g for 30 mins at 4°C to remove residual insoluble material. For

405

whole-cell samples, the cells were suspended in the original culture volume of phosphate-

406

buffered saline consisting of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4

407

(pH 7.4). Equal amounts of spent media and whole cells were boiled in SDS-PAGE loading

408

buffer for 10 min. Proteins were separated by SDS-PAGE, and Western blot analyses were

409

performed as previously described (26) except that polyvinylidene difluoride (PVDF)

410

membranes were used instead of nitrocellulose. Equal amounts of each sample based on the

411

starting material were loaded in each lane. For cell extracts this corresponded to 10 g protein,

412

whereas for spent medium this corresponded to the equivalent volume of spent medium that

413

contained 10 g cell protein before the cells were removed. For detection of mCherry by

414

Western blotting, commercially available antibodies against mCherry (0.5 mg per ml; BioVision

415

Incorporated, Milpitas, CA) were used at dilution of 1:5,000.

416

Analysis of secreted proteins by SDS-PAGE and liquid chromatography-tandem

417

mass spectrometry (LC-MS/MS). Starter cultures of F. columnare wild-type strain C#2, gldN

418

mutant, porV mutant, and complemented strains were grown in Shieh broth or Shieh broth

419

containing 2.5 μg/ml tetracycline at 25°C for 20 h. For each strain, 150 μl of starter culture was

420

inoculated into 3 ml fresh Modified Shieh broth or Modified Shieh broth containing 2.5 μg/ml

421

tetracycline and incubated at 25°C with shaking until the OD600 value reached 0.5. Before

422

collecting the spent media, the integrity of the bacterial cells for each strain was examined

19


400

microscopically. Cultures were centrifuged at 16,873 × g for 10 min at 4°C. The fluid was

424

filtered with 0.22 μm polyvinylidene difluoride filters and stored at -80°C until needed. Proteins

425

were separated by SDS-PAGE (12% polyacrylamide gel) and detected using the BioRad

426

(Hercules, CA) silver stain kit. Indicated regions of the gel were cut and peptides were analyzed

427

by enzymatic in-gel digestion and nano-LC-MS/MS at the University of WI-Madison Mass

428

Spectrometry Facility as outlined on the website

429

(https://www.biotech.wisc.edu/services/massspec), and as described previously (24) except that

430

MS/MS data were searched against a F. columnare protein database instead of against a F.

431

johnsoniae database.

432

Zebrafish challenges. All procedures utilizing fish at each research facility were approved

433

by the appropriate Institutional Animal Care and Use Committee. F. columnare wild-type,

434

mutant, and complemented strains were grown in Shieh medium at 30°C overnight. Then 250 μl

435

overnight culture were inoculated into 5 ml fresh Modified Shieh broth and shaken at 25°C until

436

the OD600 value reached 0.4. To quantify the number of cells per ml, serial dilutions were plated

437

on Shieh agar. To test the virulence of wild-type, mutant and complemented strains, the cultures

438

were diluted and used to infect adult zebrafish (Danio rerio, strain Eckwill crossed with strain

439

Tupfel Long-fin). No signs of disease were observed prior to challenge and no indications of F.

440

columnare or columnaris disease were observed in the uninfected control tanks or in the

441

maintenance tanks at any time. Serial two-fold dilutions were performed with the highest titer

442

being 1 ml of culture in 50 ml of water, and the lowest being 31 l of culture in 50 ml of water.

443

For the experiment shown in Fig. 7 the final challenge concentrations were 2.1 × 106 colony

444

forming units (CFU)/ml for C#2, 1.5 × 108 CFU/ml for the ΔgldNC#2 mutant, 2.1 × 106 CFU/ml

445

for the ΔgldNC#2 mutant complemented with pLN8, 1.5 × 107 CFU/ml for the ΔporVC#2 mutant,

20


423

and 2.1 × 106 CFU/ml for the ΔporVC#2 mutant complemented with pLN11. Zebrafish (ten fish

447

per bacterial strain for each dilution) were immersed in 50 ml of water with F. columnare at

448

28°C for 1 h. Control fish were exposed to 1 ml growth medium without F. columnare in 50 ml

449

of water. After exposure, the challenged fish were transferred to 2 l fresh water and maintained

450

for 7 days at 28°C. Mortalities were recorded daily. A minimum of 30% of the fish that died

451

during this period were examined for the presence of bacteria phenotypic of F. columnare

452

(yellow, rhizoid, adherent colonies) by swabbing gills, fins and skin and streaking on Shieh agar.

453

Rainbow trout challenges. Commercially available certified disease-free rainbow trout

454

(Oncorhynchus mykiss) eggs were acquired from Troutlodge Inc., Sumner, WA. Viable hatched

455

trout were hand fed daily to satiation using a commercially available trout feed (Ziegler Inc.,

456

PA). Trout were maintained at the USDA-ARS National Center for Cool and Cold Water

457

Aquaculture research facility in Kearneysville, WV in flow through water at a rate of 1 l/min, at

458

12.5°C, until the challenge weight of ~1.3 g was met. The fish in this facility are checked yearly

459

for multiple diseases including columnaris disease, and except for fish in the challenge room,

460

they are certified disease-free. No signs of disease were observed prior to challenge and no

461

indications of F. columnare or columnaris disease were observed in the uninfected control tanks

462

or in the maintenance tanks at any time. Fish were moved to challenge aquaria 1 week prior to

463

immersion challenge to acclimate to the elevated water temperature of 16°C.

464

The wild-type strain IA-S-4, ΔgldNIA-S-4 mutant, and ΔgldNIA-S-4 mutant complemented

465

with plasmid pLN5, were each used for immersion challenges. Frozen bacterial stocks were

466

stored at -80°C in 80% TYES broth and 20% glycerol. Bacterial cultures, for challenges, were

467

grown as previously described (11). Briefly, 100 μl of frozen stocks were inoculated into 10 ml

468

TYES broth and incubated overnight at 30°C with shaking at 200 rpm. These starter cultures

21


446

469

were used to inoculate Fernbach flasks containing 1 l TYES broth. These were incubated at 30°C

470

with shaking at 200 rpm until an optical density of 0.7 to 0.75 at 540 nm was reached. Challenges were performed using triplicate 4 l tanks with restricted water flows (~200

472

ml/min) at 16°C. Each tank contained 20 fish of approximately 1.35 g each. Water flows were

473

stopped for the immersion challenge and tanks were inoculated with bacterial cultures and

474

incubated for 1 h after which water flows were resumed. Control tanks were inoculated with

475

TYES broth. Serial dilutions of water samples from each tank after inoculation were plated on

476

TYES agar to determine CFU/ml. The final challenge concentrations for the experiment shown

477

in Fig. 8A were 1.2 × 105 CFU/ml for the wild type IA-S-4, 5.5 × 106 CFU/ml for the ΔgldNIA-S-4

478

mutant, and 2.7 × 106 CFU/ml for the ΔgldNIA-S-4 mutant complemented with gldN on pLN5.

479

Mortalities were removed and counted daily. The data for triplicate tanks of each strain were

480

pooled and survivor fractions for each strain were calculated. 20% of mortalities were randomly

481

tested by homogenizing gill tissue and streaking on TYES agar plates to determine if F.

482

columnare was present. Confirmation of F. columnare was determined by morphological

483

observation of yellow, rhizoid, adherent colonies and by amplifying and sequencing 16S rRNA

484

genes. F. columnare was detected in all mortalities. Genomovar confirmation was determined by

485

enzymatic digestion (HaeIII) of the 16S rRNA gene as previously described (7) and all were

486

genomovar I, as expected for strain IA-S-4.

487

Channel catfish challenges. Wild type F. columnare strain C#2, ∆gldNC#2 mutant, and

488

∆gldNC#2 complemented with wild type gldN on pLN8 were grown in 100 ml of Modified Shieh

489

broth for 20 h at 28°C with shaking at 150 rotations per minute (rpm). The optical densities at

490

540 nm were 1.17, 1.19, and 1.33 for strains C#2, ∆gldNC#2, and ∆gldNC#2 complemented with

491

pLN8, respectively. The number of CFU/ml was determined by spread plating 50 µl of serial

22


471

492

dilutions (in duplicate) on Modified Shieh agar plates. Plates were incubated for 48 h at 28°C,

493

and colonies were counted. Channel catfish (Ictalurus punctatus), with mean weight 4.3 g ± 0.5 g (SD) and no

495

previous history of columnaris disease, obtained from stocks held at the USDA-ARS Aquatic

496

Animal Health Research Unit in Auburn, AL were used for the bacterial challenges. Fish were

497

housed in 378 l troughs supplied with 28 ± 2 °C dechlorinated municipal water prior to

498

challenge. Fish were fed with appropriately sized fish feed (Rangen, Inc., Buhl, Idaho) at a rate

499

of 3% of body weight per day prior to challenge and to satiation following challenge. Single

500

groups of 25 fish were challenged in buckets by immersion for 15 min in 2 l of water with three

501

doses of each F. columnare strain (C#2, ∆gldN, and ∆gldNC#2 complemented with pLN8). For

502

the experiment shown in Fig. 8B the final challenge concentrations were 5.0 × 107 CFU/ml for

503

C#2, 4.6 × 107 CFU/ml for the ΔgldNC#2 mutant, and 3.4 × 107 CFU/ml for the ΔgldNC#2 mutant

504

complemented with pLN8. Following exposure to the bacteria, fish were netted from the buckets

505

and placed into pre-filled 57 l tanks supplied with water at a flow rate of approximately 0.5

506

l/min. Two additional groups of 25 fish each were mock-challenged by immersion as described

507

above by adding sterile Modified Shieh broth in a volume equal to the volume of bacterial

508

culture added for the highest dose. Tanks were observed twice daily for 14 d and dead/moribund

509

fish were removed and recorded. Re-isolation of F. columnare was attempted from 20% of the

510

daily mortalities from each tank by inoculating head kidney tissue onto Modified Shieh agar

511

containing 1 µg/ml tobramycin (49). Plates were incubated at 28°C for 48 h and then examined

512

for colonies phenotypic of F. columnare. For channel catfish, zebrafish, and rainbow trout

513

challenges, survivor fractions after exposure to each bacterial strain were calculated using the

23


494

514

product limit (Kaplan-Meier) method using GraphPad Prism (version 6.05; GraphPad Software,

515

San Diego, CA, USA). Toxicity of material secreted by F. columnare. F. columnare IA-S-4 and C#2 strains

517

were streaked from -80°C stocks onto TYES and Shieh agar respectively and incubated 48 h at

518

30°C. Strains were restreaked on fresh plates, incubated 48 h at 30°C, and used to inoculate 20

519

ml starter flasks of TYES (IA-S-4 strains) or Modified Shieh (C#2 strains). Starter cultures were

520

incubated overnight and then 6 ml volumes were used to inoculate 144 ml of media of the same

521

composition. Cultures were grown to mid-log phase (Klett of 50-60) and cells were removed by

522

centrifugation twice for 20 minutes at 3,700 × g at 4°C. The supernatants were filtered through

523

0.45 µm polyethersulfone (PES) filters to remove residual cells. 0.1 ml of the filtrate was plated

524

on TYES or Shieh agar to verify that all viable cells had been removed. The filtered supernatant

525

was kept on ice overnight before use. 100 ml of supernatant placed in a 250-ml glass beaker was

526

pre-warmed to 28°C. Six zebrafish (approximately 0.35 g each) were added and incubated for 5

527

to 7 h. Mortalities were recorded every 30 min. Control fish were exposed to TYES (IA-S-4

528

strains) or Modified Shieh medium (C#2 strains) instead of to cell-free spent media. For heat

529

inactivation experiments, cell-free spent media was heated at 60°C for 60 min prior to zebrafish

530

exposure. For trypsin inactivation experiments cell-free spent media was exposed to 50 g/ml

531

trypsin at 37oC for 12 h prior to zebrafish exposure. Samples without trypsin were also incubated

532

at 37oC for 12 h for these experiments.

533

Bioinformatic analyses. Genome sequences of F. columnare strains C#2

534

(NZ_CP015107.1) (47) and IA-S-4 (B. LaFrentz, unpublished) were analyzed. Gene clusters

535

encoding structural components of the T9SS (Table S1) were detected using MacSyFinder (55)

536

together with the TXSScan profile for the T9SS (56). Proteins predicted to be secreted by the

24


516

T9SS were detected using HmmSearch from the HMMER suite version 3.1b1

538

(http://www.hmmer.org) with TIGR04183 (referred to as type A CTD) and TIGR04131 (referred

539

to as type B CTD) HMM profiles. Hits were regarded as significant when proteins were detected

540

above HmmSearch model-specifics trusted cutoff thresholds. N-terminal cleavage sites were

541

predicted using SignalP4.1 web-server (57). Proteases and glycoside hydrolases were classified

542

using MEROPS (http://www.merops.sanger.ac.uk/) and Carbohydrate Active Enzyme (58)

543

databases, respectively.

544 545

Acknowledgements

546

The authors thank Henry Tomasiewicz, Kris Kosteretz, Rebekah Klingler, and Michael Carvan

547

at the University of Wisconsin-Milwaukee School of Freshwater Sciences Fish Facility for

548

supplying zebrafish, the University of Wisconsin-Milwaukee Animal Care Program for

549

assistance with animal care, and Greg Sabat and Greg Barret-Wilt at the University of

550

Wisconsin-Madison Mass Spectrometry Facility for LC-MS/MS analyses.

551 552

Funding Information

553

This research was supported by funds from the USDA-ARS (CRIS Project No. 6010-

554

32000-026-00D), and by grants to MJM from the USDA-ARS (Project Number 5090-31320-

555

004-03S), and from the National Science Foundation (MCB-1516990), and by grants to PN from

556

the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No.

557

XDA08010207) and from the Knowledge Innovation Program of the Chinese Academy of

558

Sciences. It was also funded by grants to MJM and DWH from the University of Wisconsin Sea

559

Grant Institute under grants from the National Sea Grant College Program, National Oceanic and

25


537

Atmospheric Administration, U.S. Department of Commerce, and the State of Wisconsin, federal

561

grant NA10OAR4170070, project R/SFA-08, and federal grant NA14OAR4170092, project

562

R/SFA-11. The funders had no role in study design, data collection and interpretation, or the

563

decision to submit the work for publication. The USDA is an Equal Opportunity Employer.

564 565

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43.

44. 45. 46. 47.

48. 49.

Slakeski N, Seers CA, Ng K, Moore C, Cleal SM, Veith PD, Lo AW, Reynolds EC. 2011. C-terminal domain residues important for secretion and attachment of RgpB in Porphyromonas gingivalis. J. Bacteriol. 193:132-142. Veith PD, Muhammad NAN, Dashper SG, Likic VA, Gorasia DG, Chen D, Byrne SJ, Catmull DV, Reynolds EC. 2013. Protein substrates of a novel secretion system are numerous in the Bacteroidetes phylum and have in common a cleavable C-terminal secretion signal, extensive post-translational modification, and cell-surface attachment. J Proteome Res 12:4449-4461. Li N, Qin T, Zhang XL, Huang B, Liu ZX, Xie HX, Zhang J, McBride MJ, Nie P. 2015. Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence. Appl Environ Microbiol 81:7394-7402. Staroscik AM, Hunnicutt DW, Archibald KE, Nelson DR. 2008. Development of methods for the genetic manipulation of Flavobacterium columnare. BMC Microbiol. 8:115. Kulkarni SS, Zhu Y, Brendel CJ, McBride MJ. 2017. Diverse C-Terminal Sequences Involved in Flavobacterium johnsoniae Protein Secretion. J Bacteriol 199:e00884-00816. Kunttu HM, Jokinen EI, Valtonen ET, Sundberg LR. 2011. Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene. J Appl Microbiol 111:1319-1326. Suomalainen LR, Tiirola MA, Valtonen ET. 2006. Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare. J. Fish Dis. 29:757-763. Newton JC, Wood TM, Hartley MM. 1997. Isolation and partial characterization of extracellular proteases produced by isolates of Flavobacterium columnare derived from catfish. J Aquat Anim Health 9:75-85. Kayansamruaj P, Dong HT, Hirono I, Kondo H, Senapin S, Rodkhum C. 2017. Comparative genome analysis of fish pathogen Flavobacterium columnare reveals extensive sequence diversity within the species. Infect Genet Evol 54:7-17. Tekedar HC, Karsi A, Reddy JS, Nho SW, Kalindamar S, Lawrence ML. 2017. Comparative Genomics and Transcriptional Analysis of Flavobacterium columnare Strain ATCC 49512. Front Microbiol 8:588. Chen YY, Peng B, Yang Q, Glew MD, Veith PD, Cross KJ, Goldie KN, Chen D, O'Brien-Simpson N, Dashper SG, Reynolds EC. 2011. The outer membrane protein LptO is essential for the Odeacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis. Mol Microbiol 79:1380-1401. Ishiguro I, Saiki K, Konishi K. 2009. PG27 is a novel membrane protein essential for a Porphyromonas gingivalis protease secretion system. FEMS Microbiol. Lett. 292:261-267. Nakane D, Sato K, Wada H, McBride MJ, Nakayama K. 2013. Helical flow of surface protein required for bacterial gliding motility. Proc. Natl. Acad. Sci. USA 110:11145-11150. Nelson SS, Bollampalli S, McBride MJ. 2008. SprB is a cell surface component of the Flavobacterium johnsoniae gliding motility machinery. J. Bacteriol. 190:2851-2857. Bartelme RP, Newton RJ, Zhu Y, Li N, LaFrentz BR, McBride MJ. 2016. Complete genome sequence of the fish pathogen Flavobacterium columnare strain C#2. Genome Announc 4:e00624-00616. Thomas-Jinu S, Goodwin AE. 2004. Morphological and genetic characteristics of Flavobacterium columnare isolates: correlations with virulence in fish. J Fish Dis 27:29-35. Decostere A, Haesebrouck F, Devriese LA. 1997. Shieh medium supplemented with tobramycin for selective isolation of Flavobacterium columnare (Flexibacter columnaris) from diseased fish. J Clin Microbiol 35:322-324.

28


647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692

50.

51. 52. 53. 54. 55.

56. 57. 58. 59. 60. 61.

LaFrentz BR, Klesius PH. 2009. Development of a culture independent method to characterize the chemotactic response of Flavobacterium columnare to fish mucus. J. Microbiological Methods 77:37-40. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST. 1994. p. 787, Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore, MD. McBride MJ, Kempf MJ. 1996. Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae. J. Bacteriol 178:583-590. Bertani G. 1951. Studies on lysogenesis I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62:293-300. Teska JD. 1993. Assay to evaluate the reaction kinetics of chondroitin AC lyase produced by Cytophaga columnaris. J Aquat Anim Health 5:259-264. Abby SS, Neron B, Menager H, Touchon M, Rocha EP. 2014. MacSyFinder: a program to mine genomes for molecular systems with an application to CRISPR-Cas systems. PLoS One 9:e110726. Abby SS, Cury J, Guglielmini J, Neron B, Touchon M, Rocha EP. 2016. Identification of protein secretion systems in bacterial genomes. Sci Rep 6:23080. Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785-786. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. 2014. The carbohydrateactive enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490-495. Juncker AS, Willenbrock H, von Heijne G, Nielsen H, Brunak S, Krogh A. 2003. Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 12:1652-1662. de Lorenzo V, Timmis KN. 1994. Analysis and construction of stable phenotypes in gramnegative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235:386-405. Agarwal S, Hunnicutt DW, McBride MJ. 1997. Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA. Proc. Natl. Acad. Sci. USA 94:12139-12144.

720

29


693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719

721

Table 1. Candidate F. columnare C#2 proteins secreted by the T9SS identified by LC-MS/MS

722

analysis of cell-free culture fluidsa.

Locus

Mole

tag/Protein

cular

name

Mass

T9SS CTDc

Lipoproteind

Predicted protein function

Spectral counts from culture fluid Wild type

gldN

gldN + pLN8

(kDa) b

AX766_RS

86.2

type A

chondroitin AC lyase

145

3

131

thiol activated cytolysin

19

2

20

peptidase M4,

13

0

9

12

0

18

CTDe

05135/ CslA AX766_RS

61.2

+

03975

AX766_RS

97.2

type A CTD

05330 AX766_RS

thermolysin 61.5

+

thiol activated cytolysin

33.9

+

metalloprotease

4

0

3

chondroitin ABC lyase

9

0

0

unknown

5

0

4

13970 AX766_RS 13405 AX766_RS

100.2

type A CTD

54.0

type A

01510/ CslB

AX766_RS 09885

+

CTDe

724

30


723

725 Proteins in cell-free culture fluid from wild type F. columnare C#2, gldN mutant, and gldN

a

727

complemented with pLN8, were separated by SDS-PAGE, silver stained, and the regions shown

728

in Figure 6 spanning approximately 20 to 200 kDa were cut from the gel and analyzed by LC-

729

MS/MS. Total/unweighted spectrum counts corresponding to total number of spectra associated

730

to a single protein and indicative of relative abundance of that protein are indicated for each of

731

the strains analyzed. Only proteins that had at least 4 'hits' for the wild type culture fluid, and had

732

at least a 5-fold reduction in the number of hits for the gldN mutant are shown.

733

b

Molecular mass as calculated for full-length protein before removal of signal peptide.

734

c

T9SS CTD-type identified by BLASTP analysis. Type A CTDs belong to TIGRFAM protein

735

domain family TIGR04183.

736

d

Lipoproteins predicted by LipoP (59). '+' indicates lipoprotein. Blank indicates not lipoprotein.

737

e

The C-terminal regions of these proteins were below the trusted cutoffs for Type A

738

(TIGR04183) CTDs but exhibited more limited similarity as detected by BLASTP analyses.

739

31


726

740

Table 2. Strains and plasmids used in this study.

741 Descriptiona

or plasmid

Source or reference

E. coli strains DH5MCR

Strain used for general cloning

Life Technologi es (Grand Island, NY)

S17-1 λ pir

Strain used for conjugation

(60)

C#2

Wild type

(47, 48)

FCC-2

∆gldN in strain C#2

This study

FCC-8

∆porV in strain C#2

This study

IA-S-4

Wild type

B.

F. columnare strains

LaFrentz ∆gldN in strain IA-S-4

This study

pCP23

E. coli-F. columnare shuttle plasmid; Apr (Tc)r

(61)

pMS75

Suicide vector carrying sacB; Apr (Tcr)

(35)

FCI-1

Plasmids

32


Strain

pLN5

Plasmid for complementation of gldN in FCI-1; IA-S-4

This study

gldN was amplified with primers 1648 and 1649 and

pLN6

2.0 kbp region upstream of C#2 gldN amplified with


cloned into BamHI and SphI sites of pCP23; Apr (Tcr) This study

primers 1618 and 1619 and cloned into BamHI and SalI sites of pMS75; Apr (Tcr) pLN7

Construct used to delete C#2 gldN; 1.7 kbp region

This study

downstream of C#2 gldN amplified with primers 1620 and 1621 and cloned into SalI and SphI sites of pLN6; Apr (Tcr) pLN8

Plasmid for complementation of gldN in FCC-2; C#2

This study

gldN was amplified with primers 1682 and 1683 and cloned into BamHI and SphI sites of pCP23; Apr (Tcr) pLN9

2.0 kbp region downstream of C#2 porV amplified with

This study

primers 1722 and 1723 and cloned into BamHI and SalI sites of pMS75; Apr (Tcr) pLN10

Construct used to delete C#2 porV; 2.0 kbp region

This study

upstream of C#2 porV amplified with primers 1724 and 1725 and cloned into SalI and SphI sites of pLN9; Apr (Tcr) pLN11

Plasmid for complementation of porV in FCC-8; C#2

This study

porV was amplified with primers 1747 and 1748 and cloned into BamHI and SphI sites of pCP23; Apr (Tcr)

33

pLN30

2.0 kbp region upstream of IA-S-4 gldN amplified with

This study

primers 1893 and 1894 and cloned into BamHI and SalI

pLN31

Construct used to delete IA-S-4 gldN; 1.7 kbp region

This study

downstream of IA-S-4 gldN amplified with primers 1895 and 1896 and cloned into SalI and SphI sites of pLN30; Apr (Tcr) pSSK52

pCP23 carrying SPChiA-mCherry-CTDChiA; Apr (Tcr)

(23)

pSSK54

pCP23 carrying SPChiA-mCherry; Apr (Tcr)

(23)

742 743

a

Antibiotic resistance phenotypes: ampicillin, Apr; tetracycline, Tcr. Unless indicated otherwise,

744

the antibiotic resistance phenotypes are those expressed in E. coli. The antibiotic resistance

745

phenotypes given in parentheses are those expressed in F. columnare but not in E. coli.

746

34


sites of pMS75; Apr (Tcr)

747

Table 3. Primers used in this study. Primers

Sequencea 5′ GCTAGGGATCCACCGACTGTAGATGCTATTGC 3′

1619

5′ GCTAGGTCGACGGCCTTGTCATTATCCTTAGTTTG 3′

1620

5′ GCTAGGTCGACGACTTCGAACAAGATATGTGG 3′

1621

5′ GCTAGGCATGCCTTTAGGTTCTAATGCGTACC 3′

1648

5′ GCTAGGGATCCAGCATCTTTCTCAGGTATTG 3′

1649

5′ GCTAGGCATGCAACGGGTAGGAGTTTTTTTA 3′

1682

5′ GCTAGGGATCCTTCAAAAAGCACAAAGAGG 3′

1683

5′ GCTAGGCATGCAAGATGAGAAAGACAGAGAAGT 3′

1722

5′ GCTAGGGATCCTCTACTGGGGCTGTTTGACC 3′

1723

5′ GCTAGGTCGACGGTGATGATTACAAACAATATTAA 3′

1724

5′ GCTAGGTCGACTAAACTAATTTTCTTCATTGGAT 3′

1725

5′ GCTAGGCATGCATGCCTTTTTGGAGATGCCT 3′

1747

5’ GCTAGGGATCCAAAATCAAACCATTTCTACAAC 3′

1748

5’ GCTAGGCATGCACTTCCTCCGATAACTCAAC 3′

1893

5′ GCTAGGGATCCACCTACTGTAGATGCTATTGC 3′

1894

5′ GCTAGGTCGACGGTTTATCATTGTCCTTAGTTTG 3′

1895

5′ GCTAGGTCGACGACTTTGAACAAGATATGTGG 3′

1896

5′ GCTAGGCATGCCTTTAGGTTCTAAAGCATACC 3′


1618

748 749

a

Underlined sequences indicate introduced restriction enzyme sites.

750

35

751

Figure legends

752 Fig. 1. The F. columnare type IX protein secretion system. Orthologs of proteins required for

754

secretion in Flavobacterium johnsoniae and Porphyromonas gingivalis are shown in orange. F.

755

johnsoniae PorV is required for secretion of many but not all proteins that are targeted to the

756

T9SS. F. johnsoniae PorU is involved in but not essential for secretion. Black lines on the

757

lipoproteins GldK and SprE indicate lipid tails. OM, outer membrane; CM, cytoplasmic

758

membrane.

759 760

Fig. 2. Maps of the regions containing F. columnare strain C#2 gldN (A) and porV (B) and

761

associated deletions. Numbers below the maps refer to kilobase pairs of sequence. Binding sites

762

for primers used in PCRs to generate deletion or complementation constructs are shown above

763

and below the maps, with the blunt ends indicating the actual binding sites. The horizontal lines

764

beneath the maps marked with open triangles denote regions deleted from the chromosome in the

765

mutants. The regions of DNA carried by complementation plasmids pLN8 and pLN11 are

766

indicated beneath the maps. The map of the gldN region of F. columnare strain IA-S-4 is

767

identical to that shown in panel A, and F. columnare IA-S-4 gldN deletion and complementation

768

constructs were constructed in the same way using the primers and plasmids listed in Table 3 and

769

Table 2 respectively.

770 771

Fig. 3. Photomicrographs of F. columnare colonies. Colonies grown from single cells were

772

incubated 48 h at 30oC on TYES agar (Row A), Shieh agar (Row B), and 10% CYE agar (Row

773

C). Row A: Wild type (WT) F. columnare IA-S-4, gldN mutant of strain IA-S-4 (gldNIA-S-4),

36


753

gldNIA-S-4 mutant complemented with wild type gldN on pLN5. Row B: Wild type F. columnare

775

C#2, gldN mutant of strain C#2 (gldNC#2), gldNC#2 mutant complemented with wild type

776

gldN on pLN8. Row C: Wild type F. columnare C#2, porV mutant of strain C#2 (porVC#2),

777

gldNC#2. Bar indicates 0.5 mm and applies to all panels.

778 779

Fig. 4. Gliding of wild type and mutant cells on glass. Cells were grown in TYES (Row A) or

780

Shieh medium (Rows B and C) at 28oC for 14 h (early stationary phase). Ten microliters of

781

cultures were introduced into tunnel slides and observed for motility using an Olympus BH-2

782

phase-contrast microscope with a heated stage set at 25°C. Row A: Wild type (WT) F.

783

columnare IA-S-4, gldN mutant of strain IA-S-4 (gldNIA-S-4), gldNIA-S-4 complemented with

784

wild type gldN on pLN5. Row B: Wild type F. columnare C#2, gldN mutant of strain C#2

785

(gldNC#2), gldNC#2 mutant complemented with wild type gldN on pLN8. Row C: Wild type F.

786

columnare C#2, porV mutant of strain C#2 (porVC#2). In each case a series of images were

787

taken for 20 s using a Photometrics Cool-SNAPcf2 camera. Individual frames were colored from

788

red (time 0) to yellow, green, cyan, and finally blue (20 s) and integrated into one image,

789

resulting in 'rainbow traces' of gliding cells. The rainbow traces correspond to the first 20 s of the

790

sequences shown in Movie S1 (top row), Movie S2 (middle row) and Movie S3 (bottom row).

791

White cells correspond to cells that exhibited little if any net movement. The first frame of each

792

movie (time 0) is shown in Fig. S1. Bar at lower right indicates 10 m and applies to all panels.

793 794

Fig. 5. Digestion of proteins and chondroitin sulfate by material secreted by wild type and

795

mutant cells. Cells examined were wild type F. columnare IA-S-4; gldN mutant in strain IA-S-

796

4 (gldNIA-S-4); gldNIA-S-4 mutant complemented with plasmid pLN5; wild type F. columnare

37


774

C#2; gldN mutant in strain C#2 (gldNC#2); gldNC#2 mutant complemented with plasmid

798

pLN8; porV mutant in strain C#2 (porVC#2); porVC#2 mutant complemented with plasmid

799

pLN11. Digestion of protein (azocasein; panel A) or of chondroitin sulfate (panel B) by cell-free

800

spent medium from each strain was determined as described in Materials and Methods. The

801

values are means and the error bars indicate standard deviations (n=3).

802 803

Fig. 6. Soluble extracellular proteins of wild-type and mutant cells. Cells of wild type F.

804

columnare C#2, gldN mutant in strain C#2 (gldNC#2), porV mutant in strain C#2 (porVC#2),

805

gldNC#2 mutant complemented with wild type gldN on pLN8, and porVC#2 mutant

806

complemented with wild type porV on pLN11, were grown in Shieh medium at 25C with

807

shaking until cells reached an OD600 of 0.5. Equal amounts of cell-free spent media of wild-type

808

and mutant cells were separated by SDS-PAGE and proteins were detected by silver staining.

809

Asterisks indicate prominent bands present in the spent media of wild-type and complemented

810

cells that were absent or reduced in intensity in the spent media of the gldNC#2 and porVC#2

811

mutant cells. The boxed regions were subjected to LC-MS/MS analysis (Table 1). The abundant

812

protein in all lanes at approximately 32 kDa was apparently secreted by a route other than the

813

T9SS and verifies approximately equivalent loading of lanes.

814 815

Fig 7. Virulence of wild type F. columnare C#2, T9SS mutants, and complemented strains

816

toward zebrafish. Zebrafish were exposed to F. columnare strains for 1 h at 28oC, transferred to

817

fresh water, and percent survival was monitored for 7 d. Cells examined were wild type F.

818

columnare C#2; gldN mutant in strain C#2 (gldNC#2); gldNC#2 mutant complemented with

819

plasmid pLN8; porV mutant in strain C#2 (porVC#2); porVC#2 mutant complemented with

38


797

plasmid pLN11. The final challenge concentrations were 2.1 × 106 CFU/ml for C#2, 1.5 × 108

821

CFU/ml for the ΔgldNC#2 mutant, 1.5 × 107 CFU/ml for the ΔporVC#2 mutant, 2.1 × 106 CFU/ml

822

for the ΔgldNC#2 mutant complemented with pLN8, and for the ΔporVC#2 mutant complemented

823

with pLN11. Ten fish were challenged with each strain as indicated in Materials and Methods.

824

Ten control fish that were exposed to an equal amount of growth medium without F. columnare

825

cells were also included, and each of these control fish survived. Challenges at additional

826

dilutions are shown in Fig. S3.

827 828

Fig 8. Challenge of rainbow trout and channel catfish with F. columnare. Fish were exposed to

829

wild type, mutant, and complemented strains and percent survival was monitored as described in

830

Materials and Methods. Panel A: Rainbow trout were exposed to wild type F. columnare IA-S-4

831

(1.2 × 105 CFU/ml), gldNIA-S-4 mutant (5.5 × 106 CFU/ml), and gldNIA-S-4 mutant

832

complemented with pLN5 (2.7 × 106 CFU/ml). 60 rainbow trout were challenged with each

833

strain as indicated in Materials and Methods. The results from triplicate tanks (20 fish each) were

834

similar, and were pooled to construct the figure. Panel B: Channel catfish were exposed to wild

835

type F. columnare C#2 (5.0 × 107 CFU/ml), gldNC#2 mutant (4.6 × 107 CFU/ml), and gldNC#2

836

mutant complemented with pLN8 (3.4 × 107 CFU/ml). 25 channel catfish were challenged with

837

each strain as indicated in Materials and Methods. Rainbow trout and channel catfish were also

838

mock challenged with growth medium lacking F. columnare cells as described in Materials and

839

Methods, and in each case these fish survived. Challenges of rainbow trout and channel catfish at

840

additional dilutions are shown in Fig. S5.

841

39


820

Fig. 9. Toxicity for zebrafish of material secreted by the T9SS. Wild type, mutant and

843

complemented cells were cultured to mid log phase (Klett of 50 to 60) in TYES (Panel A) or

844

Modified Shieh medium (Panel B). Cells were removed by centrifugation followed by filtration.

845

Zebrafish (strain Eckwill crossed with strain Tupfel Long-fin; 6 fish per bacterial strain per trial)

846

were exposed to cell-free spent medium (100 ml) for 5 to 7 h at 28oC and mortalities were

847

recorded every 30 min. Three independent trials were conducted for each strain. No mortalities

848

occurred for control fish exposed to TYES or Modified Shieh media that had not been previously

849

inoculated with F. columnare, or for fish exposed to cell-free culture fluids from the gldN or

850

porV mutants. Panel A: Toxicity of cell-free spent media from cultures of strains derived from

851

F. columnare IA-S-4. Strains used were wild type F. columnare IA-S-4; gldNIA-S-4 mutant;

852

gldNIA-S-4 mutant complemented with plasmid pLN5. Panel B: Toxicity of cell-free spent media

853

from cultures of strains derived from F. columnare C#2. Strains used were wild type F.

854

columnare C#2; gldNC#2 mutant; gldNC#2 mutant complemented with plasmid pLN8;

855

porVC#2 mutant; porVC#2 mutant complemented with plasmid pLN11.

856 857

Fig. 10. Effect of heat and trypsin treatment on toxicity of cell-free spent culture fluid for

858

zebrafish. Wild type F. columnare C#2 cells were cultured to mid log phase (Klett of 50 to 60) in

859

Modified Shieh medium. Cells were removed by centrifugation followed by filtration. Samples

860

of the cell-free culture fluid were heated (60oC, 60 min; Panel A) or treated with trypsin (50

861

g/ml, 37oC, 12 h; Panel B). In panel B, samples without trypsin (untreated trials) were

862

incubated at 37oC for 12 h. Zebrafish (strain Eckwill; 6 fish per bacterial strain per trial) were

863

exposed to untreated, heat-treated, and trypsin-treated cell-free spent medium (100 ml) for 4 h at

864

28oC and mortalities were recorded. Three independent trials for heat treatment and for trypsin

40


842

treatment are shown. No mortalities occurred for fish exposed to Modified Shieh medium that

866

had not been previously inoculated with F. columnare ('Control'), or for fish exposed to cell-free

867

spent culture fluid that had been heated or treated with trypsin (black lines).


865

41

proteases chondroitinases other soluble proteins

SprA PorV PorU

OM GldK

SprT SprE

GldN

GldM CM GldL T9SS


motility proteins adhesins other cell-surface proteins

1618

0

topA

2 AX766_RS08550 4 gldK

gldL

6

1682

gldM

gldN

1620

8

AX766_RS08575

10 AX766_RS08580

∆ pLN8 1619

B

1747

1725

0

AX766_RS02715

2

gldJ

4

porU

1683

1723

porV

6

1621

∆

pdhA

8

10

AX766_RS02685

AX766_RS02695

pLN11 1724

1748

1722


A


Mutant

Complemented

A WTIA-S-4

∆gldNIA-S-4

∆gldNIA-S-4 + pLN5

WTC#2

∆gldNC#2

∆gldNC#2 + pLN8

WTC#2

∆porVC#2

B

C


Wild type

+

IA -S -4

N 5 W T

pL

C

rV

#2

#2

C

W T

5

-4

N

IA -S

pL

+

#2

pL

N

#2

8

11

C

rV

N

C

pL

∆p o

+

C

∆ # dN g ld 2 N

+

B 100

80 IA-S-4

C#2

60

40

20

0


∆p o

∆g l

IA -S -4

IA -S -4

0

dN

10000

dN

20000

W T

30000 C#2

Percentage of Chondroitin Sulfate Degradation (%)

IA-S-4

∆g l

40000

∆g l

∆ C# dN g ld N 2 C C #2 + #2 pL N ∆p or ∆po 8 V rV C #2 + C#2 pL N 11

4

dN

IA -S -4

W T ∆g l S-

IA -

dN

∆g l

∆g l

Proteolytic Activity (U/mg dry cell)

A




B A



B A