Identification of Flagellar Motility Genes in Yersinia ruckeri by. Transposon Mutagenesis ... Water Aquaculture, Agricultural Research Service, U.S. Department of Agriculture, ... ruckeri by identifying genetic elements necessary for expression.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2009, p. 6630–6633 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.01415-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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Identification of Flagellar Motility Genes in Yersinia ruckeri by Transposon Mutagenesis䌤 Jason P. Evenhuis,1 Scott E. LaPatra,2 David W. Verner-Jeffreys,3 Inger Dalsgaard,4 and Timothy J. Welch1* National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, U.S. Department of Agriculture, Kearneysville, West Virginia 254301; Clear Springs Foods, Inc., Research Division, Buhl, Idaho2; Cefas Weymouth Laboratory, Weymouth, Dorset, United Kingdom DT4 8UB3; and National Institute of Aquatic Resources, Section for Fish Diseases Laboratory, Technical University of Denmark, Stigbøjlen 4, 1870 Frederiksberg C, Denmark4 Received 17 June 2009/Accepted 13 August 2009
Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion. FlgM, and FlhE, a protein of unknown specific function (4, 13). Immediately upstream of FlhA an additional flagellar secretion protein, FlhB, was identified. These predicted proteins were greater than 80% similar and 60% identical to analogous proteins of Yersinia enterocolitica, Yersinia pestis, and Yersinia psuedotuberculosis. An additional ORF immediately upstream
Yersinia ruckeri is the etiologic agent of enteric redmouth disease, a disease of salmonid fish species that is found worldwide in areas where salmonid fish species are farmed (3, 6, 18, 20). Vaccines for enteric redmouth disease have been used successfully for nearly 3 decades and consist of immersionapplied, killed whole-cell preparations of motile serovar 1 Y. ruckeri strains (22). Recently though, outbreaks have been reported in vaccinated fish at trout farms in the United Kingdom (2), Spain (9), and the United States (1). The Y. ruckeri strains isolated from these outbreaks are uniformly atypical serovar 1 isolates lacking both flagellar motility and secreted lipase activity. These variants have been classified as Y. ruckeri biotype 2 (BT2) and are believed to have a reduced sensitivity to immersion vaccination (2). The objective of this study was to obtain a better understanding of the emergence of BT2 Y. ruckeri by identifying genetic elements necessary for expression of the Y. ruckeri flagellum and determining the role that the flagellum plays in virulence by using a rainbow trout infection model. Identification of flagellar motility genes. To identify genes involved in flagellar motility, random mutant clones were generated using the transposon Tn5-RL27 as previously described (12) and screened on motility medium (tryptic soy broth, 0.3% agar). By using this approach, several nonmotile mutants were identified. The motility phenotype of one such mutant, designated BTF1, is shown in Fig. 1. The location of the transposon insertion in strain BTF1 was determined by transposon-directed cloning (12) and revealed a transposon insertion (Fig. 2) in an open reading frame (ORF) that encodes a predicted protein with significant homology to FlhA, an essential component of the flagellar secretion apparatus (4). Immediately downstream we identified six ORFs encoding predicted flagellar structural proteins (FlgB and FlgC), flagellar secretion chaperones (FlgN and FlgA), the flagellar regulatory protein
FIG. 1. Motility and lipase phenotypes of Y. ruckeri CSF07-82 and flhA mutant derivative BTF1. (A and B) Motility agar plates (A) and Tween 80 plates (B) show the loss of motility and lipase production in flhA mutant strain BTF1 and complementation with plasmid pJE10. Plates were incubated at 28°C for 24 h. (C) The secretion of lipase activity was confirmed by examining concentrated culture supernatants by means of a radial diffusion assay utilizing Tween 80 as a substrate (27). The development of a precipitate diffusing from the sample well indicates the presence of lipase activity. Concentrated supernatants were prepared from cells grown for 24 h at 28°C in T-medium.
* Corresponding author. Mailing address: National Center for Cool and Cold Water Aquaculture, 11861 Leetown Road, Kearneysville, WV 25430. Phone: (304) 724-8340. Fax: (304) 725-0351. E-mail: tim.welch @ars.usda.gov. 䌤 Published ahead of print on 21 August 2009. 6630
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FIG. 2. Schematic map of the flhBAE-flgNMABC gene cluster identified by transposon-directed cloning of mutant BTF1 and the corresponding regions in related Yersinia species. The point of transposon insertion in BTF1 is indicated with a lollipop symbol. The IS insertion in the invasion gene of Y. pestis Kim is also indicated.
of FlhB was identified that encodes a protein with significant similarity (59% identical, 74% similar) to HreP, a virulenceassociated protease present in Y. enterocolitica (10, 26). The organization of the flhBAE-flgNMABC cluster in Y. ruckeri is identical to that of other Yersinia species (Fig. 2) with two noteworthy exceptions. First, hreP, present upstream of flhB in Y. ruckeri, Y. enterocolitica, and Y. frederiksenii, is absent at this position in the genomes of Y. pseudotuberculosis and Y. pestis KIM (Fig. 2) as well as in the genomes of Yersinia intermedia, Yersinia bercovieri, and Yersinia mollaretii (data not shown). Furthermore, hreP-like ORFs could not be found in the genome sequences of these other Yersinia species, indicating that this gene is likely unique to Y. ruckeri, Y. enterocolitica, and Y. frederiksenii. Additionally, the inv gene encoded between flhE and flgN in Y. enterocolitica and Y. pseudotuberculosis is absent at this position in Y. ruckeri. This gene encodes a virulence factor in Y. enterocolitica and Y. pseudotuberculosis that plays a critical role in intracellular invasion (11, 14, 17). The flhA::Tn5 mutation suppresses secreted lipase production. The flagellar export apparatus of Y. enterocolitica has previously been shown to function as a secretion system for the transport of several nonflagellar proteins, in addition to flagellar secretion targets, including the virulence-associated lipase YlpA (27). To determine whether lipase production by Y. ruckeri similarly requires an intact flagellar secretion apparatus, strain BTF1 and its parent were assessed for lipase production and secretion as previously described (21, 27). Lipase activity seen in the wild-type strain was absent in mutant strain BTF1 (Fig. 1B and C). Transcomplementation experiments were performed to verify that the Tn5 insertion in BTF1 was responsible for the lack of both motility and secreted lipase activity, as opposed to the result of one or more other mutations. Plasmids were created for this analysis by directly cloning PCR products using the pBAD TOPO TA kit (Invitrogen) and transferred to Y. ruckeri by electroporation (5). Plasmids containing either flhA or flhE alone failed to restore motility or lipase production, while a construct containing both flhA and flhE (pJE10) restored
both activities (Fig. 1 and Fig. 3A). These data show that the flhA insertion in strain BTF1 exerts a downstream effect on flhE and that both of these genes are necessary for flagellar motility and lipase production. In related enteric bacteria,
FIG. 3. Genetic complementation of mutant strain BTF1 (flhA::Tn5). (A) Motility and lipase phenotypes for flhAE clones used in the complementation analysis. Clones were generated by PCR cloning into the pBAD vector and verified by DNA sequencing. (B) Western sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection of flagellin from whole-cell extracts. The following strains were analyzed: lane 1, CSF07-82 (wild type); lane 2, BTF1 (flhA::Tn5); lane 3, BTF1/pJE08 (flhA::Tn5/flhA clone); lane 4, BTF1/pJE09 (flhA::Tn5/flhE clone); lane 5, BTF1/pJE10 (flhA::Tn5/flhAE clone); lane 6, blank; lane 7, E. coli.
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FIG. 4. Survival curves for rainbow trout challenged by immersion exposure to CSF07-82 (wild type; closed circles), BTF1 (flhA::Tn5; open circles), and BTF1/pJE10 (flhA::Tn5/flhAE clone; closed diamonds). The results from two independent experiments are presented. Kaplan-Meier survival analysis indicated no significant effect of flhA::Tn5 insertion on survival after challenge.
expression of flagellar secretion targets, such as flagellin, is contingent on production of the complete flagellar secretion apparatus (19). Western sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis using an antiflagellin monoclonal antibody, specific to a conserved epitope (8), was used to investigate the effect of the flhA::Tn5 insertion on flagellin production. A band of 40 to 45 kDa was detected
in both wild-type CSF07-82 and in the complemented mutant but was absent in the mutant strain BTF1 (Fig. 3B). These results indicate that mutational loss of Y. ruckeri flagellar secretion eliminates expression of flagellin. Flagellar secretion and lipase production do not contribute to virulence in rainbow trout. Motility is clearly not required for virulence in BT2 Y. ruckeri, given that emergent BT2 strains are virulent; however, it is possible that loss of motility or lipase production could alter virulence in the BT1 background. To test this, the virulence of mutant strain BTF1 was compared to its isogenic wild-type parent by using a bath challenge model that mimics natural waterborne exposure. For each bacterial strain examined duplicate groups of 25 and 15 fish weighing approximately 7 or 10 g, respectively, were exposed to 4 ⫻ 107 cells/ml in a volume of water that was 10 times the total weight of the fish in an aerated container maintained at 15°C. After 1 h fish were transferred into aquaria supplied with 15°C flowing water, and dead fish were removed and recorded daily. Mortality due to Y. ruckeri infection was confirmed by microbiological analysis of kidney tissue on 20% of the mortalities/day. Bacterial cells for the challenges were grown for 72 h at 15°C in tryptic soy broth, and viable cell numbers were quantified using direct plate counts. Both the wild-type and complemented mutant strains were motile when grown under these conditions and therefore relevant to the infection model. The duplicated challenge groups were not significantly different, and therefore data from these experiments were pooled; the results are shown in Fig. 4. The flhA::Tn5 mutant was not significantly altered in virulence compared to its isogenic wild-type parent or the complemented strain. Additional experiments utilizing intraperitoneal injection to initiate the challenge also showed no significant difference between the
TABLE 1. Bacterial strains, plasmids, and primers used in this study Bacterial strain, plasmid, or primer
E. coli strains DH5␣pir (cloning host) BW20767
Genotype, description, or sequence
Source or reference
supE44 hsdR17 recA1 endA1 gyrA96 thi-1 ⌬lacU169 relA1(80lacZ⌬M15) pir RP4-2-Tc::Mu-1 kan::Tn7 integrant leu-63::IS10 recA1 zbf-5 creB510 hsdR17 endA1 thi uidA (⌬MluI)::pir⫹
15 12
Y. ruckeri strains CSF0782 BTF1 YRNC10 D030522-2/1 UK06020
Serovar 1, biotype 1, Idaho 1982 CSF0782-derived mini-Tn5 insertion in flhA Serovar 1, biotype 2, North Carolina 2003 Serovar 1, biotype 2, Denmark Serovar 1, biotype 2, United Kingdom
This work This work 24 25 25
Plasmids pBAD-TOPO pJE8 pJE9 pJE10
PCR cloning vector flhA cloned by PCR into pBAD-TOPO primers FlhAF/FlhAR flhE cloned by PCR into pBAD-TOPO primers FlhEF/FlhER flhAE cloned by PCR into pBAD-TOPO primers FlhAF/FlhER
Invitrogen This work This work This work
Primers FlhAF FlhAR FlhEF FlhER FlhBF FlgCR
5⬘-CCGAATTCCGATGCTGGAAGCC-3⬘ 5⬘-TTAAGCCGCTCCAATGGTAGATG-3⬘ 5⬘-GATCGTCTATTGGAACAGGCT-3⬘ 5⬘-TCACTGATAATTCACTATTACC-3⬘ 5⬘-GCTACGGCAGGTTGGTATGT-3⬘ 5⬘-CCTGATTGGCAACCTGAAAT-3⬘
This This This This This This
work work work work work work
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flhA::Tn5 mutant and the wild-type parent strain (data not shown). It is important to emphasize that bath challenge necessitates host colonization, and thus this result demonstrates that flagellar secretion is unnecessary for virulence in this organism, including early steps in the process of infection. Moreover, these results imply that motility and lipase production could be lost with no consequence to virulence in emergent BT2 strains. Identification of the flhBAE-flgNMABC gene cluster in biotype 2 strains of Y. ruckeri. Recent pulsed-field gel electrophoresis analysis suggests that the BT2 phenotype has emerged independently in the United Kingdom and Europe from indigenous motile serovar 1 isolates (25). Our results, demonstrating that the BT2 phenotype may be caused by mutational loss of the flagellar secretion apparatus in a BT1 strain, is consistent with this hypothesis, and together these observations imply that BT2 strains likely will have intact, albeit cryptic, flagellar secretion genes. Therefore, PCR was used to assess the presence of the flhBAE-flgNMABC gene cluster in representative BT2 strains from the United States and Europe. Primers flhBF and flgCR (Table 1) were used to amplify a 5,911-bp fragment using the Qiagen LongRange PCR kit for isolates of BT2 Y. ruckeri from the United States, United Kingdom, and Denmark. All three strains tested yielded PCR products identical to those of BT1 strain CSF07-82. DNA sequencing revealed that the flhBAE-flgNMABC gene cluster of these three BT2 strains was identical to that of BT1 strain CSF07-82. The discovery of this completely conserved motility gene cluster in BT2 Y. ruckeri isolates is a strong indication that they are recent variants of motile Y. ruckeri strains. This result also demonstrates that in these BT2 strains the lesion causing loss of motility and lipase production is elsewhere in the genome. Concluding remarks. The results presented here reveal a potential molecular basis for the concurrent absence of motility and lipase secretion in BT2 Y. ruckeri through the natural mutational loss of flagellar secretion. Loss of flagellar motility is not uncommon in the evolution of human pathogens. For example, Shigella species, as well as all strains of Y. pestis, are nonmotile, despite the presence of cryptic flagellar motility genes (7, 16, 23). Our results demonstrate that loss of the flagellum and flagellar secretion does not affect Y. ruckeri virulence as seen using a natural infection model, suggesting that the loss of these phenotypes in BT2 Y. ruckeri strains has likely had no deleterious effect on virulence. Finally, the correlation between vaccine failure and the recent emergence and dissemination of BT2 Y. ruckeri indicates that flagellar secretion and/or the flagellum itself may be a liability in a vaccinated host. Nucleotide sequence accession number. DNA sequences have been deposited in the GenBank database under accession number GQ217534. We thank Jennifer Harper and Travis Moorland (National Center for Cool and Cold Water Aquaculture) and Bill Shewmaker and Robin Burkhart (Clear Springs Foods, Inc.) for technical assistance and Peter Feng (U.S. Food and Drug Administration) for kindly providing antiflagellin monoclonal antibody. REFERENCES 1. Arias, C. R., O. Olivares-Fuster, K. Hayden, C. A. Shoemaker, J. M. Grizzle, and P. H. Klesius. 2007. First report of Yersinia ruckeri biotype 2 in the USA. J. Aquat. Anim. Health 19:35–40.
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