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Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and the Type IX Secretion System.

ABSTRACT: Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

SUBMITTER: Kunttu HMT

PROVIDER: S-EPMC8315173 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Flavobacterium johnsoniae SprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprB required coexpression with sprF, which lies downstream of sprB SprF is similar in sequence to Porphyromonas gingivalis PorP. Most F. johnsoniae genes encoding proteins with type B CTDs lie immediately upstream of porP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins.IMPORTANCE The F. johnsoniae gliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylum Bacteroidetes and may have roles in adhesion, motility, and virulence.

Project description:Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. IMPORTANCE Many bacteria in the Bacteroidetes phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.

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Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and the Type IX Secretion System.

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