scholarly journals Deciphering the Molecular Basis for Attenuation of Flavobacterium columnare Strain Fc1723 Used as Modified Live Vaccine against Columnaris Disease

Vaccines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1370
Author(s):  
Wenlong Cai ◽  
Covadonga R. Arias
Keyword(s):  
Vaccine Strain ◽  
Molecular Basis ◽  
Live Vaccine ◽  
Gliding Motility ◽  
Genome Comparison ◽  
Comparative Genomic ◽  
Flavobacterium Columnare ◽  
Secretion Systems ◽  
Columnaris Disease ◽  
Modified Live Vaccine

Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.

scholarly journals The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium columnare

2017 ◽  
Vol 83 (23) ◽  
Author(s):  
Nan Li ◽  
Yongtao Zhu ◽  
Benjamin R. LaFrentz ◽  
Jason P. Evenhuis ◽  
David W. Hunnicutt ◽  
...  
Keyword(s):  
Secretion System ◽  
Gliding Motility ◽  
Control Measures ◽  
Flavobacterium Columnare ◽  
Wild Type ◽  
Secretion Systems ◽  
Content Type ◽  
Freshwater Aquaculture ◽  
Columnaris Disease ◽  
Spent Media

ABSTRACT Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare. The F. columnare ΔgldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The ΔgldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare ΔporV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from ΔgldN and ΔporV mutants did not, suggesting that soluble toxins are secreted by the T9SS. IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.

scholarly journals Genomic Analysis of an Attenuated Chlamydia abortus Live Vaccine Strain Reveals Defects in Central Metabolism and Surface Proteins

2009 ◽  
Vol 77 (9) ◽  
pp. 4161-4167 ◽  
Author(s):  
L. S. Burall ◽  
A. Rodolakis ◽  
A. Rekiki ◽  
G. S. A. Myers ◽  
P. M. Bavoil
Keyword(s):  
Pyruvate Kinase ◽  
Vaccine Strain ◽  
Genomic Analysis ◽  
Live Vaccine ◽  
Surface Proteins ◽  
Comparative Genomic ◽  
Temperature Sensitive ◽  
Nucleotide Polymorphisms ◽  
Chlamydia Abortus ◽  
Virulence Attenuation

ABSTRACT Comparative genomic analysis of a wild-type strain of the ovine pathogen Chlamydia abortus and its nitrosoguanidine-induced, temperature-sensitive, virulence-attenuated live vaccine derivative identified 22 single nucleotide polymorphisms unique to the mutant, including nine nonsynonymous mutations, one leading to a truncation of pmpG, which encodes a polymorphic membrane protein, and two intergenic mutations potentially affecting promoter sequences. Other nonsynonymous mutations mapped to a pmpG pseudogene and to predicted coding sequences encoding a putative lipoprotein, a sigma-54-dependent response regulator, a PhoH-like protein, a putative export protein, two tRNA synthetases, and a putative serine hydroxymethyltransferase. One of the intergenic mutations putatively affects transcription of two divergent genes encoding pyruvate kinase and a putative SOS response nuclease, respectively. These observations suggest that the temperature-sensitive phenotype and associated virulence attenuation of the vaccine strain result from disrupted metabolic activity due to altered pyruvate kinase expression and/or alteration in the function of one or more membrane proteins, most notably PmpG and a putative lipoprotein.

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

2020 ◽  
Author(s):  
Heidi M. T. Kunttu ◽  
Anniina Runtuvuori-Salmela ◽  
Krister Sundell ◽  
Tom Wiklund ◽  
Mathias Middelboe ◽  
...  
Keyword(s):  
Phage Therapy ◽  
Secretion System ◽  
Gliding Motility ◽  
Genetic Mutations ◽  
Phage Resistance ◽  
Flavobacterium Columnare ◽  
Aquaculture Industry ◽  
Columnaris Disease ◽  
Phage Sensitivity ◽  
Type Ix Secretion System

AbstractIncreasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably 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 decrease in bacterial virulence.ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. 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 a 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-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

scholarly journals Potential Source of Francisella tularensis Live Vaccine Strain Attenuation Determined by Genome Comparison

2006 ◽  
Vol 74 (12) ◽  
pp. 6895-6906 ◽  
Author(s):  
Laurence Rohmer ◽  
Mitchell Brittnacher ◽  
Kerstin Svensson ◽  
Danielle Buckley ◽  
Eric Haugen ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Vaccine Strain ◽  
Genetic Basis ◽  
Bacterial Pathogen ◽  
Live Vaccine ◽  
Genome Comparison ◽  
Live Vaccine Strain ◽  
Loss Of Function ◽  
Coding Regions ◽  
Sequence Variations

ABSTRACT Francisella tularensis is a bacterial pathogen that causes the zoonotic disease tularemia and is important to biodefense. Currently, the only vaccine known to confer protection against tularemia is a specific live vaccine strain (designated LVS) derived from a virulent isolate of Francisella tularensis subsp. holarctica. The origin and source of attenuation of this strain are not known. To assist with the design of a defined live vaccine strain, we sought to determine the genetic basis of the attenuation of LVS. This analysis relied primarily on the comparison between the genome of LVS and Francisella tularensis holarctica strain FSC200, which differ by only 0.08% of their nucleotide sequences. Under the assumption that the attenuation was due to a loss of function(s), only coding regions were examined in this comparison. To complement this analysis, the coding regions of two slightly more distantly related Francisella tularensis strains were also compared against the LVS coding regions. Thirty-five genes show unique sequence variations predicted to alter the protein sequence in LVS compared to the other Francisella tularensis strains. Due to these polymorphisms, the functions of 15 of these genes are very likely lost or impaired. Seven of these genes were demonstrated to be under stronger selective constraints, suggesting that they are the most probable to be the source of LVS attenuation and useful for a newly defined vaccine.

scholarly journals Flavobacterium columnare: an important contributing factor to fish die-offs in southern lakes of Saskatchewan, Canada

2014 ◽  
Vol 26 (6) ◽  
pp. 832-836 ◽  
Author(s):  
Steven J. Scott ◽  
Trent K. Bollinger
Keyword(s):  
Yellow Perch ◽  
Secondary Infection ◽  
Gliding Motility ◽  
The Body ◽  
Flavobacterium Columnare ◽  
Fish Kills ◽  
Characteristic Appearance ◽  
Columnaris Disease ◽  
Prairie Lakes ◽  
Gross Lesions

During June and July 2012, Buffalo Pound Lake and Blackstrap Lake in Saskatchewan, Canada were visited biweekly and surveyed for sick and dying fish. During this investigation, 2 fish kills were identified. Buffalo Pound experienced a large die-off of yellow perch ( Perca flavascens) in June, while Blackstrap experienced a die-off of lake whitefish ( Coregonus clupeaformis) in July. In excess of 50 fish were examined for gross lesions at each lake, and dead and moribund fish consistently had 1 or more of the following lesions: multifocal petechial cutaneous hemorrhage, skin ulceration, or branchial necrosis. Of these, 17 fish were collected for necropsy, and major tissues were submitted for histology. Aerobic bacterial culture was performed on 16 out of 17 fish. In 7 out of 8 (88%) yellow perch, the body wall had multiple areas of pale discoloration that corresponded to erosion and ulceration of the skin. Seven out of 8 (88%) whitefish had severe necrotizing branchiitis, and 8 out of 8 (100%) had severe epicardial parasitism, consistent with Ichthyocotylurus erraticus. Wet mounts of skin and gill lesions demonstrated filamentous bacteria with gliding motility, which often formed haystack-like arrangements. Flavobacterium columnare and Aeromonas hydrophila were cultured from skin and gill lesions of all fish. Based on the characteristic appearance and distribution of lesions, mortality was attributed to columnaris disease with secondary infection with A. hydrophila. The current study demonstrates that columnaris disease is an important contributor to fish kills in southern Saskatchewan lakes. However, further research is needed to determine what role environmental factors play in outbreaks of columnaris disease in prairie lakes.

Type IX secretion system effectors and virulence of the model Flavobacterium columnare strain MS-FC-4

2021 ◽  
Author(s):  
Nicole C. Thunes ◽  
Rachel A. Conrad ◽  
Haitham H. Mohammed ◽  
Yongtao Zhu ◽  
Paul Barbier ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Virulence Factors ◽  
Genetic Manipulation ◽  
Secretion System ◽  
Gliding Motility ◽  
Secreted Proteins ◽  
Flavobacterium Columnare ◽  
Genes Encoding ◽  
Columnaris Disease ◽  
Type Ix Secretion System

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and for gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and to other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted ten genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE: Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding ten secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

scholarly journals Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil

2008 ◽  
Vol 68 (2) ◽  
pp. 409-414 ◽  
Author(s):  
F. Pilarski ◽  
AJ. Rossini ◽  
PS. Ceccarelli
Keyword(s):  
Fish Species ◽  
Gliding Motility ◽  
Tropical Fish ◽  
Flavobacterium Columnare ◽  
Artificial Culture ◽  
Colossoma Macropomum ◽  
Piaractus Mesopotamicus ◽  
Isolation And Characterization ◽  
Columnaris Disease

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish, implicated in skin and gill disease, often causing high mortality. The aim of this study was the isolation and characterization of Flavobacterium columnare in tropical fish in Brazil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) and cascudo (Hypostomus plecostomus) were examined for external lesions showing signs of colunmaris disease such as greyish white spots, especially on the head, dorsal part and caudal fin of the fish. The sampling comprised 50 samples representing four different fish species selected for study. Samples for culture were obtained by skin and kidney scrapes with a sterile cotton swabs of columnaris disease fish and streaked onto Carlson and Pacha (1968) artificial culture medium (broth and solid) which were used for isolation. The strains in the liquid medium were Gram negative, long, filamentous, exhibited flexing movements (gliding motility), contained a large number of long slender bacteria and gathered into ‘columns'. Strains on the agar produced yellow-pale colonies, rather small, flat that had rhizoid edges. A total of four Flavobacterium columnare were isolated: 01 Brycon orbignyanus strain, 01 Piaractus mesopotamicus strain, 01 Colossoma macropomum strain, and 01 Hypostomus plecostomus strain. Biochemical characterization, with its absorption of Congo red dye, production of flexirubin-type pigments, H2S production and reduction of nitrates proved that the isolate could be classified as Flavobacterium columnare.

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

2021 ◽  
Author(s):  
Heidi M. T. Kunttu ◽  
Anniina Runtuvuori-Salmela ◽  
Krister Sundell ◽  
Tom Wiklund ◽  
Mathias Middelboe ◽  
...  
Keyword(s):  
Protease Activity ◽  
Phage Therapy ◽  
Secretion System ◽  
Gliding Motility ◽  
Phage Resistance ◽  
Flavobacterium Columnare ◽  
Bacterial Isolates ◽  
Aquaculture Industry ◽  
Columnaris Disease ◽  
Type Ix Secretion System

Increasing problems with antibiotic resistance has directed interest towards 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 1-2 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 coding for 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 decrease in bacterial virulence. Importance Phage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing world population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. 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 a 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 a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

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