Allelic Exchange Method Research Articles

Harnessing native-cryptic plasmids for stable overexpression of heterologous genes in Clostridium butyricum DSM 10702 for industrial and medical applications

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids’ potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

Development of SacB-based Counterselection for Efficient Allelic Exchange in Fusobacterium nucleatum.

Investigations into Fusobacterium nucleatum 's role in related diseases significantly benefit from the strategies of creating unmarked gene mutations, which hinge on using a counterselective marker. Previously, the galk -based allelic exchange method, while effective, faced an inherent limitation - the need for a modified host. This study aims to surmount this limitation by substituting galK with sacB for gene modification in F. nucleatum . Our application of the sacB -based methodology successfully yielded a tonB in-frame deletion mutant and a luciferase gene knockin at the precise chromosomal location in the wild-type background. The new method augments the existing toolkit for F. nucleatum research and has far-reaching implications due to the easy accessibility to the counterselection compound sucrose. We anticipate its broader adoption in further exploration, thereby reinforcing its critical role in propelling our understanding of F. nucleatum .

Functional analysis of OmpA and its contribution to pathogenesis of Edwardsiella tarda

Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.

A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.

The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus ΔH.

This study is a continuation by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. For this, we expanded our system for genetic modification of M. thermautotrophicus ΔH using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain. IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH.

Development and potential use of Pseudomonas plecoglossicida mutant ΔOmpRΔrpoS as a live attenuated vaccine against visceral white nodules disease in large yellow croaker (Larimichthys crocea)

Pseudomonas plecoglossicida has become a major pathogen adversely affecting the farming of the large yellow croaker (Larimichthys crocea) in which it causes visceral white nodules disease (VWND), a highly devastating disease that leads to severe economic losses. The development of a live attenuated vaccine (LAV) will be an effective and beneficial strategy to control the disease in farmed L. crocea. In this study, we constructed an attenuated P. plecoglossicida mutant by knocking out the OmpR and rpoS genes using the allelic exchange method and analyzed the growth kinetics in vitro. Then, the use of the mutant ΔOmpRΔrpoS strain as a potential LAV was evaluated. We found no significant difference in the in vitro growth kinetics between the mutant and wild-type (WT) strains. The ΔOmpRΔrpoS strain showed a 1.17 × 107-fold decrease in virulence in L. crocea compared with the parental WT strain. Fish vaccinated twice via intraperitoneal (IP) injection with the ΔOmpRΔrpoS strain had a relative percent survival (RPS) of 72.25% and 64.90%, when challenged at 4 and 6 weeks post-initial vaccination (wpiv), respectively. Vaccinated fish developed a much higher P. plecoglossicida-specific antibody response than the unvaccinated fish. In ΔOmpRΔrpoS-vaccinated fish, the immune-related genes were remarkably upregulated after P. plecoglossicida challenge, and a lower bacterial load was observed in the spleen of these fish, unlike the control fish, which had high bacterial loads. Moreover, the ΔOmpRΔrpoS strain showed no adverse effects on the growth rate of large yellow croaker. Taken together, these results suggest that the ΔOmpRΔrpoS mutant is a promising LAV candidate against P. plecoglossicida infection.

Using CRISPR-Cas9-Based Methods for Genome Editing in Staphylococcus aureus.

Chromosomal mutations and targeted gene deletions and inactivations in Staphylococcus aureus are typically generated using the allelic exchange method. In recent years, however, more rapid methods have been developed, often using CRISPR-Cas9-based systems. Here, we describe recently developed CRISPR-Cas9-based plasmid systems for use in S. aureus, and discuss their use for targeted gene mutation and inactivation. First, we describe how a CRISPR-Cas9 counterselection strategy can be combined with a recombineering strategy to generate gene deletions in S. aureus We then introduce dead Cas9 (dCas9) and Cas9 nickase (nCas9) enzymes, and discuss how the nCas9 enzyme fused to different nucleoside deaminases can be used to introduce specific base changes in target genes. We then discuss how the nCas9-deaminase fusion enzymes can be used for targeted gene inactivation via the introduction of premature stop codons or by mutating the start codon. Together, these tools highlight the power and potential of CRISPR-Cas9-based methods for genome editing in S. aureus.

Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus.

Here we describe an allelic-exchange procedure for the construction of an unmarked gene deletion in the bacterium Staphylococcus aureus As a practical example, we outline the construction of a tagO gene deletion in S. aureus using the allelic-exchange plasmid pIMAY*. We first present the general principles of the allelic-exchange method, along with information on counterselectable markers. Furthermore, we summarize relevant cloning procedures, such as the splicing by overhang extension (SOE) polymerase chain reaction (PCR) and Gibson assembly methods, and we conclude by giving some general consideration to performing genetic modifications in S. aureus.

Pyoluteorin and 2,4-diacetylphloroglucinol are major contributors to Pseudomonas protegens Pf-5 biocontrol against Botrytis cinerea in cannabis.

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ∆pltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltD and ∆phlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere.

CRISPR/Cas9-mediated generation of auxotrophic Edwardsiella piscicida mutants and immunization in olive flounder (Paralichthys olivaceus)

Edwardsiella piscicida has been a cause of mass mortality in cultured fish. In this study, to produce auxotrophic E. piscicida mutants, a CRISPR/Cas9 system was used instead of the traditional sacB-based allelic exchange method. Under the optimal CRISPR engineering condition, we could efficiently produce either alr or asd gene knockout E. piscicida auxotrophic mutants, and this genome editing process was much simpler and faster than the allelic exchange method. The simultaneous knockout of double auxotrophic genes (alr and asd) and the insertion of a foreign gene expression cassette in E. piscicida chromosome were also successfully performed using the established CRISPR/Cas9 system. Furthermore, to enhance the possibility to get permission as a commercial vaccine, we produced an auxotrophic E. piscicida mutant having only one nucleotide-deleted alr gene (E. piscicida △alr-1). Olive flounder (Paralichthys olivaceus) fingerlings immunized with 1 × 106 and 1 × 105 CFU/fish of E. piscicida △alr-1 showed the superior ability in the induction of serum agglutination activity and in the protection against E. piscicida compared to killed E. piscicida. However, olive flounder immunized with 1 × 107 CFU/fish of E. piscicida △alr-1 showed high mortality far before the challenge, and the isolated E. piscicida from moribund and dead fish had the wild type alr gene, suggesting the reversion of one base-deleted alr gene to original form by a second mutation in olive flounder. Therefore, investigation on the minimum number of edited nucleotide for stable maintenance of E. piscicida mutants should be further conducted.

Inducing cellular immune responses with a marked Mycobacterium avium subsp. paratuberculosis strain in dairy calves

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a chronic granulomatous enteritis with a high global prevalence in dairy cattle. This disease causes significant economic loss in the dairy industry and has been challenging to control, as current diagnostic assays are low in sensitivity and specificity, and previously developed vaccines do not prevent infection and face regulatory concerns due to interference with bovine tuberculosis diagnostics. To remediate this issue, positive and negative immune markers were created in a MAP strain as a step towards a vaccine capable of differentiating infected from vaccinated animals (DIVA). A gene coding for an immunogenic protein (MAP1693c) in the MAP genome was replaced with a library of epitope-tagged immunogenic genes (pepA) via a stable allelic exchange method. These markers were evaluated in a calf infection trial, where Holstein-Friesian dairy calves were inoculated at two weeks of age with either the marked strain or the parent strain, or remained uninfected controls. Cellular immune responses to the markers were measured using an interferon gamma release assay (IGRA). There were no MAP1693c marker-specific differences in cellular immune responses between infection groups. A scrambled version of the HA (human influenza hemagglutinin) epitope, but not the actual HA epitope, induced a significant IFN-γ response in marker-infected calves compared to WT-infected and uninfected groups at 4.5 months post-inoculation. This scrambled HA epitope thus holds potential as a diagnostic tool as part of a DIVA vaccine for Johne’s disease.

Roles of EvpP in Edwardsiella piscicida-Macrophage Interactions.

Edwardsiella piscicida is found to be an important facultative intracellular pathogen with a broad host range. These organisms can replicate and survive within host macrophages to escape from the subversion of the immune defense. E. piscicida-macrophage interaction is very important in determining the outcome of edwardsiellasis. As an effector protein of E. piscicida T6SS, EvpP has been determined to be a very important virulence factor for E. piscicida, although its precise role in E. piscicida-macrophage interactions is not yet clear. In this study, the roles of EvpP in E. piscicida-macrophage interactions were characterized. Here, we constructed the deletion mutants of evpP (ΔevpP) and complementation (ΔevpP-C) by the allelic exchange method. Compared to wild type strain (WT), ΔevpP was found to be attenuated for growth within macrophages. In line with this observation, we found its survival capacity was lower than WT under oxidative and acid stress in vitro, which simulate conditions encountered in host macrophages. Attenuation of ΔevpP also correlated with enhanced activation of macrophages, as reflected by augmented NO production in ΔevpP-treated macrophages. Moreover, compared to WT, ΔevpP induced markedly increased apoptosis of macrophages, characterized by increased Annexin V binding and the activation of cleaved caspase-3. These findings provided strong evidence that EvpP is involved in the process of E. piscicida-macrophage interactions and is required for its survival and replication in macrophages. Thus, we propose that EvpP might be an important factor that controlling the fate of E. piscicida inside macrophages. To further exploring the underlying mechanism of EvpP action, the cDNA library was constructed from E. piscicida-infected macrophages and a yeast two-hybrid screen was performed to search for cellular proteins interacting with EvpP. Ribosomal protein S5 (RPS5) was identified as a target of EvpP. Furthermore, the interaction was validated with co-immunoprecipitation assay. This result implies that the observed effect of EvpP on macrophages might be related to RPS5-mediated regulation, contributing to a better understanding of the mechanisms of EvpP involved in E. piscicida-macrophage interactions.

Essential role of the ESX-3 associated eccD3 locus in maintaining the cell wall integrity of Mycobacterium smegmatis

Mycobacterial pathogens have evolved a unique secretory apparatus called the Type VII secretion system (T7SS) which comprises of five gene clusters designated as ESX1, ESX2, ESX3, ESX4, and ESX5. Of these the ESX3 T7SS plays an important role in the regulatory uptake of iron from the environment, thereby enabling the bacteria to establish successful infection in the host. However, ESX3 secretion system is conserved among all the mycobacterial species including the fast-growing nonpathogenic species M. smegmatis. Although the function of ESX3 T7SS is known to be absolutely critical for establishing infection by M. tuberculosis, its conserved nature in all the pathogenic and nonpathogenic mycobacterial species intrigues to explore the additional functional roles in Mycobacterium species through which potent targets for drugs can be identified and developed. In the present study, we investigated the possible role of EccD3, a transmembrane protein of the ESX3 T7SS in M. smegmatis by deleting the entire eccD3 gene by efficient allelic exchange method. The preliminary investigations through the creation of knockout mutant of the eccD3 gene indicate that this secretory apparatus has an important role in maintaining the cell wall integrity which was evident from the abnormal colony morphology, lack of biofilm formation and difference in cell wall permeability.

Establishment of a markerless gene deletion system in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25°C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37°C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.

Comparison of Protective Efficacy between Formalin‐killed and aroA Gene‐knockout Vibrio anguillarum Vaccines in Olive Flounder, Paralichthys olivaceus

AbstractThe aro genes in bacteria encode enzymes needed for the biosynthesis of aromatic amino acids, and mutant bacteria that are defective in the enzymes can replicate only a limited number in vertebrates owing to the lack or scarceness of chorismate, through which the mutant bacteria of the aro genes become attenuated. In the present study, the 5‐enolpyruvylshikimate‐3‐phosphate synthase (aroA) gene‐knockout Vibrio anguillarum (ΔaroA V. anguillarum) were generated by the allelic exchange method, and its vaccine potential was evaluated in the olive flounder, Paralichthys olivaceus, by comparing the protective efficacy of a formalin‐inactivated V. anguillarum. The LD50 (50% lethal dose) value of ΔaroA V. anguillarum was 1000 times higher than that of wild‐type V. anguillarum in olive flounder fingerlings, and the growth of ΔaroA V. anguillarum was significantly suppressed by coincubation with nonimmune olive flounder serum compared with that of wild‐type V. anguillarum. The survival rates and serum agglutination titers of fish immunized with ΔaroA V. anguillarum were significantly higher than those of fish immunized with the same amount of formalin‐inactivated V. anguillarum, suggesting that although the inactivated V. anguillarum vaccine can provide a high protection in olive flounder, the protective efficacy can be enhanced by immunization with an auxotrophic mutant ΔaroA V. anguillarum.

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4.

This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals.

Knockout of two alanine racemase genes of Vibrio anguillarum and its potential as an attenuated vaccine in olive flounder (Paralichthys olivaceus)

Vibriosis caused by Vibrio anguillarum is one of the most common bacterial diseases in cultured fish worldwide. Alanine racemases (Alrs) catalyze conversion of l-alanine to d-alanine. As d-alanine is an essential component of the peptidoglycan layer of bacteria, the alanine racemase gene (alr) knockout bacteria cannot grow without d-alanine supplementation. In the genome of V. anguillarum, two alanine racemase genes (alr1 and alr2) are present. In the present study, we produced auxotrophic V. anguillarum mutants by knockout of alr and/or alr2 genes using the allelic exchange method, and analyzed in vitro & in vivo replication ability and virulence in olive flounder (Paralichthys olivaceus) fingerlings. Furthermore, the potential of the mutant V. anguillarum (Δalr1Δalr2 V. anguillarum) as an attenuated live vaccine was evaluated. In vitro growth of Δalr1 V. anguillarum stopped in the absence of d-alanine. In the virulence experiment, the LD50 of Δalr1 V. anguillarum in olive flounder fingerlings was approximately 1000-fold lower than that of wild-type V. anguillarum. However, there were no differences between Δalr2 V. anguillarum and wild-type V. anguillarum in in vitro replication and in vivo virulence to olive flounder. These results suggest that the alr1 is the main source for the production of alanine racemase in V. anguillarum, and knockout of alr1 alone is sufficient to stop the bacterial replication. The amino acid sequence of the alr2 showed high homology to V. cholerae's broad spectrum racemase (BsrV) that was previously known as alr2 (recently redesignated as bsrV). The survival rates of V. anguillarum in low salinities were significantly reduced by the knockout of alr2, which is one of the properties of V. cholerae BsrV, suggesting that the alr2 of V. anguillarum is a bsr gene that is corresponding to bsrV of V. cholerae. Although the alr2 of V. anguillarum was involved in the resistance to osmotic challenge, the alr2 knockout was not accompanied by attenuation of the bacteria in fish. In the vaccine experiment, the survival rate and serum agglutination titer of fish immunized with 1×105CFU/fish of Δalr1Δalr2 V. anguillarum were significantly higher than fish immunized with 1×105CFU/fish of FKC V. anguillarum, suggesting that Δalr1Δalr2 V. anguillarum can elicit protective immune responses more efficiently than FKC V. anguillarum. Statement of relevanceIn this paper, we generated auxotrophic Vibrio anguillarum mutants by knockout of alanine racemase genes, and evaluated their potential as a prophylactic vaccine in olive flounder. The results of the vaccine experiment showed that the survival rate and serum agglutination titer of fish immunized with 1×105CFU/fish of Δalr1Δalr2 V. anguillarum were significantly higher than fish immunized with 1×105CFU/fish of FKC V. anguillarum, suggesting that Δalr1Δalr2 V. anguillarum can elicit protective immune responses more efficiently than FKC V. anguillarum.

Inhibition of Salmonella-induced apoptosis as a marker of the protective efficacy of virulence gene-deleted live attenuated vaccine

Vaccination is one of the best protection strategies against Salmonella infection in humans and chickens. Salmonella bacteria must induce apoptosis prior to initiating infection, pathogenesis and evasion of host immune responses. In this study, we evaluated the efficacy of vaccinating chickens against Salmonella Enteritidis (SE) using a vaccine candidate strain (JOL919), constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. In present study day old chickens were inoculated with 1×107cfu (colony forming unit) of JOL919 per os. We measured cell-mediated immunity, protective efficacy and extent of apoptosis induction in splenocytes. Seven days post-immunization, the number of CD3+CD4+ and CD3+ CD8+ T cells was significantly higher in the immunized group compared to the control group, indicating a significant augmentation of systemic immune response. The internal organs of chickens immunized with JOL919 had a significantly lower challenge-strain recovery, indicating effective protection and clearance of the challenge strain. Post-challenge, the number of apoptotic cells in the immunized group was significantly lower than in the control group. Additionally, AV/PI (Annexin V/propidium iodide) staining was performed to differentiate between apoptotic cells and necrotic cells, which corroborated TUNEL-assay (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) results. The proportions of AV+/PI− and AV+/PI+ cells, which represent the proportions of early apoptotic and late apoptotic/early necrotic cells present, respectively, were significantly lower in the immunized group. Our findings suggest that the apoptotic splenocytes in immunized chickens significantly decreased in number, which occurred concomitantly with a significant rise in systemic immune response and bacterial clearance. This suggests that inhibition of apoptosis may be a marker of protection efficacy in immunized chickens.

Generation of killed but metabolically active (KBMA) Edwardsiella tarda and evaluation of its potential as a protective vaccine.

A technology for inactivation of pathogens in human blood products by treatment with amotosalen hydrochloride (S-59) in combination with long wavelength ultraviolet light (UVA) to decrease transfusion-mediated sepsis has been applied to make safe vaccines against human pathogenic bacteria, and the resultants were called killed but metabolically active (KBMA) bacteria. In the present study, we first generated KBMA Edwardsiella tarda and evaluated its potential as a protective vaccine in olive flounder (Paralichthys olivaceus). To prevent the restoration of division ability by removal of psoralen adducts in the bacterial chromosome through the nucleotide excision repair (NER), the uvrA and uvrB genes knock-out E. tarda (ΔuvrAB E. tarda) was produced by the allelic exchange method. The optimal condition for generation of KBMA E. tarda was exposure of the ΔuvrAB E. tarda to 100 ng/ml of S-59 and 2.8 J/cm(2) of UVA irradiation. The KBMA E. tarda could not replicate but showed a high metabolic activity (measured by lactate dehydrogenase activity) that was comparable to the wild-type E. tarda. In comparison of survival rates between groups vaccinated with the same dose of bacteria, fish immunized with KBMA E. tarda showed significantly higher survival rates than fish immunized with formalin-killed cell (FKC) E. tarda. Furthermore, fish immunized with 1 × 10(7) CFU/fish of KBMA E. tarda showed no mortality, while PBS-injected fish showed 100% mortality. The serum agglutination titer was sharply increased by 10(7) CFU/fish of KBMA E. tarda compared to those of fish immunized with 10(6) CFU/fish of KBMA E. tarda or 10(7) CFU/fish of FKC E. tarda. The consistently lower serum agglutination titers against KBMA E. tarda than against FKC E. tarda in both KBMA and FKC E. tarda immunized groups suggest that some factors secreted from KBMA E. tarda might inhibit the serum agglutination activity. In conclusion, the present results showed the higher potential of KBMA E. tarda than FKC E. tarda as a prophylactic vaccine.

Mutations in the major outer membrane protein gene from Histophilus somni by an allelic exchange method

Functional roles of the major outer membrane protein (MOMP) gene from the bovine pathogen Histophilus somni have remained to be elucidated due to lack of mutagenesis methods easily applicable to this gene. In this study, the direct use of PCR-amplified mutated DNA flanking by an antibiotic selection marker for transformation of H. somni was applied to accomplish the site-directed mutagenesis via homologous recombination in H. somni non-pathogenic strain 129Pt and pathogenic strain 2336. A protocol for unmarking the antibiotic resistance gene from the created MOMP mutant by using a temperature-sensitive plasmid vector was also established. In both strains, no significant phenotypic difference was observed between the wild-type strain and its isogenic mutant expressing the exchanged MOMP in growth in broth medium and antibiotic susceptibility. However, the mutant 129Pt expressing MOMP from strain 2336 was significantly more susceptible to bacterial killing by fresh normal bovine serum than its wild-type parent strain. Serum susceptibility of the mutant 2336 expressing MOMP from strain 129Pt was significantly lower than its wild-type parent strain although the susceptibility difference was considerably less than that found between the 129Pt wild-type and MOMP mutant strains. From further assays using MOMP mutants that express chimeric MOMP consisting of the amino-terminal part that contains loops L1–L3 from one strain and the carboxyl-terminal part that contains loops L4–L8 from another strain, the C-terminal part of MOMP was found to affect serum susceptibility of H. somni.