Lambda Recombination Research Articles

Construction and evaluation of a Salmonella Paratyphi A vaccine candidate based on a poxA gene mutation

Salmonella Paratyphi A, the pathogen of paratyphoid A accounts for an obviously growing proportion of cases in many areas. Therefore, development of specific paratyphoid A vaccines is needed. In the present study, the poxA gene of Salmonella Paratyphi A, encoding the aminoacyl-tRNA synthetase, was deleted successfully by the method of lambda Red recombination system, the resulting strain, ΔpoxA was characterized in respect of growth, adhesion and invasion, virulence, immunogenicity and protective efficacy. It was found that the growth of the ΔpoxA strain was significantly delayed compared with the wild type strain, the mutant ΔpoxA was less invasive to Caco-2 BBE epithelioid cells and THP-1 macrophages than the wild type strain, strain ΔpoxA was attenuated at least 1000-fold in mice, significant immune response and efficient protection were provided by the mutant ΔpoxA after oral immunization. It is concluded that the Salmonella Paratyphi A poxA deletion mutant ΔpoxA can be used as a live oral vaccine candidate against paratyphoid A.

A genome-wide collection of barcoded single-gene deletion mutants in Salmonella enterica serovar Typhimurium.

Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences. A motility assay of a pool of the entire library, followed by a single-tube processing of the bacterial pellet, PCR, and sequencing, was used to verify the performance of the barcoded TetR collection. The new resource is useful for experiments with defined subsets of barcoded mutant strains where biological bottlenecks preclude high numbers of founder bacteria, such as in animal infections. The TetR version of the library will also facilitate the construction of triple mutants by transduction. The resource of 6197 mutants covering 3490 genes is deposited at Biological and Emerging Infections Resources (beiresources.org).

Assessment of environmental safety and protective efficacy of O-antigen deficient DIVA capable Salmonella Enteritidis against chicken salmonellosis

In this study, we incorporated deletion of the O-antigen ligase gene to an attenuated Salmonella Enteritidis (SE) strain, JOL919 (SE PS; Δlon ΔcpxR), using the Lambda-Red recombination method and evaluated the safety and immunological aspects of the novel genotype, JOL2381 (SE VS: Δlon, ΔcpxR, ΔrfaL). Assessment of fecal shedding and organ persistence following administration via oral and IM routes revealed that the SE VS was safer than its parent strain, SE PS. Immunological assays confirmed that immunization via the oral route with SE PS was superior to the SE VS. However, chickens immunized with SE PS and SE VS strains via the IM route showed higher humoral and cell-mediated immune responses. Compared to PBS control, the IM route of immunization with SE VS resulted in a higher IgY antibody titer and expansion of CD4+ and CD8+ T-cell populations, which resulted in the clearance of Salmonella from the liver and splenic tissues. Furthermore, deletion of the O-antigen ligase gene caused lower production of LPS-specific antibodies in the host, promoting DIVA functionality and making it a plausible candidate for field utilization. Due to significant protection, high attenuation, and environmental safety concerns, the present SE VS strain is an ideal choice to prevent chicken salmonellosis and ensure public health.

AI-2 quorum sensing controlled delivery of cytolysin-A by tryptophan auxotrophic low-endotoxic Salmonella and its anticancer effects in CT26 mice with colon cancer

IntroductionThe limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers. ObjectivesThis study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs. MethodsThe therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 106 CFU/mouse for cancer therapy. ResultsThe attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1β, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control. ConclusionHence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency.

High-efficiency genetic engineering toolkit for virus based on lambda red-mediated recombination.

Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development. Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication. Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication. This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.

Coupling CRISPR/Cas9 and Lambda Red Recombineering System for Genome Editing of Salmonella Gallinarum and the Effect of ssaU Knock-Out Mutant on the Virulence of Bacteria.

The poultry industry in developing countries still faces a significant threat from fowl typhoid, a disease caused by Salmonella Gallinarum that has been well contained in more economically developed countries. In addition to the virulence exhibited by large virulence plasmid (85 kb), Salmonella Pathogenicity Island 2 in S. Gallinarum plays a key role in mediating disease through its type III secretion systems (TTSS). The TTSS secrete effector protein across the Salmonella containing vacuoles and mediate the internalization of bacteria by modulating vesicular passage. In this study, candidate virulent ssaU gene (~1 kb) encoding type III secretion system was successfully deleted from indigenously isolated S. Gallinarum genome through homology-directed repair using CRISPR/Cas9 and lambda recombination systems. CRISPR/Cas9-based genome editing of poultry-derived Salmonella Gallinarum has not been previously reported, which might be linked to a lack of efficiency in its genetic tools. This is the first study which demonstrates a complete CRISPR/Cas9-based gene deletion from this bacterial genome. More importantly, a poultry experimental model was employed to assess the virulence potential of this mutant strain (ΔssaU_SG18) which was unable to produce any mortality in the experimentally challenged birds as compared to the wild type strain. No effect on weight gain was observed whereas bacteria were unable to colonize the intestine and liver in our challenge model. This in vivo loss of virulence in mutant strain provides an excellent functionality of this system to be useful in live vaccine development against this resistant and patho genic bacteria.

Systematic Review of the SARS-CoV-2 Viral Vector Vaccine AstraZeneca (Azd1222)

It has been more than two years since the spread of the COVID-19 pandemic all over the world. Scientists still are in search of a permanent treatment and cure of this infectious disease. In this regard, on 20th April, 2020, AstraZeneca in collaboration with the Oxford University came up with a recombinant adenovirus-based vaccine labeled as “ChAdOx1 nCoV-19” or “AZD1222”. Approximately, 22 viral vector-based vaccines are in the trial stage and ChAdOx1 nCoV-19 falls under the category of non-replicating viral vector-based vaccines. During vaccine development, ChAdOx-1 vector was specifically designed by red lambda recombination of Y25 serotype (chimpanzee) with HAdV-C5 serotype (human). In July 2020, clinical trials were initiated but due to some controversial side effects, these trials were halted for a while and resumed later on. AstraZeneca has been reported to generate both humoral and cell mediated immune responses. This vaccine exhibited 63% efficacy with few side effects. It also exhibited dwindling efficacy against the emerging variants of COVID-19. Since then, heterologous prime boost vaccination has been initiated, exhibiting elevated efficacy. Until now, 42.6% population of the world has been vaccinated with a single dose and this number is rising rapidly. However, due to the emerging variants of COVID-19, the efficacy of the majority of vaccines is decreasing. So, the manufacturers must work on making the vaccines more effective against these new variants as well. This review is written with the intention of summing up all the reported data regarding AZD1222 in order to provide a proper overview.

Assessing an O-antigen deficient, live attenuated Salmonella Gallinarium strain that is DIVA compatible, environmentally safe, and protects chickens against fowl typhoid

The objective of the present study was to create a highly attenuated, safe Salmonella Gallinarium (SG) vaccine strain for chicken vaccination against fowl typhoid (FT) diseases. The SG vaccine strain (SGVS) consists of three virulence-related gene deletions, namely, lon, cpxR, and rfaL. The parent strain (SGPS) with Δlon ΔcpxR genotype was utilized as the host strain for in-frame rfaL gene deletion by lambda red recombination. The SGVS was highly attenuated with improved environmental safety by zero fecal contamination beyond seven days for both oral and intramuscular immunization routes. Upon inoculation into 1-month-old young chicken, no vaccine-induced adverse behaviors were observed and did not cause a chronic state of infection as the SG wild-type strain did. Immunization of chicken elicited both humoral and cell-mediated immune responses demarcated by, IgY antibody assessment, T-cell responses in peripheral blood mononuclear cells, and the induction of immunomodulatory cytokines, IFN-γ, IL-2, IL-12, and IL-4 to resemble both Th1 and Th2 type of immune responses. The immunological assessment revealed a high level of efficacy of the SGVS when inoculated via the IM route than the oral route. The strain was less cytotoxic with reduced cytotoxicity on chicken macrophages and was DIVA capable with minimum reactivity of immunized serum with purified SG lipopolysaccharides. The challenge study could generate 70% protection in chicken for SGVS, whereas no birds were protected in the PBS challenged group. The protection levels were evident in histopathological assessment of spleen and liver specimens and also the external appearance of the spleen with reduced lesions on immunized groups. Further experiments may be warranted to dose and route optimization for further increase in the protection level derived by present SGVS.

Lambda Red Recombineering of Bacteriophage in the Lysogenic State.

We present a recombineering-based method for editing the genome of a temperate phage. The method uses the lambda Red recombination system to edit the genome of a lysogenized host with a prophage compatible with bacteriophage lambda. Linear DNA is used as the recombination substrate and antibiotic resistance is used as the basis for selection of recombinants. The method enables the genetic manipulation of a prophage in 3-5days.

Genome Editing in Klebsiella pneumoniae Using CRISPR/Cas9 Technology.

CRISPR/Cas9 systems have been widely adopted for genetic manipulation in diverse biological systems owing to the ease of use and high efficiency. We have recently developed a CRISPR/Cas9-based genome editing system (pCasKP-pSGKP) by coupling a CRISPR/Cas9 system with the lambda Red recombination system as well as a cytidine deaminase-mediated base editing system (pBECKP) in Klebsiella pneumoniae, enabling rapid, scarless, and efficient genetic manipulation in diverse K. pneumoniae strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in K. pneumoniae.

Optimization of a Lambda-RED Recombination Method for Rapid Gene Deletion in Human Cytomegalovirus.

Human cytomegalovirus (HCMV) continues to be a major cause of morbidity in transplant patients and newborns. However, the functions of many of the more than 282 genes encoded in the HCMV genome remain unknown. The development of bacterial artificial chromosome (BAC) technology contributes to the genetic manipulation of several organisms including HCMV. The maintenance of the HCMV BAC in E. coli cells permits the rapid generation of recombinant viral genomes that can be used to produce viral progeny in cell cultures for the study of gene function. We optimized the Lambda-Red Recombination system to construct HCMV gene deletion mutants rapidly in the complete set of tested genes. This method constitutes a useful tool that allows for the quick generation of a high number of gene deletion mutants, allowing for the analysis of the whole genome to improve our understanding of HCMV gene function. This may also facilitate the development of novel vaccines and therapeutics.

Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS.

We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7-10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR-Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.

Bacterial Artificial Chromosome-Based Lambda Red Recombination with the I-SceI Homing Endonuclease for Genetic Alteration of MERS-CoV.

Over the past two decades, several coronavirus (CoV) infectious clones have been engineered, allowing for the manipulation of their large viral genomes (~30 kb) using unique reverse genetic systems. These reverse genetic systems include targeted recombination, in vitro ligation, vaccinia virus vectors, and bacterial artificial chromosomes (BACs). Quickly after the identification of Middle East respiratory syndrome-CoV (MERS-CoV), both in vitro ligation and BAC-based reverse genetic technologies were engineered for MERS-CoV to study its basic biological properties, develop live-attenuated vaccines, and test antiviral drugs. Here, I will describe how lambda red recombination can be used with the MERS-CoV BAC to quickly and efficiently introduce virtually any type of genetic modification (point mutations, insertions, deletions) into the MERS-CoV genome and recover recombinant virus.

Characterization and protective efficacy of a Salmonella pathogenicity island 2 (SPI2) mutant of Salmonella Paratyphi A

Paratyphoid fever caused by Salmonella Paratyphi A is a serious public health problem in many countries. In order to and develop a live attenuated candidate vaccine of Salmonella Paratyphi A, a Salmonella pathogenicity island 2 (SPI2, approximate 40 kb) deletion mutant of Salmonella Paratyphi A was constructed by lambda Red recombination, then the biological characteristics and protective ability of the Salmonella Paratyphi A SPI2 mutant were evaluated. Our results showed that the growth and biochemical properties of the SPI2 mutant were consistent with that of its parent strain, and the mutant was stable with the loss of SPI2. The mice lethal test showed that the virulence of the SPI2 mutant was significantly decreased, it can colonize and persistent more than 14 days in the liver and spleen of mice. Vaccination with the SPI2 mutant in mice revealed no significant effect on body weight and clinical symptoms compared to control animals, and specific humoral and cellular immune responses were also significantly induced. Immunization of mice offered efficient protection against Salmonella Paratyphi A strain challenge at 14 days post vaccination based on mortality and clinical symptoms relative to control group. Overall, these findings suggested that SPI2 plays an important role in pathogenicity of Salmonella Paratyphi A, and the SPI2 mutant showed its potential to develop a live attenuated vaccine candidate.

Synonymous genome recoding: a tool to explore microbial biology and new therapeutic strategies.

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies.

Unexpected transcriptome pompTʹ contributes to the increased pathogenicity of a pompT mutant of avian pathogenic Escherichia coli

The lambda red recombination system makes it suitable for screening virulence gene utility in avian pathogenic Escherichia coli (APEC) on account of its wide applicability, simplicity and high efficiency. In APEC E058 (O2 serogroup), there are two copies of the outer membrane protease (ompT) gene, compT encoding cOmpT that is located on the chromosome and pompT encoding pOmpT that is located on a ColV plasmid. However, the relationship between pathogenesis and pompT expression in APEC E058 has yet to be elucidated. Here, we successfully constructed two pompT gene mutants: E058ΔpompT containing a chloramphenicol (cat) resistance gene and E058ΔpompTʹ without the cat gene. By RT-PCR and sequencing analysis, an unexpected transcriptome pompTʹ was detected in mutant strain E058ΔpompTʹ after deletion of the cat gene induced by the lambda red recombination system. Surprisingly, the pathogenicity of mutant E058ΔpompT was significantly attenuated compared to its parental strain in the chicken infection model and HD11 cell model then the pompT gene was knocked out, while the pathogenicity of the other mutant strain E058ΔpompTʹ had no difference. Furthermore, the presence of unexpected transcriptome pompTʹ influenced the bactericidal activity of SPF chicken serum and decreased the transcription level of TLR2 in the heart tissue of chickens. Our study identifies the pompT gene plays an important role in the virulence of APEC E058, and the unexpected transcriptome pompTʹ contributes to the increased pathogenicity of APEC E058 mutants following deletion of the cat gene induced by the lambda red recombination system, which suggests that this system still has some limitations for construction of mutant strains particularly where these are used in development of live vaccine.

Effects of dam and seqA genes on biofilm and pellicle formation in Salmonella

ABSTRACTIn this study, the effects of dam and seqA genes on the formation of pellicle and biofilm was determined using five different Salmonella serovars S. Group C1 (DMC2 encoded), S. Typhimurium (DMC4 encoded), S. Virchow (DMC11 encoded), S. Enteritidis (DMC22 encoded), and S. Montevideo (DMC89 encoded). dam and seqA mutants in Salmonella serovars were performed by the single step lambda red recombination method. The mutants obtained were examined according to the properties of biofilm on the polystyrene surfaces and the pellicle formation on the liquid medium. As a result of these investigations, it was determined that the biofilm formation properties on polystyrene surfaces decreased significantly (p < 0.05) in all tested dam and seqA mutants, while the pellicle formation properties were lost in the liquid medium. When pBAD24 vector, containing the dam and seqA genes cloned behind the inducible arabinose promoter, transduced into dam and seqA mutant strains, it was determined that the biofilm formation properties on the polystyrene surfaces reached to the natural strains’ level in all mutant strains. Also, the pellicle formation ability was regained in the liquid media. All these data demonstrate that dam and seqA genes play an important role in the formation of biofilm and pellicle structures in Salmonella serovars.

Herpes ICP8 protein stimulates homologous recombination in human cells.

Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red homologous recombination protein Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red Synaptase/Exonuclease pair (SynExo) in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a “humanized” version of Beta protein from phage λ. ICP8 significantly enhanced gene targeting rates in HEK 293T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.

Characterization of the phosphate-specific transport system in Cronobacter sakazakii BAA-894.

Characterize the phosphate-specific transport system in Cronobacter sakazakii BAA-894. The genes relevant to phosphate transfer in C. sakazakii BAA-894 were determined by using sequence alignment to the corresponding genes in Escherichia coli. Then, the determined pst operon in C. sakazakii BAA-894 was deleted using the lambda Red recombination system. Using the wild type C. sakazakii BAA-894 as a control, the membrane permeability, auto-aggregation, exopolysaccharide biosynthesis, biofilm formation, and adhesion ability of the mutant ▵pst grown in media containing high or low concentrations of phosphate were investigated; stronger auto-aggregation, less biofilm formation and higher adhesion ability were observed in ▵pst cells grown in low phosphate media. Transcriptome analysis showed that phosphate availability has a global influence to C. sakazakii BAA-894 and ▵pst cells. Phosphorus availability is important for C. sakazakii in many ways including biofilm formation and adhesion ability. This study demonstrates that phosphate availability has a global influence to C. sakazakii, expends our understanding to the phosphate transfer in C. sakazakii, and is helpful for revealing the survival mechanism of C. sakazakii under stress conditions.

Construction and characterization of a cigR deletion mutant of Salmonella enterica serovar Pullorum

ABSTRACTSalmonella enterica serovar Pullorum (S. Pullorum) is the causative agent of pullorum disease (PD) and results in severe economic losses to the poultry industry. As a Salmonella type III secretion system 2 (T3SS2) effector and predicted membrane protein, CigR is encoded by the cigR gene within Salmonella pathogenicity island 3 (SPI3). In order to research the influence of the cigR gene on S. Pullorum, a cigR mutant of S. Pullorum S06004 was constructed by the lambda Red recombination system, and then its characterization was analysed. Lack of cigR did not affect the growth and biochemical properties, but resulted in decreased biofilm formation. The mutant strain was stable with the deletion of the cigR gene. Macrophage infection assay and in vivo competition assay showed that the mutant strain increased the replication and/or survival ability in the HD11 cell line and in chickens compared to that of the parent strain, the median lethal dose (LD50) of the mutant strain was one-fifth of the parent strain for 2-day-old chickens when injected intramuscularly. These results demonstrate CigR plays roles in biofilm formation and pathogenicity of S. Pullorum, deletion of cigR can significantly decrease biofilm formation and significantly increase virulence.