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Sichuan Da Xue Xue Bao. Yi Xue Ban =... Nov 2023To construct (LM) and (LI) balanced lethal systems expressing cervical cancer antigens, to study their basic biological characteristics, and to provide reference data...
OBJECTIVE
To construct (LM) and (LI) balanced lethal systems expressing cervical cancer antigens, to study their basic biological characteristics, and to provide reference data for the immunotherapy of cervical cancer.
METHODS
Through seamless cloning via ligation kit, the HPV16 67 fusion protein antigen gene constructed in our lab was spliced to the complement plasmid pCW-LM -Amp that contained the nutritional gene . Then, we replaced the ampicillin (Amp) resistance gene of the complement plasmid with the nutrition gene. The ligation reaction mixture was transformed into () recipient bacteria DH5αΔ and the complement plasmid pCW-67-LM -Ampfree, which expressed cervical cancer antigens and had no Amp resistance, was obtained by nutrition screening from the DH5αΔ. The plasmid pCW-67-LM -Ampfree was complemented into LMΔdd and LIΔdd, the attenuated nutrition-deficient strains with the virulence genes and and nutrition genes and deleted by electroporation, thereby obtaining LM and LI balanced lethal systems expressing cervical cancer antigen genes. The growth of the strains was observed. Western blot was performed to examine the status of antigen protein expression. PCR was performed to measure the passage stability of complement plasmid pCW-67-LM -Ampfree. Their basic biological characteristics were examined by biochemical reaction tests and hemolysis assay.
RESULTS
Two balanced lethal systems expressing cervical cancer antigen were successfully constructed. The HPV16 type E6E7 fusion protein was successfully expressed in the two balanced lethal systems. pCW-67-LM Ampfree, the positive plasmid expressing cervical cancer antigen, maintained stable existence in the two balanced lethal systems. The two balanced lethal systems expressing cervical cancer antigen showed significantly better recovery growth in comparison with nutrition deficiency strains. The results of biochemical reaction tests showed that most of the biochemical reaction of the two balanced lethal systems expressing cervical cancer antigen were consistent with those of attenuated strains. The two balanced lethal systems expressing cervical cancer antigen still maintained the hemolytic ability, although their hemolytic ability was slightly inferior to that of the balanced lethal systems not expressing cervical cancer antigen and the attenuated strains.
CONCLUSION
The two balanced lethal systems expressing cervical cancer antigen genes are constructed successfully. They display normal growth. The complement plasmid pCW-67-LM -Ampfree can maintain stable existence , showing little change in its biochemical characteristics and hemolytic ability. Further research should be conducted to investigate the potential of these two recombinant strains to be used as candidate strains for cervical cancer therapeutic vaccine.
Topics: Female; Humans; Uterine Cervical Neoplasms; Escherichia coli; Listeria; Listeria monocytogenes; Recombinant Proteins
PubMed: 38162066
DOI: 10.12182/20231160210 -
MBio Feb 2024Since 2010, the genus has had the addition of 22 new species that more than tripled the number of species identified until 2010. Sixteen of these 22 new species are... (Review)
Review
Since 2010, the genus has had the addition of 22 new species that more than tripled the number of species identified until 2010. Sixteen of these 22 new species are distantly related to the type species, , and several of these present phenotypes that distinguish them from classical species (, , , , and ). These 22 newly described species also show that is more genetically diverse than previously estimated. While future studies and surveys are needed to clarify the distribution of these species, at least some of these species may not be widely spread, while other species may be frequently found spread to human-related settings (e.g., farms and processing facilities), and others may be adapted to specific environmental habitats. Here, we review the taxonomic, phylogenetic, and ecological characteristics of these new species identified since 2010 and re-iterate the suggestion of re-classification of some species into three new genera: , , and . We also provide a review of current detection issues and the relevance to food safety related to the identification of these new species. For example, several new non-pathogenic species could be misidentified as the pathogen , based on methods that do not target -specific virulence genes/factors, leading to unnecessary product recalls. Moreover, eight species in the proposed new genus are not good indicators of environmental conditions that could allow to grow since species are unable to grow at low temperatures.
Topics: Humans; Phylogeny; Listeria; Listeria monocytogenes; Virulence Factors; Food Safety
PubMed: 38126771
DOI: 10.1128/mbio.00938-23 -
Antibiotics (Basel, Switzerland) Feb 2023The essential oil of was isolated by hydrodistillation and characterized by GC-MS. The antioxidant and antimicrobial activity of essential oil was evaluated, as well...
The essential oil of was isolated by hydrodistillation and characterized by GC-MS. The antioxidant and antimicrobial activity of essential oil was evaluated, as well as its biocompatibility (LDH and MTT methods). DPPH, FRAP, and CUPRAC methods were applied for antioxidant activity evaluation, while qualitative and quantitative assays (inhibition zone diameter, minimum inhibitory concentration, and minimum fungicidal concentration), NO release (by nitrite concentration determination), and microbial adhesion capacity to the inert substrate (the biofilm microtiter method) were used to investigate the antimicrobial potential. A total of 52 compounds were identified by GC-MS in essential oil, representing 97.43% of the total area. The major constituents were borneol (32.97%), 1,8-cineole (14.94%), camphor (10.13%), artemisia ketone (4.70%), α-terpineol (3.23%), and -eudesmol (3.23%). With MICs ranging from 0.78 to 30 μg/mL, the essential oil proved to inhibit the microbial adhesion and induce the NO release. To the best of our knowledge, the present study reports for the first time the antimicrobial activity of EO against clinically and biotechnologically important microbial strains, such as , , , , , , , , and , demonstrating its antimicrobial applications beyond the clinical field.
PubMed: 36830282
DOI: 10.3390/antibiotics12020371 -
Molecules (Basel, Switzerland) Dec 2022The main target of this work is to discover new protein fractions from natural resources with high antibacterial action. The 7S and 11S globulin fractions, as well as...
The main target of this work is to discover new protein fractions from natural resources with high antibacterial action. The 7S and 11S globulin fractions, as well as the basic subunit (BS), were isolated from lupine seeds (), chemically characterized, and screened for antibacterial activity against seven pathogenic bacteria. SDS-PAGE revealed molecular weights ranging from 55 to 75 kDa for 7S globulin, 20-37 kD for 11S globulin, and 20 kD for the BS. 11S globulin and the BS migrated faster on Urea-PAGE toward the cathode compared to 7S globulin. FTIR and NMR showed different spectral patterns between the 7S and 11S globulins but similar ones between 11S globulin and the BS. The MICs of the BS were in the range of 0.05-2 μg/mL against , , , , , , and compared to higher values for 11S globulin. The BS surpassed 11S globulin in antibacterial action, while 7S globulin showed no effect. The MICs of 11S globulin and the BS represented only 5% and 2.5% of the specific antibiotic against , respectively. Scanning electron microscopy (SEM) demonstrated different signs of cellular deformation and decay in the protein-treated bacteria, probably due to interaction with the bacterial cell wall and membranes. 11S globulin and the BS can be nominated as effective food biopreservatives.
Topics: Lupinus; Globulins; Vegetables; Anti-Bacterial Agents; Seeds
PubMed: 36615230
DOI: 10.3390/molecules28010035 -
Microbes and Infection May 2023Two species of Listeria are pathogenic, Listeria monocytogenes and Listeria ivanovii. Although studies have shown that dairy ruminants shed Listeria spp. in feces, there...
Two species of Listeria are pathogenic, Listeria monocytogenes and Listeria ivanovii. Although studies have shown that dairy ruminants shed Listeria spp. in feces, there is little information about ruminants that do not shed Listeria spp. in their feces but asymptomatically carry them in organs. We evidence that ruminants can asymptomatically carry L. ivanovii in udders and L. monocytogenes and L. ivanovii in tonsils without fecal shedding. Whole-genome sequence of L. monocytogenes and L. ivanovii contained known core genes involved in virulence and antibiotic resistance. This work highlights tonsils and udders as a Listeria intra-host site of colonization.
Topics: Animals; Listeriosis; Mammary Glands, Animal; Spain; Palatine Tonsil; Listeria; Ruminants; Listeria monocytogenes; Genomics; Feces
PubMed: 36464197
DOI: 10.1016/j.micinf.2022.105079 -
Microbial Biotechnology Nov 2022Expressing heterologous antigens by plasmids may cause antibiotic resistance. Additionally, antigen expression via plasmids is unstable due to the loss of the plasmid....
Expressing heterologous antigens by plasmids may cause antibiotic resistance. Additionally, antigen expression via plasmids is unstable due to the loss of the plasmid. Here, we developed a balanced-lethal system. The Listeria monocytogenes (LM) balanced-lethal system has been previously used as an antigen carrier to induce cellular immune response. However, thus far, there has been no reports on Listeria ivanovii (LI) balanced-lethal systems. The dal and dat genes from the LI-attenuated LIΔatcAplcB (LIΔ) were deleted consecutively, resulting in a nutrient-deficient LIΔdd strain. Subsequently, an antibiotic resistance-free plasmid carrying the LM dal gene was transformed into the nutrient-deficient strain to generate the LI balanced-lethal system LIΔdd:dal. The resultant bacterial strain retains the ability to proliferate in phagocytic cells, as well as the ability to adhere and invade hepatocytes. Its genetic composition was stable, and compared to the parent strain, the balanced-lethal system was substantially attenuated. In addition, LIΔdd:dal induced specific CD4 /CD8 T-cell responses and protected mice against LIΔ challenge. Similarly, we constructed an LM balanced-lethal system LMΔdd:dal. Sequential immunization with different recombinant Listeria strains will significantly enhance the immunotherapeutic effect. Thus, LIΔdd:dal combined with LMΔdd:dal, or with other balanced-lethal systems will be more promising alternative for vaccine development.
Topics: Mice; Animals; Listeria; Tuberculosis Vaccines; Listeria monocytogenes; Vaccines, Attenuated; Anti-Bacterial Agents
PubMed: 36069650
DOI: 10.1111/1751-7915.14137 -
Microorganisms Aug 2022and , are the only pathogenic species of the genus and share many virulence factors and mechanisms of pathogenicity. shows host tropism towards small ruminants and... (Review)
Review
and , are the only pathogenic species of the genus and share many virulence factors and mechanisms of pathogenicity. shows host tropism towards small ruminants and rodents and much lower virulence for humans compared to . However, severe infections caused by , resulting in bacteremia, abortion and stillbirth, occasionally occurred in immunocompromised persons and in pregnant women, while in immunocompetent hosts can cause gastroenteritis. In this review, the updated knowledge on virulence aspects and distribution of in the environment and in food is summarized. Recent research on its virulence characters at genome level gave indications on how pathogenicity evolved in this bacterial species. As for , infections occurred after the ingestion of contaminated food, so an overview of reports regarding its distribution in food products was carried out to obtain indications on the categories of foods exposed to contamination by . It was found that a wide variety of food products can be a source of this microorganism and that, like , is able to persist in the food production environment. Studies on its ability to grow in enrichment and isolation media suggested that its occurrence in nature might be underestimated. Moreover, virulence varies among strains for differences in virulence character regulation, presence/absence of genetic regions and the possible instability of a pathogenicity genomic island, LIPI-2, which is unique to . We can conclude that , as a possible pathogen for animals and humans, requires more focused investigations regarding its occurrence in the environment and in food and on intra-species variability of pathogenic potential.
PubMed: 36014096
DOI: 10.3390/microorganisms10081679 -
Frontiers in Microbiology 2022(LM) induces efficient and specific T-cell immune responses in the host. Listeriolysin O (LLO) is the main virulence protein of LM. LLO helps LM escape from the...
(LM) induces efficient and specific T-cell immune responses in the host. Listeriolysin O (LLO) is the main virulence protein of LM. LLO helps LM escape from the lysosome. However, the pronounced pathogenicity of LM limits its practical application as a live bacterial vector. (LI) also displays intracellular parasitic abilities, cell to cell transfer, and other LM properties, with an elevated biosafety relative to LM. We have confirmed that LI can be used as a viable bacterial vaccine vector. However, we have also observed that LI vector vaccine candidates survive in the immune organ (spleen) for a shorter time compared with the survival time of LM and elicit weaker immune responses compared with LM. Studies have confirmed that hemolysin correlates with some important biological properties of , including cell invasion, intracellular proliferation, and the ability to induce immune responses. We speculated that the weaker immunogenicity of LI compared to LM may be related to the function of ivanolysin O (ILO). Here, we established a hemolysin gene deletion strain, LIΔ, and a modified strain, LIΔ:, whose was replaced by . The hemolysin-modified strain was attenuated; however, it led to significantly improved invasive and proliferative activities of antigen-presenting cells, including those of RAW 264.7 macrophages, compared with the effects of LI. Mice immunized twice with LIΔ: showed higher cytokine levels and better challenge protection rates than LI-immunized mice. This is the first description in carrier vaccine research of the modification of LI hemolysin to obtain a better vaccine carrier than LI. The recombinant strain LIΔ: showed good biosafety and immunogenicity, and thus appears to be a good vector strain for vaccine development.
PubMed: 35935244
DOI: 10.3389/fmicb.2022.962326 -
Microbial Genomics Jul 2022() is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular...
() is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular lifecycle of and facilitate cell-to-cell spread, allowing it to occupy multiple niches within the host and cross-protective barriers, including the placenta. One family of virulence factors, internalins, contributes to pathogenicity by inducing specific uptake and conferring tissue tropism. Over 25 internalins have been identified thus far, but only a few have been extensively studied. Internalins contain leucine-rich repeat (LRR) domains that enable protein-protein interactions, allowing to bind host proteins. Notably, other species express internalins but cannot colonize human hosts, prompting questions regarding the evolution of internalins within the genus . Internalin P (InlP) promotes placental colonization through interaction with the host protein afadin. Although prior studies of InlP have begun to elucidate its role in pathogenesis, there remains a lack of information regarding homologs in other species. Here, we have used a computational evolutionary approach to identify InlP homologs in additional species. We found that () and () encode InlP homologs. We also found InlP-like homologs in and the recently identified species . All newly identified homologs lack the full-length LRR6 and LRR7 domains found in 's InlP. These findings are informative regarding the evolution of one key virulence factor, InlP, and serve as a springboard for future evolutionary studies of pathogenesis as well as mechanistic studies of internalins.
Topics: Bacterial Proteins; Female; Humans; Listeria; Listeria monocytogenes; Listeriosis; Placenta; Pregnancy; Virulence Factors
PubMed: 35904424
DOI: 10.1099/mgen.0.000828 -
Biology Jun 2022A cross-sectional study was performed in 325 sheep and 119 goat dairy farms in Greece. Samples of bulk-tank milk were examined by standard microbiological techniques for...
Isolation of from Bulk-Tank Milk of Sheep and Goat Farms-From Clinical Work to Bioinformatics Studies: Prevalence, Association with Milk Quality, Antibiotic Susceptibility, Predictors, Whole Genome Sequence and Phylogenetic Relationships.
A cross-sectional study was performed in 325 sheep and 119 goat dairy farms in Greece. Samples of bulk-tank milk were examined by standard microbiological techniques for spp. was isolated from one (0.3%) and from three (0.9%) sheep farms. No associations between the isolation of or and milk quality were found. No resistance to antibiotics was identified. Three variables emerged as significant predictors of isolation of the organism: the presence of pigs, low average relative humidity and a high number of ewes on the farm. The three isolates were assessed in silico for identification of plasmids, prophages, antibiotic resistance genes, virulence factors, CRISPRs and CAS genes. Phylogenetic analysis using the core genome revealed that the three strains belonged to the subsp. branch and were especially close to the PAM 55 strain. All strains of the branch appeared to be very similar, with the distance between them being small.
PubMed: 35741392
DOI: 10.3390/biology11060871