Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo . IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to ACE2 receptor and later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the N-terminal domain (NTD) and in P1‘position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells and decrease GE:PFU ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high affinity interaction of the spikes with the ACE2 receptor.

Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo.

Bacillaene Mediates the Inhibitory Effect of Bacillus subtilis on Campylobacter jejuni Biofilms

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multi-species biofilms can affect the survival of enteric pathogens that contaminate food products, and thus investigating the underlying mechanisms of multi-species biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt pre-established C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log10 and disperses 26 h old pre-established C. jejuni biofilms. Furthermore, the co-inoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces reducing it by 2.4 log10. We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for non-ribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety. IMPORTANCE Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens), but also the most frequent cause of bacterial food-borne infection in humans. This pathogen forms biofilms which mend survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of food-borne infections. The beneficial bacterium, Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during co-culture and destroys the pre-established C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.

Efecto antimicrobiano de la tunta (Solanum juzepczukii) sobre la Salmonella enterica subespecie enterica serovar Typhimurium

The objective of the study was to investigate the antimicrobial activity of tunta on Salmonella enterica subspecies enterica serovar Typhimurium. A natural isolate of this bacterium was used that was resistant to chloramphenicol. The experiment required the preparation of a standard solution of 0,8 g / ml of tunta extract. The Kirby Bauer technique was used for the antimicrobial evaluation of the tests and the Duraffourd scale to measure the level of sensitivity. Resulting, for the ten technical repetitions of the experimental group, an average halo of 10,40 mm and a standard deviation of 0,63 of DHI (level of limit sensitivity, according to said scale). In conclusion, our work shows evidence that tunta has an antimicrobial effect at a borderline sensitivity level on Salmonella enterica subspecies enterica serovar Typhimurium. After more in-depth research, this derivative of Solanum Jueepczukii could become an alternative for the development of antibiotics of natural origin and allow a sustainable development of Andean cultures.

Rampant loss of social traits during domestication of a Bacillus subtilis natural isolate

Most model bacteria have been domesticated in laboratory conditions. Yet, the tempo with which a natural isolate diverges from its ancestral phenotype under domestication to a novel laboratory environment is poorly understood. Such knowledge, however is essential to understanding the rate of evolution, the time scale over which a natural isolate can be propagated without loss of its natural adaptive traits, and the reliability of experimental results across labs. Using experimental evolution, phenotypic assays, and whole-genome sequencing, we show that within a week of propagation in a common laboratory environment, a natural isolate of Bacillus subtilis acquires mutations that cause changes in a multitude of traits. A single adaptive mutational step in the gene coding for the transcriptional regulator DegU impairs a DegU-dependent positive autoregulatory loop and leads to loss of robust biofilm architecture, impaired swarming motility, reduced secretion of exoproteases, and to changes in the dynamics of sporulation across environments. Importantly, domestication also resulted in improved survival when the bacteria face pressure from cells of the innate immune system. These results show that degU is a target for mutations during domestication and underscores the importance of performing careful and extremely short-term propagations of natural isolates to conserve the traits encoded in their original genomes.

Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome

ABSTRACT Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo. This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.

Rescue of SARS-CoV-2 from a single bacterial artificial chromosome

ABSTRACTAn infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC).Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

Potential anti-proliferative effects of chemical constituents and hemisynthetic derivatives from Scadoxus pseudocaulus (Amarillydaceae)

Background: Biological significance of Amaryllidaceae is well advocated from the literature. In Cameroon, plants from this fam- ily are routinely used for the cure of liver, cancer and cardiovascular diseases. To date, no scientific investigation corresponding to the anti-cancer activity of extracts and isolated compounds of Scadoxus pseudocaulus is available. Objective: Current study is focused to elaborate the anti-proliferative effects of natural isolates (compounds 1-6, 9) and hemi-synthetic analogs (compounds 7-8) extracted from S. pseudocaulu. Methods: Column chromatography of the ethyl acetate extract followed by purification of different fractions led to the isolation of seven compounds (1 – 6, 9). Esterification reaction of compound 6 was carried out using butyroyl chlorides and triethylamin to produce two derivatives (7 – 8). The cytotoxic activity was performed after staining of treated cells with florescent dye propid- ium iodide. Dead cells were detected using cytometer FL2 or FL3 channels/filters. Results: Trans-derivative of narciclasine (a natural isolate from S. pseudocaulus), was found to be most potent among all tested compounds. Its effects were more significant on low malignant follicular lymphoma (DoHH2 cells) as compared to highly ma- lignant (EBV infected) Burkitts lymphoma (Raji cells). Conclusion: From our results, narciclasine appears to hold the potential of a lead molecule that can be used to bridge the ther- apeutic gaps in cancer research. Keywords: Scadoxus pseudocaulus; Amaryllidaceae; 7-deoxy-trans-dihydronarciclasin; farrerol; derivatization; cytotoxic activity.

Synthesis and antiproliferative effect of the proposed stereoisomer of the marine sponge metabolite halisphingosine A

The purported isomer of halisphingosine A was built up in 11 steps and 29% yield by catalytic Henry and hydrogenation reactions. Its 13C-NMR data differed from that of the natural isolate. It was antiproliferative in various tumour cells.

Diversity in lac Operon Regulation among Diverse Escherichia coli Isolates Depends on the Broader Genetic Background but Is Not Explained by Genetic Relatedness

ABSTRACT Transcription of bacterial genes is controlled by the coordinated action of cis- and trans-acting regulators. The activity and mode of action of these regulators can reflect different requirements for gene products in different environments. A well-studied example is the regulatory function that integrates the environmental availability of glucose and lactose to control the Escherichia coli lac operon. Most studies of lac operon regulation have focused on a few closely related strains. To determine the range of natural variation in lac regulatory function, we introduced a reporter construct into 23 diverse E. coli strains and measured expression with combinations of inducer concentrations. We found a wide range of regulatory functions. Several functions were similar to the one observed in a reference lab strain, whereas others depended weakly on the presence of cAMP. Some characteristics of the regulatory function were explained by the genetic relatedness of strains, indicating that differences varied on relatively short time scales. The regulatory characteristics explained by genetic relatedness were among those that best predicted the initial growth of strains following transition to a lactose environment, suggesting a role for selection. Finally, we transferred the lac operon, with the lacI regulatory gene, from five natural isolate strains into a reference lab strain. The regulatory function of these hybrid strains revealed the effect of local and global regulatory elements in controlling expression. Together, this work demonstrates that regulatory functions can be varied within a species and that there is variation within a species to best match a function to particular environments. IMPORTANCE The lac operon of Escherichia coli is a classic model for studying gene regulation. This study has uncovered features such as the environmental input logic controlling gene expression, as well as gene expression bistability and hysteresis. Most lac operon studies have focused on a few lab strains, and it is not known how generally those findings apply to the diversity of E. coli strains. We examined the environmental dependence of lac gene regulation in 20 natural isolates of E. coli and found a wide range of regulatory responses. By transferring lac genes from natural isolate strains into a common reference strain, we found that regulation depends on both the lac genes themselves and on the broader genetic background, indicating potential for still-greater regulatory diversity following horizontal gene transfer. Our results reveal that there is substantial natural variation in the regulation of the lac operon and indicate that this variation can be ecologically meaningful.