Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Treatment of vulvo-vaginal atrophy with hyaluronate-based gel: a randomized controlled study.

BACKGROUND: The study aimed to assess the performance and safety of single-use applicators prefilled with hyaluronic acid (HA)-based vaginal gel (Hyalo Gyn) for the treatment of vulvovaginal atrophy (VVA)-related signs and symptoms in postmenopausal women. METHODS: Eighty women were randomized, 46 to Hyalo Gyn gel and 34 to a standard treatment (water-based lubricant). Outcome data collected included: the proportion of subjects having a reduction ≥1 point in the Verbal Rating Scale (VRS) Dryness Score, VRS Global Score, vaginal pH, Vaginal Health Index, Female Sexual Function Index, Female Sexual Distress Scale-Revised, patients' global assessment, tolerability, and safety. RESULTS: Significant improvements were observed for all the assessed endpoints in both treatment groups. The perception of any improvement on the VRS Dryness Score was not statistically different between the treatment groups. A sensitivity analysis on the absolute change in VRS Dryness Score showed a statistically significant difference in favor of the Hyalo Gyn group at 3 months. CONCLUSIONS: A 3-month treatment with Hyalo Gyn gel is effective, safe, and well tolerated for VVA-related symptoms in postmenopausal women.

Antigen Localization Influences the Magnitude and Kinetics of Endogenous Adaptive Immune Response to Recombinant Salmonella Vaccines.

The use of recombinant attenuated Salmonella vaccine (RASV) strains is a promising strategy for presenting heterologous antigens to the mammalian immune system to induce both cellular and humoral immune responses. However, studies on RASV development differ on where heterologous antigens are expressed and localized within the bacterium, and it is unclear how antigen localization modulates the immune response. Previously, we exploited the plasmid-encoded toxin (Pet) autotransporter system for accumulation of heterologous antigens in cell culture supernatant. In the present study, this Pet system was used to express early secretory antigen 6 (ESAT-6), an immunodominant and diagnostic antigen from Mycobacterium tuberculosis, in Salmonella enterica serovar Typhimurium strain SL3261. Three strains were generated, whereby ESAT-6 was expressed as a cytoplasmic (SL3261/cyto), surface-bound (SL3261/surf), or secreted (SL3261/sec) antigen. Using these RASVs, the relationship between antigen localization and immunogenicity in infected C57BL/6 mice was systematically examined. Using purified antigen and specific tetramers, we showed that mice infected with the SL3261/surf or SL3261/sec strain generated large numbers of Th1 CD4+ ESAT-6+ splenic T cells compared to those of mice infected with SL3261/cyto. While all mice showed ESAT-6-specific antibody responses when infected with SL3261/surf or SL3261/sec, peak total serum IgG antibody titers were reached more rapidly in mice that received SL3261/sec. Thus, how antigen is localized after production within bacteria has a more marked effect on the antibody response than on the CD4+ T cell response, which might influence the chosen strategy to localize recombinant antigen in RASVs.

Sequencing a piece of history: complete genome sequence of the original Escherichia coli strain.

In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5 144 392 bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2 803 932 bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577 351 bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157 : H7; therefore, it is not an ancestral state for the species.