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.

Transcription of the plasmid-encoded toxin gene from enteroaggregative Escherichia coli is regulated by a novel co-activation mechanism involving CRP and Fis.

Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhoea in developing countries. EAEC 042 is the prototypical strain. EAEC 042 secretes the functionally well-characterized Pet autotransporter toxin that contributes to virulence through its cytotoxic effects on intestinal epithelial cells. Following a global transposon mutagenesis screen of EAEC 042, the transcription factors, CRP and Fis, were identified as essential for transcription of the pet gene. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation of pet. This study complements previous findings that establish Fis as a key virulence regulator in EAEC 042.

A generalised module for the selective extracellular accumulation of recombinant proteins.

BACKGROUND: It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. RESULTS: Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. CONCLUSIONS: The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.

Mutational and topological analysis of the Escherichia coli BamA protein.

The multi-protein ß-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of ß-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded ß-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a ß-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA ß-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify ß-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA ß-barrel are essential.