Type III secretion: what's in a name?

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 ϩ ESAT-6 ϩ 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 ϩ T cell response, which might influence the chosen strategy to localize recombinant antigen in RASVs.

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“…5) have been exploited to target recombinant antigens to the bacterial cell surface or extracellular milieu (13)(14)(15)(16)(17)(18)(19)(20).…”

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confidence: 99%

Controlled Human Malaria Infection: Applications, Advances, and Challenges

2018

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“…The T3SS is a transmembrane heteroprotein structure that spans both the inner and the outer plasma membranes (8,9) and secretes between 1,000 and 10,000 amino acid residues per second (10,11). Two classes of T3SS have been well characterized and are known as the injectisome and the flagella (12). Heterologous proteins secreted by the T3SS include spider silk (13), fibronectinbinding protein (14), and neuroactive peptides (10).…”

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confidence: 99%

Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

Metcalf

Finnerty

Azam

et al. 2014

Appl Environ Microbiol

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The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ؎ 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant.

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“…The term "T3S system" refers to translocation-associated and flagellar T3S systems that probably evolved from a common ancestor (24). Both T3S systems consist of a structurally conserved membrane-spanning basal body, which presumably contains two pairs of cylindrical rings in the inner and outer membrane (21,74).…”

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confidence: 99%

Functional Characterization of the Type III Secretion ATPase HrcN from the Plant Pathogen Xanthomonas campestris pv. vesicatoria

2009

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Many gram-negative plant and animal pathogenic bacteria employ a type III secretion (T3S) system to inject effector proteins into the cytosol of eukaryotic host cells. The membrane-spanning T3S apparatus is associated with an ATPase that presumably provides the energy for the secretion process. Here, we describe the role of the predicted ATPase HrcN from the plant pathogenic bacterium Xanthomonas campestris pathovar vesicatoria. We show that HrcN hydrolyzes ATP in vitro and is essential for T3S and bacterial pathogenicity. Stability of HrcN in X. campestris pv. vesicatoria depends on the conserved HrcL protein, which interacts with HrcN in vitro and in vivo. Both HrcN and HrcL bind to the inner membrane protein HrcU and specifically localize to the bacterial membranes under T3S-permissive conditions. Protein-protein interaction studies revealed that HrcN also interacts with the T3S substrate specificity switch protein HpaC and the global T3S chaperone HpaB, which promotes secretion of multiple effector proteins. Using an in vitro chaperone release assay, we demonstrate that HrcN dissociates a complex between HpaB and the effector protein XopF1 in an ATP-dependent manner, suggesting that HrcN is involved in the release of HpaB-bound effectors. Effector release depends on a conserved glycine residue in the HrcN phosphate-binding loop, which is crucial for enzymatic activity and protein function during T3S. There is no experimental evidence that T3S can occur in the absence of the ATPase, in contrast to recent findings reported for animal pathogenic bacteria.The majority of gram-negative bacteria employ a type III secretion (T3S) system to transport proteins across both bacterial membranes. The term "T3S system" refers to translocation-associated and flagellar T3S systems that probably evolved from a common ancestor (24). Both T3S systems consist of a structurally conserved membrane-spanning basal body, which presumably contains two pairs of cylindrical rings in the inner and outer membrane (21, 74). The flagellar basal body is connected to an extracellular hook and the flagellar filament, which is the key bacterial motility organelle. In contrast, the basal body of translocation-associated T3S systems is associated with an extracellular pilus (plant pathogens) or needle (animal pathogens), which serves as a conduit for secreted proteins to the host-pathogen interface (32). Translocationassociated T3S systems translocate bacterial effector proteins directly into the eukaryotic host cell cytosol and are major pathogenicity determinants of most plant and animal pathogenic bacteria. Translocation of effector proteins is mediated by the T3S translocon, a predicted transport channel that inserts into the host plasma membrane (12,20,32). Since T3S translocon mutants are completely nonpathogenic, effector protein delivery is presumably crucial for the host-pathogen interaction.The signal for T3S and translocation is often located in the N-terminal regions of secreted proteins and is not conserved on the amino acid level (...

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Type III secretion: what's in a name?

Cited by 57 publications

References 46 publications

Controlled Human Malaria Infection: Applications, Advances, and Challenges

Controlled Human Malaria Infection: Applications, Advances, and Challenges

Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

Functional Characterization of the Type III Secretion ATPase HrcN from the Plant Pathogen Xanthomonas campestris pv. vesicatoria

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