Autotransporter Pathway Research Articles

Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes.

Gingipains, extracellular cysteine proteinases of Porphyromonas gingivalis, constitute the major virulence factor of this periodontopathogenic bacterium. They are the product of three genes, two coding for an Arg-specific (RgpA and RgpB) and one for a Lys-specific proteinase (Kgp). Proteinase domains of RgpA and RgpB are virtually identical; however, the gene encoding the former enzyme is missing a large segment coding for hemaglutinin / adhesin (HA) domains. The latter domains are present also in Kgp. The tertiary structure of RgpB revealed that the proteinase domain of gingipains has a protein fold referred to as the caspase-hemoglobinase fold. On this basis, they are also evolutionary related to other highly specific proteinases including clostripain, caspases, legumains and separase (clan CD of cysteine peptidases). Gingipains are produced as large preproproteins and are subject to elaborate, not yet fully understood, secretion, glycosylation, activation, and maturation processes. How they traverse the outer membrane is unknown, although it can be hypothesized that they use an autotransporter pathway. Apparently during transport through the periplasm the LPS-like glycan moiety is added at the conserved C-terminal portion of progingipains. At the cell surface pro-gingipains fold into partially active, single-chain zymogens and undergo autocatalytic, intermolecular processing. Two sequential cleavages within the profragment domain enhance zymogen activity and in the case of RgpA and Kgp are followed by excision of the individual HA domains. These domains are further truncated at the C-terminus by concerted action of Kgp and carboxypeptidase and form a non-covalent multidomain, multifunctional complex anchored into the outer membrane by the glycated, C-terminal HA domain. This hypothetical scenario is a reasonable explanation for the occurrence of many forms of gingipains.

Functional esterase surface display by the autotransporter pathway in Escherichia coli

Bacterial surface display is a promising tool for a wide variety of biotechnological applications. In this work, a carboxylesterase, EstA from Burkholderia gladioli was translocated to the surface of Escherichia coli using the autotransporter pathway. For this purpose, an artificial gene was constructed by PCR, that encodes a fusion protein of EstA and the essential autotransporter domains. Esterase activity of whole cells expressing the EstA-autotransporter fusion protein could be detected by a filter overlay assay using α-naphthylacetate as substrate as well as by an agar plate pH-assay using p-nitrophenylacetate as a substrate. The specific esterase activity of whole cells was determined to be 1.7 mU/mg protein with p-nitrophenylacetate. After differential cell fractionation, the specific esterase activity of the outer membrane fraction was determined to be 23 mU/mg protein, using the same substrate. Western blot analysis of the different cell fractions with an EstA specific antibody yielded positive signals only in the outer membrane fraction. Furthermore, the detected protein band was in the correct size, as it was predictable from the amino acid sequence of the fusion protein. In activity staining of SDS-gels using α-naphthylacetate as a substrate, it was the identical band that exhibited esterase activity. Surface exposure could be demonstrated by proteinase K digestion of the esterase domain, whereby the protease was externally added to intact cells. These results indicated that EstA is targeted to the surface of E. coli by the autotransporter pathway in its active form.

Functional display of active bovine adrenodoxin on the surface of E. coli by chemical incorporation of the [2Fe-2S] cluster.

The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications. Up to now, however, cellular surface display was merely restricted to simple polypeptide chains. Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli. For this purpose apo-adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway. Incorporation of the iron-sulfur cluster was achieved by a single-vial, one-step titration under anaerobic conditions. The biological function of surface-displayed holo-adrenodoxin could be established through adrenodoxin-dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 10(5) per cell. Neither the expression of adrenodoxin nor the incorporation of the chemical iron-sulfur cluster reduced the viability of the bacterial cells.

The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation.

In this study, the sigA gene situated on the she pathogenicity island of Shigella flexneri 2a was cloned and characterized. Sequence analysis showed that sigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of Shigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.

Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga β autotransporter pathway

The Iga β autotransporter function of IgA1 protease from Neisseria gonorrhoeae was assessed in Escherichia coli using the Vibrio cholerae toxin B subunit (CtxB) as a heterologous passenger. N-terminal fusions with Iga β of native CtxB or mutant CtxB protein containing no cysteines were constructed and analysed in isogenic E. coli mutants carrying defects in either or both the ompT (outer membrane protease T) and dsbA (periplasmic disulfide oxidoreductase) determinants. While export of the cystein-less CtxB passenger was independent of the dsbA genotype, the native CtxB passenger was properly translocated across the outer membrane only in the dsbA mutant background. This effect was consistent in the presence and in the absence of the OmpT protease which rather determined the release of surface-bound CtxB into the medium. Therefore, in agreement with previous observations Iga β-dependent protein secretion requires an unfolded conformation of the passenger domain and can be blocked by disulfide loop formation in the presence of DsbA. Since DsbA acts in the periplasm, this provides evidence for a periplasmic intermediate in the Iga β-mediated export pathway. E. coli (dsbA ompT) is highly suitable as a strain for the surface display of recombinant proteins via Iga β, whether or not they contain cysteine residues.