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ABSTRACT: Autotransporters are widely distributed among Gram-negative bacteria. They can have a large variety of functions and many of them have a role in virulence. They are synthesized as large precursors with an N-terminal signal sequence that mediates transport across the inner membrane via the Sec machinery and a translocator domain that mediates the transport of the connected passenger domain across the outer membrane to the bacterial cell surface. Like integral outer membrane proteins, the translocator domain folds in a β-barrel structure and requires the Bam machinery for its insertion into the outer membrane. After transport across the outer membrane, the passenger may stay connected via the translocator domain to the bacterial cell surface or it is proteolytically released into the extracellular milieu. Based on the size of the translocator domain and its position relative to the passenger in the precursor, autotransporters are divided into four sub-categories. We review here the current knowledge of the biogenesis, structure and function of various autotransporters.
Research in Microbiology 04/2013; · 2.76 Impact Factor
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ABSTRACT: Various methods that are routinely used to study the subcellular localization of membrane proteins in wild-type Gram-negative bacteria fall short in genetic studies addressing the biogenesis of outer membrane proteins (OMPs). Here, we describe three biochemical methods that can be used in such studies to evaluate the proper assembly of OMPs into the outer membrane. The methods are based on (1) the differential electrophoretic mobility of folded and nonnative OMPs, (2) the intrinsically high protease resistance of folded OMPs, and (3) the observation that integral membrane proteins are not extracted from the membrane in solutions containing high concentrations of urea.
Methods in molecular biology (Clifton, N.J.) 01/2013; 966:223-37.
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ABSTRACT: Autotransporters of Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain, and the secreted passenger domain in between. The autotransporter NalP of Neisseria meningitidis includes a protease domain that facilitates the release of several immunogenic proteins from the cell surface into the extracellular milieu. Rather exceptional among autotransporters, NalP is a lipoprotein. Here, the authors investigated the role of lipidation in the biogenesis and the function of the protein. To this end, the N-terminal cysteine, which is lipidated in the wild-type protein, was substituted by alanine. Like the wild-type protein, the mutant protein was secreted into the medium demonstrating that lipidation is not required for the biogenesis of the protein. However, the non-lipidated NalP variant had a drastically reduced capacity to cleave its substrate proteins from the cell surface suggesting that the lipid moiety is important for function. Kinetic experiments demonstrated that the autocatalytic processing of the non-lipidated protein at the cell surface was much faster than that of the wild-type protein. Thus, the lipid moiety delays the release of NalP from the cell surface thereby allowing it to release other surface-exposed proteins into the milieu.
Microbiology 12/2012; · 3.06 Impact Factor
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ABSTRACT: The periplasmic chaperones Skp, SurA, and DegP are implicated in the biogenesis of outer membrane proteins (OMPs) in Escherichia coli. Here, we investigated whether these chaperones exert similar functions in Neisseria meningitidis. Although N. meningitidis does not contain a homolog of the protease/chaperone DegP, it does possess a homolog of another E. coli protein, DegQ, which can functionally replace DegP when overproduced. Hence, we examined whether in N. meningitidis, DegQ acts as a functional homolog of DegP. Single skp, surA, and degQ mutants were easily obtained, showing that none of these chaperones is essential in N. meningitidis. Furthermore, all combinations of double mutants were generated and no synthetic lethality was observed. The absence of SurA or DegQ did not affect OMP biogenesis. In contrast, the absence of Skp resulted in severely lower levels of the porins PorA and PorB but not of other OMPs. These decreased levels were not due to proteolytic activity of DegQ, since porin levels remained low in a skp degQ double mutant, indicating that neisserial DegQ is not a functional homolog of E. coli DegP. The absence of Skp resulted in lower expression of the porB gene, as shown by using a P(porB)-lacZ fusion. We found no cross-species complementation when Skp of E. coli or N. meningitidis was heterologously expressed in skp mutants, indicating that Skp functions in a species-specific manner. Our results demonstrate an important role for Skp but not for SurA or DegQ in OMP biogenesis in N. meningitidis.
Journal of bacteriology 02/2011; 193(7):1612-21. · 3.94 Impact Factor
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ABSTRACT: The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides: glucuronoxylomannan and galactoxylomannan. The presence of this polysaccharide capsule was previously shown to interfere with maturation of human dendritic cells (DCs), possibly by shielding cell-wall components from interacting with these host immune cells. Here we show that two mutant strains of C. neoformans, both lacking a visible capsule due to a defect in glucuronoxylomannan synthesis, differentially activate human monocyte-derived DCs. Cells from a cap59 mutant, but not of a cap10 mutant strain, induce maturation of DCs as indicated by an increase in the expression of the costimulatory molecules CD80 and CD86, and production of the cytokines interleukin (IL)-10, IL-12p40 and tumor necrosis factor alpha. Interestingly, cap59 mutant cells reassociated with a concentrated culture medium of wild-type C. neoformans had lost their capacity to induce DC maturation. Summarizing, our data suggest that glucuronoxylomannan confers properties to the capsule that protect the fungus against activation of DCs; however, the presence of intact glucuronoxylomannan is not an absolute requirement to prevent activation of DCs.
FEMS Immunology & Medical Microbiology 08/2009; 57(2):142-50. · 2.44 Impact Factor
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ABSTRACT: The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional (1)H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.
Eukaryotic Cell 07/2009; 8(8):1165-73. · 3.60 Impact Factor
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ABSTRACT: The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.
Eukaryotic Cell 09/2008; 7(10):1865-73. · 3.60 Impact Factor
