Dominique Missiakas

New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH

A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the σE regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA, that decreased the σE‐dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM (rfaD) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM (rfaD) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn10 insertions were isolated, which led to an increased activity of the σE regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA, fkpA and ompH/skpgenes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.

Modulation of the Escherichia coli sigmaE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins.

The σE (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The σE regulon is induced upon misfolding of proteins in the periplasm or the outer membrane. Similar to other alternative sigma factors, the activity of σE is tightly regulated in E. coli. We have previously shown that σE is positively autoregulated at the transcriptional level. DNA sequencing, coupled with transcriptional analyses, have shown that σE is encoded by the first gene of a four‐gene operon. The second gene of this operon, rseA, encodes an anti‐σE activity. This was demonstrated at both the genetic and biochemical levels. For example, mutations in rseA constitutively increase σE activity. Consistent with this, overproduction of RseA leads to an inhibitory effect on σE activity. Topological analysis of RseA suggests the existence of one transmembrane domain, with the N‐terminal part localized in the cytoplasm. Overproduction of this N‐terminal domain alone was shown to inhibit σE activity. These observations were confirmed in vitro, because either purified RseA or only its purified N‐terminal domain inhibited transcription from EσE‐dependent promoters. Furthermore, RseA and σE co‐purify, and can be co‐immunoprecipitated, and chemically cross‐linked. The σE activity is further modulated by the products of the remaining genes in this operon, rseB and rseC. RseB is a periplasmic protein, which negatively regulates σE activity and specifically interacts with the C‐terminal periplasmic domain of RseA. In contrast, RseC is an inner membrane protein that positively modulates σE activity. Most of these protein–protein interactions were verified in vivo using the yeast two‐hybrid system.

The extracytoplasmic function sigma factors: role and regulation.

Alternative sigma factors provide a means of regulating gene expression in response to various extracellular changes. One such class of sigma factors appears to control a variety of functions, including expression of heat‐shock genes in Escherichia coli, biosynthesis of alginates and carotenoids in Pseudomonas aeruginosa and Myxococcus xanthus, respectively, iron uptake in E. coli and Pseudomonas spp., nickel and cobalt efflux in Alcaligenes europhus, plant pathogenicity in Pseudomonas syringae and synthesis of outer membrane proteins in Photobacterium sp. strain SS9. Most of these activities deal with extracytoplasmic functions, and such sigmas have been designated as ECF sigma factors. They have also been characterized in Mycobacteria as well as Gram‐positive bacteria such as Streptomyces coelicolor and Bacillus subtilus and the archaea Sulpholobus acidocaldarius. ECF factors belong to a subfamily of the sigma 70 class, based on their sequence conservation and function across bacterial species. The promoter consensus sequences recognized by the ECF factors are also highly conserved. In most of the cases, the activity of these factors is modulated by a cognate inner membrane protein that has been shown, both in E. coli and in P. aeruginosa, to act as an anti‐sigma activity. This inner membrane protein is presumed to serve as a sensor and signalling molecule, allowing an adaptive response to specific environmental change. Presumably, an on‐and‐off switch of the anti‐sigma activity leads to the release of the sigma factor and thereby to the co‐ordinate transcription of the specific regulon it governs.

Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues

Staphylococcus aureus infections are associated with abscess formation and bacterial persistence; however, the genes that enable this lifestyle are not known. We show here that following intravenous infection of mice, S. aureus disseminates rapidly into organ tissues and elicits abscess lesions that develop over weeks but cannot be cleared by the host. Staphylococci grow as communities at the center of abscess lesions and are enclosed by pseudocapsules, separating the pathogen from immune cells. By testing insertional variants in genes for cell wall‐anchored surface proteins, we are able to infer the stage at which these molecules function. Fibrinogen‐binding proteins ClfA and ClfB are required during the early phase of staphylococcal dissemination. The heme scavenging factors IsdA and IsdB, as well as SdrD and protein A, are necessary for abscess formation. Envelope‐associated proteins, Emp and Eap, are either required for abscess formation or contribute to persistence. Fluorescence microscopy revealed Eap deposition within the pseudocapsule, whereas Emp was localized within staphylococcal abscess communities. Antibodies directed against envelope‐associated proteins generated vaccine protection against staphylococcal abscess formation. Thus, staphylococci employ envelope proteins at discrete stages of a developmental program that enables abscess formation and bacterial persistence in host tissues.—Cheng, A. G., Kim, H. K., Burts, M. L., Krausz, T., Schneewind, O., Missiakas, D. M. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. FASEB J. 23, 3393–3404 (2009). www.fasebj.org

IDENTIFICATION AND CHARACTERIZATION OF A NEW DISULFIDE ISOMERASE-LIKE PROTEIN (DSBD) IN ESCHERICHIA COLI

Previous studies have established that DsbA and DsbC, periplasmic proteins of Escherichia coli, are two key players involved in disulfide bond formation. A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a new gene, designated dsbD. Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin. In addition, unlike the rest of the dsb genes, dsbD is essential for bacterial growth at temperatures above 42 degrees C. Cloning of the wild‐type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein. A 138 amino acid subdomain of the protein was purified and shown to possess an oxido‐reductase activity in vitro. Expressing this subdomain in the periplasmic space helped restore the phenotypic defects associated with a dsbD null mutation. Interestingly, this domain shares 45% identity with the portion of the eukaryotic protein disulfide isomerase carrying the active site. We further show that in dsbD mutant bacteria the dithiol active sites of DsbA and DsbC proteins are mostly oxidized, as compared with wild‐type bacteria. Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining proper redox conditions. The finding that overexpression of DsbD leads to a Dsb‐ phenotype, very similar to that exhibited by dsbA null mutants, is in good agreement with such a model.

Host defenses against Staphylococcus aureus infection require recognition of bacterial lipoproteins

Toll-like receptors and other immune-signaling pathways play important roles as sensors of bacterial pattern molecules, such as peptidoglycan, lipoprotein, or teichoic acid, triggering innate host immune responses that prevent infection. Immune recognition of multiple bacterial products has been viewed as a safeguard against stealth infections; however, this hypothesis has never been tested for Staphylococcus aureus, a frequent human pathogen. By generating mutations that block the diacylglycerol modification of lipoprotein precursors, we show here that S. aureus variants lacking lipoproteins escape immune recognition and cause lethal infections with disseminated abscess formation, failing to elicit an adequate host response. Thus, lipoproteins appear to play distinct, nonredundant roles in pathogen recognition and host innate defense mechanisms against S. aureus infections.

Nontoxigenic protein A vaccine for methicillin-resistant Staphylococcus aureus infections in mice

The current epidemic of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections has caused significant human morbidity, but a protective vaccine is not yet available. Prior infection with S. aureus is not associated with protective immunity. This phenomenon involves staphylococcal protein A (SpA), an S. aureus surface molecule that binds to Fcγ of immunoglobulin (Ig) and to the Fab portion of VH3-type B cell receptors, thereby interfering with opsonophagocytic clearance of the pathogen and ablating adaptive immune responses. We show that mutation of each of the five Ig-binding domains of SpA with amino acid substitutions abolished the ability of the resulting variant SpAKKAA to bind Fcγ or Fab VH3 and promote B cell apoptosis. Immunization of mice with SpAKKAA raised antibodies that blocked the virulence of staphylococci, promoted opsonophagocytic clearance, and protected mice against challenge with highly virulent MRSA strains. Furthermore, SpAKKAA immunization enabled MRSA-challenged mice to mount antibody responses to many different staphylococcal antigens.

Contribution of Coagulases towards Staphylococcus aureus Disease and Protective Immunity

The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine.

Protein secretion and surface display in Gram-positive bacteria

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions.

Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta‐type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP‐independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.