The cell wall envelope of Gram-positive pathogens functions as a scaffold for the attachment of virulence factors and as a sieve that prevents diffusion of molecules. Here the isd genes (iron-regulated surface determinant) of Staphylococcus aureus were found to encode factors responsible for hemoglobin binding and passage of heme-iron to the cytoplasm, where it acts as an essential nutrient. Heme-iron passage required two sortases that tether Isd proteins to unique locations within the cell wall. Thus, Isd appears to act as an import apparatus that uses cell wall-anchored proteins to relay heme-iron across the bacterial envelope.

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.

Mycobacterium tuberculosis secretes ESAT-6, a virulence factor that triggers cell-mediated immune responses and IFN-␥ production during tuberculosis. ESAT-6 is transported across the bacterial envelope by a specialized secretion system with a FSD (FtsK-SpoIIIE domain) membrane protein. Although the presence of ESAT-6-like genes has been identified in the genomes of other microbes, the possibility that they may encode general virulence functions has hitherto not been addressed. Herein we show that the human pathogen Staphylococcus aureus secretes EsxA and EsxB, ESAT-6-like proteins, across the bacterial envelope. Staphylococcal esxA and esxB are clustered with six other genes and some of these are required for synthesis or secretion of EsxA and EsxB. Mutants that failed to secrete EsxA and EsxB displayed defects in the pathogenesis of S. aureus murine abscesses, suggesting that this specialized secretion system may be a general strategy of human bacterial pathogenesis.specialized secretion ͉ Gram-positive ͉ exoprotein ͉ ess

Staphylococcus aureus, a bacterial commensal of the human nares and skin, is a frequent cause of soft tissue and bloodstream infections. A hallmark of staphylococcal infections is their frequent recurrence, even when treated with antibiotics and surgical intervention, which demonstrates the bacterium’s ability to manipulate innate and adaptive immune responses. In this Review, we highlight how S. aureus virulence factors inhibit complement activation, block and destroy phagocytic cells and modify host B and T cell responses, and we discuss how these insights might be useful for the development of novel therapies against infections with antibiotic resistant strains such as methicillin-resistant S. aureus.

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 sigma E regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA, that decreased the sigma E-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 sigma E 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/skp genes. 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.

Escherichia coli responds to the accumulation of misfolded proteins by inducing the transcription of heat shock genes. E E RNA polymerase controls one of the two heat shock regulons of E. coli. This regulon is activated upon accumulation of misfolded polypeptides in the double membrane envelope of E. coli. E (RpoE) is a member of the extracytoplasmic function subfamily of sigma factors. Here we asked how many genes are activated by E E RNA polymerase and what is the identity of these genes. Using two independent genetic approaches, 20 E. coli promoters were identified which activate reporter gene transcription in a E -dependent manner. In all cases examined, a canonical E binding site could be revealed upon mapping transcriptional start sites. 10 identified promoters activated the transcription of previously identified genes with four genes acting directly on the folding of E. coli envelope proteins (dsbC, fkpA, skp, and surA). The remaining promoters transcribed genes that are presumed to encode hitherto unknown extracytoplasmic functions and were named ecf (ecfA-ecfM). Two of these ecf genes were found to be essential for E. coli growth.Heat shock and other environmental stresses result in the misfolding of polypeptides in all cells. Escherichia coli responds to the accumulation of misfolded polypeptides by activating the transcription of heat shock genes. Heat is a drastic stress that leads to protein unfolding in general and triggers two heat shock responses controlled by two distinct RNA polymerase species in E. coli 1 : ␣ 2 ␤␤Ј 32 and ␣ 2 ␤␤Ј E , E 32, and E E , respectively (1, 2). The unfolding of proteins in the envelope of E. coli uniquely induces the E regulon but not E 32 (3, 4). E (RpoE) is a member of the extracytoplasmic function (ECF) subfamily of sigma factors which function as effector molecules responding to extracytoplasmic stimuli (3, 5). Some microorganisms such as Streptomyces coelicolor harbor multiple ECFs that seem specialized in responding to different extracytoplasmic stimuli (5, 6). The E. coli E regulon is induced specifically in response to imbalanced synthesis of outer membrane proteins (7) and to misfolding of polypeptides that have been translocated across the cytoplasmic membrane (8).Previous work identified several genes that are transcribed by E E (4). E E directs its own expression. rpoE is the first gene of an operon that also contains rseA, rseB, and rseC (regulator of sigma E, genes A, B, and C (9, 10). RseA is a short hydrophobic polypeptide that integrates into the cytoplasmic membrane. The N-terminal cytoplasmic domain of RseA binds to E , sequestering the sigma factor from core RNA polymerase (E) (9, 10). The C-terminal domain of RseA protrudes into the periplasm, a compartment located between the cytoplasmic and outer membranes of E. coli. The C-terminal domain of RseA interacts with RseB, a periplasmic soluble protein (9, 10). RseB is believed to sense the concentration of misfolded polypeptides, causing RseB dissociation from RseA and liberating cytoplasmic E for i...

Previous work has established that the transcription factor sigma E (sigma 24) is necessary for maintaining the induction of the heat shock response of Escherichia coli at high temperatures. We have identified the gene encoding sigma E using a genetic screen designed to isolate trans‐acting mutations that abolish expression from either htrA or rpoHP3, two promoters recognized uniquely by sigma E‐containing RNA polymerase. Such a screen was achieved by transducing strains carrying a single copy of either phtrA‐lacZ or rpoHP3‐lacZ fusions with mutagenized bacteriophage P1 lysates and screening for Lac‐ mutant colonies at 22 degrees C. Lac‐ mutants were subsequently tested for inability to grow at 43 degrees C (Ts‐ phenotype). Only those Lac‐ Ts‐ mutants that were unable to accumulate heat shock proteins at 50 degrees C were retained for further characterization. In a complementary approach, those genes which when cloned on a multicopy plasmid led to higher constitutive expression of the sigma E regulon were characterized and mapped. Both approaches identified the same gene, rpoE, mapping at 55.5 min on the E.coli genetic map and encoding a polypeptide of 191 amino acid residues. The wild‐type and a mutant rpoE gene products were over‐expressed and purified. It was found that the purified wild‐type sigma E protein, when used in in vitro run‐off transcription assays in combination with core RNA polymerase, was able to direct transcription from the htrA and rpoHP3 promoters, but not from known sigma 70‐dependent promoters. In vivo and in vitro analyses of rpoE transcriptional regulation showed that the rpoE gene is transcribed from two major promoters, one of which is positively regulated by sigma E itself.

Bacterial invasion of host tissues triggers polymorphonuclear leukocytes to release DNA (NETs, neutrophil extracellular traps), thereby immobilizing microbes for subsequent clearance by innate defenses including macrophage phagocytosis. We report here that Staphylococcus aureus escapes these defenses by converting NETs to deoxyadenosine, which triggers the caspase-3 mediated death of immune cells. Conversion of NETs to deoxyadenosine requires two enzymes, nuclease and adenosine synthase, that are secreted by S. aureus and necessary for the exclusion of macrophages from staphylococcal abscesses. Thus, the pathogenesis of S. aureus infections has evolved to anticipate host defenses and to repurpose them for the destruction of the immune system.