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The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3[beta] to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity. type III secretion system

Helicobacter pylori persistently colonizes about half the human population and contributes to the development of peptic ulcer disease and gastric cancer. This organism has evolved means to structurally alter its surface characteristics to evade innate and adaptive immune responses. H. pylori produces LPS O-antigen units that can be posttranslationally fucosylated to generate Lewis antigens, structures also found on human epithelial cells. We demonstrate an extensive diversity of Lewis x and Lewis y expression in LPS O-antigen units, occurring over time and in different regions of the human stomach. Lewis expression patterns were correlated with the on/off status of the three fucosyltransferases (FucT), FutA, FutB, and FutC, which are regulated via slipped-strand mispairing in intragenic polyC tract regions of the corresponding genes. The [alpha]1,3-FucT, FutA and FutB, each contain a C-terminal heptad repeat region, consisting of a variable number of DD/NLRV/INY tandem repeats. Variations in the number of heptad repeats correlated to the sizes of O-antigen polymers to become decorated by fucose residues. Our data support a molecular ruler mechanism for how H. pylori varies its LPS fucosylation pattern, where one heptad repeat in the enzyme corresponds to one N-acetyl-[beta]-lactosamine unit in the O-antigen polysaccharide. chronic | human stomach | phase variation

The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O--dominant human populations., The gastric pathogen Helicobacter pylori causes chronic inflammation, which may progress to peptic ulceration and gastric cancer (1). H. pylori expresses adhesins that confer intimate adherence to the gastric epithelium, [...]

For many pathogens, the ability to regulate their replication in host cells is a key element in establishing persistency. Here, we identified a single point mutation in the gene for polynucleotide phosphorylase (PNPase) as a factor affecting bacterial invasion and intracellular replication, and which determines the alternation between acute or persistent infection in a mouse model for Salmonella enterica infection. In parallel, with microarray analysis, PNPase was found to affect the mRNA levels of a subset of virulence genes, in particular those contained in Salmonella pathogenicity islands 1 and 2. The results demonstrate a connection between PNPase and Salmonella virulence and show that alterations in PNPase activity could represent a strategy for the establishment of persistency.

Amyloid is associated with debilitating human ailments including Alzheimer's and prion diseases. Biochemical, biophysical, and imaging analyses revealed that fibers produced by Escherichia coil called cure were amyloid. The CsgA curlin subunit, purified in the absence of the CsgB nucleator, adopted a soluble, unstructured form that upon prolonged incubation assembled into fibers that were indistinguishable from curli. In vivo, curli biogenesis was dependent on the nucleation-precipitation machinery requiring the CsgE and CsgF chaperone-like and nucleator proteins, respectively. Unlike eukaryotic amyloid formation, curli biogenesis is a productive pathway requiring a specific assembly machinery., Bacteria express a variety of cell-surface proteinacious filaments that can promote colonization of an epithelial surface, entry into host cells, exchange of DNA between bacteria, and development of bacterial communities [...]

Author(s): Per Uhlén [1, 2]; Ãsa Laestadius [1, 2]; Timo Jahnukainen [1]; Tomas Söderblom [3]; Fredrik Bäckhed [3]; Gianni Celsi [1]; Hjalmar Brismar [1]; Staffan Normark [3]; Anita Aperia [1]; [...]

The mppA genetic locus was isolated and characterized. The periplasmic binding protein was found to be essential for import of the bacterial cell wall peptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate. Two mutants, one with a mutation in groESL and the other with a mutation in oppB, were shown to be deficient in Opp function due to their rejection of triornithine toxicity. The oppB mutation suggested that murein tripeptide is transferred from MppA into the cytoplasm through membrane components of Opp.

An experiment designed to clone and characterize two operons for curli biogenesis from Salmonella typhimurium SR-11 was conducted. Results show similarities in the gene order and flanking genes of the curli fibers compared with their Escherichia coli counterparts. Biogenesis regulation was the same between S. typhimurium's Sr-11 and ATCC 14028-1 and E. coli's MC4100 and Y Mel.

The genetic characteristics of the novel TlpA protein was analyzed to characterize its thermosensing capacity in bacterial cells. Analysis of transcriptional fusion activity after the induction of tlpA in bacterial cells indicated the role of the protein as a novel gene regulator that exhibited a built-in thermosensing capacity. The sensor-regulator activity of TlpA was also based on a monomer-to-coiled-coil equilibrium which represented a unique adaptation of the coiled-structure for the regulation of gene function.

The potential interactions of the transcriptional regulator AmpR with intermediates of the degradative and/or biosynthetic pathways of murein metabolism in gram negative bacteria were investigated. Transcription assay results revealed that AmpR activates ampC beta-lactamase synthesis. The murein precursor, UDP-MurNAc-pentapeptide, on the other hand, inhibits AmpR-mediated transcriptional activation in vitro but such inhibition can be offset by the action of the muropeptide, anhMurNAc-tripeptide which accumulates in beta-lactamase-overproducing mutants. It is thus evident that cytosolic intermediates of murein biosynthesis and degradation operate as a cell-wall sensing device as they control beta-lactamase production.

Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins. We have found that polymerization of curlin to fimbriae-like structures (curli) on the surface of Escherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein. The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleator-presenting cell or by adjacent cells. The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation.

The pilC1 gene, present in the MS11 strain of Neisseria gonorrhoeae, contains amino acid sequences similar to those present in the opa and pil gene locus. These amino acid sequences are repetitive in nature and are probably involved in recombination during the formation of pili, an antigen. The amino acid structure of pilC1 is 90% similar to that of pilC2 gene. The pilC1 gene is a part of the chromosome that encodes for pili and is formed by DNA duplication. Pili controls attachment of the bacterial cell to the host cell. The amino acid sequence of pilC1 is given.

The research of the role of asparagine 152 in the catalytic process of Escherichia coli AmpC beta-lactamase was done by site-directed mutagenesis. The substrate hydrolysis rate decreases by factors of 500-5000. The rate of acylation registers a 2-50-fold decrease and deacylation registers a 50-500-fold decrease. The wild-type AmpC beta-lactamase exists as an equilibrium mixture of the two forms.

An investigation of the requirement of the GII adhesin for the induction of acute pyelonephritis and/or cystitis by P-fimbriated Escherichia coli using an isogenic papG mutant DS17-8 reveals that the wild-type DS17 and DS17-8 induce bladder infection in cynomolgus monkeys while DS17 results in pyelonephritis. The absence of the Gal(alpha1-4)Gal-specific PapG adhesin in the mutant DS17-8 indicates that the incidence of acute pyelonephritis requires the ability of P-fimbriae to bind carbohydrates.

In beta lactam hydrolysis, serine hydroxyl groups occupy the position of the hydroxyl group of Try 150 in class A enzymes and the two hydroxyl groups function as a general base in catalysis or at least protonate the nitrogen as it is exuded during the formation of acyl intermediate. Tyr 150 action is similar to the action of essential histidine of serine proteases to enhance protein bond hydrolysis. The effects of mutagenesis of the serine residue on the activity of class A enzyme are different from those with AmpC enzyme due to perturbation of structure by the residue.

Site-directed mutagenesis enables the investigation of the function of conserved Lys315 residue in the catalytic process of class C beta-lactamase. Histidine replaces Lys315 in Enterobacter cloacae. The replacement causes introduction of a titratable group in the probe to help determine the function of Lys315.

The degree of Neisseria meningitidis adhesion to cells is influenced by PilC antigenic variation through changes in pilin glycosylation. PilC acts as an adhesive through a mechanism unrelated to pilus biogenesis. Neisseria meningitidis has two PilC genes termed PilC1 and PilC2, which influence pilus biogenesis.

Helicobacter pylori is associated with development of gastritis, gastric ulcers, and adenocarcinomas in humans. The [Lewis.sup.b] ([Le.sup.b]) blood group antigen mediates H. pylori attachment to human gastric mucosa. Soluble glycoproteins presenting the [Le.sup.b] antigen or antibodies to the [Le.sup.b] antigen inhibited bacterial binding. Gastric tissue lacking [Le.sup.b] expression did not bind H. pylori. Bacteria did not bind to [Le.sup.b] antigen substituted with a terminal GaINAcα 1-3 residue (blood group A determinant), suggesting that the availability of H. pylori receptors might be reduced in individuals of blood group A and B phenotypes, as compared with blood group O individuals., Helicobacter pylori, a prevalent human-specific pathogen, is a causative agent in chronic active gastritis (1), gastric and duodenal ulcers (2), and gastric adenocarcinoma (3), one of the most common forms [...]

The only family of bacterial proteins identified to have an immunoglobulin fold is needed for the assembly of adhesive pili. These proteins employ their unique immunoglobulin features in recognition processes that are being studied. Investigations into the action of periplasmic chaperones also reveal a great deal about the basic biological processes of assembly functions. Not all bacterial adhesins are assembled into pilus rods, and the ones that cannot are called nonpilus adhesins.

An in situ assay for Helicobacter pylori adhesion to gastric epithelium was developed to elucidate the cell lineage-specific patterns of binding during infection. The results showed that H. pylori adhesins interact specifically with surface mucous cells in the gastric epithelium. The interaction is mediated by fucosylated rather than sialylated glycoprotein receptors located on the surface of mucous cells.

A new mechanism for the phagocytic processing of bacterial antigens by class I major histocompatibility complex I molecules has been discovered. This mechanism, which works for antigens lacking cytosolic penetration, produces resistance to the drugs cycloheximide and Brefeldin A. In contrast, these drugs are able to inhibit the classical class I pathway. This pathway may allow CD8 T-cells to be used against intracellular pathogens.

The pili of Neisseria gonnorhoeae are virulence factors that undergo both phase and structural variation. The phase variation results from alterations in the G-rich tract of the signal-peptide coding region of pilC. The basis of the structural variation in N. gonnorhoeae pili was elucidated by mutational analysis. The results showed that PilC- mutants contain sequence alterations in pilE, resulting in production and processing of soluble pilin. The results further show that structural variation is the result of a selective mechanism in which the selective pressure is exerted by an on/off switch in PilC expression.

The in vivo processing of lipase in Staphylococcus aureus was elucidated by analysis of lipase gene products and culture supernatants. The results indicated that S. aureus lipase is secreted as an 82 KDa prolipase having full lipolytic activity, which is subsequently processed outside the cell. The processing is hypothesized to occur in a stepwise manner with the intermediates having full lipolytic activity as the mature 45 to 46 KDa lipase. A metallocysteine protease seems to be involved in the initial step, while other different proteases contribute to successive processing.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.04.032 Byline: Robert Daniels (1)(2), Staffan Normark (1)(2) Abstract: The chaperone/usher pathway is responsible for the assembly of adhesive pili on the surface of gram-negative pathogenic bacteria. In this issue, present the crystal structure of the PapC usher translocation domain and images of the FimD usher bound to a pilus translocation intermediate. These new structures provide the first detailed view of a translocase in action. Author Affiliation: (1) Swedish Institute for Infectious Disease Control, 171 82 Solna, Sweden (2) Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden

Author(s): Staffan Normark [1]; Birgitta Henriques Normark [1]; Mathias Hornef [1] As recently as 1936, pneumonia was the major cause of death in the United States. Today, the bacterium Streptococcus [...]

Research has been conducted on Escherichia coli asr gene. The authors report the characterization of this gene and its product, discuss its role in stress response, and compare asr mutant strain and a wild-type strain under different conditions.

Research has been conducted on Escherichia coli BarA and UvrY proteins. Results demonstrate that mutations in E. coli sensor kinase gene barA or response regulator gene uvrY affect survival in long-term competition cultures .

Many Gram-negative bacteria interact with human, animal, or plant hosts by injecting effector proteins into the cytosol of host cells through the so-called type III secretion system (injectisome) (1). The [...]

Author(s): Birgitta Henriques Normark [1]; Staffan Normark [1]; Anna Norrby-Teglund [1] Staphylococcus aureus colonizes the noses of about 30% of healthy humans, normally without causing disease [1]. But it can [...]

Author(s): Birgitta Henriques Normark [1]; Staffan Normark (corresponding author) [1] Mild infections, such as streptococcal throat infection, as well as life-threatening illnesses can be caused by group A streptococci. This [...]

The bacterium Helicobacter pylori possesses antibacterial activity to which it is itself resistant. This activity has been traced to cecropin-like amino-terminal peptides derived from the ribosomal protein L1 (RpL1). It appears that bacterial lysis on the stomach may release L1-derived cecropin-like peptides that are active against faster-growing microorganisms located there. It also seems that cecropins have evolved from an early rpl1 gene in a prokaryote that was first an intracellular parasite, then a symbiont, then an organelle.

A new gene of Escherichia coli induced by low external pH has been identified. The gene named, asr, (for acid shock RNA), has been mapped at 35.98 min on the E. coli chromosome. Its DNA sequence shows an open reading frame for a 111-amino-acid polypeptide with a molecular mass of approximately 11.6 kDa. Its transcript contains a region similar to the Pho box upstream from the determined transcription start. The DNA region was observed to bind PhoB proteins in vitro.