Gunna Christiansen

Involvement of N‐acyl‐l‐homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens

Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer‐cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high‐performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N‐butanoyl‐l‐homoserine lactone (BHL) and N‐hexanoyl‐l‐homoserine lactone (HHL) in cell‐free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.

Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae

SummaryThree novel fim genes of Escherichia coli, fimF, fimG and fimH, were characterized. These genes were not necessary for the production of fimbriae but were shown to be involved in the adhesive property and longitudinal regulation of these structures. Complementation experiments indicated that both the major fimbrial subunit gene, fimA, and the fimH gene in combination with either the fimF or the fimG gene were required for mannose-specific adhesion. The fimF, fimG and fimH gene products were likewise shown to play a major role in the fimbrial morphology as longitudinal modulators. The amount of FimF, FimG and FimH proteins appeared to control the length and number of the fimbriae. The DNA sequence of a 2050 bp region containing the three genes was determined. The corresponding protein sequences all exhibited homology with the fimbrial subunit protein, FimA.

Functional amyloid in Pseudomonas

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid‐like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA–F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.

SDS-Induced Fibrillation of α-Synuclein: An Alternative Fibrillation Pathway

A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in Parkinsons disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per alphaSN molecule, which is also the range where SDS binding is associated with the highest enthalpy. Fibrillation is only reduced in proportion to the fraction of SDS below 25 mol% SDS in mixed surfactant mixtures with nonionic surfactants and is inhibited by formation of bulk micelles and induction of alpha-helical structure. In this fibrillogenic complex, 4 alphaSN molecules initially associate with 40-50 SDS molecules to form a shared micelle that gradually grows in size. The complex initially exhibits a mixture of random coil and alpha-helix, but incubation results in a structural conversion into beta-sheet structure and concomitant formation of thioflavin-T-binding fibrils over a period of several hours. Based on small-angle X-ray scattering, the aggregates elongate as a beads-on-a-string structure in which individual units of ellipsoidal SDS-alphaSN are bridged by strings of the protein, so that aggregates nucleate around the surface of protein-stabilized micelles. Thus, fibrillation in this case occurs by a process of continuous accretion rather than by the rate-limiting accumulation of a distinct nucleus. The morphology of the SDS-induced fibrils does not exhibit the classical rod-like structures formed by alphaSN when aggregated by agitation in the absence of SDS. The SDS-induced fibrils have a flexible worm-like appearance, which can be converted into classical straight fibrils by continuous agitation. SDS-induced fibrillation represents an alternative and highly reproducible mechanism for fibrillation where protein association is driven by the formation of shared micelles, which subsequently allows the formation of beta-sheet structures that presumably link individual micelles. This illustrates that protein fibrillation may occur by remarkably different mechanisms, testifying to the versatility of this process.

Interleukin‐1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection

Chlamydia trachomatis infection is associated with severe Fallopian tube tissue damage leading to tubal infertility and ectopic pregnancy. To explore the molecular mechanisms behind infection an ex vivo model was established from human Fallopian tubes and examined by scanning electron microscopy and immunohistochemistry. Extensive tissue destruction affecting especially ciliated cells was observed in C. trachomatis infected human Fallopian tube organ culture. Interleukin‐1 (IL‐1) produced by epithelial cells was detected after infection. Addition of IL‐1 receptor antagonist (IL‐1RA) completely eliminated tissue destruction induced by C. trachomatis. The anti‐inflammatory cytokine IL‐10 reduced the damaging effect of C. trachomatis infection, however, to a lesser extent than IL‐1RA. Furthermore, IL‐1 was found to induce IL‐8, a neutrophil attractant, using a signal transduction pathway involving p38 MAP kinase. Consequently, IL‐1 has the potential to generate a cellular infiltrate at the site of infection in vivo. Blocking the IL‐1 receptors by IL‐1RA eliminated tissue destruction and cytokine production. Hence, these studies show the importance of IL‐1 in initiating the tissue destruction observed in the Fallopian tube following C. trachomatis infection. Because leukocytes are absent in the ex vivo model, this study strongly indicates that IL‐1 is the initial proinflammatory cytokine activated by C. trachomatis infection.

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection.

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia‐containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule‐associated Chlamydia vesicles also collocated with tyrosine‐phosphorylated McCoy cell proteins. After migration, the Chlamydia‐containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule‐dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule‐dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR‐1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR‐1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.

Proteome analysis of the Chlamydia pneumoniae elementary body.

Chlamydia pneumoniaeis an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniaecultivated in Hep‐2 cells were 35S‐labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two‐dimensional gel electrophoresis. Excised protein spots were in‐gel digested with trypsin and peptides were concentrated on reverse‐phase chromatographic beads for identification analysis by matrix‐assisted laser desorption/ionization‐mass spectrometry. In the pH range from 3–11, 263 C. pneumoniaeprotein spots encoded from 167 genes were identified. These genes constitute 15 % of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.

Branching in Amyloid Fibril Growth

Using the peptide hormone glucagon and Abeta(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40 degrees . Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Abeta(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Abeta(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth.

The fimD gene required for cell surface localization of Escherichia coli type 1 fimbriae

SummaryThe fimD gene of Escherichia coli K12 was shown to be necessary for surface localization of type 1 fimbriae, since deletion of the gene resulted in a virtually bald phenotype. The FimD protein was found to be located in the outer membrane. Expressed alone, this protein had a very deleterious effect on cell growth. The DNA sequence of the fimD gene was determined; the corresponding amino acid sequence of the FimD protein was compared with those of the PapC and FaeD proteins. A deletion derivative of FimD gave clues as to which parts of the protein were necessary for outer membrane integration.

Induction of phospholipase- and flagellar synthesis in Serratia liquefaciens is controlled by expression of the flagellar master operon flhD.

When a liquid culture of Serratia spp. reaches the last part of the logarithmic phase of growth it induces the synthesis of several extracellular hydrolytic enzymes. In this communication we show that synthesis and secretion of the extracellular phospholipase is coupled to expression of flagella. Expression of flagella is demonstrated to follow a growth‐phase‐dependent pattern. Cloning, complementation studies and DNA‐sequencing analysis has identified a genetic region in Serratia liquefaciens which exhibits extensive homology to the Escherichia coli flhD flagellar master operon. Interruption of the chromosomal flhD operon in S. liquefaciens results in non‐flagellated and phospholipase‐negative cells, but the synthesis of other exoenzymes is not affected. By placing the flhD operon under the control of a foreign inducible promoter we have shown that increased transcription through the flhD operon leads to induction of flagellar synthesis and phospholipase expression.