Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element.

Cell surface-located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface-located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild-type bacteria to a capsule-negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule-negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element-based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.     

Capsule phase variation in Neisseria meningitidis serogroup B by slipped-strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule-deficient variants indicated that the capsular polysaccharide was re-expressed at a frequency of 10^?3. Measurement of enzymatic activities involved in the biosynthesis of the α-2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule-negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)₇ residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood-culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo-(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the out-break of meningococcal disease.     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Metagenomic analysis of a stable trichloroethene-degrading microbial community

Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.     

Molecular divergence of the sia locus in different serogroups of Neisseria meningitidis expressing polysialic acid capsules

The serogroups B, C, W135 and Y of Neisseria meningitidis express chemically and immunologically distinct capsular polysaccharides containing sialic acid. In the case of serogroup B meningococci sialic acid is synthesized by the gene products of a locus termed sia and forms the homopolymers of the capsule. The organization of the genes required for sialic acid synthesis in serogroups B, C, W135 and Y was elucidated by PCR technology. Cloning, sequencing and the functional expression of the polysialyltransferase (PST) genes of serogroups B and C demonstrated that the difference in capsule composition derives from the presence of related, but distinct siaD genes coding for PSTs. Analysis of meningococci of serogroups W135 and Y expressing sialic acid heteropolymers revealed that the DNA sequences of the corresponding genetic loci in these serogroups were highly homologous, but differed completely from the siaD genes of serogroups B and C. This finding suggests that enzymes unrelated to those of serogroups B and C are required for the formation of sialic acid heteropolymers characteristic of the capsules of serogroups W135 and Y.     

Complex O-acetylation in Legionella pneumophila serogroup 1 lipopolysaccharide. Evidence for two genes involved in 8-O-acetylation of legionaminic acid.

A putative gene encoding an O-acetyl transferase, lag-1, is involved in biosynthesis of the O-polysaccharide (polylegionaminic acid) in some Legionella pneumophila serogroup 1 strains. To study the effect of the presence and absence of the gene on the O-polysaccharide O-acetylation, lag-1 from strain Philadelphia 1 was expressed in trans in the naturally lag-1-negative OLDA strain RC1, and immunoblot analysis revealed that the lag-1-encoded O-acetyl transferase is active. O-Polysaccharides of different size were prepared from the lipopolysaccharides of wild-type and transformant strains by mild acid degradation followed by gel-permeation chromatography. Using NMR spectroscopy and MALDI-TOF mass spectrometry, it was found that O-acetylation of the first three legionaminic acid residues next to the core occurs in the short-chain O-polysaccharide (<10 sugars) from both strains. Hence, there is another O-acetyl transferase encoded by a gene different from lag-1. In the longer-chain O-polysaccharide, a legionaminic acid residue proximal to the core is N-methylated and could be further 8-O-acetylated in the lag-1-dependent manner. Only strains expressing a functional lag-1 gene were recognized in Western blot analysis by monoclonal antibody 3/1 requiring 8-O-acetylated polylegionaminic acid for binding. The highly O-acetylated outer core region of the lipopolysaccharide is involved in the epitope of another serogroup 1-specific monoclonal antibody termed LPS-1. The O-acetylation pattern of the L. pneumophila serogroup 1 core oligosaccharide was revised using MALDI-TOF mass spectrometry. lag-1-independent O-acetylation of the core and short-chain O-polysaccharide was found to be a common feature of L. pneumophila serogroup 1 strains. The biological importance of conserved lag-1-independent and variable lag-1-dependent O-acetylation is discussed.     

Neisseria meningitidis Induces Brain Microvascular Endothelial Cell Detachment from the Matrix and Cleavage of Occludin: A Role for MMP-8

Disruption of the blood-brain barrier (BBB) is a hallmark event in the pathophysiology of bacterial meningitis. Several inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), nitric oxide and matrix metalloproteinases (MMPs), contribute to this disruption. Here we show that infection of human brain microvascular endothelial cells (HBMEC) with Neisseria meningitidis induced an increase of permeability at prolonged time of infection. This was paralleled by an increase in MMP-8 activity in supernatants collected from infected cells. A detailed analysis revealed that MMP-8 was involved in the proteolytic cleavage of the tight junction protein occludin, resulting in its disappearance from the cell periphery and cleavage to a lower-sized 50-kDa protein in infected HBMEC. Abrogation of MMP-8 activity by specific inhibitors as well as transfection with MMP-8 siRNA abolished production of the cleavage fragment and occludin remained attached to the cell periphery. In addition, MMP-8 affected cell adherence to the underlying matrix. A similar temporal relationship was observed for MMP activity and cell detachment. Injury of the HBMEC monolayer suggested the requirement of direct cell contact because no detachment was observed when bacteria were placed above a transwell membrane or when bacterial supernatant was directly added to cells. Inhibition of MMP-8 partially prevented detachment of infected HBMEC and restored BBB permeability. Together, we established that MMP-8 activity plays a crucial role in disassembly of cell junction components and cell adhesion during meningococcal infection.