Crystal structure and standardized geometric analysis of InlJ, a listerial virulence factor and leucine-rich repeat protein with a novel cysteine ladder

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-Å resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR-defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.     

Glycosylphosphatidylinositol-specific phospholipase D of human serum--activity modulation by naturally occurring amphiphiles

The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6,000-fold purified enzyme. This was obtained in 100 microg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) co-chromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidyl-serine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.     

Polarized packaging of bacteriophage lambda chromosomes.

Packaging of chromosomes during lytic growth of cohesive end-site (cos site) duplication strains of phage lambda is strikingly asymmetric; the duplication segment is generally at the left chromosome end (Emmons, 1974). In the present study, the packaging of non-replicating cos duplication chromosomes is shown to be similarly asymmetric. It is, therefore, likely that the packaging process itself is polarized, in an A to R direction. This conclusion is based on the study of packaging of repressed prophage chromosomes of dilysogenic strains of Escherichia coli by a heteroimmune helper. In these strains one of the two prophages contains a cos duplication (see Fig. 2). The frequency with which helper-packaged chromosomes carry the cos duplication segment agrees well with expectations derived from lytically grown phage.Haploid segregants are produced from the cos duplication strain at a lower level (35%) during lytic growth than during packaging of repressed prophage chromosomes (50%). This is expected if chromosomes are packaged processively (in sequence) during lytic growth.Packaging of repressed cos triplication chromosomes by a heteroimmune helper also yields a distribution of haploid and duplication chromosomes that agrees with expectations from lytically grown cos duplication phage and the assumption that the initial cutting of a cos site to initiate a packaging sequence is made at random.Polarized, processive packaging and random initial cutting are elements of a model of lambda chromosome packaging proposed by Emmons (1974), for which our experiments provide support.     

Effect of Coronatine on Ethylene Release and ATPase Activity of Tomato Cell Cultures

The effects of the phytotoxin coronatine formed by pathovars of Pseudomonas syringae on coronatine sensitive cell cultures of Lycopersicon peruvianum and Lycopersicon esculentum were investigated. The studied parameters were ethylene release, activity of membrane-bound ATPase and vitality using the TTC test. The cells of L. esculentum responded with an increase, the cells of L. peruvianum with a decrease of ethylene formation after application of different coronatine concentrations. These results indicate that the effect on ethylene release is not the primary effect of the toxin. The activity of membrane-bound ATPase was not affected by coronatine.     

Epidemiology of Pathogenic Enterobacteria in Humans,Livestock, and Peridomestic Rodents in Rural Madagascar

Background

Among the families of enteric bacteria are globally important diarrheal agents. Despite their potential for zoonotic and environmental transmission, few studies have examined the epidemiology of these pathogens in rural systems characterized by extensive overlap among humans, domesticated and peridomestic animals. We investigated patterns of infection with Enterotoxigenic Escherichia coli, Shigella spp., Salmonella enterica, Vibrio cholerae, and Yersinia spp. (enterocolitica, and pseudotuberculosis) in Southeastern Madagascar where the potential for the aforementioned interactions is high. In this pilot project we conducted surveys to examine behaviors potentially associated with risk of infection and if infection with specific enterobacteria species was associated with diarrheal disease.

Methodology/Principal Findings

PCR was conducted on DNA from human, livestock, and rodent fecal samples from three villages. Overall, human prevalence was highest (77%), followed by rodents (51%) and livestock (18%). Rodents were ∼2.8 times more likely than livestock to carry one of the bacteria. The incidence of individual species varied between villages, with the observation that, E. coli and Shigella spp. were consistently associated with co-infections. As an aggregate, there was a significant risk of infection linked to a water source in one village. Individually, different pathogens were associated with certain behaviors, including: those who had used medication, experienced diarrhea in the past four weeks, or do not use toilets.

Conclusions/Significance

Different bacteria were associated with an elevated risk of infection for various human activities or characteristics. Certain bacteria may also predispose people to co-infections. These data suggest that a high potential for transmission among these groups, either directly or via contaminated water sources. As these bacteria were most prevalent in humans, it is possible that they are maintained in humans and that transmission to other species is infrequent. Further studies are needed to understand bacterial persistence, transmission dynamics, and associated consequences in this and similar systems.     

Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes

During a bacterial infection, each successive step is orchestrated by a dedicated set of virulence factors. In Gram-positive bacteria, the presentation or release of such factors is crucially dependent on the continual remodelling of the cell wall. We have investigated the autolysin or peptidoglycan hydrolase Auto (Lmo1076) from the human pathogen Listeria monocytogenes to structurally and biochemically underpin its role in host cell invasion. We demonstrate that Auto is an N -acetylglucosaminidase, that it is autoinhibited when newly secreted but activated by proteolytic cleavage, that it has an acidic pH optimum and that it preferentially cleaves acetylated over de-acetylated peptidoglycan. The crystal structure of Auto, the first for glycoside hydrolase family 73, and the first for a listerial autolysin, indicates that autoinhibition is due to an N-terminal α-helix unique to Auto that physically blocks the substrate-binding cleft. We identify Glu122 and Glu156 as the two catalytically essential carboxylate groups. The physical properties of Auto as well as its localization to lipoteichoic acid by its four C-terminal GW modules imply cell wall degradation by Auto to be highly co-ordinated. Its spatio-temporally controlled activation and localized activity in an acidified environment indicate that it facilitates remodelling of the cell wall and may be involved in co-ordinating the release of virulence factors at specific stages of an infection.     

Levels of glucose dehydrogenases with different substrate specificities in rat and beef livers

The relative substrate specificities of glucose dehydrogenases (E.C. 1.1.1.47) from beef liver and rat liver are very different. The beef enzyme oxidizes glucose more rapidly than either glucose-6-phosphate or galactose-6-phosphate. On the other hand, the dehydrogenase from rat liver prefers the hexose phosphates to glucose.A procedure for estimating the level of glucose dehydrogenase in rat and beef liver is described. The glucose-6-phosphate dehydrogenase activity attributed to glucose dehydrogenases is estimated to be about one-fifth and one-third that of cytoplasmic glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) in female and male rat liver respectively.A fluorometric adaptation of the less sensitive spectrophotometric assay for glucose dehydrogenase is described.     

Pan‐Arctic sea ice‐algal chl a biomass and suitable habitat are largely underestimated for multiyear ice

There is mounting evidence that multiyear ice (MYI) is a unique component of the Arctic Ocean and may play a more important ecological role than previously assumed. This study improves our understanding of the potential of MYI as a suitable habitat for sea ice algae on a pan‐Arctic scale. We sampled sea ice cores from MYI and first‐year sea ice (FYI) within the Lincoln Sea during four consecutive spring seasons. This included four MYI hummocks with a mean chl a biomass of 2.0 mg/m², a value significantly higher than FYI and MYI refrozen ponds. Our results support the hypothesis that MYI hummocks can host substantial ice‐algal biomass and represent a reliable ice‐algal habitat due to the (quasi‐) permanent low‐snow surface of these features. We identified an ice‐algal habitat threshold value for calculated light transmittance of 0.014%. Ice classes and coverage of suitable ice‐algal habitat were determined from snow and ice surveys. These ice classes and associated coverage of suitable habitat were applied to pan‐Arctic CryoSat‐2 snow and ice thickness data products. This habitat classification accounted for the variability of the snow and ice properties and showed an areal coverage of suitable ice‐algal habitat within the MYI‐covered region of 0.54 million km² (8.5% of total ice area). This is 27 times greater than the areal coverage of 0.02 million km² (0.3% of total ice area) determined using the conventional block‐model classification, which assigns single‐parameter values to each grid cell and does not account for subgrid cell variability. This emphasizes the importance of accounting for variable snow and ice conditions in all sea ice studies. Furthermore, our results indicate the loss of MYI will also mean the loss of reliable ice‐algal habitat during spring when food is sparse and many organisms depend on ice‐algae.