Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

Chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation.     

Structure and mechanism of the lipooligosaccharide sialyltransferase from Neisseria meningitidis

The first x-ray crystallographic structure of a CAZY family-52 glycosyltransferase, that of the membrane associated α2,3/α2,6 lipooligosaccharide sialyltransferase from Neisseria meningitidis serotype L1 (NST), has been solved to 1.95 Å resolution. The structure of NST adopts a GT-B-fold common with other glycosyltransferase (GT) families but exhibits a novel domain swap of the N-terminal 130 residues to create a functional homodimeric form not observed in any other class to date. The domain swap is mediated at the structural level by a loop-helix-loop extension between residues Leu-108 and Met-130 (we term the swapping module) and a unique lipid-binding domain. NST catalyzes the creation of α2,3- or 2,6-linked oligosaccharide products from a CMP-sialic acid (Neu5Ac) donor and galactosyl-containing acceptor sugars. Our structures of NST bound to the non-hydrolyzable substrate analog CMP-3F_(axial)-Neu5Ac show that the swapping module from one monomer of NST mediates the binding of the donor sugar in a composite active site formed at the dimeric interface. Kinetic analysis of designed point mutations observed in the CMP-3F_(axial)-Neu5Ac binding site suggests potential roles of a requisite general base (Asp-258) and general acid (His-280) in the NST catalytic mechanism. A long hydrophobic tunnel adjacent to the dimer interface in each of the two monomers contains electron density for two extended linear molecules that likely belong to either the two fatty acyl chains of a diglyceride lipid or the two polyethylene glycol groups of the detergent Triton X-100. In this work, Triton X-100 maintains the activity and increases the solubility of NST during purification and is critical to the formation of ordered crystals. Together, the mechanistic implications of the NST structure provide insight into lipooligosaccharide sialylation with respect to the association of substrates and the essential membrane-anchored nature of NST on the bacterial surface.     

Sequence analysis of porothramycin biosynthetic gene cluster

The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.     

Radiation protection from whole-body gamma irradiation (6.7 Gy): behavioural effects and brain protein-level changes by an aminothiol compound GL2011 in the Wistar rat

GL2011 is a naturally occurring thiol compound and a series of thiol compounds have been proposed as radioprotectors. Radioprotective efficacy of a triple intraperitoneal dose of GL2011 of 100 mg/kg body weight of Wistar rats, 30 min prior to and 3 and 6 h following irradiation (6.7 Gy) was evaluated. Four groups of animals were used, vehicle-treated non-irradiated (VN), GL2011-treated and irradiated (GI), GL2011-treated and non-irradiated (GN) and vehicle-treated and irradiated (VI) (n = 30 per group). The radioprotective efficacy of GL2011 was determined by measuring 28-day survival and intestinal crypt cell survival. Neuroprotection in terms of behaviour was evaluated using the behavioural observational battery, open field test and elevated plus maze paradigm. An RNA microarray was carried out in order to show differences at the RNA level between VI and VN groups. Brain protein changes were identified using a gel-based proteomics method and major brain receptor complex levels were determined by blue-native gels followed by immunoblotting. 28-Day survival rate in VI was 30 %, in GI survival was 93 %, survival of VN and GN was 100 %. Jejunal crypt cell survival was significantly enhanced in GI. Protein-level changes of peroxiredoxin-5, Mn-superoxide dismutase 2, voltage-dependent anion-selective channel protein 1, septin 5 and dopamine D2 receptor complex levels were paralleling radiation damage and protection. Taken together, the findings demonstrate that GL2011 improves survival rates and jejunal crypt survival, provides partial neuroprotection at the behavioural level and modulates proteins known to be involved in protection against oxidative stress-mediated cell damage.     

Colostrum of Healthy Slovenian Mothers: Microbiota Composition and Bacteriocin Gene Prevalence

Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial ''inoculum'' for their infants’ gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of Staphylococcus and Gemella in most of the samples and exposed 4 clusters based on the abundance of 3 bands: Staphylococcus epidermidis/Gemella, Streptococcus oralis/pneumonia and Streptococcus salivarius. Bacilli represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), Clostridia (95.6%), Bacteroides-Prevotella group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml^-1) varied greatly among the samples; Staphylococcus epidermidis and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus Staphylococcus was represented by the species Staphylococcus epidermidis (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence.     

Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.