Adelfia Talà

The Neisseria meningitidis Capsule Is Important for Intracellular Survival in Human Cells

ABSTRACT While much data exist in the literature about how Neisseria meningitidis adheres to and invades human cells, its behavior inside the host cell is largely unknown. One of the essential meningococcal attributes for pathogenesis is the polysaccharide capsule, which has been shown to be important for bacterial survival in extracellular fluids. To investigate the role of the meningococcal capsule in intracellular survival, we used B1940, a serogroup B strain, and its isogenic derivatives, which lack either the capsule or both the capsule and the lipooligosaccharide outer core, to infect human phagocytic and nonphagocytic cells and monitor invasion and intracellular growth. Our data indicate that the capsule, which negatively affects bacterial adhesion and, consequently, entry, is, in contrast, fundamental for the intracellular survival of this microorganism. The results of in vitro assays suggest that an increased resistance to cationic antimicrobial peptides (CAMPs), important components of the host innate defense system against microbial infections, is a possible mechanism by which the capsule protects the meningococci in the intracellular environment. Indeed, unencapsulated bacteria were more susceptible than encapsulated bacteria to defensins, cathelicidins, protegrins, and polymyxin B, which has long been used as a model compound to define the mechanism of action of CAMPs. We also demonstrate that both the capsular genes (siaD and lipA) and those encoding an efflux pump involved in resistance to CAMPs (mtrCDE) were up-regulated during the intracellular phase of the infectious cycle.

Epibiotic Vibrio Luminous Bacteria Isolated from Some Hydrozoa and Bryozoa Species

Luminous bacteria are isolated from both Hydrozoa and Bryozoa with chitinous structures on their surfaces. All the specimens of the examined hydroid species (Aglaophenia kirchenpaueri, Aglaophenia octodonta, Aglaophenia tubiformis, Halopteris diaphana, Plumularia setacea, Ventromma halecioides), observed under blue light excitation, showed a clear fluorescence on the external side of the perisarc (chitinous exoskeleton) around hydrocladia. In the bryozoan Myriapora truncata, luminous bacteria are present on the chitinous opercula. All the isolated luminous bacteria were identified on the basis of both phenotypic and genotypic analysis. The isolates from A. tubiformis and H. diaphana were unambiguously assigned to the species Vibrio fischeri. In contrast, the isolates from the other hydroids, phenotypically assigned to the species Vibrio harveyi, were then split into two distinct species by phylogenetic analysis of 16S rRNA gene sequences and DNA–DNA hybridization experiments. Scanning electron microscopy analysis and results of culture-based and culture-independent approaches enabled us to establish that luminous vibrios represent major constituents of the bacterial community inhabiting the A. octodonta surface suggesting that the interactions between luminous bacteria and the examined hydrozoan and bryozoan species are highly specific. These interactions might have epidemiological as well as ecological implications because of the opportunistic pathogenicity of luminous Vibrio species for marine organisms and the wide-distribution of the hydrozoan and bryozoan functioning as carriers.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

Activation of Dormant Bacterial Genes by Nonomuraea sp. Strain ATCC 39727 Mutant-Type RNA Polymerase

There is accumulating evidence that the ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to the presence of cryptic gene clusters. The activation of dormant genes is therefore one of the most important areas of experimental research for the discovery of drugs in these organisms. The recent observation that several actinomycetes possess two RNA polymerase beta-chain genes (rpoB) has opened up the possibility, explored in this study, of developing a new strategy to activate dormant gene expression in bacteria. Two rpoB paralogs, rpoB(S) and rpoB(R), provide Nonomuraea sp. strain ATCC 39727 with two functionally distinct and developmentally regulated RNA polymerases. The product of rpoB(R), the expression of which increases after transition to stationary phase, is characterized by five amino acid substitutions located within or close to the so-called rifampin resistance clusters that play a key role in fundamental activities of RNA polymerase. Here, we report that rpoB(R) markedly activated antibiotic biosynthesis in the wild-type Streptomyces lividans strain 1326 and also in strain KO-421, a relaxed (rel) mutant unable to produce ppGpp. Site-directed mutagenesis demonstrated that the rpoB(R)-specific missense H426N mutation was essential for the activation of secondary metabolism. Our observations also indicated that mutant-type or duplicated, rpoB often exists in nature among rare actinomycetes and will thus provide a basis for further basic and applied research.

Regulation and differential expression of gdhA encoding NADP‐specific glutamate dehydrogenase in Neisseria meningitidis clinical isolates

Meningococcal gdhA, encoding the NADP‐specific l‐glutamate dehydrogenase (NADP‐GDH), is essential for systemic infection in an infant rat model. In this paper, a limited transcriptional analysis detected differences in gdhA expression among clinical isolates. In strains expressing high levels of gdhA mRNA, two promoters, gdhA P1 and gdhA P2, initiated transcription of gdhA. In contrast, in strains expressing low mRNA levels, gdhA P2 was not active because of weak expression of gdhR, an associated regulatory gene. Gene knock‐out and complementation of a gdhR‐defective mutant confirmed that GdhR is a positive regulator for gdhA P2. Trans‐activation of gdhA P2 was maximal in complex medium during late logarithmic growth phase and in chemical defined medium (MCDA) when glucose (MCDA‐glucose) instead of lactate (MCDA‐lactate) was used as a carbon source in the presence of glutamate. gdhR knock‐out mutants lost both growth phase and carbon source regulation, and exhibited a growth defect more severe in MCDA‐glucose than in MCDA‐lactate. DNA–protein interaction studies demonstrated that 2‐oxoglutarate, a product of the catabolic reaction of the NADP‐GDH and an intermediate of the tricarboxylic acid (TCA) cycle, inhibits binding of GdhR to gdhA P2.

The HrpB–HrpA two‐partner secretion system is essential for intracellular survival of Neisseria meningitidis

In this study we used HeLa cells to investigate the role of the HrpB–HrpA two‐partner secretion (TPS) system in the meningococcal infection cycle. Although there is evidence that several pathogenic microorganisms may use TPS systems to colonize epithelial surfaces, the meningococcal HrpB–HrpA TPS system was not primarily involved in adhesion to or invasion of HeLa cells. Instead, this system was essential for intracellular survival and escape from infected cells. Gentamicin protection assays, immunofluorescence and transmission electron microscopy analyses demonstrated that, in contrast to the wild‐type strain, HrpB–HrpA‐deficient mutants were primarily confined to late endocytic vacuoles and trapped in HeLa cells. Haemolytic tests using human erythrocytes suggested that the secreted HrpA proteins could act as manganese‐dependent lysins directly involved in mediating vacuole escape. In addition, we demonstrated that escape of wild‐type meningococci from infected cells required the use of an intact tubulin cytoskeleton and that the hrpB–hrpA genes, which are absent in other Neisseria spp., were upregulated during infection.

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

BackgroundThere is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.ResultsSpontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.ConclusionRifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

Efficacy of silver treated catheters for haemodialysis in preventing bacterial adhesion

The growing resistance of many strains of bacteria to antibiotics and antiseptics is becoming a serious problem in medicine. Nano-silver is one of the most prominent products in medicine because it exhibits unusual physicochemical properties and a strong biological activity. In this work an innovative silver deposition technology was applied to temporary polyurethane catheters for haemodialysis. The working conditions of catheters were reproduced through laboratory equipment that ensured the flow of deionized water and simulated body fluid inside the lumina at corporeal temperature. The growth and the adhesion of Staphylococcus aureus on the surface of the device were studied through fluorescence microscopy. ICP-AES was adopted to calculate the amount of silver released from the substrate. The stability of the coating during the whole working life of the device was demonstrated through thermo-gravimetric analysis.

Identification of a Meningococcal l-Glutamate ABC Transporter Operon Essential for Growth in Low-Sodium Environments

ABSTRACT GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific l-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in l-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of l-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing l-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the l-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.

Comparative genomics and transcriptional profiles of Saccharopolyspora erythraea NRRL 2338 and a classically improved erythromycin over-producing strain

BackgroundThe molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood although this information is essential to design rational strategies for industrial strain improvement. In this study, we applied comparative genomics to identify all genetic changes occurring during the development of an erythromycin overproducer obtained using the traditional mutate-and- screen method.ResultsCompared with the parental Saccharopolyspora erythraea NRRL 2338, the genome of the overproducing strain presents 117 deletion, 78 insertion and 12 transposition sites, with 71 insertion/deletion sites mapping within coding sequences (CDSs) and generating frame-shift mutations. Single nucleotide variations are present in 144 CDSs. Overall, the genomic variations affect 227 proteins of the overproducing strain and a considerable number of mutations alter genes of key enzymes in the central carbon and nitrogen metabolism and in the biosynthesis of secondary metabolites, resulting in the redirection of common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, affect global expression profile as demonstrated by a comparative analysis of the parental and overproducer expression profiles. Genomic data, finally, suggest that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes.ConclusionsThis study helps to clarify the mechanisms underlying antibiotic overproduction providing valuable information about new possible molecular targets for rationale strain improvement.