Caterina Pagliarulo

Whole-genome organization and functional properties of miniature DNA insertion sequences conserved in pathogenic Neisseriae

The chromosome of pathogenic Neisseriae is peppered by members of an abundant family of small DNA sequences known as Correia elements. These DNA repeats, that we call nemis (for neisseria miniature insertion sequences) can be sorted into two major size classes. Both unit-length (154-158 bp) and internally rearranged (104-108 bp) elements feature long terminal inverted repeats (TIRs), and can potentially fold into robust stem-loop structures. Nemis are (or have been) mobile DNA sequences which generate a specific 2-bp target site duplication upon insertion, and strictly recall RUP, a repeated DNA element found in Streptococcus pneumoniae. The subfamilies of 26L/26R, 26L/27R, 27L/27R and 27L/26R elements, found by wide-genome computer surveys in both the Neisseria meningitidis and the Neisseria gonorrhoeae genomes, originate from the combination of TIRs which vary in length (26-27 bp) as in sequence content (L and R types). In both species, the predominant subfamily is made by the 26L/26R elements. The number of nemis is comparable in the N. meningitidis Z2491 (A serogroup) and the MC58 (B serogroup) strains, but is sharply reduced in the N. gonorrhoeae strain F1090. Consequently, several genes which are conserved in the two pathogens are flanked by nemis DNA in the meningococcus genome only. More than 2/3 of nemis are interspersed with single-copy DNA, and are found at close distance from cellular genes. Both primer extension and RNase protection data lend support to the notion that nemis are cotranscribed with cellular genes and subsequently processed, at either one or both TIRs, by a specific endoribonuclease, which plausibly corresponds to RNase III.

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.

Inhibitory effect of pomegranate (Punica granatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli

In the present study major polyphenols of pomegranate arils and peel by-products were extracted in 50% (v/v) aqueous ethanol, characterized and used in microbiological assays in order to test antimicrobial activity against clinically isolated human pathogenic microorganisms. Total concentration of polyphenols and in vitro antioxidant properties were determined by the Folin-Ciocalteu and DPPH methods, respectively. The most abundant bioactive molecules, including anthocyanins, catechins, tannins, gallic and ellagic acids were identified by RP-HPLC-DAD, also coupled to off-line matrix assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS). The inhibitory spectrum of extracts against test microorganisms was assessed by the agar well-diffusion method. Data herein indicated that both pomegranate aril and peel extracts have an effective antimicrobial activity, as evidenced by the inhibitory effect on the bacterial growth of two important human pathogens, including Staphylococcus aureus and Escherichia coli, which are often involved in foodborne illness.

Detrimental effects of Bartonella henselae are counteracted by L-arginine and nitric oxide in human endothelial progenitor cells.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor l-arginine (l-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l-arg (1–30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Evolution and function of the neisserial dam-replacing gene.

Phase variation through slippage‐like mechanisms involving homopolymeric tracts depends in part on the absence of Dam‐methylase in several pathogenic isolates of Neisseria meningitidis. In Dam‐defective strains drg (dam‐replacing gene), flanked by pseudo‐transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5′‐GmeATC‐3′. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE‐providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction‐modification system (RM) specific for 5′‐GATC‐3′, similar to Sau3AI‐RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.

CXCR4-CXCL12-Dependent Inflammatory Network and Endothelial Progenitors

The endothelial progenitor cells (EPCs) are angiogenic cells having properties similar to those of embryonal angioblasts. The number and function of EPCs are affected by a variety of conditions, including cytokines and chemokines, which are pivotal inflammatory signaling molecules. The purpose of this paper is to review current knowledge about the role of these progenitor in different vascular diseases, emphasizing the important biological role played from the CXCR4-CXCL12 axis in the cellular trafficking. Indeed, as described in detail in this review, the CXCR4/CXCL12 interaction produces pleiotropic effects in stem cells and plays a pivotal role in several processes related to development, tissue regeneration and development/progression of malignancies.

Therapeutic angiogenesis in diabetic apolipoprotein E-deficient mice using bone marrow cells, functional hemangioblasts and metabolic intervention.

OBJECTIVE Peripheral arterial disease (PAD) is a major health problem especially when associated to concomitant diabetes and hypercholesterolemia. Hyperglycemia with an overwhelming generation of oxygen radicals and formation of glycation end-products exacerbates oxidation-sensitive mechanisms activated by tissue ischemia. Administration of autologous bone marrow cells (BMC) is an increasing notable intervention to induce therapeutic angiogenesis, ameliorated by metabolic intervention (MT). Recently, hemangioblasts (HS) with functional properties were isolated. METHODS The effects of integrate regimen with intravenous BMC, HS, and MT (1.0% vitamin E, 0.05% vitamin C, and 6% l-arginine) were examined in the ischemic hindlimb of ApoE(-/-) diabetic and non-diabetic. Blood flow ratio was monitored by use of a laser Doppler blood flowmeter. Capillary density was determined in sections of the adductor and semimembranous muscles with antibody against CD31. RESULTS BMC or HS alone, and BMC plus HS increased blood flow and capillary densities and decreased interstitial fibrosis. These effects were amplified by additional MT, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage infiltration. Investigation of molecular mechanisms in bone marrow (BM)-derived progenitor cells from mice revealed that BMC therapy and, more consistently, in combination with MT ameliorated functional activity via decreased cellular senescence and increased telomerase and chemokine CXCR4 activities. Telomerase activity was also increased by HS alone or HS+MT and, more consistently, by BMC+HS alone or in combination with MT. CONCLUSIONS/INTERPRETATION Intravenous autologous BMC and HS intervention together with MT increased therapeutic angiogenesis in the ApoE(-/-) diabetic mouse hindlimb.

Phenotypes of a Naturally Defective recB Allele in Neisseria meningitidis Clinical Isolates

ABSTRACT Neisseria meningitidis strains belonging to the hypervirulent lineage ET-37 and several unrelated strains are extremely UV sensitive. The phenotype is consequent to the presence of a nonfunctional recBET-37 allele carrying multiple missense mutations. Phenotypic analysis has been performed with congenic meningococcal strains harboring either the wild-type recB allele or the recBET-37 allele. Congenic recBET-37 meningococci, in addition to being sensitive to UV, were defective both in repair of DNA lesions induced by UV treatment and, partially, in recombination-mediated transformation. Consistently, the wild-type, but not the recBET-37, allele was able to complement the Escherichia coli recB21 mutation to UV resistance and proficiency in recombination. recBET-37 meningococci did not exhibit higher frequencies of spontaneous mutation to rifampin resistance than recB-proficient strains. However, mutation rates were enhanced following UV treatment, a phenomenon not observed in the recB-proficient counterpart. Interestingly, the results of PCR-based assays demonstrated that the presence of the recBET-37 allele considerably increased the frequency of recombination at the pilin loci. The main conclusion that can be drawn is that the presence of the defective recBET-37 allele in N. meningitidis isolates causes an increase in genetic diversity, due to an ineffective RecBCD-dependent DNA repair and recombination pathway, and an increase in pilin antigenic variation.

Identification, Characterization, and Variable Expression of a Naturally Occurring Inhibitor Protein of IS1106 Transposase in Clinical Isolates of Neisseria meningitidis

ABSTRACT Transposition plays a role in the epidemiology and pathogenesis ofNeisseriameningitidis. Insertion sequences are involved in reversible capsulation and insertional inactivation of virulence genes encoding outer membrane proteins. In this study, we have investigated and identified one way in which transposon IS1106 controls its own activity. We have characterized a naturally occurring protein (Tip) that inhibits the transposase. The inhibitor protein is a truncated version of the IS1106 transposase lacking the NH2-terminal DNA binding sequence, and it regulates transposition by competing with the transposase for binding to the outside ends of IS1106, as shown by gel shift and in vitro transposition assays. IS1106Tip mRNA is variably expressed among serogroup B meningococcal clinical isolates, and it is absent in most collection strains belonging to hypervirulent lineages.

The meningococcal ABC-type L-glutamate transporter GltT is necessary for the development of experimental meningitis in mice

ABSTRACT Experimental animal models of bacterial meningitis are useful to study the host-pathogen interactions occurring at the cerebral level and to analyze the pathogenetic mechanisms behind this life-threatening disease. In this study, we have developed a mouse model of meningococcal meningitis based on the intracisternal inoculation of bacteria. Experiments were performed with mouse-passaged serogroup C Neisseria meningitidis. Survival and clinical parameters of infected mice and microbiological and histological analysis of the brain demonstrated the establishment of meningitis with features comparable to those of the disease in humans. When using low bacterial inocula, meningococcal replication in the brain was very efficient, with a 1,000-fold increase of viable counts in 18 h. Meningococci were also found in the blood, spleens, and livers of infected mice, and bacterial loads in different organs were dependent on the infectious dose. As glutamate uptake from the host has been implicated in meningococcal virulence, mice were infected intracisternally with an isogenic strain deficient in the ABC-type l-glutamate transporter GltT. Noticeably, the mutant was attenuated in virulence in mixed infections, indicating that wild-type bacteria outcompeted the GltT-deficient meningococci. The data show that the GltT transporter plays a role in meningitis and concomitant systemic infection, suggesting that meningococci may use l-glutamate as a nutrient source and as a precursor to synthesize the antioxidant glutathione.