inducing-meningococcal-meningitis-serogroup-c-mice-via-intracisternal

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

University of Naples Federico II

CEINGE – Advanced Biotechnology

Puroindolines are antimicrobial peptides that occur in wheat seed, and are characterized by broad antimicrobial activity. We describe the heterologous expression of puroindoline A and B in the Origami strain of Escherichia coli. The recombinant puroindolines showed the same antimicrobial activity on Staphylococcus epidermidis as compared to the native peptides (MIC(90)=30microgml(-1)). The bactericidal activity was 125microgml(-1) for recombinant puroindoline A and 42microgml(-1) for recombinant puroindoline B. Neither protein shows in vitro haemolytic activity or toxicity towards the murine macrophage cell line J774, but they are able to kill intracellular staphylococci. Our preliminary results suggest that recombinant puroindolines deserve further attention as alternatives to the conventional antibiotics in the control of S. epidermidis skin infections.

Expression of recombinant puroindolines for the treatment of staphylococcal skin infections (acne vulgaris).

The present study describes a bacteriophage (M(Sa)) active against Staphylococcus aureus, including methicillin-resistant staphylococcal strains. When inoculated into mice simultaneously with S. aureus A170 (10(8) CFU/mouse), phage (10(9) PFU) rescued 97% of the mice; when applied to nonlethal (5 x 10(6) CFU/mouse) 10-day infections, the phage also fully cleared the bacteria. The phage M(Sa), delivered inside macrophages by S. aureus, kills the intracellular staphylococci in vivo and in vitro. The phage can also prevent abscess formation and reduce the bacterial load and weight of abscesses. These results suggest a potential use of the phage for the control of both local and systemic human S. aureus infections.

Experimental phage therapy against Staphylococcus aureus in mice.

Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.

Impairment of circulating endothelial progenitors in Down syndrome.

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenylated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes--possibly novel miRNA targets or regulatory sites for gene transcription--were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders.

Massive-scale RNA-Seq analysis of non ribosomal transcriptome in human trisomy 21.

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.

Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage.

Cardiovascular disease is still the major cause of death worldwide despite the remarkable progress in its prevention and treatment. Endothelial progenitor cells (EPCs) have recently emerged as key players of vascular repair and regenerative medicine applied to cardiovascular disease. A large amount of effort has been put into discovering the factors that could aid or impair the number and function of EPCs, and also into characterizing these cells at the molecular level in order to facilitate their therapeutic applications in vascular disease. Interestingly, the major cardiovascular risk factors have been associated with reduced number and function of EPCs. The bacterial contribution to cardiovascular disease represents a long-standing controversy. The discovery that Bartonella henselae can infect and damage EPCs revitalizes the enduring debate about the microbiological contribution to atherosclerosis, thus allowing the hypothesis that this infection could impair the cardiovascular regenerative potential and increase the risk for cardiovascular disease. In this review, we summarize the rationale suggesting that Bartonella henselae could favour atherogenesis by infecting and damaging EPCs, thus reducing their vascular repair potential. These mechanisms suggest a novel link between communicable and non-communicable human diseases, and put forward the possibility that Bartonella henselae could enhance the susceptibility and worsen the prognosis in cardiovascular disease. 

Infections and cardiovascular disease: is Bartonella henselae contributing to this matter?

Rifampin chemoprophylaxis against Neisseria meningitidis infections led to the onset of rifampin resistance in clinical isolates harboring point mutations in the rpoB gene, coding for the RNA polymerase β chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistant rpoB mutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strain in vitro. Our results demonstrate that different rpoB mutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitness in vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates. 

Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation.

Celiac disease (CD)-associated duodenal dysbiosis has not yet been clearly defined, and the mechanisms by which CD-associated dysbiosis could concur to CD development or exacerbation are unknown. In this study, we analyzed the duodenal microbiome of CD patients.

Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients.

No Change in the Mucosal Gut Mycobioma Is Associated with Celiac Disease-Specific Microbiome Alteration in Adult Patients.

 is a gram-negative facultative intracellular bacterium and is the causative agent of cat-scratch disease. Our previous data have established that  colonization is able to prevent  damages through the polysaccharide A (PSA) in an experimental murine model. In order to determine whether the PSA is essential for the protection against pathogenic effects of  in immunocompromised hosts, SCID mice were co-infected with  wild type or its mutant  ΔPSA and the effects of infection on murine tissues have been observed by High-Frequency Ultrasound (HFUS), histopathological examination, and Transmission Electron Microscopy (TEM). For the first time, echostructure, hepatic lobes length, vascular alterations, and indirect signs of hepatic dysfunctions, routinely used as signs of disease in humans, have been analyzed in an immunocompromised murine model. Our findings showed echostructural alterations in all infected mice compared with the Phosphate Buffer Solution (PBS) control group; further, those infected with  and co-infected with  ΔPSA presented the major echostructural alterations. Half of the mice infected with  and all those co-infected with  ΔPSA have showed an altered hepatic echogenicity compared with the renal cortex. The echogenicity score of co-infected mice with  ΔPSA differed significantly compared with the PBS control group (p < 0.05). Moreover the inflammation score of the histopathological evaluation was fairly concordant with ultrasound findings. Ultrastructural analysis performed by TEM revealed no significant alterations in liver samples of SCID mice infected with  wild type while those infected with  ΔPSA showed the presence of collagen around the main vessels compared with the PBS control group. The liver samples of mice infected with  showed macro-areas rich in collagen, stellate cells, and histiocytic cells. Interestingly, our data demonstrated that immunocompromised SCID mice infected with  and co-infected with  ΔPSA showed the most severe morpho-structural liver damage. In addition, these results suggests that the HFUS together with histopathological evaluation could be considered good imaging approach to evaluate hepatic alterations.

Novel Approach for Evaluation of  Protective Role against  Liver Damage in Immunocompromised Murine Model.

This report describes a case of congenital toxoplasmosis in a newborn in Southern Italy. A pregnant mother had been admitted at the 20th week of her pregnancy on account of pharyngodynia and laterocervical lymphadenopathy. Although serological testing of the mother's serum documented a seroconversion with positive IgG and IgM anti-Toxoplasma antibodies during II trimester, the woman refused to perform prenatal diagnosis for congenital toxoplasmosis. Fetal ultrasound scan already showed mild asymmetrical triventricular hydrocephaly and cerebral calcifications. After birth, real-time PCR on cerebrospinal fluid and blood samples of the newborn showed a positive result for 529bp-repeat element DNA of T. gondii, In addition brain magnetic resonance imaging and computed tomography showed a characteristic diffuse brain tissue loss associated with hydrocephalus. For the first time molecular characterization of T. gondii isolate was performed directly from the newborn's CSF samples by using nested-PCR-RFLP of sag-2 and pk1 genes. The PCR-RLFP analysis revealed that the isolate belongs to the clonal type II, the predominant lineage causing human toxoplasmosis, as confirmed by DNA sequencing.

Genotyping of Toxoplasma gondii strain directly from human CSF samples of congenital toxoplasmosis clinical case.

To evaluate the antimicrobial activity of hydroalcoholic extracts of pomegranate ( L.) peel and juice, against the microorganisms considered the main etiologic agents of dental caries.

 Antibacterial Activity of Pomegranate Juice and Peel Extracts on Cariogenic Bacteria.

We previously profiled duodenal microbiome in active (a-), gluten-free diet (GFD) celiac disease (CD) patients and controls finding higher levels of the Proteobacterium Neisseria flavescens in a-CD patients than in the other two groups. Here, we investigate the oropharyngeal microbiome in CD patients and controls to evaluate whether this niche share microbial composition with the duodenum. We characterized by 16S rRNA gene sequencing the oropharyngeal microbiome in 14 a-CD, 22 GFD patients and 20 controls. Bacteroidetes, Proteobacteria and Firmicutes differed significantly between the three groups. In particular, Proteobacteria abounded in a-CD and Neisseria species mostly accounted for this abundance (p < 0.001), whereas Bacteroidetes were more present in control and GFD microbiomes. Culture-based oropharyngeal microbiota analysis confirmed the greater abundance of Proteobacteria and of Neisseria species in a-CD. Microbial functions prediction indicated a greater metabolic potential for degradation of aminoacids, lipids and ketone bodies in a-CD microbiome than in control and GFD microbiomes, in which polysaccharide metabolism predominated. Our results suggest a continuum of a-CD microbial composition from mouth to duodenum. We may speculate that microbiome characterization in the oropharynx, which is a less invasive sampling than the duodenum, could contribute to investigate the role of dysbiosis in CD pathogenesis.

Oropharyngeal microbiome evaluation highlights Neisseria abundance in active celiac patients.

In serogroup C , the  () gene codes for an UDP--acetylglucosamine 2-epimerase that catalyzes the conversion of UDP--acetyl-α-d-glucosamine into -acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogenic  knockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of the  mutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in the , indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of the  in the mouse model of meningococcal meningitis.

Article	Year
Expression of recombinant puroindolines for the treatment of staphylococcal skin infections (acne vulgaris). Journal of biotechnology\| PubMed ID: 17157405	2007
Experimental phage therapy against Staphylococcus aureus in mice. Antimicrobial agents and chemotherapy\| PubMed ID: 17517843	2007
Impairment of circulating endothelial progenitors in Down syndrome. BMC medical genomics\| PubMed ID: 20836844	2010
Massive-scale RNA-Seq analysis of non ribosomal transcriptome in human trisomy 21. PloS one\| PubMed ID: 21533138	2011
Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage. PloS one\| PubMed ID: 23166739	2012
Infections and cardiovascular disease: is Bartonella henselae contributing to this matter? Journal of medical microbiology\| PubMed ID: 26066633	2015
Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation. Antimicrobial agents and chemotherapy\| PubMed ID: 26416867	2015
Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients. The American journal of gastroenterology\| PubMed ID: 27045926	2016
No Change in the Mucosal Gut Mycobioma Is Associated with Celiac Disease-Specific Microbiome Alteration in Adult Patients. The American journal of gastroenterology\| PubMed ID: 27808136	2016
Novel Approach for Evaluation of Protective Role against Liver Damage in Immunocompromised Murine Model. Frontiers in microbiology\| PubMed ID: 27872616	2016
Genotyping of Toxoplasma gondii strain directly from human CSF samples of congenital toxoplasmosis clinical case. The new microbiologica\| PubMed ID: 28368078	2017
Antibacterial Activity of Pomegranate Juice and Peel Extracts on Cariogenic Bacteria. BioMed research international\| PubMed ID: 29209624	2017
Oropharyngeal microbiome evaluation highlights Neisseria abundance in active celiac patients. Scientific reports\| PubMed ID: 30038321	2018
Virulence Traits of a Serogroup C Meningococcus and Isogenic Mutant, Defective in Surface-Exposed Sialic Acid, in a Murine Model of Meningitis. Infection and immunity\| PubMed ID: 30718288	2019

Paola Salvatore

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