Vaginal microbiome and metabolome highlight specific signatures of bacterial vaginosis

In this study, we sought to find novel bacterial and metabolic hallmarks for bacterial vaginosis (BV). We studied the vaginal microbiome and metabolome of vaginal fluids from BV-affected patients (n = 43) and healthy controls (n = 37) by means of an integrated approach based on quantitative polymerase chain reaction (qPCR) and proton nuclear magnetic resonance ((1)H-NMR). The correlations between the clinical condition and vaginal bacterial communities were investigated by principal component analysis (PCA). To define the metabolomics signatures of BV, 100 discriminant analysis by projection on latent structure (PLS-DA) models were calculated. Bacterial signatures distinguishing the health condition and BV were identified by qPCR. Lactobacillus crispatus strongly featured the healthy vagina, while increased concentrations of Prevotella, Atopobium and Mycoplasma hominis specifically marked the infection. (1)H-NMR analysis has led to the identification and quantification of 17 previously unreported molecules. BV was associated with changes in the concentration of metabolites belonging to the families of amines, organic acids, short chain fatty acids, amino acids, nitrogenous bases and monosaccharides. In particular, maltose, kynurenine and NAD(+) primarily characterised the healthy status, while nicotinate, malonate and acetate were the best metabolic hallmarks of BV. This study helps to better understand the role of the vaginal microbiota and metabolome in the development of BV infection. We propose a molecular approach for the diagnosis of BV based on quantitative detection in the vaginal fluids of Atopobium, Prevotella and M. hominis, and nicotinate, malonate and acetate by combining qPCR and (1)H-NMR

Ruolo dell'Enantiomero (R)-9-HSA nel controllo della proliferazione in una linea di Adenocarcinoma del Colon umano

9-hydroxystearic acid (9-HSA) is an endogenous lipoperoxidation product and its administration to HT29, a colon adenocarcinoma cell line, induced a proliferative arrest in G0/G1 phase mediated by a direct activation of the p21WAF1 gene, bypassing p53. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity, showing interesting features as a new anticancer drug. The interaction of 9-HSA with the catalytic site of the 3D model has been tested with a docking procedure: noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one. Thus, in this study, (R)- and (S)-9-HSA were synthesized and their biological activity tested in HT29 cells. At the concentration of 50 M (R)-9-HSA showed a stronger antiproliferative effect than the (S) isomer, as indicated by the growth arrest in G0/G1. The inhibitory effect of (S)-9-HSA on HDAC1, HDAC2 and HDAC3 activity was less effective than that of the (R)-9-HSA in vitro, and the inhibitory activity of both the (R)- and the (S)-9-HSA isomer, was higher on HDAC1 compared to HDAC2 and HDAC3, thus demonstrating the stereospecific and selective interaction of 9-HSA with HDAC1. In addition, histone hyperacetylation caused by 9-HSA treatment was examined by an innovative HPLC/ESI/MS method. Analysis on histones isolated from control and treated HT29 confirmed the higher potency of (R)-9-HSA compared to (S)-9-HSA, severely affecting H2A-2 and H4 acetylation. On the other side, it seemed of interest to determine whether the G0/G1 arrest of HT29 cell proliferation could be bypassed by the stimulation with the growth factor EGF. Our results showed that 9-HSA-treated cells were not only prevented from proliferating, but also showed a decreased [3H]thymidine incorporation after EGF stimulation. In this condition, HT29 cells expressed very low levels of cyclin D1, that didn’t colocalize with HDAC1. These results suggested that the cyclin D1/HDAC1 complex is required for proliferation. Furthermore, in the effort of understanding the possible mechanisms of this effect, we have analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. EGF/EGFR/HDAC1 complex quantitatively increases in 9-HSA-treated cells but not in serum starved cells after EGF stimulation. Our data suggested that 9-HSA interaction with the catalytic site of the HDAC1 disrupts the HDAC1/cyclin D1 complex and favors EGF/EGFR recruitment by HDAC1, thus enhancing 9-HSA antiproliferative effects. In conclusion 9-HSA is a promising HDAC inhibitor with high selectivity and specificity, capable of inducing cell cycle arrest and histone hyperacetylation, but also able to modulate HDAC1 protein interaction. All these aspects may contribute to the potency of this new antitumor agent

IDENTIFICATION OF HISTONE MODIFICATIONS INVOLVED IN HDAC1 INHIBITION BY 9-HSA

9-hydroxystearic acid (9-HSA) is a product of endogenous lipoperoxidation identified in several human cell lines; its administration to HT29, a colon adenocarcinoma cell line, causes growth arrest in G0/G1 and differentiation toward a benign phenotype. The relevant molecular events are the increased transcription of p21WAF1, TGFbeta1, alkaline phosphatase, and subunit alfa5 of integrin. The number of genes whose activity is modified is indeed rather restricted: in particular variations of the activity of genes that regulate the cell cycle, apoptosis and differentiation have been evidenced for several HDAC1 inhibitors, but the mechanisms that induce or repress the selective activation of genes, beginning from the acetylation state of chromatin or of nuclear proteins, are not yet clear. In HT29, both histones H3 and H4 appear to be hyperacetylated as a consequence of 9-HSA administration. The isolation of histones, the identification of their isoforms as well as the determination of their modifications are essential steps of our study. Reverse phase, ion exchange or hydrophylic-interaction liquid chromatography and subsequent identification of each fraction by electrophoresis or mass spectrometry have become urepleaceble approaches for the analysis of protein modification sites. This work proposes an experimental approach for studying the different isoforms through the injection of the mixture of histones, extracted and solubilized in water, in a liquid chromatograph Jasco PU-1585 equipped with a Rheodyne Model 7725i injector, connected to a UV (JascoUV-1575) detector and to a LCQDuo (Thermo Finnigan) mass spectrometer, that is in turn interfaced to an electrospray ionization source (ESI), and equipped with an ion trap analyzer. The analysis of the peaks so obtained has been carried out by employing deconvolution programs that allow the determination of the MW of proteins and characterize and quantify also the acetylated and methylated isoforms

Cell cycle arrest induced by sulforaphane in human colon carcinoma cells HT29 is associated with the hyperacetylation of histone H4.

Introduction. Sulforaphane (SFN), a dietary isothiocyanate isolated from Brassicaceae, has been shown to prevent different cancers in laboratory animals [1]. In particular, SFN protects against colon carcinogenesis in vivo and causes a G0/G1 growth arrest and apoptosis in human colon cancer cells, by inducing p21Cip1/Waf1[2]. SFN also acts as an inhibitor of histone deacetylases (HDACs) in human embryonic kidney 293 cells and HCT116 colon cancer cells [3]. The objective of this study is to evaluate the ability of the HDAC inhibitor SFN to induce cytotoxic and cytostatic effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. The cells were plated in 6-well dishes at equal density, grown for 24 hours, and then treated with 5 M SFN. 3H-thymidine (1 μCi/mL) was added for the last 6 hours of the incubation. The cells were washed in ice-cold PBS and 5 % trichloroacetic acid was added for 30 minutes at 4 °C, and then incubated with 0.5 N NaOH for 1 hour at 50 °C. Cell lysates were assayed for protein content by Bio Rad assay kit and measured for incorporated radioactivity. Counts were normalized for total cellular protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and the acetylation status of the histone classes were assayed by Western Blot and HPLC/MS. Results. SFN negatively affects HT29 growth in a dose- and time-dependent manner with a 24h IC50 equal to 22.3 M 2.4. At concentrations as low as 5 M, a significant inhibition of cell proliferation is observed, accompanied by a 47% decrease of the mitotic index, with respect to the control. Histones extracted from SFN treated HT29 cells show a 63% increase in the acetylation status; in particular SFN markedly prolonges the half- life of the acetyl groups on histone H4

Inhibitory activity of vaginal lactobacilli towards Candida spp.

Objectives. Lactobacilli are the dominant bacteria of healthy vaginal microbiota and their principal function is to maintain an environment that restricts the growth of pathogenic and opportunistic microorganisms, as fungi belonging to the genus Candida. Lactobacilli form a critical line of defence against potential pathogens by lowering the environmental pH through lactic acid production and producing antimicrobial compounds, or through competitive exclusion. Anyway, the mechanisms underlying antifungal activity against Candida spp. are still not fully understood. In this study, the potential activity against Candida spp. of different strains of vaginal lactobacilli was analysed, focusing on hydrogen peroxide generation, lactic acid production and antimicrobial supernatant fluids activity. Methods. Seventeen strains of lactobacilli were isolated from vaginal swabs collected from pre-menopausal healthy women. They were taxonomically identified by sequencing the 16S ribosomal RNA gene. Hydrogen peroxide generation was tested in a semi-quantitative assay on de Man, Rogosa, Sharpe (MRS) agar plates containing tetramethylbenzidine and horseradish peroxidase in anaerobic conditions. Isolates were scored as low, medium and high producing strains. Lactic acid production was measured in cell free supernatants of Lactobacillus cultures by 1H-nuclear magnetic resonance (NMR) analysis. Lactobacillus culture supernatants were tested for their fungistatic or fungicidal activity against 9 Candida strains isolated from vaginal swabs submitted to the Microbiology Laboratory of Sant\u2019Orsola-Malpighi University Hospital of Bologna for routine diagnostic procedures, belonging to C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, and C. lusitaniae species. The in vitro activity of free-cell supernatants was determined by broth microdilution assay in accordance with EUCAST guidelines. To determine if Lactobacillus strains supernatants had a killing effect, samples from wells exhibiting less than 50% of growth were taken and spotted onto SD agar plates. Fungicidal activity was defined as a 653 log10 reduction from the starting inoculum. Results. The Lactobacillus isolates were taxonomically identified as follows: 8 strains of L. crispatus (BC1-BC8), 6 strains of L. gasseri (BC9-BC14), and 3 strains of L. vaginalis (BC15-BC17). All Lactobacillus strains exhibited a good generation of hydrogen peroxide, while the production of lactic acid, even if recorded for all the strains tested, showed concentrations ranging from 4.8 to 50.9 mM. When the anti-fungal activity of Lactobacillus was assessed, L. crispatus supernatants were the most effective, especially versus C. albicans and C. lusitaniae. None of the Lactobacillus strains was able to interfere with C. krusei and C. parapsilosis. Detailed results of fungistatic or fungicidal activity are shown in figure 1. Conclusion. A major potential application of this study concerns the identification of active Lactobacillus strains that could be administered as probiotics for prophylaxis and/or adjuvant therapy of vulvovaginal candidiasis. Further studies are ongoing to elucidate the mechanisms by which lactobacilli exert their protective functions against Candida

Accuratezza della spettrometria di massa MALDI-TOF nell’identificazione di specie di lattobacilli

INTRODUZIONE. I lattobacilli rappresentano un vasto genere di batteri che colonizzano diverse sedi dell’organismo umano, dove svolgono un ruolo chiave nel mantenimento dell’omeostasi microbica. Sebbene il loro ruolo come microrganismi ‘health-promoting’ sia ben riconosciuto, la loro identificazione a livello di specie pone ancora numerose difficoltà e si basa sulla combinazione di differenti approcci. Scopo del presente lavoro è stato quello di valutare l’accuratezza della spettrometria di massa MALDI-TOF per l’identificazione di specie di ceppi di lattobacilli, comparando tale metodica con il classico approccio basato sul sequenziamento del gene 16s rRNA. MATERIALI E METODI. Sono stati inclusi nello studio 40 ceppi di lattobacilli, isolati da campioni clinici (n=27), presenti in formulazioni di probiotici (n=8) o di origine alimentare (n=5). L’identificazione di specie è stata ottenuta mediante sequenziamento del gene 16s rRNA e il relativo albero filogenetico è stato costruito mediante software MEGA 6. L’analisi spettrometrica è stata condotta sia direttamente da colonia batterica su terreno solido sia dopo estrazione proteica con acido formico/acetonitrile. I campioni sono stati analizzati con lo strumento Bruker MicroFlex MALDI-TOF MS e l’identificazione è stata ottenuta confrontando lo spettro proteico con il database di riferimento. Inoltre, a partire dallo spettro medio di ogni ceppo, è stato costruito un dendrogramma, tramite il software Biotyper 3.1. RISULTATI. Il sequenziamento del gene 16s rRNA ha portato alle seguenti identificazioni: 7 L. crispatus, 7 L. gasseri, 5 L. acidophilus, 5 L. delbrueckii, 2 L. vaginalis, 2 L. reuteri, 6 L. plantarum, 1 L. pentosus, 2 L. rhamnosus, 2 L. casei/paracasei e 1 L. brevis. L’analisi MALDI-TOF ha evidenziato elevati score identificativi (score medio≥1,9) sia in caso di analisi diretta, sia dopo estrazione proteica. Tale approccio ha consentito di identificare correttamente a livello di specie tutti i ceppi di lattobacilli tranne uno (L. pentosus vs L. plantarum), con una percentuale di concordanza con il sequenziamento del 97,5%. A tale riguardo, la stretta relazione filogenetica fra le due specie spiega questo risultato. In alcuni casi, a differenza del sequenziamento, la spettrometria di massa ha addirittura consentito l’identificazione batterica a livello di sub-specie (es. L. delbrueckii). Infine, il dendrogramma proteomico ha mostrato un’elevata sovrapponibilità con quello costruito mediante analisi genomica. CONCLUSIONI. La spettrometria di massa MALDI-TOF si è dimostrata altamente affidabile nell’identificazione di specie di diversi lattobacilli, rappresentando un’ottima alternativa al sequenziamento genico, grazie alla sua rapidità e semplicità di utilizzo. Inoltre, potrebbe rappresentare un metodo innovativo per studi tassonomici dei lattobacilli

Lactobacillus crispatus interferes with Chlamydia trachomatis infectivity through modulation of integrin exposure in cervical cells

In women, urogenital CT infections are often asymptomatic, thus remaining unnoticed and untreated. This can lead to complications and sequelae including pelvic inflammatory disease, tubal infertility and ectopic pregnancy (1, 2). A normal vaginal microbiota, dominated by lactobacilli, is crucial for the prevention of several urogenital and sexually transmitted infections, including Chlamydia (3, 4, 5). This aspect is strengthened by the demonstration that in case of bacterial vaginosis, a clinical condition characterized by the depletion of lactobacilli, a higher risk of STI transmission and acquisition is reported (6). This study aimed to elucidate the molecular bases of the interaction among lactobacilli, Chlamydia trachomatis and epithelial cells. We evaluated the capacity of lactobacilli cells and supernatants to interfere with C. trachomatis infectivity in HeLa cells, by means of competition, exclusion and displacement mechanisms. Lactobacilli cells were the most active fraction, by means of an exclusion strategy. We investigated the potential mechanism of protection in Lactobacillus crispatus BC5 (model strain), and we demonstrated that the incubation of HeLa cell line with BC5 cells induces important modifications al the level of the epithelial plasma membrane, by altering lipid composition and α5 integrin subunit exposure. When α5 integrin subunits were masked by a specific blocking antibody, Chlamydia infection was precluded. α5 integrin subunit is thus crucial for the pathogen penetration into HeLa cells, and the anti-Chlamydia activity of BC5 can be directly linked to membrane properties modifications in epithelial cells. In conclusion, we identified a potential molecular mechanism at the basis of the protection exerted by Lactobacillus against the sexually transmitted pathogen Chlamydia trachomatis, getting insights into the role of the vaginal microbiota for the woman’s health