Caratterizzazione di una famiglia di geni codificanti proteine formanti poro nel protozoo patogeno Trichomonas vaginalis

Trichomonas vaginalis è un protozoo flagellato responsabile della tricomoniasi, una delle malattie a trasmissione sessuale più diffuse al mondo. L’infezione è caratterizzata da un importante effetto citopatico a carico dell’epitelio vaginale. Evidenze sperimentali indirette indicano che nel meccanismo di citopatogenicità del protozoo sono coinvolte proteine formanti poro (PFP), suggerendo un’omologia con altri protozoi, quali Entamoeba histolytica e Naegleria fowleri, che producono PFP appartenenti alla famiglia delle saposin-like proteins. Tuttavia in T.vaginalis non è stata finora dimostrata la presenza di geni codificanti per proteine formanti poro. Scopo del lavoro di dottorato è dimostrare la presenza di geni specifici per PFP anche in T.vaginalis, e caratterizzarne gli eventuali prodotti. Il lavoro svolto ha permesso di individuare, mediante analisi del genoma di T.vaginalis, la presenza di 12 geni (TvSaplip1-12) che mostrano un altissimo grado di similarità con i geni delle saposin-like proteins di E.histolytica e di N.fowleri. E’ noto che il meccanismo di patogenicità di T.vaginalis è fortemente dipendente dall’adesione del protozoo alla cellula bersaglio e da valori di pH intorno a 6.0. E’ stato perciò valutato se tali fattori fossero coinvolti nella modulazione della trascrizione dei geni TvSaplip 1-12. I risultati ottenuti hanno messo in evidenza un incremento dei livelli di trascrizione in seguito alla stimolazione per i geni TvSaplip 5, TvSaplip 6, TvSaplip 10 e TvSaplip 12. In particolare il gene TvSaplip 12 subisce una modulazione dei livelli di trascrizione in seguito al contatto del protozoo con le membrane delle cellule bersaglio, mentre i geni TvSaplip 6 e TvSaplip 5 mostrano un significativo aumento dei livelli di trascrizione a pH 6.0. Le proteine TvSaplip5 e TvSaplip6 sono state ottenute in forma ricombinante. Sono risultate essere dei buoni immunogeni, e sono state quindi utilizzate per la produzione di anticorpi specifici monoclonali e policlonali. Gli anticorpi α-TvSaplip6 hanno permesso di localizzare la proteina in immunofluorescenza su protozoi interi. I dati ottenuti dimostrano che essa è confinata all'interno di granuli intracitoplasmatici. La proteina è stata localizzata anche sulle membrane degli eritrociti dopo contatto con T.vaginalis. L’identificazione e la caratterizzazione dei geni TvSaplip nel genoma di T.vaginalis e l’osservazione che i geni TvSaplip 6, TvSaplip 5, TvSaplip 10 e TvSaplip 12, subiscono un incremento dei livelli di trascrizione determinato da fattori che influenzano la patogenicità del protozoo, ha permesso di confermare le evidenze sperimentali indirette sul coinvolgimento di proteine formanti poro nel meccanismo di patogenicità del microrganismo. Si tratta della prima caratterizzazione di geni direttamente coinvolti nella lisi della cellula bersaglio da parte di T.vaginalis

Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics

<p>Abstract</p> <p>Background</p> <p><it>Trichomonas vaginalis </it>is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of <it>T. vaginalis </it>genome identified a plethora of candidate extracellular proteins.</p> <p>Results</p> <p>Data mining of the <it>T. vaginalis </it>genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these <it>T. vaginalis </it>is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from <it>Clostridium difficile </it>pathogenic strains and <it>C. difficile </it>phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male <it>T. vaginalis </it>infected patients were also demonstrated.</p> <p>Conclusions</p> <p>The biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of <it>T. vaginalis </it>pathobiology at the host-pathogen interface.</p

<i>Trichomonas vaginalis</i> vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics

Background. Trichomonas vaginalis is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of T. vaginalis genome identified a plethora of candidate extracellular proteins. Results. Data mining of the T. vaginalis genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these T. vaginalis is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from Clostridium difficile pathogenic strains and C. difficile phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male T. vaginalis infected patients were also demonstrated. Conclusions. The biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of T. vaginalis pathobiology at the host-pathogen interface

Genotypic and phenotypic features of enteropathogenic <i>Escherichia coli</i> isolated in industrialized and developing countries

Introduction: Typical EPEC are considered a leading cause of diarrhoea in developing countries, while atypical EPEC have been isolated more frequently in developed areas. The actual geographic distribution of the two EPEC subgroups is controversial, since data can be highly influenced by laboratory resources. This study aimed to compare the distribution of typical and atypical EPEC among children in developed and developing countries, and to characterize the bacterial isolates, using a unique methodological approach. Methodology: A total of 1,049 E. coli were isolated from faeces of children with acute diarrhoea in Mozambique, Angola and Italy, and processed by PCR to assess the presence of a large panel of virulence genes. All isolates classified as EPEC were further characterized by evaluating adherence and capability to induce actin rearrangement on Hep-2 cells. Results: Overall we isolated 59 EPEC, likewise distributed in the three countries, representing the 5.04%, 4.44% and 6.97% of all Mozambican, Angolan and Italian isolates, respectively. Nevertheless, the geographic distribution of the two EPEC subgroups was not homogeneous: in Italy we isolated 28 aEPEC but no tEPEC, while in Angola and Mozambique the percentage of the two subgroups was comparable. Twelve atypical EPEC were FAS positive and able to induce localized-like adherence on Hep-2 cells, but no correlation with the geographic origin of isolates was observed. Conclusion: Atypical EPEC are present in sub-Saharan areas in a percentage similar to that of typical strains, and are not mainly restricted to industrialized countries, as it was previously supposed.</br

Biological activities of essential oil extracted from leaves of Atalantia sessiflora Guillauminin Vietnam

Introduction: The present study aimed to determine the chemical compositions and bioactivities of the essential oil of Atalantia sessifloraGuillaumin (A. sessiflora), including antibacterial, antimycotic, antitrichomonas, anti-inflammatory and antiviral effects. Methodology: The essential oil from leaves of A. sessiflora was extracted by hydrodistillation using a Clevenger apparatus. Chemical compositions of oil were identified by GC/MS. Antimicrobial and antitrichomonas activity were determined by the microdilution method; anti-inflammatory and antiviral were determined by the MTT method. Results: The average yield of oil was 0.46 ± 0.01% (v/w, dry leaves). A number of 45 constituents were identified by GC/MS. The essential oil comprised four main components. The oil showed antimicrobial activities against Gram-positive strains as Staphylococcus; Gram-negative bacteria such as Klebsiella pneumoniae and Escherichia coli; and finally four Candidaspecies. Enterococcus faecalis and Pseudomonas aeruginosawere least susceptible to the oil of A. sessiflora, as seen in their MIC and MLC values over 16% (v/v). Activity against Trichomonas vaginalis was also undertaken, showing IC50, IC90 and MLC values of 0.016, 0.03 and 0.06% (v/v) respectively, after 48 hours of incubation. The oil of A. sessiflora displayed activity against the nitric oxide generation with the IC50 of 95.94 ± 6.18 μg/mL. The oil was completely ineffective against tested viruses, ssRNA+, ssRNA-, dsRNA, and dsDNA viruses. Conclusions: This is the first yet comprehensive scientific report about the chemical compositions and pharmacological properties of the essential oil of A. sessiflora. Further studies should be done to evaluate the safety and toxicity of A. sessiflora oil

Myrtle-Functionalized Nanofibers Modulate Vaginal Cell Population Behavior While Counteracting Microbial Proliferation

Vaginal infections affect millions of women annually worldwide. Therapeutic options are limited, moreover drug-resistance increases the need to find novel antimicrobials for health promotion. Recently phytochemicals were re-discovered for medical treatment. Myrtle (Myrtus communis L.) plant extracts showed in vitro antioxidant, antiseptic and anti-inflammatory properties thanks to their bioactive compounds. The aim of the present study was to create novel nanodevices to deliver three natural extracts from leaves, seeds and fruit of myrtle, in vaginal milieu. We explored their effect on human cells (HeLa, Human Foreskin Fibroblast-1 line, and stem cells isolated from skin), resident microflora (Lactobacillus acidophilus) and on several vaginal pathogens (Trichomonas vaginalis, Escherichia coli, Staphylococcus aureus, Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei). Polycaprolactone-Gelatin nanofibers encapsulated with leaves extract and soaked with seed extracts exhibited a different capability in regard to counteracting microbial proliferation. Moreover, these nanodevices do not affect human cells and resident microflora viability. Results reveal that some of the tested nanofibers are interesting candidates for future vaginal infection treatments

Rapid detection of coinfections by <i>Trichomonas vaginalis, Mycoplasma hominis</i>, and <i>Ureaplasma urealyticum</i> by a new multiplex polymerase chain reaction

We developed a multiplex polymerase chain reaction (M-PCR) assay to simultaneously detect Trichomonas vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum. The test is extremely specific and has a sensitivity of 10 cells for T. vaginalis and U. Urealyticum and of 1 cell for M. hominis. The technique was validated on vaginal swabs from 240 women presenting symptoms of vaginitis, and results were compared with data obtained using microscopic and culture techniques on the same patients. The M-PCR revealed to be greatly more sensitive and specific than traditional techniques. It has been well demonstrated, in vitro, that T. vaginalis can establish a symbiosis with M. hominis; our data confirm in vivo this strict association: in fact, M. hominis has been detected in 78.6% of all samples positive for T. vaginalis, as compared to only 4.8% of women without trichomoniasis. The species specificity of this association has been confirmed by the absence of any significant correlation between T. vaginalis and U. urealyticum

<i>Mycoplasma hominis</i> and <i>Candidatus</i> Mycoplasma girerdii in <i>Trichomonas vaginalis</i>: Peaceful Cohabitants or Contentious Roommates?

Trichomonas vaginalis is a pathogenic protozoan diffused worldwide capable of infecting the urogenital tract in humans, causing trichomoniasis. One of its most intriguing aspects is the ability to establish a close relationship with endosymbiotic microorganisms: the unique association of T. vaginalis with the bacterium Mycoplasma hominis represents, to date, the only example of an endosymbiosis involving two true human pathogens. Since its discovery, several aspects of the symbiosis between T. vaginalis and M. hominis have been characterized, demonstrating that the presence of the intracellular guest strongly influences the pathogenic characteristics of the protozoon, making it more aggressive towards host cells and capable of stimulating a stronger proinflammatory response. The recent description of a further symbiont of the protozoon, the newly discovered non-cultivable mycoplasma Candidatus Mycoplasma girerdii, makes the picture even more complex. This review provides an overview of the main aspects of this complex microbial consortium, with particular emphasis on its effect on protozoan pathobiology and on the interplays among the symbionts

Patterns of antibiotic resistance of Mycoplasma hominis endosymbiont of Trichomonas vaginalis and the influence of bacterial intracellular location on drug susceptibility

ABSTRACT: Objectives: Mycoplasma hominis, an opportunistic pathogen of the human lower urogenital tract, can survive and replicate within the protozoan Trichomonas vaginalis, establishing an endosymbiotic relationship. The intracellular location may provide a means for the bacteria to evade the immune system and protection from antimicrobial activities. Our aim was to investigate the influence of the endosymbiotic association of M. hominis with trichomonad cells on bacterial antibiotic susceptibility. Methods: We evaluated antibiotic resistance patterns in a group of M. hominis isolated from T. vaginalis clinical specimens as well as in M. hominis isolated from patients without trichomoniasis. Using an experimental model system, we compared the minimum inhibitory concentration (MIC) and lethal concentration (MLC) of tetracycline on M. hominis endosymbionts of T. vaginalis and extracellular bacteria. Results: The incidence rate of M. hominis strains resistant to C14 and C15 macrolide antibiotics was higher in intracellular strains associated with T. vaginalis compared with extracellular bacteria isolated from women not affected by trichomoniasis. However, sensitivity to tetracycline and quinolones was similar in both groups. In vitro experiments demonstrated that M. hominis strains, when isolated as endosymbionts from T. vaginalis, exhibited reduced sensitivity to tetracycline when cultured extracellularly for at least eight weeks. Conclusion: The intracellular localization of bacteria within trichomonad cells may affect antibiotic susceptibility

<i>Mycoplasma hominis</i> and <i>Trichomonas vaginalis</i>: a unique case of symbiotic relationship between two obligate human parasites

Mollicutes are the smallest and simplest selfreplicating microorganisms. Despite the minimal genome and apparent lack of complexity, mycoplasmas show a high degree of adaptation to the most diverse environments. Mycoplasma hominis is a human sexually transmitted mycoplasma which is able to establish a biological association with Trichomonas vaginalis, a pathogenic flagellated protist. M.hominis and T.vaginalis share the same specific natural niche, the human genitourinary tract. Symbiotic relationships between unicellular eukaryotes and bacteria are well known and have been extensively studied, providing interesting insights into the biology of one or both the symbionts. The relationship between T.vaginalis and M.hominis is unique in that it was the first described association of two obligated human parasites. Several aspects of this relationship have been investigated, showing how the trichomonad may be viewed not only as a new niche for M.hominis, but also as a "Trojan horse" for the transmission of the bacterial infection to the human host