The protozoan parasite Trichomonas vaginalis is the causative agent of trichomonosis
which is – with more than 170 million new cases each year – the most prevalent
non-viral sexually transmitted disease (STD) worldwide. Although trichomonosis is
not a primarily lethal disease, the clinical picture can include severe urogenital
inflammations. Chronic infections have been associated with cervical/prostate cancer
and a predisposition of HIV infections. In case of pregnancy, chronic infections can
also lead to preterm delivery and low birth weight. For more than 50 years,
metronidazole, a nitroimidazole antibiotic, has been in use for the treatment of
trichomonosis. It is applied orally and although it is mostly compliant, it can have
serious side effects. It is also not applicable for pregnant women due to its ability to
pass the placenta. Furthermore, an increasing number of emerging metronidazoleresistant
T. vaginalis strains has lead to more treatment failures in the last few years.
To this day, however, there is no effective alternative drug against trichomonosis
available.
Pentamycin is a polyene antimycotic and has been in use in the treatment of
candidiasis, in preliminary studies it also turned out to be effective against
trichomonads.
The aim of this study was to evaluate the efficacy of pentamycin against T. vaginalis
and the ability to develop resistances in vitro. For these purposes, the dose-effect
relationship between pentamycin and four differently metronidazole-sensitive T.
vaginalis strains was investigated. Moreover, the protein composition before and after
the treatment was compared. To induce resistance, strains were treated with
sublethal concentrations of pentamycin within a time of six months.
It could be shown that pentamycin is highly effective against T. vaginalis. A 100%
eradication of trichomonads was reached with a concentration of 15 ;g/ml and an
incubation time of 1h. All four differently metronidazole-sensitive strains showed
almost the same sensitivity to pentamycin. The comparison of the protein profiles of
untreated and treated cells analysed by SDS-PAGE showed that the mode of action
of pentamycin is based on an interaction and subsequent damage of the cell
membrane which consequently leads to total lysis and death of the cell. [...]
Table of contents
Abstracts
1 Introduction
1.1 Trichomonads
1.1.1 Parasitism andprotozoa
1.1.2 Research history
1.1.3 Systematics andevolution
1.1.4 TheTrichomonadea
1.2 Trichomonas vaginalis
1.2.1 Morphology
1.2.2 Distribution
1.2.3 Lifecycle
1.2.4 Metabolism
1.2.5 Genetics
1.3 Trichomonosis
1.3.1 Epidemiology
1.3.2 Clinical manifestations
1.3.3 Pathomechanism
1.3.4 Diagnostics
1.3.5 Therapy
1.3.6 Immunity andprophylaxis
1.4 Scientific background
1.4.1 Pentamycin - a polyene antibiotic
1.4.2 The field of proteomic research
1.5 Aimsof thestudy
2 Materials and Methods
2.1 Strains
2.2 Cultivation of Trichomonas vaginalis
2.3 Preparation of drugs
2.4 Treatment of trichomonads
2.5 Staining with Giemsa stain
2.6 Staining with Trypan Blue stain
2.7 Counting cells with the Fuchs-Rosenthal Hemacytometer
2.8 1DSDS-PAGE
2.8.1 Sample preparation
2.8.2 Gel preparation
2.8.3 Electrophoresis
2.8.4 Staining with Coomassie stain
2.8.5 Protein diminishment assay
2.9 2D SDS-PAGE
2.9.1 Sample preparation
2.9.2 First dimension - Isoelectric focussing
2.9.3 Gel preparation
2.9.4 Equibrilation
2.9.5 Second dimension - Electrophoresis
2.9.6 Staining with Coomassie stain
2.9.7 Staining with Silver stain
2.10 Microtiter assays
2.11 Long-termtreatment
2.12 Lactate dehydrogenase enzyme activity assay
2.13 Cellpermeability assay
2.14 Nile red stain and fluorescence microscopy
3 Results
3.1 Morphology of pentamycin-treated trichomonads
3.2 Correlation between cell density and dose rate
3.3 Effective and inhibitory concentrations (EC50/90, IC50/90) of pentamycin on four T. vaginalis strains
3.4 Protein composition pre and post treatment
3.4.1 Comparison of treated versus untreated cells
3.4.2 Protein diminishment assay
3.5 Long-term treatment and adaption
3.5.1 Morphology of trichomonads in long-term treatment
3.5.2 Recordable adaption to pentamycin
3.5.3 Cross resistance to amphotericin B
3.5.4 Comparison of the protein profile of wildtype and adapted strains
3.5.5 Lactate dehydrogenase enzyme activity assay
3.6 Cell permeability assay
3.7 Nile Red stain
4 Discussion
4.1 Mode of action of pentamycin
4.1.1 Phenotype of pentamycin-treated trichomonads
4.1.2 Protein profiles of pentamycin-treated trichomonads
4.1.3 Mode of action of pentamycin at molecular level and compared to metronidazole
4.2 Dose-response relationship
4.2.1 Microscopic observations
4.2.2 Efficacy of pentamycin
4.3 Resistance
4.3.1 Phenotype of adapted trichomonads
4.3.2 Establishment of an adaption to pentamycin and cross resistance to amphotericin B
4.3.3 Protein profile of long-term treated trichomonads
4.3.4 Mechanisms of adaption
4.4 Relevance of temperature
4.5 Advantages of pentamycin as a pharmaceutical drug
5 Summary
6 Appendix
6.1 Glossary
6.2 Abbreviations
6.3 Chemical reagents
6.4 Equipment
6.5 Literature
6.6 Acknowledgments
Abstract (deutsch)
Der einzellige, humanpathogene Parasit Trichomonas vaginalis ist der Erreger der Trichomonose, die mit rund 170 Millionen Neuinfektionen pro Jahr die weltweit häufigste nicht-virale Geschlechtskrankheit darstellt. Die Trichomonose ist zwar nicht primär lebensbedrohlich, ist aber eine sehr unangenehme Infektion und kann mitunter auch schwer verlaufen. Chronische Infektionen gelten als Risikofaktor für verschiedene Krebserkrankungen und begünstigen eine HIV-Infektion. Im Falle einer Schwangerschaft führen sie meist zu einer Frühgeburt und niedrigem Geburtsgewicht. Zur Behandlung der Trichomonose wird seit nunmehr 50 Jahren Metronidazol eingesetzt. Dieses zu den Nitroimidazolen zählende Antibiotikum wird oral verabreicht und im Allgemeinen auch gut vertragen, hat jedoch zum Teil schwere Nebenwirkungen und ist für Schwangere wegen seiner Plazentagängigkeit ungeeignet. Außerdem führt die wachsende Zahl an Metronidazol-resistenten Trichomonaden-Stämmen immer wieder zu Therapieversagen. Bisher steht allerdings kein gutes Alternativpräparat zur Behandlung der Trichomonose zur Verfügung.
Pentamycin, ein Antimykotikum, das zu den Polyenen zählt, wurde bislang hauptsächlich gegen Infektionen mit Candida-Pilzen angewendet, erwies sich allerdings in Vorversuchen auch als wirksam gegen Trichomonaden.
Das Ziel der vorliegenden Arbeit war die genaue Untersuchung der Wirksamkeit von Pentamycin gegen T. vaginalis. Hierzu wurden sowohl die inkubationszeit- und dosisabhängige Effektivität von Pentamycin gegen vier unterschiedlich Metronidazolsensitive Stämme als auch die Zusammensetzung des Proteoms vor und nach der Behandlung untersucht. Außerdem wurde über einen Zeitraum von sechs Monaten die Ausbildung von Resistenzen überwacht.
Es konnte gezeigt werden, dass Pentamycin außerordentlich effektiv gegen T. vaginalis ist. Eine hundertprozentige Abtötung der Trichomonaden wurde bereits mit einer Konzentration von 15 gg/ml und einer Behandlungsdauer von 1h erreicht. Alle vier Trichomonas-Stämme zeigten eine annähernd gleiche Sensitivität. Die mittels SDS-PAGE analysierten Proteinprofile sprechen dafür, dass die Wirkung von Pentamycin auf einer Schädigung der Zellmembran beruht, die in weiterer Folge zu einer totalen Lyse der Trichomonaden führt. Die vier untersuchten T. vaginalis- Stämme haben auch nach sechsmonatiger Dauerbehandlung mit niedrigen Dosen von Pentamycin keine Resistenzen ausgebildet, es konnte allerdings ein reversibler Gewöhnungseffekt beobachtet werden. Ebenso konnte eine Kreuzresistenz mit dem verwandten Antibiotikum Amphotericin B, das vor allem gegen Leishmanien angewendet wird, ausgeschlossen werden; allerdings war ein synergistischer Effekt bei gleichzeitiger Anwendung beider Wirkstoffe feststellbar.
Die Ergebnisse dieser Studie sprechen für eine hohe Wirksamkeit von Pentamycin gegenüber T. vaginalis, besonders vielversprechend ist das gute Ansprechen von Metronidazol-resistenten Stämmen auf die Behandlung.
Abstract (english)
The protozoan parasite Trichomonas vaginalis is the causative agent of trichomono- sis which is - with more than 170 million new cases each year - the most prevalent non-viral sexually transmitted disease (STD) worldwide. Although trichomonosis is not a primarily lethal disease, the clinical picture can include severe urogenital inflammations. Chronic infections have been associated with cervical/prostate cancer and a predisposition of HIV infections. In case of pregnancy, chronic infections can also lead to preterm delivery and low birth weight. For more than 50 years, metronidazole, a nitroimidazole antibiotic, has been in use for the treatment of trichomonosis. It is applied orally and although it is mostly compliant, it can have serious side effects. It is also not applicable for pregnant women due to its ability to pass the placenta. Furthermore, an increasing number of emerging metronidazole- resistant T vaginalis strains has lead to more treatment failures in the last few years. To this day, however, there is no effective alternative drug against trichomonosis available.
Pentamycin is a polyene antimycotic and has been in use in the treatment of candidiasis, in preliminary studies it also turned out to be effective against trichomonads.
The aim of this study was to evaluate the efficacy of pentamycin against T vaginalis and the ability to develop resistances in vitro. For these purposes, the dose-effect relationship between pentamycin and four differently metronidazole-sensitive T vaginalis strains was investigated. Moreover, the protein composition before and after the treatment was compared. To induce resistance, strains were treated with sublethal concentrations of pentamycin within a time of six months.
It could be shown that pentamycin is highly effective against T. vaginalis. A 100% eradication of trichomonads was reached with a concentration of 15 gg/ml and an incubation time of 1h. All four differently metronidazole-sensitive strains showed almost the same sensitivity to pentamycin. The comparison of the protein profiles of untreated and treated cells analysed by SDS-PAGE showed that the mode of action of pentamycin is based on an interaction and subsequent damage of the cell membrane which consequently leads to total lysis and death of the cell. After six months of long-term treatment, no establishment of resistance but a partial, reversible adaptation to low doses was recordable. Moreover, T. vaginalis did not express cross resistance to amphotericin B - a pentamycin related polyene antibiotic that is mostly applied against leishmaniosis - but both drugs acted synergistically when administered simultaneously.
The results of this study confirm the high efficacy of pentamycin against T. vaginalis, and particuarly the susceptibility of metronidazole-resistant strains to pentamycin is promising.
1 Introduction
This diploma thesis deals with the unicellular human parasite Trichomonas vaginalis, the disease caused by it, the treatment with metronidazole, and, which is the main part of the thesis, the investigation of the effect of the polyene drug pentamycin on T. vaginalis.
1.1 Trichomonads
1.1.1 Parasitismandprotozoa
The term “parasitology” is defined as the work with parasites, parasitism and parasitoses. The word “parasite” has its origin in Greek (“para” for besides, next to; “sitos” for eating). Originally it described people who participated in sacrificial feasts by tasting the slaughtered animals to avoid food poisoning. This was an honourable occupation and did not have any negative meaning yet. The term later received negative connotation when rich people invited such parasites not only to eat with them but also to entertain them, whereupon the parasites became dependent on their hosts. So the understanding of a parasite became established as someone who lives at the expense of someone else (Hiepe, 2006).
In the biological-medical context a parasite is defined as an organism that lives obligatorily or temporarily in or on a foreign, usually larger organism (host), harming it as a consequence of depriving life energy for its benefit but without killing it. In the narrower context it defines exclusively organisms that belong to the arthropods, helminths, or protozoans, of these only the arthropods representing a true phylum (Aspöck & Walochnik, 2002; Deplazes & Eckert, 2005).
The term “protozoa” has been introduced by the German biologist Georg August Goldfuss in 1818 from the Greek (“proto” for first; “zoa” for animals) and was considered as a term for a discrete and monophyletic systematic group for a long time, although it comprises a variety of organisms that are not really related to each other. Today the term is used as a collective for unicellular, heterotrophic eukaryotes without a cell wall and is only based on morphological characteristics (Hemphill & Gottstein, 2006).
1.1.2 Research history
The first isolation of trichomonads, which were classified as Trichomonas tenax by Clifford Dobell later in 1939, was achieved in the year 1773 by the Danish scientist Otho Fridericus Müller. 63 years later, in 1836, Alfred Donné succeeded in the first isolation of T vaginalis. He named it Trichomonas as he believed to recognize properties of the two protozoa Tricodes and Monas, and vaginalis because he had found it in a woman suffering from vaginitis. Nevertheless, he mentioned that the organism can occur in the genital secretion of both women and men. Around the turn of the 19th to the 20th century, Ernst Haeckel published the classic “Kunstformen der Natur”, in which he precisely described the morphology of Trichomonas and painted it on page 13, figure 4 (Fig.1). He named it Trichomonas intestinalis because he observed mainly Trichomonas species living in the intestines of vertebrates (Ackers, 2001; Walochnik & Aspöck, 2002).
In the 1880s, Otto Bütschli (1848-1920), a German zoologist who is honoured as the “architect of protozoology”, published the standard work “Protozoa” which has been released in three editions and stands out as a very precise description of families and genera of trichomonads. Moreover, Bütschli tried to understand the evolution of eukaryotic cells and to relate the different groups. So he created the term “undulipodia” which is not correct regarding to systematics but should clarify the common origin of evolution between pseudopodia and flagellata (Bütschli, 18801889).
From the date of Donné’s discovery, T. vaginalis was not considered as a human pathogenic organism until 1916, when Höhne proved it to be the causative agent in some cases of vaginitis (Höhne, 1916). As recently as in 1978, this concept became universally accepted by the work of Honigberg (Honigberg, 1978).
The invention of the electronic microscope around 1940 revolutionized the field of biological sciences and allowed the elucidation of small cellular compartments due to its higher resolution compared to light microscopy. The anatomy of eukaryotic flagella was discovered and described as well as the existence of the hydrogenosomes, organella for anaerobic metabolism and characteristic for trichomonads (Walochnik & Aspöck, 2002).
Before the discovery of metronidazole, the human veneral disease caused by Trichomonas vaginalis was treated with natural remedies like garlic, cranberries and other red berries in naturopathy - today some authors still believe in that way of treatment - (Hemerka, 2008) and typically with substances like acetarsol, trichomycin and potassium iodide in orthodox medicine, which may have relieved the symptoms but did not cure the disease as they are unable to take effect in the genitourinary region (Durel et al., I960). In 1959, the 5-nitro-imidazole drug metronidazole was introduced for treatment of trichomonosis and it has been the standard drug until now, although metronidazole-resistant trichomonads were already discovered and described in 1979 (Cosar & Julou, 1959; Kulda, 1999).
illustration not visible in this excerpt
Fig-1 : Trichomonas intestinalis from Ernst Haeckel, Kunstformen der Natur; page 13, fig. 4: Die Geißelthierchen.
1.1.3 Systematics and evolution
Ernst Haeckel, the man who made Darwin’s theory of evolution popular, did not only describe the morphological properties of trichomonads very precisely, he also established a phylogenetic system in which he classified the trichomonads as members of the phylum Protozoa, the order of the Infusoria and the class of the Flagellata. The taxon Flagellata had already been established in 1853 by Cohn for unicellular organisms (including also bacteria) with one or more flagella. Later, Otto BÜTSCHLI added the criterion that these organisms have flagella only in the main stage of their life cycle. So it was possible to distinguish between “real flagellates” and “unreal flagellates” like gametes (Walochnik&Aspöck, 2002).
This system was later replaced by a more precise but still morphology-based one that divided the eukaryotes into the Protozoa and the Metazoa, the Protozoa including six groups: the Flagellata, the Rhizopodia, the Sporozoa, the Microspora, the Myxozoa and the Ciliata. Amongst six other orders, the order Trichomonidida belonged to the class of the Flagellates (Storch & Welsch, 1997).
This widely used classification system was more or less kept up until 2005. Currently, the most widely accepted classification scheme is the one by Adl et al., who classified the kingdom of the eukaryotes into six supergroups: The Amoebozoa (Tubulinea, Flabellinaea, Stereomyxida, Acanthamoebidae, Entamoebida, Mastigamoebida, Pelomyxa and Eumycetozoa), the Opisthokonta (Fungi, Mesomycetozoa, Chonomonada, and Metazoa, including all multi-cellular animals), the Rhizaria (Cercozoa, Haplosporidia, Foraminifera, Gromia and Radiolaria), the Archaeplastida (Glaucophyta, Rhodophyceae and Chloroplastida, including the foliage plants), the Chromalveolata (Cryptophyceae, Haptophyta, Stramenopyles and Alveolata) and the Excavata (Fornicata, Malawimonas, Parabasala, Praexostylata, Jakobida, Heterolobosea and Euglenozoa) (Tab.1). In this system, Trichomonas vaginalis is one of four species of the genus Trichomonas in the family Trichomonadida and the order Trichomonadidae. Further families are Calonymphidae, Cochlosomatidae, Devescovinidae and Monocercomonadidae, which all belong to the group of the Parabasalia within the Excavata supergroup (Adl et al., 2005).
A characteristic of the Excavata is their cytostome, a sort of mouth. Additionally their subgroup - the Parabasalids - is characterised by an anaerobic metabolism, lack of mitochondria, amoeboid-like cell surface and complex microtubule structures. They can exist only in a community with animals, whether by symbiosis, commensalism, or parasitism, like T. vaginalis (Cavalier-Smith, 2002).
As Trichomonas - as well as diplomonads, microsporidians and Entamoeba - do not possess mitochondria, they have special organella, the so-called hydrogenosomes, for their anaerobic metabolism. Initially, it was thought that eukaryotes without mitochondria are primitive and had already developed before endosymbiotic bacteria led to the evolution of mitochondria. In the 1980s, Cavalier-Smith created a new term for this group, the Archezoa, including diplomonads, microsporidians and trichomonads, but not Entamoeba due to their higher evolutionary level. This has led to a theory of secondary loss of mitochondria (Cavalier-Smith, 1987; Tovar et al., 1999).
Later, in 1998, Cavalier-Smith published “A revised six-kingdom system of life”, in which he divided the kingdom Archezoa into two phyla, the Metamonada, including Giardia and Chylomastix, and the Trichozoa. The Trichozoa were divided further into the Anaeromonada, including Trimastix, and the Parabasala, including the families Trichomonadida and Trichonympha (Cavalier-Smith, 1998).
Tab. 1 : Systematic table of the eukaryotes, modified after Adl etal., 2005.
illustration not visible in this excerpt
However, more recent molecularbiological studies have shown that trichomonads as well as the other members of the Archezoa primarily did have mitochondria but either lost them by adaptation to their anaerobic mode of life, as in diplomonads and microsporidians, or were converted to hydrogenosomes, as in trichomonads (Embley et al., 2003). Also, genes that seem to have a mitochondrial origin were found in their nuclei. These genes code for adenylate kinase, heat-shock proteins 10, 60, and 70 kDa, triosephosphate isomerase, and the valyl-tRNA synthetase (Embley & Hirt, 1998). Because organisms with mitochondria possess genes that were transferred from mitochondria into the nucleus during the evolution (“numts”), this concedes the case that the mitochondrial genes of trichomonads have their origin in true mitochondria (Lopez et al., 1994). Nevertheless, trichomonads seem to have diverged from the eukaryotic tree of life very early. This theory is supported by many molecular biological studies and 18S rRNA analyses that have also shown that the Trichomonadida are a monophyletic lineage and represent a deep branch of the eukaryotic evolutionary tree (Gunderson et al., 1995). Apparently, the lack of mitochondria does not mean an ancestral role in the tree of life implicitly, but may be a result of adaptations to environment. Data from phylogenetical analyses of fungi and ciliates possessing hydrogenosomes and lacking mitochondria have also proved that these organella must have evolved from mitochondria and also many times amongst the eukaryotes (Embley et al., 2003).
Further molecular biological and phylogenetical studies have confirmed that the family of the Trichomonadidae represents a monophyletic group (Delgado- Viscogliosi et al., 2000). Even all investigated strains within the species Trichomonas vaginalis are monophyletic although there are some differences in their metabolism. Nevertheless, or even for this reason, there is a positive correlation between the degree of relationship and the susceptibility to metronidazole. On the other hand, the relationship does not correlate with virulence or geographic origin (Hampl et al., 2001).
18S rRNA analyses indicate that the radiation of the recent trichomonadiae occurred rather recently in the history of Earth, maybe in connection with the radiation of their animal hosts which are mostly mammals and birds (Gunderson et al., 1995).
A recent study assumes that even dinosaurs may have had trichomonads, which could have harmed them severely. In 2009, scientists found out that lesions in the jaw bones of a Tyrannosaurus rex had been caused by trichomonads. These infections might have led to starvation as the dinosaur could not move its jaws anymore. Interestingly, these trichomonads are closely related to recent Trichomonas galinae that are specialised on birds and can harm them profoundly in a similar way. This does not only show that trichomonads have existed before their current hosts but also supports the relationship between dinosaurs and birds on the level of their parasites (Wolff et al., 2009).
There are still some inclaritites regarding the role of Trichomonas in the tree of life. The early branching of Trichomonas and also Giardia in 18S rRNA trees can also be explained by the “long branch attraction”, which says that long branches always form a group, neglecting the phylogenetic relationship. So these two groups could also have developed simultaneously with all other eukaryonts. Moreover, analyses of RBP1 as well as EF-1<x and EF-2 could not confirm an early diverging of Trichomonas and Giardia. The “hydrogen hypothesis”, a theory that supports a sister group relationship between Trichomonas and Giardia, assumes that proteobacteria became incorporated first by an archaebacterium, evolving to mitochondria later, or to hydrogenosomes in anaerobians, respectively. A relationship between Trichomonas and Giardia would also support the theory of modified mitochondria (Trichomonas) and secondary loss of mitochondria (Giardia) (Embley & Hirt, 1998).
1.1.3 The Trichomonadea
Tab. 2 shows the phylogenetic position of T. vaginalis. Basing on recent ultrastructural and molecular phylogenetic studies, the parabasalids consist of six main lineages at the rank of a class: Hypotrichomonadea, Trichomonadea, Tritrichomonadea, Cristamonadea, Trichononymphea, and Spirotrichonymphea. The presence of a single mastigont with 2-6 flagella and the absence of a comb-like structure as well as an infrakinetosomal body are characteristic for the Trichomonadea. The class of the Trichomonadea is dividided into two orders which are monophyletic: Trichomonadida, containing the family Trichomonadidae (with a costa), and Honigbergiellida, containing the families Honigbergiellidae,
Hexamastigidae, and Tricercomitidae (without a costa).
The order Trichomonadida is further subdivided into the family Trichomonadidae which splits into two groups: The Trichomonas group and the Pentatrichomonas group. Within the Trichomonas group, four genera branch: Trichomonas, Trichomonoides, Tetratrichomonas, and Pentatrichomonas. Representatives of Trichomonas colonize the urogenital tract, intestine, and oral cavity of birds and mammals and comprise some true pathogens (Cepicka et al., 2010):
Tab. 2: Classification of Trichomonas vaginalis (after Cepcicka et al., 2010).
illustration not visible in this excerpt
Trichomonas galinae parasitizes birds, especially pigeons, and can lead to severe decimations in the populations. The trichomonads colonize the mucosa of the pharynx, the oesophagus and the struma, causing yellow accretions that disable ingestion and lead to starvation (Robinson et al., 2010).
Trichomonas tenax, sometimes also referred to as T. buccalis or T. elongata, colonizes the oral cavity, especially the enamel and gingival, and feeds on the bacterial flora. Cases of a T. tenax infestation of workers in Eastern Europe and the Former Soviet Union are linked to periodontal disease (Ackers, 2001). It is also reported that T. tenax can habitate the nasopharyngeal region and can cause pulmonal trichomonosis, when the patients had a pulmonal disease (Stratakis et al., 1999).
T. vaginalis is the only representative of the genus Trichomonas which is pathogenic for humans, and therefore the most relevant for human medical research. The genus Tetratrichomonas is the most diversified one of the Trichomonas group, with representatives living in vertebrates, molluscs, and leeches, and includes also one newly described species - T undula sp. nov. -, which is the only known free-living species of the order Trichomonadida. The genus Trichomonoides contains only one member, T. trypanoides, which has been found in termites. Further species colonising Homo sapiens are Pentatrichomonas hominis (formerly referred as Trichomonas hominis) of the genus Pentatrichomonas, which is a non-pathogenic commensal of the ileum although it was reported to be associated with cases of diarrhea, and Dientamoeba fragilis of the genus Dientamoeba, family Dientamoebidae, order Tritrichomonadida, class Tritrichomonadea (thus not belonging to the Trichomonadea, although formerly it had been classified as member of the family Monocercomonadidae, order Trichomonadida after Cavalier-Smith, 1998), which is assumed to evoke gastrointestinal symptoms, however, of low pathogenic potential (Ackers, 2001; Cepicka et al., 2010).
All other members of the Trichomonadea are associated with animals and of different pathogenic potential.
1.2 Trichomonas vaginalis
1.2.1 Morphology
T. vaginalis is approximately 10 pm (5-15 pm) wide and 15 pm (8-25 pm) long. It appears as pear-shaped and oval, however, it also shows an amorphous-amoeboid morphology especially when attached to epithelial cells of the host (Arroyo et al., 1993). It is also known that the parasite can appear as an over-dimensioned sphere, with or without flagella, and often with multiple nuclei. The reason for this phenomenon is not yet completely understood, but it may be a reaction to physical discomfort or a cellular developmental stage (Abonyi, 1995).
The cell has four anterior flagella which are fixed on the apical pole. The undulant membrane runs along one side for about two-thirds of the length and is confined by the costa on the proximal side, which functions as a balancer, and by the fifth flagella on the distal side, which generates the wave-like motion of the membrane. The end of the flagellum is fixed in the pellicula. By flapping the free flagella, the parasite generates a hectic, staggering, and characteristic movement. The cell consists of plasma, nucleus, cytoskeleton, parabasal bodies, basal apparatus, axostyle, and hydrogenosomes. There are also granula for glycogen-storage, phagosomes and organelles, similar to lysosomes. The axostyle, a microtubular rod, divides the cell into two longitudinal halves and runs from the anterior to the posterior pole of the cell, where it breaks out as a sharp pinnacle. The purpose of this structure may be cell stabilization and for docking onto vaginal epithelial cells. Around the nucleus, the axostyle forms a collar-like pelta, which makes a cavity surrounding the nucleus and two parabasal bodies. The parabasal bodies consist of a modified Golgi apparatus and two filamentous elements containing dictyosomes and fibrillae (Fig. 2). The cytoplasm contains glycogen granules, large vacuoles as well as free and membrane-bound ribosomes. Furthermore, T. vaginalis has no mitochondria, but hydrogenosomes which are analogous organella for an anaerobic metabolism. Mainly they are arranged alongside the costa and the axostyle (Thomason & Gelbart, 1989; Ackers, 2001).
illustration not visible in this excerpt
Fig- 2 : Schematic view of Trichomonas vaginalis (Original).
T. vaginalis is distributed worldwide, occurs in every climate zone with no seasonal variability and was found in every racial group and socio-economical strata, with differences in incidence, though. In Central Europe, it is one of the three most frequent pathogenic protozoa, besides Toxoplasma gondii and Giardia lamblia. It cannot form cysts, hence, it is dependent on environmental factors like temperature and humidity for its survival. The only way for transmission is direct contact. Other ways of transmission as the common use of towels are only theoretically possible because the parasite can exist outside the human body only for a short time. Swimming pool water as a transmitter can be excluded (Ackers, 2001).
As a microaerophilic anaerobian with a pH optimum of 5.4-6.0, which correlates with the pH of the human vaginal milieu, the parasite is adapted for living in the genitourinary tract. There it lives on the epithelial layer of the anterior fornix vaginae, skene glands and urethra in women, and in urethra, prostate and occasionally under the foreskin in men. Results from in vitro studies have indicated a pH optimum of 6.0-6.3 and a decrease of motility below 4.5 (Diamond, 1986). At 0°C, the organism can survive for up to 5 days, however, at 60°C and above, it is killed within 4 minutes (Thomason & Gelbart, 1989). Crucially, T. vaginalis is very sensitive to dehydration and can survive in bath waters with its different osmotic value from 30 minutes to 3 hours and in chlorinated swimming pool waters (44 mg/l) only for a few seconds to minutes. In supply water, the survival span can reach up to 24 hours. As mentioned before, Homo sapiens is the only natural host of T. vaginalis, however, experimentally it can infect and breed it in the urogenital tracts as well as in the peritoneum of mice (Ackers, 2001; Walochnik&Aspöck, 2002).
T. vaginalis is an extracellular parasite and does only exist in the trophozoite stage. Vectors or cysts for transmission are not needed as it is directly transmitted from human to human during sexual intercourse. The trophozoite feeds by pinocytosis, a specification of phagocytosis, and ingests bacteria, yeasts, and erythrocytes, whereas liquids and solved nutrients are taken off by pseudopodia-like extensions (Thomason & Gelbart, 1989).
T. vaginalis reproduces by longitudinal binary fission every four to six hours, but in contrast to other eukaryotes the nucleus membrane does not disappear during this event. The nucleus divides by kryptopleuromitosis in which the spindle is located outside the nucleus. After disaggregation of the axostyle, the flagella double and the mitosis starts by forming two attractophores on both sides of the nucleus that become the poles of mitosis. From there, microtubules reach into the nucleus and attach on the centromeres. The extranuclear spindle between the attractophores, called paradesmose, elongates and the cell divides when nuclei and flagella migrate to separate poles. Subsequently, daughter cells produce the missing organella. Round forms - as mentioned in 1.2.1 - are assumed to divide by amitotic budding. Some species of the Trichomonadida are also able to form pseudocysts that are not protective but allow a faeco-oral transmission. Hence, it is assumed that T. vaginalis has lost its ability to form cysts as a result of its obligatory parasitic lifestyle and the direct way of transmission (Petrin et al., 1998).
Interestingly, T. vaginalis also seems to have a meiotic recombination machinery as 27 of 29 resembling meiotic genes were found as ortholog genes. Due to the presence of 21 of these genes also in Giardia intestinalis, the meiosis machinery seems to be present in a common ancestor of both organisms and, hence, occurred early in the evolution. This theory is supported by the high genetic variation between T. vaginalis strains (Malik et al., 2008).
1.2.4 Metabolism
The energy metabolism of T. vaginalis is, along with those of Giardia and Enteromonas, more similar to anaerobic bacteria than to eukaryotes. Trichomonads lack mitochondria and peroxisomes, thus they can oxidize glucose only partially and cannot catabolise H2O2. Moreover, their parasitic habitus also seems to result from their disability to synthesize nucleic acids, so that they have to incorporate purines and pyrimidines from their host cells. Adenine and guanine salvage is mediated by nucleoside phosphorylases and kinases, whereas thymine, cytidine and uracil are metabolized by phosphoriboysltransferases and nucleoside kinases. Nucleosides are transferred through the cell membrane by active carrier transport and are converted into nucleotides by specific kinases (Petrin et al., 1998).
De novo lipid biosynthesis is confined to the phospholipid phospatidylethanolamine, a component of the cell membrane. Other lipids, including cholesterol, are most likely aquired from exogenous factors because of the absence of their enzyme-coding genes and degradation of the metabolic pathways (Carlton et al., 2007).
Amino acids are consumed in higher amounts when the main energy source, carbohydrates, is not available or limited (Petrin et al., 1998). Carbohydrate metabolism takes place in the cytoplasm, where glucose is converted to phosphoenolpyruvate and pyruvate via glycolysis (Emden-Meyerhoff-Parnas pathway), and subsequently in the hydrogenosomes, where pyruvate is oxidatively decarboxylated to acetyl-coA by the enzyme PFOR. In the respiratory chain, acetyl- coA is conversed to acetate, coenzyme A, and ATP. Under physiological stress, T. vaginalis decreases the activity of hydrogenosomal enzymes and changes to cytosolic conversion of pyruvate to lactate by enhanced activity of the enzyme LDH. The catabolic end products of T. vaginalis are acetate, lactate, malate, glycerol, CO2, and H2 (Kim et al., 2006; Leitsch et al., 2010; Petrin et al., 1998).
Hydrogenosomes are organelles with a size of 0.5-1 pm and represent an analogue to mitochondria in anaerobic organisms. Contrary to them, they lack cristae, cytochromes, and also own DNA, (except in the ciliate Nyctotherus ovalis) - which corroborates the theory of a common ancestor of hydrogenosomes and mitochondria (the hypotheses on the origin of hydrogenosomes range from modified or degenerated mitochondria to the endosymbiosis of anaerobic bacteria in eukaryotic cells). The main function of the hydrogenosomes is the generation of energy by substrate-levelphosphorylation of acetyl-coA to ATP (Hackstein et al., 2001; Petrin et al., 1998).
The ATP-generating pump works identically as those in mitochondria, however, it loses its function in the presence of oxygen as it is catalyzed by the strictly anaerobic enzyme hydrogenase. Due to the production of hydrogen and the consecutive conversion to methane by methanogenic bacteria, anaerobic protozoa with hydrogenosomes also contribute to the greenhouse effect significantly. PFOR is also supposed to play a role in the resistance mechanism against metronidazole (Müller, 1986; Müller, 1990; Kulda, 1999).
The karyotype of the Trichomonadida depends on the species and varies from three to twelve chromosomes. T. vaginalis contains six chromosomes, comprising of three maxichromosomes, two intermediate chromosomes, and one minichromosome. The same number of chromosomes was found in 15 isolates of T. vaginalis from different geographic regions, which supports the theory of a highly conserved karyotype within the genus Trichomonas (Lekher &Alderte, 1999).
In 2007, the “T. vaginalis Genome Project” was completed by Carlton et al. (2007), and the draft genome sequence was published. The whole size of the genome was found to be 160 megabases (Mb) in size, which is about tenfolds larger than originally estimated by Lekher & Alderte (1999). About 60.000 protein-coding genes were identified, which is more than in Homo sapiens with “only” 30-40.000 coding genes. Moreover, more than two thirds of these genes contain repeats and transposable elements. It is assumed that this enlargement of the genome occurred by lateral gene transfer from bacteria, viruses, and phagocytosis from host proteins as well as by the parasite’s relocation from the digestive into the urogenital tract. An enlargement of the genome might correlate with an enlargement of the cell (T. vaginalis cells are distinctly larger than those of other Trichomonadida) - probably a benefit at colonizing new habitats. Numerous of the identified genes are responsible for specific metabolic pathways and pathomechanism. About 800 genes stand by for the process of attachment on host cells, about 400 are reponsible for the degradome (proteindegrading peptidases) (Carlton et al., 2007).
A recent study has shown that in case of the transmembrane adenylyl cyclase genes (TMAC genes), a high percentage are pseudogenes (46%) and the high number of gene copies result from a recent duplication of a small ancestral gene family.
Especially mavericks - giant transposable elements - are abundant and constitute 30% of the total genome and 50% of the putative protein-coding genes (Pritham et al., 2006; Cui et al., 2010). Introns and non-coding elements can be no explanation for the large-scale genome amplification as they are rare and short. In T. vaginalis, only 65 introns have been identified. Additionally, the size of the genome appears not to be correlated to pathogenicity although the largest genomes were found in the pathogenic species T. vaginalis and T. foetus (177 Mb). However, a similar genome size was found in the non-pathogenic species T. augusta, a harmless commensal in reptile intestines, (165 Mb) and T. tenax (133 Mb), whereas the smallest genome was found in the bird-pathogen T. gallinarum (86 Mb) (Zubáčová et al., 2008).
Arguably, a high polymorphism between microsatellite markers of a population of common laboratory strains indicates that T. vaginalis is a genetically diverse organism, however, sequencing of 5.8S rDNA and the flanking ITS regions of T. vaginalis isolates from sex workers on the Philippines showed low genetic polymorphism between the strains (Rivera et al., 2009; Conrad et al., 2010).
The identfication of genes for the biosynthesis of common cytosolic FeS proteins gives a distinct support of the hypothesis that hydrogenosomes may have their origin in mitochondria. Furthermore, hydrogenosomes may also be able to metabolize amino acids. The existence of an RNA interference pathway might be a possible target for developing drugs manipulating the parasite’s gene expression (Carlton et al., 2007).
1.3 Trichomonosis
1.3.1 Epidemiology
T. vaginalis is the causative agent of trichomonosis and therefore of high medical and socio-economical relevance. Trichomonosis is the most frequent non-viral sexually transmitted disease, with 174 million cases globally and an incidence of 2-3 million new infections annually (WHO, 2001; NIAID, 2003). Prevalence varies from region to region and ranges from 3% in the USA (Allsworth et al., 2009), over 10-20% in Europe and the Republic of Korea (Aspöck, 1994; Ryu & Min, 2006) up to 31% in Mozambique (Menéndez et al., 2010) and 40% in Papua New Guinea (Vallely et al., 2010). From Mongolia, a prevalence of 67% was reported (Schwebke et al., 1998). In Vienna, Austria, the prevalence of T. vaginalis in prostitutes was found to be approximately 5% (Stary et al., 1991).
Studies from the USA have shown that trichomonosis is 12 times more prevalent among black women, besides it correlates also positively with young age, high frequency of changing sexual partners (especially with older partners), high poverty level, other genital infections (especially Neisseria gonorrhoeae, Chlamydia, herpes, warts), and low educational and hygienic level (Allsworth et al., 2009; Krashin et al, 2010). A much higher prevalence - from 8.5% to 47% - was also found in female US jail and prison inmates (Sutcliffe et al., 2010). Moreover, in Greece T. vaginalis was more frequently detected in immigrants (8%) than in native women (3%) (Piperaki et al., 2010).
The maximum prevalence is between 20 and 45 years, which is of higher age than for the most sexually transmitted diseases. However, this correlates with the age of maximal sexual activity. High-risk groups are prostitutes and promiscuous individuals. Besides, an altered vaginal pH milieu, caused by hormonal changes like birth control pill or pregancy, leads to a stronger incidence of infections with T. vaginalis. With the beginning of the menopause, the disease often heals spontaneously (Petrin et al., 1998). Infected mothers can transmit trichomonads to their child during birth (perinatal mode of infection) with a risk of approximately 5%. In this case, the child can aquire an infection of trichomonads in the urogenital tract, however, the infection heals spontaneously a few weeks after birth and requires normally no treatment (Walochnik & Aspöck, 2004). Trichomonads can even be detected in infants - in this case, it is mainly an evidence for sexual child abuse (Lewin, 2007).
1.3.2 Clinicalmanifestations
The habitat of T. vaginalis is the human urogenital tract where it colonizes the mucosal epithel cells. Both women and men can be infected and act as a vector for transmission. In women, the disease occurs mainly between adolescence and menopause. Primary affected organs are vagina, cervix and - additionally - urethra in 75-90% of the cases, whereas bladder and uterus are not affected normally. In 50% of infected and symptomatic women, the incubation time is between 4 and 28 days (Thomason & Gelbart, 1989; Petrin et al., 1998). 10-50% of infected women show no symptoms and also have a normal vaginal flora and pH. However, 50% of these women will develop symptoms within the next six months (Perazzi et al., 2010).
In contrast to women, infections with T. vaginalis are asymptomatic in men in 90% of the cases. The parasite could be detected in 72% of male partners of infected women, of whom 78% were asymptomatic. These cases may lead to misdiagnosis which supports further distribution (Seña et al., 2007). The incubation time in symptomatic men is approximately 10 days, in which the trichomonads colonize the foreskin, urethra, and prostate gland. In about 10% of the cases, urethritis and prostatitis can occur. In women, the symptoms are more severe and include mostly considerable irritations with a fulminant vaginitis. The first symptoms are strong urgency, pain at urinating, and itching of the vagina. Typical symptoms are a sore and reddend vulva, an edematous and reddend vagina, “fluor vaginalis” (a foamy, malodorous, viridescent discharge), and a “strawberry-cervix” (punctual, hemorrhagic lesions in the vaginal cervix). Furthermore, the number of leukocytes in and the pH- value of the vagina can be increased dramatically, thus the vaginal flora is altered and susceptible for further infections. Dyspareunia and postcoital hemorrhage can also occur. When therapy fails, the acute stage fades into a chronic stage, in which the symptoms continue but in a far milder way, being ignored often. This is also an important factor for the transmission of the disease (Petrin et al., 1998).
In many cases, the symptoms are unspecific and can be confounded with those of other veneral diseases. The “strawberry cervix” is observed in 2% of cases and “fluor vaginalis” in 12% of cases only (Fouts & Kraus, 1980).
Even though trichomonosis is not a lethal disease, an infection can impair health and life quality severly. Besides the above mentioned symptoms, it is also believed that an infection with T. vaginalis can cause complications during pregnancy including damage of the placenta, higher risk of premature birth and a low birth weight (Cotch et al., 1997). Furthermore, there seems to be a coherence of trichomonosis and cancer, especially when the infection has passed into the chronic stage. Steadily inflamed urogenital organs like ovaries and prostate gland have a higher risk to develop neoplasia and cancer (Sutcliffe et al., 2009). In rare cases, a fulminant infection may also lead to temporary sterility (El-Shazly et al., 2001).
In 2008, an acquisition of trichomonads in neonatals during their passage through the birth canal and conseqently evoked infections of the childrens’ respiratory tract could be detected (Carter & Whithaus, 2008).
Finally, it is reported that trichomonosis may increase the risk for co-infections, not only for fungi (Candida) and bacteria (Mycoplasma), but also for HIV as an infection with T. vaginalis leads to a weaker integrity of the epithelial cells, to a decreased innate immunity and an accumulation of HIV-affine cells like lymphocytes and macrophages (Laga et al., 1993; Sorvillo et al., 2001; Thurman & Doncel, 2011).
1.3.3 Pathomechanism
T. vaginalis is an extracellular parasite and primary affine to erythrocytes and vaginal epithelial cells but also to immune cells and vaginal bacteria. Additionally, T. vaginalis internalizes also viruses like HIV and human papillomavirus and can act as a Trojan horse for bacteria such as Mycoplasma that survives and multiplies inside the trichomonad cell. Similarly, after being tranferred from one person to another, T. vaginalis can support the spread of HIV by this way (Taylor-Robinson, 1998; Hirt et al., 2007).
The mechanism of pathogenesis is complex - as well not fully understood yet - and includes cell-cell-adhesion, haemolysis, excretion of lytic factors, endocytosis of host cell content, and degradation of immunoglobulins and complement proteins (Hirt et al., 2007).
Adhesion to epithelial cells is an essential step in pathogenesis. T. vaginalis attaches with the side opponent to the undulant membrane, changing from its ovoid shape to an amoeboid form. This step depends on temperature, pH value, and time span of contact. A key protein of this action is alpha-actinin, an actin-binding protein that participates in the morphological transformation by the redistribution of actin. Hence, the parasite’s shape becomes amoeboid-amorphous and capable for adhesion onto host cells. In vitro studies have shown that motile parasites form large aggregates and recruit other parasites. After a few hours, retraction, rounding, and detachment of the epithelial cells were observed (Alderete & Garza, 1985; Fiori et al., 1999). Adhesion seems to be dependent on adhesion proteins (AP65, AP51, AP33, AP120, AP23) and certain components of the glycocalyx of the cell membrane, especially its main component, lipophosphoglycan (LPG). In vitro studies have shown that LPG defective mutants have less adherence and cytotoxicity for human ectocervical cells (Bastida-Corcuera et al., 2005; Zhang et al., 2010).
Regarding the adhesion proteins, it is reported that lectins, considered as important virulence factors also in other pathogens (f.e. Entamoeba histolytica), could be involved in the adhesion process via binding to sugar moieties from glycoconjugates of host cells. Furthermore, hydrogenosomal enzymes are claimed to have dual cellular localizations as surface proteins, where they function as adhesins (so called “moonlighting proteins”). Although these data are controversial, it could be proved that the adhesion protein AP120 is identical to the hydrogenosomal enzyme PFOR. In this context, an outstanding feature is the absence of glycoyslphosphatidylinositol- anchored surface proteins which are characteristic for eukaryotes (Moreno-Brito et al., 2005; Hirt et al., 2007).
Haemolysis is dependent on temperature, pH value, Ca2+, effector concentration, and includes several steps. After adhesion, cysteine proteinases - transmembrane poreforming proteins - induce lysis of host cells by pore formation and can also manipulate the host’s immune systeme by degrading antibodies. For example, CP39, one of the best characterized cystein proteinases, is able to degrade collagens I, III, IV, and V, human fibronectin, hemoglobin, and immunoglobulins A and G (Hernández-Gutiérrez et al., 2004).
During this step of haemolysis, the parasite detaches from the cell via a specific celldetaching factor (CDF) and the contents of the lysed cell are phagocytosed. Red blood cells are a means for acquiring lipids and iron, essential nutrients for the parasite. It was also found that spectrin, the main protein of the cytoskeleton of the membrane of red blood cells, is totally absent after exposure to T. vaginalis. This may be a strategy of the parasite to increase the sensivity of host cells to pore-forming proteins (Fiori et al., 1999). A decrease of cysteine proteinase expression also correlates with a low virulence phenotype (De Jesus et al., 2008).
Recently, it was found that not only cysteine proteinases may be involved in pathogenicity; the first putative serine proteinase (SUBI; subtilisin-like serine protease) was identified and characterized (Hernández-Romano et al., 2010). The emergence of inflammation may be contributed to the recruitment and accumulation of neutrophils by the release of chemokines by T. vaginalis like IL-8 and GRO-a (Ryu & Min, 2006).
An additional important factor in regulating virulence is iron, as trichomonads exhibit less cystein proteinase activity and therefore low cytotoxicity levels at highest iron concentrations and vice versa (Alvarez-Sánchez et al., 2007).
T. vaginalis has allocated a great many of gene clans and families and proteins for its pathomechanism. There are about 800 genes for the process of cell-cell-adhesion and about 23 different proteinase activities (León-Sicairos et al., 2004; Carlton et al., 2007). Curiously, proteins like CDF can even act independently from direct cell contact. It could be shown that epithelial cells in a trichomonad-free filtrate containing CDF react by rounding and detaching from the culture flask (Garber et al., 1989).
An important role in pathogenesis accords to the interactions of T. vaginalis with the vaginal flora. The population of Lactobacillus acidophilus, an important symbiont of the vaginal milieu, can be reduced or disappear following on a massive reproduction of trichomonads. Consequently, the pH value - normally at 4.5 - can be deranged as trichomonads prefer a pH >5 which is disadvantageous for vaginal bacteria. Further co-infections can also appear, especially with Candida and Mycoplasma hominis (Petrin et al., 1998; van Belkum et al., 2001).
1.3.4 Diagnostics
Due to the mentioned unspecific symptoms of trichomonosis, laboratory diagnosis is of major importance and focused on the direct detection of parasites in vaginal smears. If diagnosis is based only upon the classic clinical symptoms (“strawberry cervix”, frothy discharge), 88% of infections cannot be diagnosed, and 29% of uninfected women would be false positive in being infected (Fouts & Kraus, 1980).
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