The human vagina & associated microorganisms

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Vaginal infections

Although the vaginal mucosa is considered a tightly balanced barrier against pathogen invasion, one million people nonetheless acquire sexually transmitted infections (STI) every day, according to the world health organisation (WHO; WHO, 2018). Roughly 37 million people worldwide were living with the human immunodeficiency virus (HIV) at the end of 2017 (WHO, 2018). The worldwide prevalence of genital human papillomavirus (HPV) infection is estimated at 440 million people. The Herpes simplex virus type 2 causes 23.6 million new genital infections/year (Saslow et al., 2007; Looker et al., 2008; Nardis et al., 2013). The prevalence of bacterial sexual infections is also alarming. Neisseria gonorrheae infects an estimated 78 million people per year, while Chlamydia trachomatis causes 131 million new infections annually (WHO, 2018). Syphilis, caused by the bacterium Treponema pallidum, affects an estimated 5.6 million people per year worldwide (WHO, 2018; Nardis et al., 2013). A common genital infection, which is often neglected for not causing mortality, is trichomoniasis (Secor et al., 2014; Menezes et al., 2016). This is caused by the protozoal pathogen Trichomonas vaginalis, the main focus of this thesis. Aside from viruses, trichomoniasis is the most common STI worldwide, accounting for almost a quarter of a billion infections annually (more than N. gonorrheae and C. trachomatis together) (Ginocchio et al., 2012). Finally, but still important, a sexual condition known as bacterial vaginosis (BV) does affect up to 3 out of 4 women in America (Koumans et al., 2007). While sexual transmission of BV is disputed, this polymicrobial infection has gyneco-obstetric consequences (Minkoff et al., 1984; Tabatabaei et al., 2018). In the following sections of this chapter, T. vaginalis and BV will be further discussed.


Trichomoniasis is an STI caused by infection by the extracellular protozoan parasite T. vaginalis, the only protozoan of the human urogenital tract (Petrin et al., 1998). Assessment of epidemiological data of trichomoniasis is difficult as it is not a reportable disease in any country (Poole & McClelland, 2013). Hence, the best estimate is likely that of the WHO, which indicates a figure of 143 million new cases of infections per year worldwide (WHO, 2018).
With a higher incidence than C. trachomatis and N. gonorrhoeae infections combined, these numbers imply that trichomoniasis is the most common non-viral STI worldwide (WHO, 2018; Edwards et al., 2014). However, several studies showed a rather high variation in prevalence within countries, which might be due to differ testing for the infection, but also between countries most like due to the lack of reporting. In the US, prevalence of trichomonas infections is estimated at 2.5-26.2 % (Menezes et al., 2016), while in Asia trichomoniasis prevalence is 7.8 % in South Korea and 8.5 % in India (Madhivanan et al. 2009; Kim, 2013). Among indigenous Australians, prevalence varies between 8.5 % and 48 % (Ryder et al., 2012; Bygott & Robson, 2013). Nordic European countries show a prevalence of 1.5 % (Faber et al., 2011).
In South Africa, a variation of 6.5 % to 46 % of T. vaginalis infections has been reported (Naidoo & Wand, 2013). In South America, similar prevalence of trichomoniasis was observed in Argentina (7.6 %), Chile (7.8 %) and Peru (9.1%) (Salomon et al., 2011; Neira et al., 2005; Leon et al., 2009). However, in Brazil there was again a variation of 2.6 % to 20 % in the prevalence of trichomoniasis (Rafael et al., 2006; Luppi et al., 2011; Grama et al., 2013).
Another problem in collecting data is that up to 85 % of cases can be asymptomatic with a healthy vaginal pH of 3.8 to 4.2 (Petrin et al., 1998), although a third of these cases will develop symptoms within six months (Edwards et al., 2014). Due to the underestimation of prevalence and the absence of a surveillance system to determine actual prevalence and treatment success, Secor et al. classified trichomonasis as a neglected disease for which the impact has been nderestimated; it is therefore deserving more attention (Secor et al., 2014).

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Chapter 1 – Introduction
General Introduction
The human vagina & associated microorganisms
Vaginal Infections
Clinical manifestation
The (patho)biology of T. vaginalis
Bacterial vaginosis
Clinical aspects, diagnosis and treatment
Polymicrobial infections
When our Microbiota goes rogue
Hypothesis and Aim of the project
Chapter 2 – Materials and Methods
Equipment, Software and Databases
Dyes, Loading dyes and Ladders
Assay Kits
Buffer and Solutions
Sera and antibodies
Cells and Organisms
Cell Culture
Culture of T. vaginalis
Culture of Bacterial strains
Culture of human vaginal cells
Flow Cytometry (FCM)
Cell staining
T. vaginalis cell staining
Bacterial cell staining
Flow Cytometry (FCM) analysis
Cell-based assays
Biofilm formation: quantitative and qualitative
Analyzing resistance of T. vaginalis to metronidazole
Migration of T. vaginalis towards a biofilm
Examining survival of T. vaginalis in the presence of metronidazole
and a bacterial biofilm
Growth curve of T. vaginalis
Nutrient deficient medium growth experiments of T. vaginalis
Collection of GC-MS samples
Binding of T. vaginalis to an artificial mucus layer
Migration of T. vaginalis through an artificial mucus layer
Assessing metabolic activity of hVECs
Analyzing early apoptosis of hVECs
Measuring Paracellular permeability
Measuring Phosphatase activity
Measuring phosphatase inhibition
Molecular Biology
Genomic DNA extractions
RNA extraction and cDNA conversion
qPCR analysis
Primer design
Polymerase Chain Reaction
Agarose gel electrophoresis
Phylogenetic analysis
Protein Analysis
Human cell lysis
Determination of Protein concentrations
SDS-Polyacrylamide gel electrophoresis
Western Blotting
Gas chromatography-Mass spectrometry
Methyl chloroformate extraction
Trimethylsilyl extraction
Chapter 3 – CST-IV bacteria and Trichomonas vaginalis affect the integrity of the human vaginal epithelial cell layer by dysregulating tight junctions
3.1 Introduction
3.2 CST-IV and T. vaginalis synergistically increase the paracellular permeability of hVECs
3.3 Increased paracellular permeability of hVECs is due to phosphatase activity
3.4 Tight Junction integrity is affected by CST-IV and T. vaginalis
3.5 Discussion
Chapter 4 – Identifying novel binding substrates for Trichomonas vaginalis and the effect of parasitic adhesion in the presence of CST-IV bacteria
4.1 Introduction
4.2 T. vaginalis uses the bacteria biofilm created by Gardnerella vaginlis as
initial binding substrate
4.3 T. vaginalis can bind to and migrate through an artificial mucus layer
4.4 Bioinformatic analysis revealed 11 putative proteins as potential candidates
for mucin binding proteins in T. vaginalis
4.5 CST-IV bacteria increase T. vaginalis cytoadherence to hVECs in
concentration and contact dependent manner
4.6 T. vaginalis and CST-IV bacteria decrease metabolic activity and cause early
apoptosis in hVECs
4.7 Discussion
Chapter 5 – Metabolic changes as a result of CST-IV and Trichomonas vaginalis coincubation
contribute to parasitic growth in nutrient deprived media and resistance of
T. vaginalis to the drug metronidazole
Chapter 6 – Discussion
Appendix I – MBP sequence alignment
Appendix II – Permissions for reprint of Figures

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When our Microbiota goes rogue: A possible inter-domain microbial association between Trichomonas vaginalis and bacteria of the human vaginal microbiota

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