An improved quantitative method to assess adhesive properties of Trichomonas vaginalis to host vaginal ectocervical cells using flow cytometry

https://doi.org/10.1016/j.mimet.2012.10.011Get rights and content

Abstract

Microbial adhesion is a critical step for infection and colonization of the host. Trichomonas vaginalis, a human urogenital extracellular parasite, relies on host cell adhesion for infection and pathogenesis. Although host cell adhesion of T. vaginalis is strain-dependent and it may be influenced by many environmental factors, a technical limitation to quantify T. vaginalis adhesion falls upon a laborious and time-consuming protocol of fluorescent microscopy. This technical limitation reduces the ability of screening multiple parameters or detecting multiple cell types simultaneously. Here we tested the capability of using flow cytometry as a qualitative and quantitative method to measure adhesion of this human infectious microorganism to vaginal ectocervical cells. Various strains of T. vaginalis with different adhesion properties were stained with CellTracker Orange (CMTMR) prior to incubation with host cells. Analyses by flow cytometry revealed that adhered CMTMR-stained parasites were clearly distinguishable from the host cells and also enabled absolute cell counts to be determined. This method was validated with the comparison of parasite strains that display variable degrees of host cell adhesion. This assay can now be applied to test many variables and environmental factors simultaneously that may affect T. vaginalis adhesion.

Highlights

► Flow cytometry (FC) was used to discriminate T. vaginalis and host vaginal cells. ► CellTracker CMTMR allowed absolute quantification of adhered parasites via FC. ► The method was validated using parasites that display a variable degree of adhesion. ► This method is reliable and provides a better solution than fluorescent microscopy. ► This method is accurate and robust and many variables can be tested simultaneously.

Introduction

Adhesion is an inherent characteristic of microorganisms and it is implicated into various biological processes. For instance, microbial adhesion to a host cell or substrate is central to infection. Infectious microorganisms depend on surface receptors that mediate binding to specific molecules present in the host mucus, extracellular matrix or cell surface (Juge, 2012, Ryan et al., 2011a). Particularly for obligate extracellular microbial pathogens, adhesion is critical for survival in the host.

Trichomonas vaginalis is an extracellular parasite and the causative agent of the most common non-viral sexually transmitted disease worldwide known as trichomoniasis (WHO, 2005). T. vaginalis is capable of binding to a variety of host cell substrates including components of the extracellular matrix and numerous cell types (Addis et al., 2000, Alderete et al., 2002, Ryan et al., 2011a). Adhesion of T. vaginalis to host cells is strain-dependent and the level of adhesion is directly associated with the virulence properties of the strains (Petrin et al., 1998, Rojas et al., 2004, Ryan et al., 2011a). Nevertheless, a technical challenge to study the parasite–host interaction has been the ability to measure host cell adhesion both qualitatively (discriminate cell types) and quantitatively. To date, conventional fluorescent microscopy has been used to measure the ability of T. vaginalis to attach to host cells (Bastida-Corcuera et al., 2005). However, this technique is laborious and it does not provide the actual total number of adhered cells in a sample.

For the purpose of studying microorganisms and their interaction with eukaryotic host cells, the practicality of a universal and semi-automated approach that can discriminate different cell types (host and microbe) and count the number of cells in the whole sample makes flow cytometry a very attractive method. Flow cytometry has commonly been used to quantify and characterise eukaryotic cells. In addition, some microbiologic applications have been described, for instance, for the detection and determination of viability of microbes (Barbosa et al., 2008, Pina-Vaz et al., 2004, Pina-Vaz et al., 2005, Pina-Vaz and Rodrigues, 2010). Although flow cytometry has proved to be advantageous when compared to conventional methods, its potential use for more complex applications in microbiology has been underestimated. More recently, flow cytometry was used to measure the interaction between bacteria, fungi and other eukaryotic cells as well as adhesion of yeast to various surfaces (Hollmer et al., 2006, Hytonen et al., 2006, Polacheck et al., 1995).

Here we report the development of a flow cytometry-based approach to measure the adhesion of T. vaginalis to vaginal ectocervical cells. This method provides qualitative and quantitative data and it is more efficient and less subjective than conventional fluorescent microscopy. Using a tissue culture model of human vaginal ectocervical cells, the validity of this technique was confirmed when different strains of T. vaginalis, including strains that are chemically defective on adhesion, were compared. This study demonstrates the application of flow cytometry to quickly quantify microbial adhesion from a multi-cellular sample. The practicality of this approach makes it more robust to screen several variables that may affect T. vaginalis adhesion simultaneously. We will now be able to further explore the adhesive behaviour of this parasite.

Section snippets

Cell culture

Human vaginal ectocervical cells or VECs (ATCC CRL-2614) were grown on 24-well plates in keratinocyte-SFM (K-SFM) complemented with growth factor and penicillin/streptomycin (pen/strep; GIBCO) and cultured at 37 °C/5% CO2 (Fichorova et al., 1997). VECs were grown to 80–90% confluence prior to use in the adhesion assay.

T. vaginalis reference strains F1623, G3, B7RC2 and its chemical mutants 2E2 and 4–12 were a gift from Dr Peter and Jacqui Upcroft, Queensland Institute of Medical Research

Results and discussion

A flow cytometric assay was investigated as an alternative approach to accurately quantify parasite adhesion to its host cell in a more robust, multi-well format. This assay involved optimising flow cytometry settings and cellular dyes, in combination with liquid counting beads, to enable the detection and enumeration of the parasite T. vaginalis following a tissue culture infection model. We tested various T. vaginalis strains with known host cell adhesive properties (Bastida-Corcuera et al.,

Acknowledgements

The authors want to thank Dr Peter and Jacqui Upcroft (Queensland Institute of Medical Research, Australia) and Prof. Patricia J Johnson (University of California Los Angeles, USA) for sharing T. vaginalis strains. This work was supported by the Faculty Research Development Fund (University of Auckland), the Health Research Council of New Zealand (reference 11/314) and the Maurice and Phyllis Paykel Trust in New Zealand.

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