Nuno Alexandre
Pedro
...
DP_AEM student
Institute for Bioengineering and Biosciences (IBB)
Doctoral Program in Biotechnology and Biosciences (Técnico)
2018-2022
Nuno Mira
Nuno Alexandre
0
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Phd Thesis

A molecular overview on the interference effect established between the commensal vaginal species Lactobacillus gasseri and the pathogenic yeasts Candida albicans and Candida glabrata

FTC Scholarship: PD/BD/143026/2018

Abstract

Candida albicans and Candida glabrata are common human commensals but under certain conditions can cause mucosal infections and, in more serious cases, life-threatening systemic candidiasis. The essential role of commensal microflora in shaping the equilibrium between health and disease through the control of pathogens is being increasingly recognized, supported by extensive metagenomic analyses. Lactobacilli predominate in the vaginal microbiome tract and this predominance was linked with reduced activity of multiple vaginal pathogens although the mechanisms underlying this interference effect remain elusive. In vitro vaginal lactobacilli strains were found to inhibit growth of Candida and taking advantage of this a few commercial probiotic formulations have already been used for the treatment of vaginal candidiasis, with various degree of success. However, both the in vitro studies and the clinical trials have explored species other than those that are indigenous to the vaginal tract. In this PhD program the interaction established between vaginal species Lactobacillus gasseri and C. albicans and C. glabrata, the top two causative agents of vaginal candidiasis, will be examined at multiple cellular levels resorting to a combination of OMICS analyses. It is expected that this knowledge might provide seminal insights into this relevant equilibrium between the vaginal commensal microbiota and vaginal pathogens fostering the utilization of L. gasseri (and eventually other vaginal lactobacilli species) as probiotics. In a time of awareness due to the constant emergence of Candida strains resistant to currently used antifungals, the development of therapeutic approaches based on alternative targets is essential. 

State of the art

Candida albicans (Ca) and C. glabrata (Cg) colonize the human host as commensals but under certain conditions this colonization progresses to superficial infections or deathly disseminated mycoses[1]. This pathobiont behaviour of commensal populations was clearly shown for intestinal populations that were found to be able to invade epithelial tissue, further disseminating in the bloodstream[2]. A similar behaviour was suggested for vaginal populations, however, no studies were performed to confirm or deny this hypothesis. Life-threatening candidiasis is mostly observed in susceptible populations but superficial infections, like vulvovaginal candidiasis (VVC), are common among the healthy population[1,3,4]. About 75% of women are estimated to experience VVC during their life-time, a substantial percentage recurrently[4,5]. Ca and Cg are the more common causative agents of both superficial and invasive candidiasis[3–5]. Due to their aggressiveness and recurrence Candida infections represent a massive financial burden for healthcare systems, spent in preventive and active therapies[6]. However, the success of these therapies is hampered by the continuous emergence of resistant strains, particularly within the Cg species[7]. This, along with the delay in the development of a fungal vaccine, requires the development of non-conventional therapeutic approaches aiming for biological targets other than those targeted by currently used antifungals. 

To successfully thrive in the different infection sites, Candida spp must cope with a competing microbiota. Lactobacilli predominate in the vaginal tract, with L. crispatus, L. jensenii and L. gasseri (Lg) being the more abundant species, as unveiled by several metagenomic analyses[8,9]. Predominance of lactobacilli is a recognized hallmark of vaginal health and a decrease in the abundance of these bacteria was linked to the development of candidiasis[10]. A few probiotic products based on lactobacilli were developed to treat VVC with varying degrees of success; nonetheless, these products were developed using species other than those present in the vaginal microbiome. It has been suggested (but not clearly demonstrated) that lactobacilli restrain the overgrowth of Candida spp by positively modulating the host immune system[11][15], by competing for nutrients and adhesion sites, by producing bacteriocins[12] and, mostly, by producing lactic and, less significantly, acetic acid [13]. Recent results obtained by the iBB-IST team show that Ca and Cg tolerate well concentrations of these acids (specially of lactic acid) similar to those found in conditions of vaginal eubiosis (when lactobacilli predominate)[14]. This observation strongly suggests that the interference effect of lactobacilli goes beyond the mere production lactic acid. 

Objectives

The results obtained so far (by our lab and others) render clear the essential role played by lactobacilli in modulating activity of vaginal pathogens, including of Candida. However, this intimate relationship has not been thoroughly investigated and in this PhD program it is intended to fulfil that gap in knowledge exploring for that a combination of OMICS approaches, in a collective PathoGENOMICs plan. A thorough characterization of the molecular mechanisms underlying the lactobacilli-Candida interference in the vaginal tract, focusing specifically the endogenous species L. gasseri is aimed including characterization of: i) the effect exerted by Lg in the transcriptome-wide alterations of Cg and Ca and vice-versa; ii) the set of genes determining survival of Ca and Cg in the presence of Lg, these being an interesting and unexplored set of novel therapeutic targets by mediating competitiveness of these two pathogenic yeasts in the vaginal tract; iii) the impact of the presence of lactobacilli in virulence of Ca and Cg against vaginal cells; iv) the effect exerted by the bacteria and by the two Candida spp. in reprogramming of the genomic expression of vaginal cells. 

On the overall it is expected that the knowledge herein gathered could foster the development of probiotic formulations, based on Lg, that could be used as novel treatments of candidiasis, clearly contributing to upgrade the technological capacity of pharmaceutical industries developing these types of treatments.

Tasks

To accomplish the proposed goals for this PhD program 3 well inter-connected tasks are planned: 

Task 1. Transcriptomic response of the Candida-Lg interaction 

Using an optimized experimental setup (essentially cultivating cells of both species in MRS medium) in our laboratory for co-cultivation of Cg and Lg[17] a transcriptomics study, based on RNA-seq, is planned. For that, two time-points are expected to be analysed along the co-cultivation, an early time-point after a few hours of co-cultivation and a later time point, in which visible reduction of Candida CFUs in the presence of Lg could be observed. As controls it will be used the transcriptome of cells of the different species in single-culture. This approach will be employed to study both the interaction of Lg with C. albicans and C. glabrata and we plan to leverage on the available genomic sequences for the two yeast species as well as for the L. gasseri type strain (which is the one we are also using in the co-cultures). This RNA-seq will be outsourced and the data will be analysed together with the sequencing company in order to take into account the necessary normalization procedures that are required in a co-cultivation. Quantification of transcript levels will be performed using the pipeline available at the CLC Genomics Workbench software which we have available in the laboratory.  

Task 2. Identification of competitiveness genes associated to the Candida-lactobacilli interaction 

T1 will provide sets of genes activated when Ca/Cg are cultivated in the presence of Lg. These genes are candidates to mediate survival of the yeasts in the presence of the bacteria. In this task, we aim to examine the effect of deleting these activated genes in competitiveness of Candida in the presence of the Lg. Two commercial collections of Ca and Cg haploid deletion mutants (available in the lab) will be used as sources of necessary mutant strains. In case the strains needed are not in the collections, the mutants will be engineered using homologous recombination[16]. No more than 40 strains will be used (assuming that at least 2/3 will be available in the commercial collections; if this is not the case the number of examined strains will be reduced concomitantly). Further investigations will be performed to characterize the molecular mechanism by which these genes confer protection against Lg. At this point, it is difficult to detail the experimental approaches that will be explored since the gene set is not known, nevertheless the previous experience of the group in gene functional analysis in Yeasts (e.g.[16]), including in Cg, assures the necessary expertise to pursue the most appropriate strategies. Not all genes determining competitiveness between Candida and Lg have to be transcriptionally activated and thus we will expand the phenotypic analyses to the entire collection of deletion strains available. However, co-cultivation with the bacteria is difficult with this many strains and we will use as a proxy the cultivation in the presence of Lg supernatants (also found to inhibit growth of Candida(Fig.4)[17,18,19]. High-throughput screenings are well implemented in the iBB laboratory[20]. The assays will be conducted in 96-multiwell plates and growth of the mutants in the presence of the supernatants will be monitored (based on OD) and compared with the one of wt. More susceptible mutants will be profiled for their growth in co-culture with the live lactobacilli using the already established experimental setting[17]. The congregation of the results obtained will provide a genome-wide view of the Cg and Ca genes required for ability to survive in the presence of Lg. 

A similar approach will be performed in order to investigate a set of Lg genes activated in response to Cg or Ca. For that we plan to delete and/or over-express the top 5 genes found in the dataset of activated genes and examine the effect of these genetic modifications in the capability of the bacteria to restrain growth of Candida. For the genetic engineering of the lactobacilli cells the student will spend some time in the laboratory of Prof Sarah Lebeer, University of Antwerp, an experienced researcher in biology and genetics of Lactobacillus species. 

Task 3. Effects of lactobacilli in Candida virulence against vaginal epithelial cells

To get a clearer picture of how the Candida-bacteria system interacts with the vaginal cells, a system for co-cultivation of lactobacilli/Candida/reconstituted vaginal human epithelium (RHVE) will be established. This ex-vivo system has been used before by the group to investigate virulence traits in Cg considering the difficulties associated with manipulation of animal models and also because lactobacilli cannot naturally colonize mice vaginal tract[23]. The assays will be conducted using a co-infection setup (in RPMI, at pH 4, ~24h), in which Ca/Cg are co-inoculated with the lactobacilli in flasks containing the RVHE. The progress of the infection will be followed by histological studies and confocal microscopy analysis using fluorescent reports to detect the microbial and mammalian cells, as before[16]. The ability of Ca/Cg to colonize and invade the vaginal cells will be examined as well as the immune response triggered by the vaginal cells. This tri-culture system will also be used to get a snapshot of the transcriptomic response of the human cells to the presence of the Candida and of the bacteria. For this, after short periods of incubation in the tri-species setting (e.g. 4 or 8h of incubation), the microbial cells will be removed from the surface of RHVE and the human cells will be processed for RNA extraction and subsequent sequencing. 

References

 

1. Gonçalves, B. et al. (2016). Vulvovaginal candidiasis: Epidemiology, microbiology and risk factors. Crit. Rev. Microbiol. 42, pp.905–927.

2. Koh, A. Y. (2013). Murine models of Candida gastrointestinal colonization and dissemination. Eukaryot. Cell 12, pp.1416–1422.

3. Richter, S. S. et al. (2005). Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. J. Clin. Microbiol. 43, pp.2155–2162.

4. Silva, S. et al. (2012). Candida glabrata, Candida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol. Rev. 36, pp.288–305.

5. Sobel, J. D. (2016). Recurrent vulvovaginal candidiasis. Am. J. Obstet. Gynecol. 214, pp.15–21.

6. Lewis, R. E. (2011). Pharmacokinetic – pharmacodynamic optimization of triazole antifungal therapy. Curr. Opin. Infect. Dis. 24, pp.14–29.

7. Chakrabarti, A. et al. (2009). Recent experience with fungaemia: change in species distribution and azole resistance. Scand. J. Infect. Dis. 41, pp.275–284.

8. Zhou, X. et al. (2010). The vaginal bacterial communities of Japanese women resemble those of women in other racial groups. FEMS Immunol. Med. Microbiol. 58, pp.169–181.

9. Ravel, J. et al. (2011). Vaginal microbiome of reproductive-age women. Proc. Natl. Acad. Sci. 108, pp.4680–4687.

10. Bradford, L. L. et al. (2017). The vaginal mycobiome: A contemporary perspective on fungi in women’s health and diseases. Virulence 8, pp.342–351.

11. Romani, L. et al.(2014). Microbiota control of a tryptophan – AhR pathway. Eur. J. Immunol. 0, pp.1–9. 

12. Morales, D. K. et al. (2010). Candida albicans Interactions with Bacteria in the Context of Human Health and Disease. PLoS Pathog. 6.

13. Hickey, R. J., et al. (2012). Understanding vaginal microbiome complexity from an ecological perspective. Transl. Res. 160, pp.267–282.

14 . Lourenço, A., et al. (2019). Effect of Acetic Acid and Lactic Acid at Low pH in Growth and Azole Resistance of Candida albicans and Candida glabrata. Frontiers in Microbiology, 9:3265.

15. Ballou, E. et al. (2016). Lactate signalling regulates fungal ?-glucan masking and immune evasion. Nature Microbiology, 2:16238.

16. Bernardo, R. T. et al. (2017). The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata. G3 Genes|Genomes|Genetics 7, pp.1–18.

17. Pedro, N. (2017). Study of the interaction between vaginal Lactobacilli , Candida albicans and Candida glabrata?: from physiological aspects to transcriptomic analyses. (Master's thesis). Instituto Superior Técnico da Universidade de Lisboa, Lisboa, Portugal. 

18. Parolin, C. et al. (2015). Isolation of vaginal lactobacilli and characterization of anti-candida activity. PLoS One 10, pp.1–17.

19. Matsubara, V. H., et al. (2016). Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth, cell adhesion, and filamentation. Appl. Microbiol. Biotechnol. 100, pp.6415–6426.

20. Mira, N. P. et al. (2010). Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid. Microb. Cell Fact. 9, 79.

21. Pereira, C. A. et al. (2015). Candida albicans and virulence factors that increases its pathogenicity. Biology. pp.631–636.

22 . Ramos, L. de S. et al. (2015). Protease and phospholipase activities of Candida spp. isolated from cutaneous candidiasis. Rev. Iberoam. Micol. 32, pp.122–125.

23 . Ma, B., Forney, et al. (2012). Vaginal Microbiome: Rethinking Health and Disease. Annu. Rev. Microbiol. 66, pp.371–389.

 

 

Publication and Meetings

2017-2018 - Luísa M. Marques, Marta M. Alves, Sónia Eugénio, Sara B. Salazar, Nuno Pedro, Liliana Grenho, Nuno P. Mira, Maria H. Fernandes, Maria F. Montemor, “Potential anti-cancer and anti-Candida activity of Zn-derived foams” Journal of Materials Chemistry B, 6, 2821-2830, (2018)

2017-2018 - Lourenço, A., Pedro, N., Salazar, S.B., & Mira, N. P. "Effect of acetic acid and lacitc acid at low pH in growth and azole resistance of Candida albicans and Candida glabrata". Frontiers in Microbiology, 9, 3265. (2019)

2018-2019 - Bruna Gonçalves, Ruben Bernardo, Can wang, Nuno A. Pedro, Geraldine Butler, Joana Azeredo, mariana Henriques, Nuno Pereira Mira, Sónia Silva. "Effect of progesterone on Candida Albicans biofilm formation under acidic conditions: a transcriptomic anaysis". International Journal of Medical Microbiology (2019)

2019-2020 - Salazar SB, Simões RS, Pedro NA, Pinheiro MJ, Carvalho MF, Mira NP. An overview on conventional and non-conventional therapeutic approaches for the treatment of candidiasis and underlying resistance mechanisms in clinical strains. Journal of Fungi. 2020 Mar;6(1):23.

7th Workshop of the PhD Program in Applied and Environmental Microbiology (DP_AEM) in University of Minho, Braga from 24-27 September 2018

8th Workshop of the PhD Program in Applied and Environmental Microbiology (DP_AEM) in University of Minho, Braga from 10-13 February 2020

Seminar of the PhD Program in Applied and Environmental Microbiology (DP_AEM) at 17 November 2020, Virtual meeting.

Posters in conferences:

1. Nuno A. Pedro, Marta Alves and Nuno P. Mira. Study of the interaction between vaginal lactobacilli, Candida albicans and Candida glabrata: from physiological aspects to transcriptomic analyses. Poster presented at 14th ASM Conference on Candida and Candidiasis, Providence, USA, 15-19, April (2018).

2. Nuno A Pedro, Catarina Lima, Andreia Pimenta, Sandra C Pinto, Marta Alves, Arsénio Fialho and Nuno P Mira. Study of the interaction between vaginal lactobacilli, Candida albicans and Candida glabrata: from physiological aspects to transcriptomic analyses. Poster presented at HFP2019: Human Fungal Pathogens: Molecular Mechanisms of Host-Pathogen Interactions and Virulence, Nice, France, 18-24, May (2019).

3. Nuno A Pedro, Catarina Lima, Andreia Pimenta, Dalila Mil-Homens, Sandra C Pinto, Marta Alves, Arsénio Fialho, Nuno Mira. Study of the interaction between vaginal and intestinal lactobacilli, Candida albicans and Candida glabrata. Abstract submitted to Microbiology Society Conference on Candida and Candidiasis 2021, 21-27 March (2021), Virtual meeting.

4. Nuno Pedro, Catarina Lima, Andreia Pimenta, Dalila Mil-Homens, Sandra C Pinto, Marta Alves, Arsénio Fialho, Nuno Mira. Study of the interaction between vaginal and intestinal lactobacilli, Candida albicans and Candida glabrata. Poster presented at Microbiotec’21, Portugal 2021, 23-26 March (2021), Virtual meeting.

Abstracts in conferences:

1. Nuno A. Pedro and Nuno P. Mira. Study of the interaction between vaginal lactobacilli, Candida albicans and Candida glabrata: from physiological aspects to transcriptomic analyses. Abstract submitted to XXI Jornadas de Biologia de Leveduras "Professor Nicolau van Uden", Braga, Campus de Gualtar, University of Minho, 8-9, June (2018)

2. Nuno A Pedro, Catarina Lima, Andreia Pimenta, Sandra C Pinto, Marta Alves, Arsénio Fialho and Nuno P Mira. Study of the interaction between vaginal lactobacilli, Candida albicans and Candida glabrata: from physiological aspects to transcriptomic analyses. Abstract submitted to HFP2019: Human Fingal Pathogens: Molecular Mechanisms of Host-Pathogen Interactions and Virulence, Nice, France, 18-24, May (2019).

3. Nuno A Pedro, Catarina Lima, Andreia Pimenta, Dalila Mil-Homens, Sandra C Pinto, Marta Alves, Arsénio Fialho, Nuno Mira. Study of the interaction between vaginal and intestinal lactobacilli, Candida albicans and Candida glabrata. Abstract submitted to Microbiology Society Conference on Candida and Candidiasis 2021, 21-27 March (2021), Virtual meeting.

4. Nuno Pedro, Catarina Lima, Andreia Pimenta, Dalila Mil-Homens, Sandra C Pinto, Marta Alves, Arsénio Fialho, Nuno Mira. Study of the interaction between vaginal and intestinal lactobacilli, Candida albicans and Candida glabrata. Poster presented at Microbiotec’21, Portugal 2021, 23-26 March (2021), Virtual meeting.

Oral Presentation in Conferences:

1. Nuno A. Pedro and Nuno P. Mira. Study of the interaction between vaginal lactobacilli, Candida albicans and Candida glabrata: from physiological aspects to transcriptomic analyses. Presented to XXI Jornadas de Biologia de Leveduras "Professor Nicolau van Uden", Braga, Campus de Gualtar, University of Minho, 8-9, June (2018)

2. Nuno Pedro, Catarina Lima, Andreia Pimenta, Dalila Mil-Homens Sandra C Pinto, Marta Alves, Arsénio Fialho and Nuno Mira. Study of the interaction between vaginal and intestinal lactobacilli, Candida albicans and Candida glabrata. Presented to 15th International Congress on Yeasts (ICY), 23 -27August (2021), Virtual meeting

 

Training

Good computer skills as an office user and other software. Notions of Matlab and easy learning ability of other types of programming or software.