Final Activity Report Summary - DonNA-R (Natural Antimony Resistance in Leishmania donovani: A fundamental study to guide future epidemiological strategies.)
Antimonials (SbV) are the first line drug used in many parts of the world. In the Indian sub-continent, leishmania donovani has developed drug resistance over the past 10 years. This problem is now being countered on a clinical level by changing first line treatment from SbV to miltefosine in the frame of the kala-azar elimination programme. Within this context, two questions were addressed in the course of this project.
Firstly, a biological question was posed, related to how the Nepalese l. donovani populations responded to past years of clinical SbV pressure and what was the putative epidemiological impact of SbV-adapted strains. The action mechanism of SbV was complex and involved several activities targeting the parasite redox system. This suggested that SbV-resistant parasites might have adapted their redox capacity, and as such be cross-resistant to oxidative or nitrosative stress, one of the central defence tools of the host immune system. This hypothesis could have severe epidemiological implications, as parasites armoured against host defence tools were likely to be harder to combat and contain, and thus continue to threaten visceral leishmaniasis (VL) control.
The validity of this hypothesis was verified within this project by characterising the oxidative stress defence mechanisms of SbV-resistant l. donovani using a targeted approach on the following three different levels:
1. targeted transcriptomic or proteomic profiling. Putative markers found on gene expression level could not be confirmed on protein level and this questioned the biological significance of those gene expression markers. Putative markers on protein level suggested that in some genetic subgroups SbV-resistance was related to decreased oxidative stress resistance, thus countering the original hypothesis.
2. quantitation of parasite thiols, an approach that yielded a conclusion similar to that of the protein profiling.
3. assessment of parasite survival exposed to oxidative or nitrosative stress . This final approach demonstrated that SbV-R parasites had different responses to different types of oxidative or nitrosative stress. Hence, we hypothesised that the relation between SbV-resistance and oxidative or nitrosative stress depended on the exact nature of the stress, which in natural conditions could greatly vary with the type of host cell, the phase of infection, the activity of the immune system, co-infections etc. As a result, it was difficult to draw general conclusions on how well the SbV-R parasites would thrive in host macrophages, or on exactly what the epidemiological impact of the SbV-R parasites would be.
We concluded that:
1. parasites might develop different natural adaptations under antimonial treatment pressure, depending on the genetic background of the parasite. This should pull up a red flag for all leishmania drug surveillance programmes, as resistance surveillance tools should be able to detect most frequent drug resistant modifications.
2. the used targeted approach yielded fragmentary biological information and was not the most adequate to investigate drug resistance in all genetic subgroups. This compromised the understanding of the impact of SbV-R parasites globally and highlighted the need for untargeted global approaches in this research field.
Secondly, a research approach question was addressed, related to how the newly developed omics technologies could be integrated in fundamental studies to guide epidemiological strategies of infectious diseases. In this project, we explored the potential of these new omics technologies that were specific for the biological research question at hand and concluded that they were powerful tools to:
1. identify global differential genomic and metabolic properties between various phenotypes which had the potential to highlight multi-factorial changes.
2. identify the various phenotypic clusters in a population without having prior knowledge of the nature of the different phenotypes present in the population.
Our future research strategy would focus on the integration of global genomic and metabolomic parasite diversity information which we believed that would enable the much needed advance in parasite population characterisation, and as such contribute greatly to design of epidemiological surveillance strategies.
Firstly, a biological question was posed, related to how the Nepalese l. donovani populations responded to past years of clinical SbV pressure and what was the putative epidemiological impact of SbV-adapted strains. The action mechanism of SbV was complex and involved several activities targeting the parasite redox system. This suggested that SbV-resistant parasites might have adapted their redox capacity, and as such be cross-resistant to oxidative or nitrosative stress, one of the central defence tools of the host immune system. This hypothesis could have severe epidemiological implications, as parasites armoured against host defence tools were likely to be harder to combat and contain, and thus continue to threaten visceral leishmaniasis (VL) control.
The validity of this hypothesis was verified within this project by characterising the oxidative stress defence mechanisms of SbV-resistant l. donovani using a targeted approach on the following three different levels:
1. targeted transcriptomic or proteomic profiling. Putative markers found on gene expression level could not be confirmed on protein level and this questioned the biological significance of those gene expression markers. Putative markers on protein level suggested that in some genetic subgroups SbV-resistance was related to decreased oxidative stress resistance, thus countering the original hypothesis.
2. quantitation of parasite thiols, an approach that yielded a conclusion similar to that of the protein profiling.
3. assessment of parasite survival exposed to oxidative or nitrosative stress . This final approach demonstrated that SbV-R parasites had different responses to different types of oxidative or nitrosative stress. Hence, we hypothesised that the relation between SbV-resistance and oxidative or nitrosative stress depended on the exact nature of the stress, which in natural conditions could greatly vary with the type of host cell, the phase of infection, the activity of the immune system, co-infections etc. As a result, it was difficult to draw general conclusions on how well the SbV-R parasites would thrive in host macrophages, or on exactly what the epidemiological impact of the SbV-R parasites would be.
We concluded that:
1. parasites might develop different natural adaptations under antimonial treatment pressure, depending on the genetic background of the parasite. This should pull up a red flag for all leishmania drug surveillance programmes, as resistance surveillance tools should be able to detect most frequent drug resistant modifications.
2. the used targeted approach yielded fragmentary biological information and was not the most adequate to investigate drug resistance in all genetic subgroups. This compromised the understanding of the impact of SbV-R parasites globally and highlighted the need for untargeted global approaches in this research field.
Secondly, a research approach question was addressed, related to how the newly developed omics technologies could be integrated in fundamental studies to guide epidemiological strategies of infectious diseases. In this project, we explored the potential of these new omics technologies that were specific for the biological research question at hand and concluded that they were powerful tools to:
1. identify global differential genomic and metabolic properties between various phenotypes which had the potential to highlight multi-factorial changes.
2. identify the various phenotypic clusters in a population without having prior knowledge of the nature of the different phenotypes present in the population.
Our future research strategy would focus on the integration of global genomic and metabolomic parasite diversity information which we believed that would enable the much needed advance in parasite population characterisation, and as such contribute greatly to design of epidemiological surveillance strategies.