Acanthamoeba are ubiquitous protists that play an environmental role in regulating microbial diversity; they also occasionally cause infections of the eye (Acanthamoeba keratitis) and brain (granulomatous amoebic encephalitis). These organisms exhibit two distinct phenotypes. The trophozoite form dominates in favorable conditions, in which the Acanthamoeba move through the extension of pseudopodia, engulfing microbes and other particles. During stressful conditions, the Acanthamoeba undergo a process of encystment, in which they build a double cell wall and become relatively inactive. The cyst form can survive years until more favorable conditions arise, at which point they may excyst. For this study, multiple laboratory encystment methods were compared to determine the percent encystment and the different viabilities of laboratory-produced cysts. Furthermore, four different encystment genes were targeted for development of a primer library for reverse-transcription, polymerase chain reaction expression studies. The library was developed using sequences accessed from various databases, including NCBI and EMBL; primers were screened through polymerase chain reaction, and those primers producing positive results were used to further screen cellular RNA that was extracted from encysting cells over various time points during the encystment process, and using various encystment media. Using these methods, target gene involvement in the encystment process was compared between species and encystment methods. These studies lay the foundation for quantitative gene expression analysis, and provide the basis for comparison of various encystment methods.

Les amibes appartenant au genre Acanthamoeba sont ubiquitaires et responsables de diverses infections, en particulier de kératite amibienne. Par ailleurs elles sont résistantes à de nombreux traitements de désinfection, aussi bien sous leur forme libre (trophozoite) que sous leur forme enkystée. La plupart des données d’efficacité disponibles ont évalué des biocides utilisés pour la désinfection de l’eau potable et/ou des lentilles de contact, mais peu de données sont disponibles concernant le traitement des surfaces ou des matériels médicaux. Ces amibes sont également capables de servir de réservoir à des bactéries pathogènes, notamment des nouvelles espèces de Chlamydia regroupées sous le terme « Chlamydia-like ». Il s’agit de bactéries découvertes récemment et potentiellement responsables d’infections respiratoires et d’avortements spontanés. Malgré leur importance, peu de données sont disponibles concernant la survie et la résistance aux biocides de ces bactéries. L’objectif de ce travail a été dans un premier temps d’évaluer la résistance des amibes aux traitements de désinfections, aussi bien pour les trophozoites que pour les kystes. Dans un second temps, nous avons étudié la survie et la résistance des Chlamydia-like à la désinfection, ainsi que leurs interactions avec les amibes et avec différentes lignées cellulaires ; Chlamydia trachomatis a servi de contrôle dans cette deuxième série d’expérimentations. Les méthodes de culture et d’enkystement des trophozoites ainsi que le choix des souches testées se sont avérés critiques pour l’évaluation des biocides. Certains traitements de désinfection généralement réputés efficaces contre la plupart des micro-organismes ont montré une efficacité limitée vis-à-vis des kystes amibiens ainsi que des trophozoites (glutaraldehyde). Les Chlamydia-like se sont avérées capables de survivre dans l’environnement pendant de longues périodes mais sont globalement sensibles aux désinfectants. Certaines de ces bactéries sont également capables de survivre dans les kystes d’amibes, ce qui peut leur conférer une résistance accrue vis-à-vis des biocides. / Acanthamoebae are ubiquitous amoebae responsible for several infections, mostly amoebic keratitis. They are also resistant to numerous disinfection treatments, as well under their free shape (trophozoite ) as under their encysted shape. Most of the available data of efficiency estimated biocides used for the disinfection of the drinking water and\or the contact lenses, but few data are available concerning the treatment of surfaces or medical devices. These amoebae are also capable of serving as reservoir for pathogenic bacteria, in particular for new species of Chlamydia named " Chlamydia-like ". These new bacteria recently discovered are potentially responsible for respiratory infections and miscarriages. Despite their importance, only few data are available concerning the survival and the resistance to biocides. The objective of this work was at first time to evaluate the resistance of amoebae to disinfection treatments, as well for the trophozoites as for the cysts. Secondly, we studied the survival and the resistance of Chlamydia-like to disinfection, as well as their interactions with amoebae and with various cellular lineages; Chlamydia trachomatis served as control in this second series of experiments. The methods of culture and encystement of trophozoites as well as the choice of selected strains turned out critical for the evaluation of biocides. Some treatments generally considered as effective treatments of disinfection against most of the microorganisms showed an efficiency limited towards the amoebic cysts as well as the trophozoites ( glutaraldehyde ). Chlamydia-like turned out capable of surviving in the environment during long periods but are globally sensitive to disinfectants. Some of these bacteria are also capable to surve in amoebal cysts, what can confer them a resistance increased towards biocides.

Legionella est une bactérie pathogène, ubiquitaire des environnements aqueux. L'utilisation des eaux de surface, par les centrales nucléaires, provoque un réchauffement de ces eaux, qui associé aux conditions de fonctionnement peut entraîner sa prolifération. Afin de limiter le développement de cette bactérie, en deçà des seuils réglementaires, EDF met en œuvre un traitement à la monochloramine dans ses circuits. Notre étude a eu pour but d’améliorer les connaissances sur les associations entre les légionelles et leur principal hôte : les amibes, et sur les facteurs susceptibles d’influencer leur sensibilité au traitement biocide. Nous avons, tout d’abord, mis en évidence la permissivité de 12 souches d’amibes à 2 souches de L. pneumophila. La température est apparue comme le paramètre le plus influant, la prolifération de Legionella semble favorisée à 40°C. Puis, nous avons cherché à développer un protocole pour séparer Legionella selon son état d’association. Ce protocole n’est pas abouti à l’heure actuelle. Finalement, nous avons comparé la sensibilité, de Legionella, dans différents états physiologiques et étudié l’impact de la qualité de l’eau et des MES. Les cellules post-amibes et dans une moindre mesure, les cellules en phases stationnaires sont les moins sensibles. L’effet de la matrice a été mis en évidence pour l’eau de Vienne, par contre la sensibilité n’a pas été modifiée suite à l’élimination ou l’enrichissement en MES. Cette étude constitue une base de connaissances nouvelles pour orienter les choix de gestion du traitement anti-Legionella à la monochloramine et d’exploitation des circuits. / Legionella is a pathogenic gram negative bacterium, ubiquitously found in waterborne environment. The use of surface water, in open recirculating cooling circuits of nuclear power plants, causes a warming of these waters, which may cause proliferation of Legionella. To limit the development of this bacterium, under the legal thresholds, EDF implements a monochloramine treatment. The aim of our study was to improve the knowledge on the association between Legionella and their main host : amoebae, and on the factors that may influence their susceptibility to biocide treatment. We first showed the permissiveness of 12 strains of amoebae to 2 strains of L. pneumophila. Temperature is appeared as the most influential parameter, the proliferation of Legionella seems favored at 40°C. Then, we searched to develop a protocol to quantify Legionella according to its state of association. This protocol is not operational, yet. Finally, we compared the sensitivity of Legionella, under different physiological states and studied the impact of water quality and suspended matter. Post-amoeba cells and at a lesser extent, stationary cells, are the less sensitive. The effect of the matrix was demonstrated for the Vienne river water, on the other side the elimination or enrichment in suspended matter has not modified the sensitivity. This study provides a basis for a better knowledge to manage monochloramine treatment against Legionella, and for operating cooling systems.

Les amibes appartenant au genre Acanthamoeba sont ubiquitaires et responsables de diverses infections, en particulier de kératite amibienne. Par ailleurs elles sont résistantes à de nombreux traitements de désinfection, aussi bien sous leur forme libre (trophozoite) que sous leur forme enkystée. La plupart des données d'efficacité disponibles ont évalué des biocides utilisés pour la désinfection de l'eau potable et/ou des lentilles de contact, mais peu de données sont disponibles concernant le traitement des surfaces ou des matériels médicaux. Ces amibes sont également capables de servir de réservoir à des bactéries pathogènes, notamment des nouvelles espèces de Chlamydia regroupées sous le terme " Chlamydia-like ". Il s'agit de bactéries découvertes récemment et potentiellement responsables d'infections respiratoires et d'avortements spontanés. Malgré leur importance, peu de données sont disponibles concernant la survie et la résistance aux biocides de ces bactéries. L'objectif de ce travail a été dans un premier temps d'évaluer la résistance des amibes aux traitements de désinfections, aussi bien pour les trophozoites que pour les kystes. Dans un second temps, nous avons étudié la survie et la résistance des Chlamydia-like à la désinfection, ainsi que leurs interactions avec les amibes et avec différentes lignées cellulaires ; Chlamydia trachomatis a servi de contrôle dans cette deuxième série d'expérimentations. Les méthodes de culture et d'enkystement des trophozoites ainsi que le choix des souches testées se sont avérés critiques pour l'évaluation des biocides. Certains traitements de désinfection généralement réputés efficaces contre la plupart des micro-organismes ont montré une efficacité limitée vis-à-vis des kystes amibiens ainsi que des trophozoites (glutaraldehyde). Les Chlamydia-like se sont avérées capables de survivre dans l'environnement pendant de longues périodes mais sont globalement sensibles aux désinfectants. Certaines de ces bactéries sont également capables de survivre dans les kystes d'amibes, ce qui peut leur conférer une résistance accrue vis-à-vis des biocides.

Heat shock proteins (Hsps) are a class of molecular chaperones which were first discovered as proteins up-regulated in response to heat stress in Drosophila. Later, it was found that these set of proteins get up-regulated as a general stress response associated with destabilization of native protein structures. Over a period of time, intricate involvement of Hsps in various biological processes has been well established. Heat shock protein 90 (Hsp90) is one of the important representative of this class of proteins. Hsp90 is an essential molecular chaperone which is evolutionarily conserved. It has a selective set of proteins to chaperone called as clients, which majorly include transcription factors and protein kinases. Through its interaction with its clients it modulates cell cycle, signal transduction, differentiation, development and evolution. Previous studies from Candida, Leishmania and Plasmodium have implicated Hsp90 to be involved in stage transition and growth. It is also critically involved in regulating growth of other protozoans such as Dictyostelium, Entamoeba and Trypanosoma. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. In Plasmodium falciparum, Hsp90 plays a critical role in stage transition. The parasite inside the human RBC develops from ring to trophozoite to schizont stage and inhibition of Hsp90 using specific pharmacological inhibitor arrests the growth of parasite at ring stage. In Dictyostelium, it has been observed that Hsp90 function is required for development. Inhibition of Hsp90 causes mound arrest and stops the cells from entering to its next developmental stage, fruiting bodies. In parallel, Hsp90 in Candida has been shown to be involved in morphogenesis. In nature Candida exists as a single cell yeast form and upon entry into the human host these yeast forms undergo morphogenesis to form virulent filamentous fungi. Inhibition of Hsp90 mimics temperature mediated morphogenesis. All together, these studies suggest that Hsp90 functions in a context dependent manner and each biological system explored has given new insights into the Hsp90 biology. Giardia lamblia, a protozoan parasite of humans and animals, is an important cause of diarrheal disease causing significant morbidity and also mortality in tropical countries. In the present study we focus on the biology of Hsp90 from Giardia lamblia. Giardia has a biphasic life cycle with infective cyst stage and pathogenic trophozoite stage. These cysts are present in the environment and enter mammalian host through oral route. They undergo a process called as excystation in the intestine giving rise to trophozoites. The trophozoites so formed colonize the upper part of the small intestine which causes the symptoms of giardiasis. Some of the trophozoites escape from the nutrition rich milieu of the upper part of small intestine to the lower part. In this region, trophozoites undergo a process called as encystation, wherein each trophozoite forms a cyst which escapes through faeces back into the environment. As seen in the life cycle of Giardia there are two major biological transitions, excystation and encystation; and till date no definitive player or pathway is known to regulate these processes. With the knowledge of Hsp90 playing an important role in similar biological transitions in other organisms we were encouraged to study role of Hsp90 in Giardia lamblia. Trans-splicing based generation of a full length Hsp90 in Giardia lamblia To understand the role of Hsp90, we first carried out sequence alignment of Hsp90 predicted ORFs in Giardia genome with yeast Hsp90. On alignment we observed that Hsp90 in Giardia is discontinuous and is annotated to be encoded by two different ORFs. Hsp90 in most organisms is coded by a single ORF with none to many cis-spliced introns. In a relatively intron poor organism G. lamblia, cytosolic Hsp90 is coded by two different ORFs separated by 777 kb in the genome. On multiple sequence alignment, we noticed that these two ORFs correspond to two independent regions of the Hsp90 protein. The ORFs are designated as hspN and hspC, containing the N-terminal and the C-terminal region of the protein respectively. We began our study by sequencing whole genome of Giardia lamblia clinical strain. Our genome sequencing confirmed the split nature of hsp90 and showed high ‘synteny’ between the other sequenced isolates. Using PCR based approach we have ruled out the possibility of having a full length gene in the genome. In contradiction to the genome result, we have observed a higher molecular weight protein in the lysate on proteomic analysis which was further confirmed by western blotting. The protein was observed to have a molecular weight of 80 kDa which could be a resultant of combination of two ORFs, suggesting the presence of a full length mRNA for Hsp90. PCR amplification using primers against both the fragments resulted in amplification of 2.1 kb product from the RNA pool of Giardia. Sequencing of this product showed that hspN and hspC were stitched together to form a mature messenger for full length Hsp90. In total our results suggest a post transcriptional process, trans-splicing, to be involved in the construction of Hsp90. The transition marked by this fusion coincides with the canonical GU¬AG splice site transitions as observed in other eukaryotes. Interestingly, a 26 nt near-complementary region was observed inside and upstream of hspN and hspC ORFs respectively. Put together these results suggest that the 26 nt complementary region acts as the positioning element to bring these two precursors in spatial proximity. With efficient spliceosomal activity these two precursor forms are trans-spliced to generate a full length cytosolic Hsp90 in Giardia. There are only four genes which have cis-spliced introns in the Giardia genome and the core components of the spliceosomal machinery are also present. The presence of canonical splice site in both the transcripts suggests that these transcripts are fused together by the spliceosomal machinery by the phenomenon of trans-splicing. The formation of full length Hsp90 RNA by its fragmented gene is the first example of trans-splicing in Giardia. To understand, are there any other genes which are also similarly trans-spliced we have carried out shotgun proteomic analysis of the total cell lysate obtained from Giardia trophozoites. Using Hsp90 as template, in our proteomic datasets, we have designed an algorithm for identification of additional trans-spliced gene products at the protein level. We have identified a total of 476 proteins of which hypothetical proteins constitute the major class followed by metabolic enzymes. We have compared the theoretical molecular weights for the identified proteins with the experimentally determined mass. Any discrepancy in the molecular mass was further analyzed and we assigned a gene to be potentially trans-spliced based on three criteria: if they were encoded by two or more different ORFs (loci), absence of a single full length counterpart and presence of splice sites with branch point and positional elements. Using this algorithm we were able to identify dynein as a potential candidate of trans-splicing reaction which was confirmed by the nucleotide sequence analysis of the predicted ORFs. Interestingly, dynein gene fragments were observed to be scattered on different chromosomes with minor splice sites unlike hsp90 genes. In vivo Expression of Hsp90 sub-fragments, HspN and HspC In the mature Hsp90 mRNA formed upon trans-splicing, 33 additional codons are present right between hspN and hspC sequences and they were acquired from the upstream region of hspC ORF. The 33 codons encode for an important region of Hsp90 which harbours the conserved catalytic “Arg” residue; suggesting that the full length Giardia Hsp90 (GlHsp90) formed could be an active ATPase. To confirm the same we have carried out in vitro characterization of trans-spliced Hsp90. Towards this, we have cloned, expressed and purified His tag-GlHsp90. As a first step, highly purified protein was used to assess its efficiency in binding to it cognate ligand, ATP, and the known inhibitors. Our binding studies show that GlHsp90 binds to ATP with a dissociation constant of 628 M and to its inhibitors, GA and 17AAG with 1.5 μM and 17.5 μM respectively. The bound ATP will be subsequently cleaved by Hsp90 which is an essential step in the chaperone cycle. As determined in our ATPase assay we observed that GlHsp90 hydrolyzes bound ATP with the catalytic efficiency of 4.4 × 10-5μM-1.min-1which confirms that Hsp90 generated upon trans-splicing is an active ATPase. The uniqueness of the hsp90 gene arrangement in Giardia posed a new question. Do these gene fragments also get translated? Our results suggest that HspN and HspC are poly¬adenylated. In order to determine the levels of these transcripts we performed qRT-PCR using primers specific to HspN, HspC and GlHsp90. We have observed that, in comparison with HspN transcript level, HspC and GlHsp90 transcripts are 15 and 75 folds higher respectively. To check for the presence of translation products of these transcripts, we have re-analyzed our proteomic datasets wherein we could identify peptides corresponding to HspN and HspC in their respective molecular weight region, 45 to 35 kDa. To confirm the proteomic data, western blot analysis was performed for trophozoite lysate on both 1D and 2D gels using anti-HspN antibody. Two specific bands (1D) / spots (2D) corresponding to the full length Hsp90 and HspN were identified. Gel filtration analysis revealed that HspN co¬eluted with full length Hsp90 thereby suggesting that both the proteins are in a same complex. With the background that HspN and HspC are present at the protein level, we asked if these fragments in combination can hydrolyse ATP. We reconstituted recombinant HspN and HspC in equimolar amounts and scored for the hydrolysis of ATP. However, no Pi release was observed. To determine whether HspN and HspC could modulate Hsp90 function, ATPase activity was monitored in the presence of HspN or HspC, in vitro. It was observed that ATPase activity was inhibited by both the fragments thus suggesting that HspN and HspC negatively regulate Hsp90 ATPase activity. Role of Hsp90 in Giardia encystation Giardia has a biphasic life cycle with proliferative trophozoites and latent cyst stage. In Giardia, in vitro encystation was established nearly two decades back by modulating the medium conditions. However, the mechanism and triggers underlying this transition are not well characterized. To understand whether Hsp90 has any role in this transition, in vitro conversion of trophozoites to cysts was achieved. The cysts obtained showed all the characteristic features of mature Giardia cyst with cyst wall protein 1 (CWP1) on the cyst wall and four nuclei as determined by immunofluorescence analysis. Further, the levels of Hsp90 in trophozoites were compared with mature cysts at both transcript and protein levels and it was found that cysts show more than 50% reduction in the level of Hsp90 in comparison with normal trophozoites. In accordance, exogenous inhibition of Hsp90 using 17AAG promoted the formation of cysts in vitro by 60 folds in a dose dependent manner; however, the window period of Hsp90 function compromise plays an important role in this process. Higher numbers of cysts were obtained from the cells treated with inhibitors during pre-encystation condition but inhibition of Hsp90 during encystation did not affect the formation of cysts, suggesting that Hsp90 down-regulation plays an important role during commitment towards encystation. To further show that cyst formation is a specific response to Hsp90 inhibition we have carried out encystation in the presence of metranidazole and from heat shocked cells; however, in both the conditions we did not observe any significant change in cyst formation, thus confirming that Hsp90 plays an important role during encystation in Giardia lamblia. Summary In Conclusion, Our study throws light on a unique aspect of Hsp90 biology in Giardia Lamblia, wherein the formation of the full length protein is dependent on a unique trans splicing reaction of its gene components representing different domains. We have also shown that HsP90 fragments, HspN and HspC, are also expressed in Trophozoites. Our in vitro data suggests that these fragments possibly regulate the function of Hsp90. Furthermore, the full length of Hsp90 plays an important role in stage transition in Giardia wherein inhibition of Hsp90 induces encystations. The study has opened many new avenues for research. Understanding the exact role of HspN and HspC in vivo will provide better appreciation for the evolution of such a complex biogenesis of an essential protein.