Presence of Non-Tuberculous Mycobacteria Including Mycobacterium avium subsp. paratuberculosis Associated with Environmental Amoebae.

One of the obstacles to eradicating paratuberculosis or Johne's Disease (JD) seems to be the persistence of Mycobacterium avium subsp. paratuberculosis (Map) in the environment due to its ability to survive alone or vectorized. It has been shown that Map is widely distributed in soils and water. Previously, we isolated amoebae associated with Map strains in the environment of bovines from an infected herd. This work aims to verify our working hypothesis, which suggests that amoebae may play a role in the transmission of JD. In this study, we sampled water in the vicinity of herds infected with Map or Mycobacterium bovis (M. bovis) and searched for amoebae and mycobacteria. Live amoebae were recovered from all samples. Among these amoebae, four isolates associated with the presence of mycobacteria were identified and characterized. Map and other mycobacterial species were detected by qPCR and, in some cases, by culture. This study suggests that amoebae and Map may be found in the same environment and might represent a risk of exposure of animals to pathogenic mycobacteria. These data open up new perspectives on the control measures to be put in place to prevent contamination by Map.

Proteomic analysis of Acanthamoeba castellanii response to Legionella pneumophila infection.

Legionella pneumophila is an opportunistic pathogen responsible for Legionnaires' disease or Legionellosis. This bacterium is found in the environment interacting with free-living amoebae such as Acanthamoeba castellanii. Until now, proteomic analyses have been done in amoebae infected with L. pneumophila but focused on the Legionella-containing vacuole. In this study, we propose a global proteomic analysis of the A. castellanii proteome following infection with L. pneumophila wild-type (WT) or with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We found that infection with L. pneumophila WT leads to reduced levels of A. castellanii proteins associated with lipid homeostasis/metabolism, GTPase regulation, and kinase. The levels of organelle-associated proteins were also decreased during infection. Legionellapneumophila WT infection leads to increased levels of proteins associated with polyubiquitination, folding or degradation, and antioxidant activities. This study reinforces our knowledge of this too little explored but so fundamental interaction between L. pneumophila and A. castellanii, to understand how the bacterium could resist amoeba digestion.

Here, there and everywhere: Ecology and biology of the Dependentiae phylum.

Our view of bacterial diversity has been dramatically impacted by cultivation-independent approaches such as metagenomics and 16S rRNA gene sequencing. Consequently, most bacterial phyla known to date are only documented by the presence of DNA sequences in databases and lack cultivated representatives. This bacterial majority that is yet-to-be cultivated, is forming the 'Microbial Dark Matter', (MDM) a consortium, whose ecology and biology remain largely unexplored. The Candidatus Dependentiae stands as one of many phyla within this MDM, found worldwide in various environments. Genomic evidence suggests ancestral, unusual adaptations of all Ca. Dependentiae to a host dependent lifestyle. In line with this, protists appear to be important for Ca. Dependentiae biology, as revealed by few recent studies, which enabled their growth in laboratory through host cultivation. However, the Ca. Dependentiae still remain to this day a poorly documented phylum. The present review aims to summarize the current knowledge accumulated on this often found, but rarely highlighted, bacterial phylum.

Characterization of Rosculus vilicus sp. nov., a rhizarian amoeba interacting with Mycobacterium avium subsp. paratuberculosis.

Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. It has been hypothesized that this might be the case for Mycobacterium avium subsp. paratuberculosis, the bacterium responsible for paratuberculosis. In a previous work, we isolated an amoeba from a water sample in the environment of infected cattle and showed that this amoeba was associated with Mycobacterium avium subsp. paratuberculosis. While a partial 18S rRNA gene has allowed us to suggest that this amoeba was Rosculus-like, at that time we were not able to sub-cultivate it. In the present study, we succeeded in cultivating this strain at 20-25°C. This amoeba is among the smallest (5-7 µm) described. The sequencing of the whole genome allowed us to extract the full 18S rRNA gene and propose this strain as a new species of the Rosculus genus, i.e., R. vilicus. Of note, the mitochondrial genome is particularly large (184,954 bp). Finally, we showed that this amoeba was able to phagocyte Mycobacterium avium subsp. paratuberculosis and that the bacterium was still observed within amoebae after at least 3 days. In conclusion, we characterized a new environmental amoeba species at the cellular and genome level that was able to interact with Mycobacterium avium subsp. paratuberculosis. As a result, R. vilicus is a potential candidate as environmental reservoir for Mycobacterium avium subsp. paratuberculosis but further experiments are needed to test this hypothesis.

Identifying group-specific primers for environmental Heterolobosa by high-throughput sequencing.

Diversity of Heterolobosea (Excavata) in environments is poorly understood despite their ecological occurrence and health-associated risk, partly because this group tends to be under-covered by most universal eukaryotic primers used for sequencing. To overcome the limits of the traditional morpho-taxonomy-based biomonitoring, we constructed a primer database listing existing and newly designed specific primer pairs that have been evaluated for Heterolobosea 18S rRNA sequencing. In silico taxonomy performance against the current SILVA SSU database allowed the selection of primer pairs that were next evaluated on reference culture amoebal strains. Two primer pairs were retained for monitoring the diversity of Heterolobosea in freshwater environments, using high-throughput sequencing. Results showed that one of the newly designed primer pairs allowed species-level identification of most heterolobosean sequences. Such primer pair could enable informative, cultivation-free assays for characterizing heterolobosean populations in various environments.

A time-resolved multi-omics atlas of Acanthamoeba castellanii encystment.

Encystment is a common stress response of most protists, including free-living amoebae. Cyst formation protects the amoebae from eradication and can increase virulence of the bacteria they harbor. Here, we mapped the global molecular changes that occur in the facultatively pathogenic amoeba Acanthamoeba castellanii during the early steps of the poorly understood process of encystment. By performing transcriptomic, proteomic, and phosphoproteomic experiments during encystment, we identified more than 150,000 previously undescribed transcripts and thousands of protein sequences absent from the reference genome. These results provide molecular details to the regulation of expected biological processes, such as cell proliferation shutdown, and reveal new insights such as a rapid phospho-regulation of sites involved in cytoskeleton remodeling and translation regulation. This work constitutes the first time-resolved molecular atlas of an encysting organism and a useful resource for further investigation of amoebae encystment to allow for a better control of pathogenic amoebae.

Morphology and Ecology of Two New Amoebae, Isolated From a Thalassohaline Lake, Dziani Dzaha.

Dziani Dzaha is a hypersaline lake (Mayotte island), whose microbial community is dominated by photosynthetic microorganisms. Here, we describe two new free-living heteroloboseans. One belonging to the Pharyngomonas genus and the other, whose 18S rRNA gene sequence shares only 85% homology to its closest relatives Euplaesiobystra hypersalinica, was proposed as a new species of this genus being called Euplaesiobystra dzianiensis. Both strains were salt tolerant to 75‰ and grew between 25 and 37°C. Their distribution patterns varied seasonally and depended also on depth. Noticeably, both free-living amoebae isolates were able to graze on Arthrospira filaments, which are found within the same water layer. In conclusion, we document for the first time the presence and ecology of free-living amoebae in the thalassohaline lake Dziani Dzaha, and describe a new species of the Euplaesiobystra genus.

Delayed cytokinesis generates multinuclearity and potential advantages in the amoeba Acanthamoeba castellanii Neff strain.

Multinuclearity is a widespread phenomenon across the living world, yet how it is achieved, and the potential related advantages, are not systematically understood. In this study, we investigate multinuclearity in amoebae. We observe that non-adherent amoebae are giant multinucleate cells compared to adherent ones. The cells solve their multinuclearity by a stretchy cytokinesis process with cytosolic bridge formation when adherence resumes. After initial adhesion to a new substrate, the progeny of the multinucleate cells is more numerous than the sibling cells generated from uninucleate amoebae. Hence, multinucleate amoebae show an advantage for population growth when the number of cells is quantified over time. Multiple nuclei per cell are observed in different amoeba species, and the lack of adhesion induces multinuclearity in diverse protists such as Acanthamoeba castellanii, Vermamoeba vermiformis, Naegleria gruberi and Hartmannella rhysodes. In this study, we observe that agitation induces a cytokinesis delay, which promotes multinuclearity. Hence, we propose the hypothesis that multinuclearity represents a physiological adaptation under non-adherent conditions that can lead to biologically relevant advantages.

Encystment Induces Down-Regulation of an Acetyltransferase-Like Gene in Acanthamoeba castellanii.

Acanthamoeba castellanii is a ubiquitous free-living amoeba. Pathogenic strains are causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. In response to adverse conditions, A. castellanii differentiate into cysts, which are metabolically inactive and resistant cells. This process, also named encystment, involves biochemical and genetic modifications that remain largely unknown. This study characterizes the role of the ACA1_384820 Acanthamoeba gene during encystment. This gene encodes a putative N-acetyltransferase, belonging to the Gcn5-related N-acetyltransferase (GNAT) family. We showed that expression of the ACA1_384820 gene was down-regulated as early as two hours after induction of encystment in A. castellanii. Interestingly, overexpression of the ACA1_384820 gene affects formation of cysts. Unexpectedly, the search of homologs of ACA1_384820 in the Eukaryota gene datasets failed, except for some species in the Acanthamoeba genus. Bioinformatics analysis suggested a possible lateral acquisition of this gene from prokaryotic cells. This study enabled us to describe a new Acanthamoeba gene that is down-regulated during encystment.

Resistance of the oyster pathogen Vibrio tasmaniensis LGP32 against grazing by Vannella sp. marine amoeba involves Vsm and CopA virulence factors.

Vibrios are ubiquitous in marine environments and opportunistically colonize a broad range of hosts. Strains of Vibrio tasmaniensis present in oyster farms can thrive in oysters during juvenile mortality events and behave as facultative intracellular pathogen of oyster haemocytes. Herein, we wondered whether V. tasmaniensis LGP32 resistance to phagocytosis is specific to oyster immune cells or contributes to resistance to other phagocytes, like marine amoebae. To address this question, we developed an integrative study, from the first description of amoeba diversity in oyster farms to the characterization of LGP32 interactions with amoebae. An isolate of the Vannella genus, Vannella sp. AP1411, which was collected from oyster farms, is ubiquitous, and belongs to one clade of Vannella that could be found associated with Vibrionaceae. LGP32 was shown to be resistant to grazing by Vannella sp. AP1411 and this phenotype depends on some previously identified virulence factors: secreted metalloprotease Vsm and copper efflux p-ATPase CopA, which act at different steps during amoeba-vibrio interactions, whereas some other virulence factors were not involved. Altogether, our work indicates that some virulence factors can be involved in multi-host interactions of V. tasmaniensis ranging from protozoans to metazoans, potentially favouring their opportunistic behaviour.

Free-living amoebae and squatters in the wild: ecological and molecular features.

Free-living amoebae are protists frequently found in water and soils. They feed on other microorganisms, mainly bacteria, and digest them through phagocytosis. It is accepted that these amoebae play an important role in the microbial ecology of these environments. There is a renewed interest for the free-living amoebae since the discovery of pathogenic bacteria that can resist phagocytosis and of giant viruses, underlying that amoebae might play a role in the evolution of other microorganisms, including several human pathogens. Recent advances, using molecular methods, allow to bring together new information about free-living amoebae. This review aims to provide a comprehensive overview of the newly gathered insights into (1) the free-living amoeba diversity, assessed with molecular tools, (2) the gene functions described to decipher the biology of the amoebae and (3) their interactions with other microorganisms in the environment.

Spatio-temporal survey of opportunistic premise plumbing pathogens in the Paris drinking water distribution system.

Opportunistic premise plumbing pathogens present in drinking water are linked to a significant number of infections for health compromised patients. However, their monitoring is not required in current water potability standards and they have been poorly studied in a full-scale network. In this study, we quantified, by qPCR, three opportunistic pathogens, Mycobacterium spp., Legionella pneumophila, Pseudomonas aeruginosa throughout the Paris drinking water network over a one-year sampling campaign. While Mycobacteria spp. seemed ubiquitous whatever the distribution system and the time of the year, the occurrence of L. pneumophila and P. aeruginosa showed seasonal variations. Unlike L. pneumophila and P. aeruginosa, the concentration (copies number/L) of Mycobacterium spp. varied between sampling sites. The variation in microbial numbers did not demonstrate any correlations with temperature, pH, chlorine, conductivity, orthophosphate or nitrate levels. In conclusion, Mycobacterium spp. are common inhabitants of the Paris network while L. pneumophila and P. aeruginosa presence fluctuate over space and time. Such qPCR approach would help to better understand the behaviour of opportunistic premise plumbing pathogens.

Targeted Metagenomics of Microbial Diversity in Free-Living Amoebae and Water Samples.

The presence of Legionella spp. in natural and man-made water systems is a great public health concern and heavily depends on the presence of free-living amoebae. Taking advantage of the development and affordability of next-generation sequencing technology, we present here a method to characterize the whole bacterial community directly from water samples, as well as from isolated free-living amoebae.

Microbiome of drinking water: A full-scale spatio-temporal study to monitor water quality in the Paris distribution system.

The microbiological water quality of drinking water distribution system (DWDS) is of primary importance for human health. High-throughput sequencing has gained more and more attention in the last decade to describe this microbial diversity in water networks. However, there are few studies describing this approach on large drinking water distribution systems and for extended periods of time. To fill this gap and observe the potential subtle variation in microbiota of a water network through time and space, we aimed to apply high-throughput sequencing of the 16S rRNA gene approach to characterize bacterial communities of the Paris' DWDS over a one-year period. In this study, the Paris network, composed of four different DWDSs, was sampled at 31 sites, each month for one year. The sampling campaign was one of the largest described so far (n = 368) and the importance of key spatio-temporal and physico-chemical parameters was investigated. Overall, 1321 taxa were identified within the Paris network, although only fifteen of them were found in high relative abundance (>1%) in all samples. Two genera, Phreatobacter and Hyphomicrobium were dominant. The whole bacterial diversity was not significantly affected between the four DWDSs (spatial parameter) and by physico-chemical parameters. However, the bacterial diversity was slightly modified over the one-year period (temporal parameter) as we were able to observe DWDS microbiome perturbations, presumably linked to a preceding flood event. Comparison of high-throughput sequencing of the 16S rRNA gene amplicons vs. cultivation-based techniques showed that only 1.8% of bacterial diversity was recovered through cultivation. High throughput sequencing has made it possible to monitor DWDS more accurately than conventional methods by describing the whole diversity and detecting slight fluctuations in bacterial communities. This method would be further used to supervise drinking water networks, to follow any perturbations due to internals events (such as treatments) or external events (such as flooding).

Environmental Mycobacterium avium subsp. paratuberculosis Hosted by Free-Living Amoebae.

Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of M. avium subsp. paratuberculosis is poorly understood and several studies suggest that free-living amoebae (FLA) might be a potential environmental host. FLA are protozoa found in water and soil that are described as reservoirs of pathogenic and non-pathogenic bacteria in the environment. Indeed, bacteria able to survive within these amoebae would survive phagocytosis from immune cells. In this study, we assessed the in vitro interactions between several strains of M. avium subsp. paratuberculosis and Acanthamoeba castellanii. The results indicate that the bacteria were able to grow within the amoeba and that they can survive for several days within their host. To explore the presence of M. avium subsp. paratuberculosis in environmental amoebae, we sampled water from farms positive for paratuberculosis. A M. avium subsp. paratuberculosis strain was detected within an environmental amoeba identified as related to the poorly described Rosculus genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various M. avium subsp. paratuberculosis strains were able to grow within amoebae and that these bacteria could be found on farm within amoebae isolated from the cattle environment. It validates that infected amoebae might be a reservoir and vector for the transmission of M. avium subsp. paratuberculosis.

Legionella pneumophila decreases velocity of Acanthamoeba castellanii.

Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila. Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila. Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii.

Mycobacterium llatzerense, a waterborne Mycobacterium, that resists phagocytosis by Acanthamoeba castellanii.

Nontuberculous mycobacteria (NTM) are environmental bacteria increasingly associated to public health problems. In water systems, free-living amoebae (FLA) feed on bacteria by phagocytosis, but several bacteria, including many NTM, are resistant to this predation. Thus, FLA can be seen as a training ground for pathogenic bacteria. Mycobacterium llatzerense was previously described as frequently associated with FLA in a drinking water network. The present study aimed to characterize the interactions between M. llatzerense and FLA. M. llatzerense was internalised by phagocytosis and featured lipid inclusions, suggesting a subversion of host resources. Moreover, M. llatzerense survived and even multiplied in presence of A. castellanii. Using a genomic-based comparative approach, twelve genes involved in phagocytosis interference, described in M. tuberculosis, were identified in the M. llatzerense genome sequenced in this study. Transcriptomic analyses showed that ten genes were significantly upregulated during the first hours of the infection, which could partly explain M. llatzerense resistance. Additionally, M. llatzerense was shown to actively inhibit phagosome acidification. In conclusion, M. llatzerense presents a high degree of resistance to phagocytosis, likely explaining its frequent occurrence within FLA in drinking water networks. It underscores that NTM should be carefully monitored in water networks to prevent human health concerns.

Amoebicidal Activity of Caffeine and Maslinic Acid by the Induction of Programmed Cell Death in Acanthamoeba.

Free-living amoebae of the genus Acanthamoeba are the causal agents of a sight-threatening ulceration of the cornea called Acanthamoeba keratitis, as well as the rare but usually fatal disease granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba infections, they are generally lengthy and/or have limited efficacy. For the best clinical outcome, treatments should target both the trophozoite and the cyst stages, as cysts are known to confer resistance to treatment. In this study, we document the activities of caffeine and maslinic acid against both the trophozoite and the cyst stages of three clinical strains of Acanthamoeba These drugs were chosen because they are reported to inhibit glycogen phosphorylase, which is required for encystation. Maslinic acid is also reported to be an inhibitor of extracellular proteases, which may be relevant since the protease activities of Acanthamoeba species are correlated with their pathogenicity. We also provide evidence for the first time that both drugs exert their anti-amoebal effects through programmed cell death.

Legionella pneumophila prevents proliferation of its natural host Acanthamoeba castellanii.

Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b.

Identification of Atg8 from Acanthamoeba castellanii by genetic complementation in Saccharomyces cerevisiae.

Autophagy is a eukaryotic process responsible for the degradation of intracellular content such as damaged organelles. Several putative autophagy-related genes have been identified within the annotated genome of the free-living amoeba Acanthamoeba castellanii. However, the involvement of the corresponding proteins in the autophagy pathway had not been formerly established. Here, we report that AcAtg8 cDNA can complement ATG8-deficient Saccharomyces cerevisiae.