Oxidase enzyme genes are differentially expressed during Acanthamoeba castellanii encystment.

Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A crucial survival strategy of A. castellanii involves the formation of highly resistant cysts during adverse conditions. This study delves into the cellular processes underpinning encystment, focusing on gene expression changes related to reactive oxygen species (ROS) balance, with a particular emphasis on mitochondrial processes. Our findings reveal a dynamic response within the mitochondria during encystment, with the downregulation of key enzymes involved in oxidative phosphorylation (COX, AOX, and NADHalt) during the initial 48 h, followed by their overexpression at 72 h. This orchestrated response likely creates a pro-oxidative environment, facilitating encystment. Analysis of other ROS processing enzymes across the cell reveals differential expression patterns. Notably, antioxidant enzymes, such as catalases, glutaredoxins, glutathione S-transferases, peroxiredoxins, and thioredoxins, mirror the mitochondrial trend of downregulation followed by upregulation. Additionally, glycolysis and gluconeogenesis are downregulated during the early stages in order to potentially balance the metabolic requirement of the cyst. Our study underscores the importance of ROS regulation in Acanthamoeba encystment. Understanding these mechanisms offers insights into infection control and identifies potential therapeutic targets. This work contributes to unraveling the complex biology of A. castellanii and may aid in combatting Acanthamoeba-related infections. Further research into ROS and oxidase enzymes is warranted, given the organism's remarkable respiratory versatility.

Can Amphotericin B-mediated effects be limited using intranasal versus intravenous route?

Aim: CNS infections due to parasites often prove fatal. In part, this is due to inefficacy of drugs to cross the blood-brain barrier. Methods: Here, we tested intranasal and intravenous route and compared adverse effects of Amphotericin B administration, through blood biochemistry, liver, kidney and brain histopathological evidence of toxicities in vivo post-administration. Results: It was observed that intranasal route limits the adverse side effects of Amphotericin B, in contrast to intravenous route. Conclusion: As parasites such as Naegleria fowleri exhibit unequivocal affinity toward the olfactory bulb and frontal lobe in the central nervous system, intranasal administration would directly reach amoebae bypassing the blood-brain barrier selectivity and achieve the minimum inhibitory concentration at the target site.

Brain infections due to parasites are often fatal. One of the reasons is the inability of drugs to get to the brain. When given in large dose to reach the brain, the drug can cause serious side effects. Here, we tested the side effects of Amphotericin B (drug of choice against brain-eating amoebae), when given intranasally versus intravenous. Our findings clearly show that intranasal route limits the side effects of Amphotericin B. These are important findings and should serve as an important step in the development of effective therapy against parasitic infections affecting the brain.

Gut microbiome of Crocodylus porosus and cellular stress: inhibition of nitric oxide, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells.

Crocodiles are renowned for their resilience and capacity to withstand environmental stressors, likely influenced by their unique gut microbiome. In this study, we determined whether selected gut bacteria of Crocodylus porosus exhibit anti-inflammatory effects in response to stress, by measuring nitric oxide release, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells. Using the Griess assay, the findings revealed that among several C. porosus gut bacterial isolates, the conditioned media containing the metabolites of two bacterial strains (CP27 and CP36) inhibited nitric oxide production significantly, in response to the positive control, i.e., taxol-treatment. Notably, CP27 and CP36 were more potent at reducing nitric oxide production than senloytic compounds (fisetin, quercetin). Using enzyme linked immunosorbent assays, the production of pro-inflammatory cytokines (IL-1ß, TNF-α, PGE2), was markedly reduced by treatment with CP27 and CP36, in response to stress. Both CP27 and CP36 contain a plethora of metabolites to exact their effects [(3,4-dihydroxyphenylglycol, 5-methoxytryptophan, nifedipine, 4-chlorotestosterone-17-acetate, 3-phenoxypropionic acid, lactic acid, f-Honaucin A, l,l-Cyclo(leucylprolyl), 3-hydroxy-decanoic acid etc.], indicative of their potential in providing protection against cellular stress. Further high-throughput bioassay-guided testing of gut microbial metabolites from crocodiles, individually as well as in combination, together with the underlying molecular mechanisms, in vitro and in vivo will elucidate their value in the rational development of innovative therapies against cellular stress/gut dysbiosis.

Dogs' health and demographics in wildlife-populated and tsetse-infested villages of Mambwe district, eastern Zambia.

Good dog-keeping practices and access to veterinary care are essential for the well-being of dogs. As the main causes of morbidity and mortality in the rural canine population in Zambia are poorly understood, we followed a cohort of 162 indigenous dogs for six months in wildlife-populated and tsetse-infested villages of Mambwe district, eastern Zambia to gain deeper insights. Dogs lacked basic home and veterinary care, they were often starved and burdened with ticks, and some passed live adult worms in their stool. The frequent exposure of dogs to tsetse bites and consumption of fresh raw game meat and bones puts them at greater risk of acquiring African trypanosomiasis. Nearly 20 % of dogs were lost to follow-up, with the main causes being poor health (58.1 %), predation by wild carnivores (29 %), and owner culling or euthanasia (12.9 %). We observed that indigenous dogs' general well-being and survival were largely influenced by their environment, infectious diseases, injuries sustained during interaction with conspecifics and wildlife, and community attitudes and practices associated with dog ownership.

Bacterial flora varies throughout the saltwater crocodile (Crocodylus porosus) gastrointestinal tract.

OBJECTIVE: The objective of this study was to determine bacterial flora throughout the gastrointestinal tract of a saltwater crocodile (Crocodylus porosus) using 16S rRNA gene analysis. ANIMALS: A convention on international trade in endangered species (CITES) of wild fauna and flora registered crocodile farm, provided a healthy male saltwater crocodile, Crocodylus porosus for this study. PROCEDURES: Three samples were taken from the oral cavity, 3 samples from the proximal region of the small intestine (jejunum), and 3 samples from the distal part of the large intestine of the gastrointestinal tract of C. porosus were obtained using sterile cotton swabs. Next, swabs were placed in 15 mL sterile centrifuge tubes, individually, and kept on ice for immediate transportation to the laboratory. This was followed by 16S rRNA gene analysis using specific primers (341F-CCTAYGGGRBGCASCAG, and 806R-GGACTACNNGGGTATCTAAT). Amplicons were sequenced on Illumina paired-end platform, and bacterial gastrointestinal communities, the relative abundance of taxa, and principal component and coordinate analysis were performed. RESULTS: The findings revealed that bacterial community structures from differing regions exhibited several differences. The number of observed bacterial operational taxonomic units (OTUs) was 153 in the oral cavity, 239 in the small intestine, and 119 in the large intestine of C. porosus. The small intestine reflects the highest richness. In contrast, the large intestine exhibited the least richness of microbial communities. Relative abundance of taxa showed that Proteobacteria, Bacteroidetes, and Firmicutes were dominant in all 3 sample sites. Pseudomonas differed in the oral cavity and the large intestine, with the latter exhibiting less distribution of Pseudomonas. Stenotrophomonas and Castellaniella were higher in the oral cavity, while the relative abundance of Comamonas and Salmonella was higher in the small intestine. Conversely, the relative abundance of Salmonella and Pannonibacter was augmented in the large intestine. CLINICAL RELEVANCE: For the first time, this study demonstrates the bacterial diversity along the segments of the gastrointestinal tract of C. porosus. Bacterial flora varies throughout the gastrointestinal tract. Although further studies using large cohorts are warranted; however, our findings suggest that microbiome composition may have the potential as a biomarker in determining the overall health and well-being of C. porosus.

Mass spectrometric analysis of bioactive conditioned media of bacteria isolated from reptilian gut.

Aim: To determine whether selected gut bacteria of crocodile exhibit antibacterial properties. Materials & methods: Two bacteria isolated from Crocodylus porosus gut were used, namely: Pseudomonas aeruginosa and Aeromonas dhakensis. Conditioned media were tested against pathogenic bacteria and metabolites were analyzed using liquid chromatography-mass spectrometry. Results & conclusion: Antibacterial assays revealed that conditioned media showed potent effects against pathogenic Gram-positive and Gram-negative bacteria. LC-MS revealed identity of 210 metabolites. The abundant metabolites were, N-Acetyl-L-tyrosine, Acetaminophen, Trans-Ferulic acid, N, N-Dimethylformamide, Pyrocatechol, Cyclohexanone, Diphenhydramine, Melatonin, Gamma-terpinene, Cysteamine, 3-phenoxypropionic acid, Indole-3-carbinol, Benzaldehyde, Benzocaine, 2-Aminobenzoic acid, 3-Methylindole. These findings suggest that crocodile gut bacteria are potential source of novel bioactive molecules that can be utilized as pre/post/antibiotics for the benefit of human health.

Crocodiles thrive in unsanitary conditions, feed on rotten meat, and endure conditions that are detrimental to human health. In addition to their immune system, we speculate that their microbial gut flora produce substances contributing to their "hardiness" and "longevity". Herein, we showed that selected bacteria isolated from crocodile gut produced potent antibacterial properties against multiple drug-resistant pathogenic Gram-negative and Gram-positive bacteria. LC­MS/MS revealed the identity of gut microbial metabolites. These findings suggest that analyses of crocodile gut bacteria may reveal potential drug leads that can be utilized as probiotics/pre/post/antibiotics for the benefit of human health, however intensive future research is needed to realize these expectations.

mRNA Sequencing Reveals Upregulation of Glutathione S-Transferase Genes during Acanthamoeba Encystation.

Some members of the genus Acanthamoeba are facultative pathogens typically with a biphasic lifestyle: trophozoites and cysts. Acanthamoeba is capable of infecting the cornea, resulting in Acanthamoeba keratitis. The cyst is one of the key components for the persistence of infection. Gene expression during Acanthamoeba encystation showed an upregulation of glutathione S-transferase (GST) genes and other closely related proteins. mRNA sequencing showed GST, and five genes with similar sequences were upregulated after 24 h of inducing encystation. GST overexpression was verified with qPCR using the HPRT and the cyst-specific protein 21 genes as controls. The GST inhibitor ethacrynic acid was found to decrease cell viability by 70%. These results indicate a role of GST in successful encystation, possibly by maintaining redox balance. GST and associated processes could be targets for potential treatments alongside regular therapies to reduce relapses of Acanthamoeba infection.

Modular nanotheranostic agents for protistan parasitic diseases: Magic bullets with tracers.

Protistan parasitic infections contribute significantly to morbidity and mortality, causing more than 2 billion human infections annually. However, current treatments are often limited; due to ineffective drugs and drug resistance, thus better options are urgently required. In the present context, theranostics agents are those that offer simultaneous detection, diagnosis and even treatment of protistan parasitic diseases. "Nanotheranostics" is the term used to describe such agents, that are around 100 nm or less in size. Anti-parasitic activity of nanoparticles (NPs) has been reported, and many have useful intrinsic imaging properties, but it is perhaps their multifunctional nature that offers the greatest potential. NPs may be used as adapters onto which various subunits with different functions may be attached. These subunits may facilitate targeting parasites, coupled with toxins to eradicate parasites, and probe subunits for detection of particles and/or parasites. The modular nature of nano-platforms promises a "mix and match" approach for the construction of tailored agents by using combinations of these subunits against different protistan parasites. Even though many of the subunits have shown promise alone, these have not yet been put together convincingly enough to form working theranostics against protistan parasites. Although the clinical application of nanotheranostics to protistan parasitic infections in humans requires more research, we conclude that they offer not just a realisation of Paul Ehrlich's long imagined "magic bullet" concept, but potentially are magic bullets combined with tracer bullets.

Acanthamoeba castellanii exhibits intron retention during encystment.

Intron retention (IR) refers to the mechanism of alternative splicing in which an intron is not excised from the mature transcript. IR in the cosmopolitan free-living amoeba Acanthamoeba castellanii has not been studied. We performed an analysis of RNA sequencing data during encystment to identify genes that presented differentially retained introns during this process. We show that IR increases during cyst formation, indicating a potential mechanism of gene regulation that could help downregulate metabolism. We identify 69 introns from 67 genes that are differentially retained comparing the trophozoite stage and encystment after 24 and 48 h. These genes include several hypothetical proteins. We show different patterns of IR during encystment taking as examples a lipase, a peroxin-3 protein, an Fbox domain containing protein, a proteasome subunit, a polynucleotide adenylyltransferase, and a tetratricopeptide domain containing protein. A better understanding of IR in Acanthamoeba, and even other protists, could help elucidate changes in life cycle and combat disease such as Acanthamoeba keratitis in which the cyst is key for its persistence.

Epigenetic-Mediated Antimicrobial Resistance: Host versus Pathogen Epigenetic Alterations.

Since the discovery of antibiotics, humans have been benefiting from them by decreasing the morbidity and mortality associated with bacterial infections. However, in the past few decades, misuse of antibiotics has led to the emergence of bacterial infections resistant to multiple drugs, a significant health concern. Bacteria exposed to inappropriate levels of antibiotics lead to several genetic changes, enabling them to survive in the host and become more resistant. Despite the understanding and targeting of genetic-based biochemical changes in the bacteria, the increasing levels of antibiotic resistance are not under control. Many reports hint at the role of epigenetic modifications in the bacterial genome and host epigenetic reprogramming due to interaction with resistant pathogens. Epigenetic changes, such as the DNA-methylation-based regulation of bacterial mutation rates or bacteria-induced histone modification in human epithelial cells, facilitate its long-term survival. In this review article, epigenetic changes leading to the development of antibiotic resistance in clinically relevant bacteria are discussed. Additionally, recent lines of evidence focusing on human host epigenetic changes due to the human-pathogen interactions are presented. As genetic mechanisms cannot explain the transient nature of antimicrobial resistance, we believe that epigenetics may provide new frontiers in antimicrobial discovery.

Crocodylus porosus Sera a Potential Source to Identify Novel Epigenetic Targets: In Silico Analysis.

We have previously found that sera from Crocodylus porosus contain anticancer agents and the treatment of MCF7 cells with this serum resulted in the differential expression of 51 genes. The purpose of this study was to use in silico analysis to identify genes that might be epigenetically modulated in cells treated with crocodile serum and to understand the role of potential genes as novel candidates with epigenetic therapeutic potential. The findings report five proto-oncogenes (TUBA1B, SLC2A1, PGK1, CCND1, and NCAPD2) and two tumor suppressor genes (RPLP2, RPL37) as novel therapeutic targets. Furthermore, we present a comprehensive overview of relevant studies on epigenetic regulation of these genes along with an insight into their clinical implications. Therefore, elucidating the molecules present in the serum and gut bacteria of reptiles such as crocodiles may offer insights into the role of these genes on longevity, health, disease, and life expectancy.

Statins Induce Actin Cytoskeleton Disassembly and an Apoptosis-Like Process in Acanthamoeba spp.

Acanthamoeba is a ubiquitous opportunistic protozoan pathogen that is known to cause blinding keratitis and rare, but usually fatal, granulomatous encephalitis. The difficulty in treating infections and the toxicity issues of the current treatments emphasize the need to use alternative agents with amoebicidal activity. The aim of this study was to evaluate the in vitro antiamoebic activity of three third-generation statins-cerivastatin, pitavastatin and rosuvastatin-against both cysts and trophozoites of the following four strains of Acanthamoeba: A. castellanii Neff, A. polyphaga, A. griffini and A. quina. Furthermore, programmed cell death (PCD) induction traits were evaluated by measuring chromatin condensation, damages at the mitochondrial level, production of reactive oxygen species (ROS) and the distribution of actin cytoskeleton fibers. Acanthamoeba castellanii Neff was the strain most sensitive to all the statins, where cerivastatin showed the lowest amoebicidal activity for both trophozoite and cyst forms (0.114 ± 0.050 and 0.704 ± 0.129 µM, respectively). All the statins were able to cause DNA condensation, collapse in the mitochondrial membrane potential and a reduction in ATP level production, and disorganization of the total actin fibers in the cytoskeleton of all the evaluated Acanthamoeba strains. Our results showed that the tested statins were able to induce PCD compatible events in the treated amoebae, including chromatin condensation, collapse in the mitochondrial potential and ATP levels, cytoskeleton disassembly and ROS generation.

Gut microbiome-immune system interaction in reptiles.

Reptiles are ectothermic amniotes in a world dominated by endotherms. Reptiles originated more than 300 million years ago and they often dwell in polluted environments which may expose them to pathogenic micro-organisms, radiation and/or heavy metals. Reptiles also possess greater longevity and may live much longer than similar-sized land mammals, for example, turtles, tortoises, crocodiles and tuatara are long-lived reptiles living up to 100 years or more. Many recent studies have emphasized the pivotal role of the gut microbiome on its host; thus, we postulated that reptilian gut microbiome and/or its metabolites and the interplay with their robust immune system may contribute to their longevity and overall hardiness. Herein, we discuss the composition of the reptilian gut microbiome, immune system-gut microbiome cross-talk, antimicrobial peptides, reptilian resistance to infectious diseases and cancer, ageing, as well the current knowledge of the genome and epigenome of these remarkable species. Preliminary studies have demonstrated that microbial gut flora of reptiles such as crocodiles, tortoises, water monitor lizard and python exhibit remarkable anticancer and antibacterial properties, as well as comprise novel gut bacterial metabolites and antimicrobial peptides. The underlying mechanisms between the gut microbiome and the immune system may hold clues to developing new therapies overall for health, and possible extrapolation to exploit the ancient defence systems of reptiles for Homo sapiens benefit.

Opportunistic free-living amoebal pathogens.

Pathogenic free-living amoebae affecting the central nervous system are known to cause granulomatous amoebic encephalitis (GAE) or primary amoebic meningoencephalitis (PAM). Although hosts with impaired immunity are generally at a higher risk of severe disease, amoebae such as Naegleria fowleri and Balamuthia mandrillaris can instigate disease in otherwise immunocompetent individuals, whereas Acanthamoeba species mostly infect immunocompromised people. Acanthamoeba also cause a sight-threatening eye infection, mostly in contact lens wearers. Although infections due to pathogenic amoebae are considered rare, recently, these deadly amoebae were detected in water supplies in the USA. This is of particular concern, especially with global warming further exacerbating the problem. Herein, we describe the epidemiology, presentation, diagnosis, and management of free-living amoeba infections.

Crocodylus porosus Gut Bacteria: A Possible Source of Novel Metabolites.

Crocodiles are remarkable animals that have the ability to endure extremely harsh conditions and can survive up to a 100 years while being exposed to noxious agents that are detrimental to Homo sapiens. Besides their immunity, we postulate that the microbial gut flora of crocodiles may produce substances with protective effects. In this study, we isolated and characterized selected bacteria colonizing the gastrointestinal tract of Crocodylusporosus and demonstrated their inhibitory effects against three different cancerous cell lineages. Using liquid chromatography-mass spectrometry, several molecules were identified. For the first time, we report partial analyses of crocodile's gut bacterial molecules.

Development of anti-acanthamoebic approaches.

Acanthamoeba keratitis is a sight-endangering eye infection, and causative organism Acanthamoeba presents a significant concern to public health, given escalation of contact lens wearers. Contemporary therapy is burdensome, necessitating prompt diagnosis and aggressive treatment. None of the contact lens disinfectants (local and international) can eradicate Acanthamoeba effectively. Using a range of compounds targeting cellulose, ion channels, and biochemical pathways, we employed bioassay-guided testing to determine their anti-amoebic effects. The results indicated that acarbose, indaziflam, terbuthylazine, glimepiride, inositol, vildagliptin and repaglinide showed anti-amoebic effects. Compounds showed minimal toxicity on human cells. Therefore, effects of the evaluated compounds after conjugation with nanoparticles should certainly be the subject of future studies and will likely lead to promising leads for potential applications.

Longevity, cellular senescence and the gut microbiome: lessons to be learned from crocodiles.

Crocodiles are flourishing large-bodied ectotherms in a world dominated by endotherms. They survived the Cretaceous extinction event, that eradicated the dinosaurs who are thought to be their ancestral hosts. Crocodiles reside in polluted environments; and often inhabit water which contains heavy metals; frequent exposure to radiation; feed on rotten meat and considered as one of the hardy species that has successfully survived on this planet for millions of years. Another capability that crocodiles possess is their longevity. Crocodiles live much longer than similar-sized land mammals, sometimes living up to 100 years. But how do they withstand such harsh conditions that are detrimental to Homo sapiens? Given the importance of gut microbiome on its' host physiology, we postulate that the crocodile gut microbiome and/or its' metabolites produce substances contributing to their "hardiness" and longevity. Thus, we accomplished literature search in PubMed, Web of Science and Google Scholar and herein, we discuss the composition of the crocodile gut microbiome, longevity and cellular senescence in crocodiles, their resistance to infectious diseases and cancer, and our current knowledge of the genome and epigenome of these remarkable species. Furthermore, preliminary studies that demonstrate the remarkable properties of crocodile gut microbial flora are discussed. Given the profound role of the gut microbiome in the health of its' host, it is likely that the crocodile gut microbiome and its' metabolites may be contributing to their extended life expectancy and elucidating the underlying mechanisms and properties of these metabolites may hold clues to developing new treatments for age-related diseases for the benefit of Homo sapiens.

Is Naegleria fowleri an Emerging Parasite?

Naegleria fowleri causes an uncommon but deadly disease called primary amoebic meningoencephalitis (PAM). There has been an increase of reported PAM cases, particularly since 2000. Although water is the dominant route of transmission of PAM, infection through soil/dust is a possible alternative route. We have observed differences in epidemiology between the southern states in the USA and the Indian subcontinent (ISC). The patient age range is greater in the ISC than in the USA, and there are more infections in the ISC which are not water-associated. We show that PAM is under-reported and argue that climate change will increase the incidence of PAM, and that the geographic range of N. fowleri will spread polewards.

Ancestral Eukaryotes Reproduced Asexually, Facilitated by Polyploidy: A Hypothesis.

The notion that eukaryotes are ancestrally sexual has been gaining attention. This idea comes in part from the discovery of sets of "meiosis-specific genes" in the genomes of protists. The existence of these genes has persuaded many that these organisms may be engaging in sex, even though this has gone undetected. The involvement of sex in protists is supported by the view that asexual reproduction results in the accumulation of mutations that would inevitably result in the decline and extinction of such lineages. It is argued that this phenomenon can be obviated by polyploidy and that the "meiosis-specific genes" are used in other processes, including polyploidy control and homologous recombination, independent of meiosis. These phenomena account for the finding that these genes are expressed in cultures devoid of apparent cell fusion events. Hence, it is also proposed that asexual, and not sexual, reproduction is the ancestral condition.