Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.

Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays.

Background: In vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess Acanthamoeba cytopathogenicity. Methods: The viability of human corneal epithelial cells (HCECs) after co-culture with Acanthamoeba was evaluated in phase contrast microscopy. Results: It was shown that Acanthamoeba is unable to considerably reduce the tetrazolium salt and the NanoLuc® Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify Acanthamoeba cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of Acanthamoeba on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity. Discussion: Our findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc® Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of Acanthamoeba with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests.

Transcriptional changes of proteins of the thioredoxin and glutathione systems in Acanthamoeba spp. under oxidative stress - an RNA approach.

The thioredoxin (Trx) and the glutathione (GSH) systems represent important antioxidant systems in cells and in particular thioredoxin reductase (TrxR) has been shown to constitute a promising drug target in parasites. For the facultative protozoal pathogen Acanthamoeba, it was demonstrated that a bacterial TrxR as well as a TrxR, characteristic of higher eukaryotes, mammals and humans is expressed on the protein level. However, only bacterial TrxR is strongly induced by oxidative stress in Acanthamoeba castellanii. In this study, the impact of oxidative stress on key enzymes involved in the thioredoxin and the glutathione system of A. castellanii under different culture conditions and of clinical Acanthamoeba isolates was evaluated on the RNA level employing RT-qPCR. Additionally, the effect of auranofin, a thioredoxin reductase inhibitor, already established as a potential drug in other parasites, on target enzymes in A. castellanii was investigated. Oxidative stress induced by hydrogen peroxide led to significant stimulation of bacterial TrxR and thioredoxin, while diamide had a strong impact on all investigated enzymes. Different strains displayed distinct transcriptional responses, rather correlating to sensitivity against the respective stressor than to respective pathogenic potential. Culture conditions appear to have a major effect on transcriptional changes in A. castellanii. Treatment with auranofin led to transcriptional activation of the GSH system, indicating its role as a potential backup for the Trx system. Altogether, our data provide more profound insights into the complex redox system of Acanthamoeba, preparing the ground for further investigations on this topic.

Title: Modifications transcriptionnelles des protéines des système thiorédoxine et glutathion chez Acanthamoeba spp. sous stress oxydatif ­ une approche par l'ARN. Abstract: Les systèmes de la thiorédoxine (Trx) et du glutathion (GSH) représentent des systèmes antioxydants importants dans les cellules et, en particulier, la thiorédoxine réductase (TrxR) s'est avérée constituer une cible médicamenteuse prometteuse chez les parasites. Pour le pathogène protozoaire facultatif Acanthamoeba, il a été démontré qu'une TrxR bactérienne ainsi qu'une TrxR, caractéristique des eucaryotes supérieurs, des mammifères et des humains, s'expriment au niveau protéique. Cependant, seule la TrxR bactérienne est fortement induite par le stress oxydatif chez Acanthamoeba castellanii. Dans cette étude, l'impact du stress oxydatif sur les enzymes clés impliquées dans la thiorédoxine et le système glutathion d'A. castellanii dans différentes conditions de culture et d'isolats cliniques d'Acanthamoeba a été évalué au niveau de l'ARN en utilisant la RT-qPCR. De plus, l'effet de l'auranofine, un inhibiteur de la thiorédoxine réductase déjà établi comme médicament potentiel chez d'autres parasites, a été étudié sur les enzymes cibles chez A. castellanii. Le stress oxydatif induit par le peroxyde d'hydrogène a conduit à une stimulation significative du TrxR bactérien et de la thiorédoxine tandis que le diamide a eu un fort impact sur toutes les enzymes étudiées. Différentes souches ont affiché des réponses transcriptionnelles distinctes, plutôt corrélées à la sensibilité contre le facteur de stress respectif qu'à leur potentiel pathogène respectif. Les conditions de culture semblent avoir un effet majeur sur les changements transcriptionnels chez A. castellanii. Le traitement à l'auranofine a conduit à une activation transcriptionnelle du système GSH, indiquant son rôle de sauvegarde potentielle pour le système Trx. Dans l'ensemble, nos données fournissent des informations plus approfondies sur le système redox complexe d'Acanthamoeba, préparant le terrain pour de nouvelles investigations sur ce sujet.

Antimicrobial effect of auranofin against Acanthamoeba spp.

Acanthamoebae are opportunistic pathogens that cause serious infections, including Acanthamoeba keratitis, a sight-threatening disease affecting mainly contact lens wearers, and granulomatous amoebic encephalitis, an infection of the central nervous system that occurs mostly in immunocompromised individuals. Although these infections are rare, they are a challenge for healthcare providers. In the last decade, the search for and implementation of novel treatment approaches against these parasites and the infections they cause have intensified, but current options are still unsatisfactory. The aim of this study was to investigate the in vitro activity of the gold-based compound auranofin against Acanthamoeba spp. The study showed that auranofin has potent antimicrobial activity against Acanthamoeba spp., with an IC50 ranging from 2.9 to 3.48 µM, and thus may be useful in the prevention and control of Acanthamoeba infections.

The elusive parasite: comparing macroscopic, immunological, and genomic approaches to identifying malaria in human skeletal remains from Sayala, Egypt (third to sixth centuries AD).

Although malaria is one of the oldest and most widely distributed diseases affecting humans, identifying and characterizing its presence in ancient human remains continue to challenge researchers. We attempted to establish a reliable approach to detecting malaria in human skeletons using multiple avenues of analysis: macroscopic observations, rapid diagnostic tests, and shotgun-capture sequencing techniques, to identify pathological changes, Plasmodium antigens, and Plasmodium DNA, respectively. Bone and tooth samples from ten individuals who displayed skeletal lesions associated with anaemia, from a site in southern Egypt (third to sixth centuries AD), were selected. Plasmodium antigens were detected in five of the ten bone samples, and traces of Plasmodium aDNA were detected in six of the twenty bone and tooth samples. There was relatively good synchronicity between the biomolecular findings, despite not being able to authenticate the results. This study highlights the complexity and limitations in the conclusive identification of the Plasmodium parasite in ancient human skeletons. Limitations regarding antigen and aDNA preservation and the importance of sample selection are at the forefront of the search for malaria in the past. We confirm that, currently, palaeopathological changes such as cribra orbitalia are not enough to be certain of the presence of malaria. While biomolecular methods are likely the best chance for conclusive identification, we were unable to obtain results which correspond to the current authentication criteria of biomolecules. This study represents an important contribution in the refinement of biomolecular techniques used; also, it raises new insight regarding the consistency of combining several approaches in the identification of malaria in past populations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12520-021-01350-z.