Correlation of Redondovirus and Entamoeba gingivalis Detections in the Human Oral Cavity Suggests That This Amoeba Is Possibly the Redondovirus Host.

The virome of the human oral cavity and the relationships between viruses and diseases such as periodontitis are scarcely deciphered. Redondoviruses were reported in the human oral cavity in 2019, including in periodontitis patients. Here, we aimed at detecting redondoviruses and at searching for a potential viral host in human saliva. Non-stimulated saliva was collected between December 2020 and June 2021. These samples were tested using real-time PCR regarding the presence of redondovirus and Entamoeba gingivalis DNA. Similarity searches were performed using BLAST against eukaryotic and prokaryotic sequences from GenBank. The redondovirus DNA was detected in 46% of the 28 human saliva samples. In addition, short fragments of redondovirus genomes were detected in silico within Entamoeba sequences. Finally, Entamoeba gingivalis DNA was detected in 46% of the 28 saliva samples, with a strong correlation between redondovirus DNA and E. gingivalis DNA detections, in 93% of the cases. Regarded together, these findings and previous ones strongly support the presence of redondoviruses in the human oral cavity and their association to E. gingivalis as their likely host.

Inhibition of MMPs supports amoeboid angiogenesis hampering VEGF-targeted therapies via MLC and ERK 1/2 signaling.

BACKGROUND: In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the "amoeboid angiogenesis" in the absence of proteases activity. METHODS: We first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies. RESULTS: Our data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn't boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF. CONCLUSIONS: We underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.

SARS-CoV-2 surrogate (Phi6) environmental persistence within free-living amoebae.

The reported persistence of SARS-CoV-2 virions in aquatic environments highlights the need to better understand potential mechanisms that may prolong its dissemination. We evaluated the possibility that amoebae might serve as transport hosts by studying the interaction of the enveloped bacteriophage Phi6, as a potential surrogated along with one of the most common amoebae in engineered aquatic environments, Vermamoeba vermiformis. Using microscopy, imaging flow cytometry and bacteriophage cell culture, our results imply that the SARS-CoV-2 surrogate triggers amoebic mitochondria and induced apoptosis to promote viral persistence in trophozoites. Furthermore, virus-infected amoebae were still infectious after 2 months within FLA cysts. These results suggest that amoebae could contribute to the environmental persistence of SARS-CoV-2, including disinfection processes. In addition, amoebae could be a successful model system for understanding respiratory virus-eukaryotic biology at the cellular and molecular levels.

Can Neurotropic Free-Living Amoeba Serve as a Model to Study SARS-CoV-2 Pathogenesis?

Of the single-celled eukaryotic microbes, Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba spp. are known to cause fatal encephalitis in humans. Being eukaryotes, these cells have been used as a model for studying and understanding complex cellular processes in humans like cell motility, phagocytosis, and metabolism. The ongoing pandemic caused by SARS-CoV-2 that infects multiple organs has emerged as a challenge to unravel its mode of infection and the pathogenicity resulting in eukaryotic cell death. Working with these single-celled eukaryotic microbes provided us the opportunity to plan bioinformatic approaches to look into the likelihood of studying the known and alternative mode of infection of the SARS-CoV-2 in eukaryotic cells. Genome databases of N. fowleri, B. mandrillaris, and Acanthamoeba spp. were used to explore the expression of angiotensin-converting enzyme 2 (ACE2), androgen-regulated serine protease precursor (TMPRSS2), CD4, CD147, and furin that are known to be cardinal for SARS-CoV-2 in recognition and binding to human cells. It was hypothesized that if a receptor-dependent or phagocytosis-assisted SARS-CoV-2 uptake does occur in free-living amoebae (FLA), this model can provide an alternative to human cells to study cellular recognition and binding of SARS-CoV-2 that can help design drugs and treatment modalities in COVID-19. We show that, of the FLA, ACE2 and TMPRSS2 are not expressed in Acanthamoeba spp. and B. mandrillaris, but primitive forms of these cell recognition proteins were seen to be encoded in N. fowleri. Acanthamoeba spp. and N. fowleri encode for human-like furin which is a known SARS-CoV-2 spike protein involved in host cell recognition and binding.