Entamoeba histolytica is a pathogen that during its infective process confronts the host defenses, which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Acid sphingomyelinases (aSMases) have been reported in mammalian cells to promote endocytosis and removal of PM lesions. In this work, six predicted amoebic genes encoding for aSMases were found to be transcribed in the HM1:IMSS strain, finding that the EhaSM6 gene is the most transcribed in basal growth conditions and rendered a functional protein. The secreted aSMase activity detected was stimulated by Mg +2 and inhibited by Co +2 . Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase of 2-fold in the secreted aSMase activity. This transfectant trophozoites exposed to pore-forming molecules (SLO, Magainin, β-Defensin 2 and human complement) exhibited an increase from 6 to 25-fold in the secreted aSMase activity which correlated with higher amoebic viability in a Ca +2 dependent process. However, other agents that affect the PM such as hydrogen peroxide also induced an increase of secreted aSMase, but to a lesser extent. The aSMase6 enzyme is N- and C-terminal processed. Confocal and transmission electron microscopy showed that trophozoites treated with SLO presented a migration of lysosomes containing the aSMase towards the PM, inducing the formation of membrane patches and endosomes in the control strain. These cellular structures were increased in the overexpressing strain, indicating the involvement of the aSMase6 in the PM injury repair. The pore-forming molecules induced an increase in the expression of EhaSM1 , 2 , 5 and 6 genes, meanwhile, hydrogen peroxide induced an increase in all of them. In all the conditions evaluated, the EhaSM6 gene exhibited the highest levels of induction. Overall, these novel findings show that the aSMase6 enzyme from E . histolytica promotes the repair of the PM damaged with pore-forming molecules to prevent losing cell integrity. This novel system could act when encountered with the lytic defense systems of the host.

The presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

Entamoeba histolytica virulence results from complex host–parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica.

The present study describes the antibacterial behavior and the bacterial resistance analysis of extremophile Pseudomonas aeruginosa in contact with copper nanoparticles (CuNPs). For this purpose, green synthesis of CuNPs was performed by combined ultrasound-assisted and chemical reduction methods, obtaining semispherical CuNPs ranging from ca. 4-9 nm. Antibacterial activity (AA) of biosynthesized CuNPs demonstrates an antibacterial inhibition of 85 % (LD85) at 400 μg/mL and a minimum bactericidal concentration (MBC) of 800 μg/mL after 3 h of contact. Bacterial adaptation in contact with CuNPs was observed through the consecutive exposition of microorganisms, presenting a significant increase of LD85 values from 400 μg/mL to 6400 μg/mL after 11 expositions. This behavior demonstrates the bacterial growth adaptation with high-dose of CuNPs. The bacterial resistance mechanism was determined through the overproduction of pyocyanin, associated with oxidative stress events, the genomic polymorphism of resistant bacteria obtained by PCR-RAPDs, and the morphological interaction between P. aeruginosa and CuNPs evidenced by transmission electron microscopy (TEM) micrographs. Our results suggest that under controlled CuNPs exposition, extremophile P. aeruginosa can generate bacterial resistance mechanisms, an important issue for the effective design of antimicrobial nanomaterials.

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