Theoretical study of ArcB and its dimerization, interaction with anaerobic metabolites, and activation of ArcA.

The complex metabolism of Escherichia coli has been extensively studied, including its response to oxygen availability. The ArcA/B two-component system (TCS) is the key regulator for the transition between these two environmental conditions and has been thoroughly characterized using genetic and biochemical approaches. Still, to date, limited structural data is available. The breakthrough provided by AlphaFold2 in 2021 has brought a reliable tool to the scientific community for assessing the structural features of complex proteins. In this report, we analyzed the structural aspects of the ArcA/B TCS using AlphaFold2 models. The models are consistent with the experimentally determined structures of ArcB kinase. The predicted structure of the dimeric form of ArcB is consistent with the extensive genetic and biochemical data available regarding mechanistic signal perception and regulation. The predicted interaction of the dimeric form of ArcB with its cognate response regulator (ArcA) is also consistent with both the forward and reverse phosphotransfer mechanisms. The ArcB model was used to detect putative binding cavities to anaerobic metabolites, encouraging testing of these predictions experimentally. Finally, the highly accurate models of other ArcB homologs suggest that different experimental approaches are needed to determine signal perception in kinases lacking the PAS domain. Overall, ArcB is a kinase with features that need further testing, especially in determining its crystal structure under different conditions.

Two-Component System Sensor Kinases from Asgardian Archaea May Be Witnesses to Eukaryotic Cell Evolution.

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

Attenuation of In Vitro and In Vivo Virulence Is Associated with Repression of Gene Expression of AIG1 Gene in Entamoeba histolytica.

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.

Escherichia coli transcription factors of unknown function: sequence features and possible evolutionary relationships.

Organisms need mechanisms to perceive the environment and respond accordingly to environmental changes or the presence of hazards. Transcription factors (TFs) are required for cells to respond to the environment by controlling the expression of genes needed. Escherichia coli has been the model bacterium for many decades, and still, there are features embedded in its genome that remain unstudied. To date, 58 TFs remain poorly characterized, although their binding sites have been experimentally determined. This study showed that these TFs have sequence variation at the third codon position G+C content but maintain the same Codon Adaptation Index (CAI) trend as annotated functional transcription factors. Most of these transcription factors are in areas of the genome where abundant repetitive and mobile elements are present. Sequence divergence points to groups with distinctive sequence signatures but maintaining the same type of DNA binding domain. Finally, the analysis of the promoter sequences of the 58 TFs showed A+T rich regions that agree with the features of horizontally transferred genes. The findings reported here pave the way for future research of these TFs that may uncover their role as spare factors in case of lose-of-function mutations in core TFs and trace back their evolutionary history.

Chromogenic Escherichia coli reporter strain for screening DNA damaging agents.

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.

Refinement of the Griess method for measuring nitrite in biological samples.

Nitric oxide (NO) is a reactive gas that participates in many physiological as well as pathogenic processes in higher eukaryotic organisms. Inflammatory responses elicit higher levels of this molecule. Nevertheless, there are many technical challenges to accurately measure the amount of NO produced. Previously, a method using whole-cell extracts from Escherichia coli was able to generate the conversion of nitrate into nitrite to measure the amount of nitrate or indirectly the NO present in a sample using the Griess reaction. Here we present an improvement to this method, by using E. coli whole-cell extracts lacking one of the two nitrite reductases, rendered a more precise measurement when coupled with the Griess reaction than our previous report. Alternatively, osmotic stress showed to downregulate the expression of both nitrate reductases, which can be an alternative for indirect nitrate and NO reduction. The results presented here show an easy method for nitrate and NO reduction to nitrite and avoid the reconversion to nitrate, also as an alternative for other analytical methods that are based on cadmium, purified nitrate reductase enzyme, or salicylic methods to reduce NO. This method can be widely used for measuring NO production in living organisms, soil, and other relevant microbiological samples.

Possible role played by the SINE2 element in gene regulation, as demonstrated by differential processing and polyadenylation in avirulent strains of E. histolytica.

Entamoeba histolytica represents a useful model in parasitic organisms due to its complex genomic organization and survival mechanisms. To counteract pathogenic organisms, it is necessary to characterize their molecular biology to design new strategies to combat them. In this report, we investigated a less-known genetic element, short interspersed nuclear element 2 (SINE2), that is present in this ameba and is highly transcribed and polyadenylated. In this study, we show that in two different nonvirulent strains of E. histolytica, SINE2 is differentially processed into two transcript fragments, that is, a full-length 560-nt fragment and a shorter 393-nt fragment bearing an approximately 18-nt polyadenylation tail. Sequence analysis of the SINE2 transcript showed that a Musashi-like protein may bind to it. Also, two putative Musashi-like sequences were identified on the transcript. Semiquantitative expression analysis of the two Musashi-like proteins identified in the E. histolytica genome (XP_648918 and XP_649094) showed that XP_64094 is overexpressed in the nonvirulent strains tested. The information available in the literature and the results presented in this report indicate that SINE2 may affect other genes, as observed with the epigenetic silencing of the G3 strain, by an antisense mechanism or via RNA-protein interactions that may ultimately be involved in the phenotype of nonvirulent strains of E. histolytica.

aeBlue Chromoprotein Color is Temperature Dependent.

BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.

Plasma membrane damage repair is mediated by an acid sphingomyelinase in Entamoeba histolytica.

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, ß-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.

Flotillin homologue is involved in the swimming behavior of Escherichia coli.

Cellular membrane is a key component for maintaining cell shape and integrity. The classical membrane structure and function by Singer and Nicolson groundbreaking model has depicted the membrane as a homogeneous fluid structure. This view has changed by the discovery of discrete domains containing different lipid compositions, called lipid rafts, which play a key role in signal transduction in eukaryotic cells. In the past few years, lipid raft-like structures have been found in bacteria also, constituted by cardiolipin and other modified lipids, perhaps involved in generating a specific site for protein clustering. Here, we report the analysis of a protein termed YqiK from Escherichia coli, a prohibitin homolog that has been implicated in stress sensing by the formation of membrane-associated microdomains. The E. coli yqiK-deficient mutant strain showed an enhanced swimming behavior and was resistant to ampicillin but its response to other stressing conditions was similar to that of the wild-type strain. The abnormal swimming behavior is reversed when the protein is expressed in trans from a plasmid. Also, we demonstrate that YqiK is not redundant with QmcA, another flotillin homolog found in E. coli. Our results, along with the data available in the literature, suggest that YqiK may be involved in the formation of discrete membrane-associated signaling complexes that regulate and agglomerate signaling proteins to generate cell response to chemotaxis.

Contribution of neutral sphingomyelinases to in vitro virulence of Entamoeba histolytica.

Amoebiasis is a worldwide health problem caused by the pathogen Entamoeba histolytica. Several virulence factors have been implicated in host invasion, immune evasion, and tissue damage. There are still new factors that remain to be elucidated and characterized. In this work, we obtained amoebic transfectants overexpressing three of the neutral sphingomyelinase enzymes encoded in the E. histolytica genome. The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities, besides an increase in gene expression of amoebapore A, B, and C. Meanwhile the EhnSM1 and EhnSM2 overexpression caused an increase in cytopathic activity. In all the neutral sphingomyelinases overexpressing strains, the gene expression levels for cysteine proteinase 5, adhesin 112 and, heavy and light Gal/GalNAc lectin subunits were not affected. We propose that the increase of cytotoxic and lytic effect of EhnSM3 overexpressed strain can be related to the sum of the effect of EhnSM3 plus amoebapores, in a process cell contact-dependent or as mediator by inducing the gene expression of amoebapores enabling a link between EhnSM3 with the virulence phenotype in E. histolytica. Our results suggest a differential role for neutral sphingomyelinases in E. histolytica virulence.

Replacing Standard Reporters from Molecular Cloning Plasmids with Chromoproteins for Positive Clone Selection.

Cloning and expression plasmids are the workhorses of modern molecular biology. Despite the pathway paved by synthetic biology, laboratories around the globe still relay on standard cloning techniques using plasmids with reporter proteins for positive clone selection, such as ß-galactosidase alpha peptide complementation for blue/white screening or ccdB, which encodes for a toxic DNA gyrase. These reporters, when interrupted, serve as a positive clone detection system. In the present report, we show that molecular cloning plasmids bearing the coding sequence for a 25.4 kDa protein, AmilCP, encoded by a 685 bp gene, that is well expressed in Escherichia coli, render blue-purple colonies. Using this reporter protein, we developed and tested a cloning system based on the constitutive expression of the non-toxic AmilCP protein, that once interrupted, the loss of purple color serves to facilitate positive clone selection. The main advantage of this system is that is less expensive than other systems since media do not contain chromogenic markers such as X-gal, which is both expensive and cumbersome to prepare and use, or inductors such as IPTG. We also designed an inducible expression plasmid suitable for recombinant protein expression that also contains AmilCP cloning selection marker, a feature not commonly found in protein expression plasmids. The use of chromogenic reporters opens an important avenue for its application in other organisms besides E. coli for clone selection or even for mutant selection.

Red fluorescent protein (DsRFP) optimization for Entamoeba histolytica expression.

Entamoeba histolytica genetic organization and genome structure is complex and under intense research. The genome is fully sequenced, and several tools have been developed for the molecular study of this organism. Nevertheless, good protein tracking tags that are easy to measure and image, like the fluorescent proteins are lacking. In this report, we codon-optimized the red fluorescent protein from the coral Discosoma striata (DsRFP) for its use in E. histolytica and demonstrated functionality in vivo. We envision that this protein can be widely used for the development of transcriptional reporter systems and protein-tagging applications.

In Vitro Studies of Chromone-Tetrazoles against Pathogenic Protozoa, Bacteria, and Fungi.

In vitro studies to fourteen previously synthesized chromone-tetrazoles and four novel fluorine-containing analogs were conducted against pathogenic protozoan (Entamoeba histolytica), pathogenic bacteria (Pseudomonas aeruginosa, and Staphylococcus aureus), and human fungal pathogens (Sporothrix schenckii, Candida albicans, and Candida tropicalis), which have become in a serious health problem, mainly in tropical countries.

Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays.

Bacterial reporter assays are powerful tools used to study the effect of different compounds that affect the physiology of cellular processes. Most bacterial reporters are luciferase based and can be monitored in real time. In the present study we designed and implemented two sets of Escherichia coli bacterial reporter assays, using a multicopy plasmid system. Each reporter strain was constructed using either green fluorescent protein or ß-galactosidase (LacZ) proteins. The designed reporter strains are capable of responding in a specific manner to molecules that either oxidative stress, or membrane, protein, or DNA damage. In order to respond to the desired stimulus, promoter sequences from E. coli were used. These sequences correspond to the promoter of the major catalase (KatG) activated with cellular oxidative damage, the promoter of the ß-hydroxydecanoyl-ACP dehydrase (FabA) which is activated with membrane perturbation, the promoter of DNA recombinase (RecA) which is activated by DNA lesions. For protein misfolding, the promoter of the heat-shock responsive chaperon (DnaK) was used. Our constructs displayed activation to damage from specific stimuli, and low response to nonspecific stimuli was detected. Our results suggest that these types of bacterial reporter strains can be used in semiquantitative (fluorometric) and qualitative (ß-galactosidase activity) studies of different xenobiotic substances and pollutants.

Entamoeba histolytica: effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055.

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing(R) technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.