Frequency of Toxoplasma gondii and Risk Factors Associated with the Infection in Stray Dogs and Cats of Panama

Stray animals such as dogs and cats have an important role in maintaining the transmission cycles and dissemination of Toxoplasma gondii. Therefore, the objective of this study was to evaluate the frequency of T. gondii in stray dogs and cats in six different regions of Panama and determine risk factors associated with the dynamics of infection in each of the studied regions. Data were obtained using serological tests for the detection of anti-T. gondii IgG and IgM antibodies. The results of this study revealed an overall infection frequency of 23.73%. The infection frequencies found in dog and cat populations were 25.70% and 21.93% respectively, showing no statistically significant difference. Risk factor correlations suggested different infection dynamics depending on the region analyzed. The San Miguelito, North and West regions were more associated with positive cases in dogs with an age range greater than 13 months. Conversely, the Metro, Central and East regions were more associated with negative cases in cats with age ranging between 0 and 5 months. Infection of the parasite in stray animals can be influenced by intrinsic characteristics of each region, which can potentiate different risk factors associated with the different routes of transmission.Stray animals such as dogs and cats have an important role in maintaining the transmission cycles and dissemination of Toxoplasma gondii. Therefore, the objective of this study was to evaluate the frequency of T. gondii in stray dogs and cats in six different regions of Panama and determine risk factors associated with the dynamics of infection in each of the studied regions. Data were obtained using serological tests for the detection of anti-T. gondii IgG and IgM antibodies. The results of this study revealed an overall infection frequency of 23.73%. The infection frequencies found in dog and cat populations were 25.70% and 21.93% respectively, showing no statistically significant difference. Risk factor correlations suggested different infection dynamics depending on the region analyzed. The San Miguelito, North and West regions were more associated with positive cases in dogs with an age range greater than 13 months. Conversely, the Metro, Central and East regions were more associated with negative cases in cats with age ranging between 0 and 5 months. Infection of the parasite in stray animals can be influenced by intrinsic characteristics of each region, which can potentiate different risk factors associated with the different routes of transmission

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells. RESULTS: Here we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections. CONCLUSIONS: Sat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype

Outer membrane vesicles from the probiotic Escherichia coli Nissle 1917 and the commensal ECOR12 enter intestinal epithelial cells via clathrin-dependent endocytosis and elicit differential effects on DNA damage.

Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs) are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN) and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether colibactin is secreted via OMVs is an open question that needs further study

Establishing a cell-free transcription-translation platform for Cutibacterium acnes to prototype engineered metabolic and synthetic biology

Data de publicació electrònica: 31-12-2021In the past few years, new bacterial-cell-free transcription-translation systems have emerged as potent and quick platforms for protein production as well as for prototyping of DNA regulatory elements, genetic circuits, and metabolic pathways. The Gram-positive commensal Cutibacterium acnes is one of the most abundant bacteria present in the human skin microbiome. However, it has recently been reported that some C. acnes phylotypes can be associated with common inflammatory skin conditions, such as acne vulgaris, whereas others seem to play a protective role, acting as possible "skin probiotics". This fact has made C. acnes become a bacterial model of interest for the cosmetic industry. In the present study we report for the first time the development and optimization of a C. acnes-based cell-free system (CFS) that is able to produce 85 μg/mL firefly luciferase. We highlight the importance of harvesting the bacterial pellet in mid log phase and maintaining CFS reactions at 30 °C and physiological pH to obtain the optimal yield. Additionally, a C. acnes promoter library was engineered to compare coupled in vitro TX-TL activities, and a temperature biosensor was tested, demonstrating the wide range of applications of this toolkit in the synthetic biology field.This work was funded by the Office of Naval Research (Award N62909-18-1-2155). M.-J.F was funded by a Juan de la Cierva Fellowship (Spanish Government). N.K. was funded by a Maria Maetzu-UPF Fellowship (Catalan Government). G.N. was funded by an FI Fellowship (AGAUR-Catalan Govern- ment). This project received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie Grant Agreement 882387 (to J.S.-M.)

New insights into the role of Cutibacterium acnes-derived extracellular vesicles in inflammatory skin disorders

Abstract Cutibacterium acnes (C. acnes) is one of the most prevalent bacteria that forms the human skin microbiota. Specific phylotypes of C. acnes have been associated with the development of acne vulgaris, while other phylotypes have been linked to healthy skin. In this scenario, bacterial extracellular vesicles (EVs) play a role in the interkingdom communication role with the human host. The purpose of this study was to examine the impact of EVs generated by various phylotypes of C. acnes on inflammation and sebum production using different in vitro skin cell types. The main findings of this study reveal that the proteomic profile of the cargo embodied in the EVs reflects distinct characteristics of the different C. acnes phylotypes in terms of life cycle, survival, and virulence. The in vitro skin cell types showed an extended pro-inflammatory modulation of SLST A1 EVs consistently triggering the activation of the inflammation-related factors IL-8, IL-6, TNFα and GM-CSF, in comparison to SLST H1 and SLST H2. Additionally, an acne-prone skin model utilizing PCi-SEB and arachidonic acid as a sebum inducer, was employed to investigate the impact of C. acnes EVs on sebum regulation. Our findings indicated that all three types of EVs significantly inhibited sebum production after a 24-h treatment period, with SLST H1 EVs exhibiting the most pronounced inhibitory effect when compared to the positive control. The results of this study highlight the protective nature of C. acnes SLST H1 EVs and their potential use as a natural treatment option for alleviating symptoms associated with inflammation and oily skin

Effect of EcN and ECOR12 OMVs on proliferation and viability of HT-29 cells.

<p><b>(A)</b> Cell viability, <b>(B)</b> Mean proliferation index, <b>(C)</b> Population doubling level of HT-29 cells exposed to OMVs (5 μg/ml) from EcN (squares) or ECOR12 (triangles) for up to 7 days, measured by the trypan blue exclusion assay. In panel <b>(C)</b>, control cells are indicated by circles. <b>(D)</b> MTT reduction activity measured along the experiment. Values are means ± standard error from four independent experiments (<i>P</i><0.01, versus untreated control cells).</p

Outer membrane vesicles from the probiotic Escherichia coli Nissle 1917 and the commensal ECOR12 enter intestinal epithelial cells via clathrin-dependent endocytosis and elicit differential effects on DNA damage.

Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs) are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN) and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether colibactin is secreted via OMVs is an open question that needs further study

Uptake of EcN and ECOR12 OMVs in HT-29 cells.

<p><b>(A)</b> HT-29 cells were incubated at 37°C with rhodamine B-R18-labeled OMVs (2 μg/well) from strains EcN and ECOR12 (squares) and fluorescence was measured over time with a microplate reader. OMVs (circles) and cells (triangles) alone were analyzed in parallel as controls. Fluorescence intensity was normalized by fluorescence detected at the indicated time points by labeled OMVs in the absence of cells. Data are presented as means ± standard error from four independent experiments. Results significantly different from that of untreated control cells are indicated by an asterisk (<i>P</i><0.006). <b>(B, C)</b> Visualization of internalized OMVs by florescence microscopy. HT-29 cells were incubated with rhodamine B-R18-labeled OMVs <b>(B)</b> or with unlabeled OMVs <b>(C)</b> for 1 and 3 h at 37°C. When indicated incubations were performed at 4°C as a control. The cell membrane was stained with WGA (green) and nuclei with DAPI (blue). Internalized rhodamine B-R18-labeled OMVs are visualized in red. In <b>(C)</b> vesicles were immunostained with <i>E</i>. <i>coli</i> anti-LPS antibody and Alexa Fluor 546-conjugated secondary antibody. Analysis was performed in a Leica TCS SP5 laser scanning confocal spectral microscope with 63x oil immersion objective lens, and images were captured with a Nikon color camera (16 bit). Scale bar: 20 μm.</p

Colocalization of EcN and ECOR12 OMVs with clathrin (A), endosomes (B) and lysosomes (C).

<p>HT-29 cells were incubated with rhodamine B-R18-labeled OMVs (2 μg) for the indicated times and analyzed using laser scanning confocal spectral microscope. Scale bar: 20 μm. Clathrin was stained using anti-clathrin mouse monoclonal antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Endosomes were labeled with a rabbit polyclonal antibody against the endosome-associated protein EEA1 and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Lysosomes were detected using LysoTracker Green DND-26 at 300 nM (green). Images are from a single representative experiment (n = 4). Colocalization of the green (clathrin, EEA1 or the LysoTracker probe)) and red (vesicles) signals was confirmed by histogram analysis of the fluorescence intensities along the yellow lines. Analysis by laser scanning confocal spectral microscope was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160374#pone.0160374.g001" target="_blank">Fig 1</a>.</p

EcN and ECOR12 OMVs induce cell cycle arrest in HT-29 cells.

<p>Cell cycle analysis of HT-29 cells challenged with OMVs (5 μg/ml) from strains EcN (gray bars) or ECOR12 (black bars) for up to 3 days. Non-treated cells were analyzed as a control (white bars). Cells were gated based on FSC and SSC and then by their area <i>vs</i> peak fluorescence signal for propidium iodide. A total of 10,000 events were analyzed for each sample. Values are means ± standard error from three independent experiments (<i>P</i><0.02, versus untreated control cells).</p