Effect of Clindamycin on Intestinal Microbiome and Miltefosine Pharmacology in Hamsters Infected with Leishmania infantum.

Visceral leishmaniasis (VL), a vector-borne parasitic disease caused by Leishmania donovani and L. infantum (Kinetoplastida), affects humans and dogs, being fatal unless treated. Miltefosine (MIL) is the only oral medication for VL and is considered a first choice drug when resistance to antimonials is present. Comorbidity and comedication are common in many affected patients but the relationship between microbiome composition, drugs administered and their pharmacology is still unknown. To explore the effect of clindamycin on the intestinal microbiome and the availability and distribution of MIL in target organs, Syrian hamsters (120-140 g) were inoculated with L. infantum (108 promastigotes/animal). Infection was maintained for 16 weeks, and the animals were treated with MIL (7 days, 5 mg/kg/day), clindamycin (1 mg/kg, single dose) + MIL (7 days, 5 mg/kg/day) or kept untreated. Infection was monitored by ELISA and fecal samples (16 wpi, 18 wpi, end point) were analyzed to determine the 16S metagenomic composition (OTUs) of the microbiome. MIL levels were determined by LC-MS/MS in plasma (24 h after the last treatment; end point) and target organs (spleen, liver) (end point). MIL did not significantly affect the composition of intestinal microbiome, but clindamycin provoked a transient albeit significant modification of the relative abundance of 45% of the genera, including Ruminococcaceae UCG-014, Ruminococcus 2; Bacteroides and (Eubacterium) ruminantium group, besides its effect on less abundant phyla and families. Intestinal dysbiosis in the antibiotic-treated animals was associated with significantly lower levels of MIL in plasma, though not in target organs at the end of the experiment. No clear relationship between microbiome composition (OTUs) and pharmacological parameters was found.

Leishmania infantum infection does not affect the main composition of the intestinal microbiome of the Syrian hamster.

BACKGROUND: Visceral leishmaniasis (VL) is the most severe form of all leishmanial infections and is caused by infection with protozoa of Leishmania donovani and Leishmania infantum. This parasitic disease occurs in over 80 countries and its geographic distribution is on the rise. Although the interaction between the intestinal microbiome and the immune response has been established in several pathologies, it has not been widely studied in leishmaniasis. The Syrian hamster is the most advanced laboratory model for developing vaccines and new drugs against VL. In the study reported here, we explored the relationship between the intestinal microbiome and infection with L. infantum in this surrogate host. METHODS: Male Syrian hamsters (120-140 g) were inoculated with 108 promastigotes of a canine-derived L. infantum strain or left as uninfected control animals. Infection was maintained for 19 weeks (endpoint) and monitored by an immunoglobulin G (IgG) enyzme-linked immunosorbent assay throughout the experiment. Individual faecal samples, obtained at weeks 16, 18 and 19 post-inoculation, were analysed to determine the 16S metagenomic composition (the operational taxonomic units [OTUs] of the intestinal microbiome and the comparison between groups were FDR (false discovery rate)-adjusted). RESULTS: Leishmania infantum infection elicited moderate clinical signs and lesions and a steady increase in specific anti-Leishmania serum IgG. The predominant phyla (Firmicutes + Bacteriodetes: > 90%), families (Muribaculaceae + Lachnospiraceae + Ruminococcaceae: 70-80%) and genera found in the uninfected hamsters showed no significant variations throughout the experiment. Leishmania infantum infection provoked a slightly higher-albeit non-significant-value for the Firmicutes/Bacteriodetes ratio but no notable differences were found in the relative abundance or diversity of phyla and families. The microbiome of the infected hamsters was enriched in CAG-352, whereas Lachnospiraceae UCG-004, the [Eubacterium] ventriosum group and Allobaculum were less abundant. CONCLUSIONS: The lack of extensive significant differences between hamsters infected and uninfected with L. infantum in the higher taxa (phyla, families) and the scarce variation found, which was restricted to genera with a low relative abundance, suggest that there is no clear VL infection-intestinal microbiome axis in hamsters. Further studies are needed (chronic infections, co-abundance analyses, intestinal sampling, functional analysis) to confirm these findings and to determine more precisely the possible relationship between microbiome composition and VL infection.

SARS-CoV-2 Infection in One Cat and Three Dogs Living in COVID-19-Positive Households in Madrid, Spain.

In this study, we describe SARS-CoV-2 infection dynamics in one cat and three dogs from households with confirmed human cases of COVID-19 living in the Madrid Community (Spain) at the time of expansion (December 2020 through June 2021) of the alpha variant (lineage B.1.1.7). A thorough physical exam and nasopharyngeal, oropharyngeal, and rectal swabs were collected for real-time reverse-transcription PCR (RT-qPCR) SARS-CoV-2 testing on day 0 and in successive samplings on days 7, 14, 21, and 47 during monitoring. Blood was also drawn to determine complete blood counts, biochemical profiles, and serology of the IgG response against SARS-CoV-2. On day 0, the cat case 1 presented with dyspnea and fever associated with a mild bronchoalveolar pattern. The dog cases 2, 3, and 4 were healthy, but case 2 presented with coughing, dyspnea, and weakness, and case 4 exhibited coughing and bilateral nasal discharge 3 and 6 days before the clinical exam. Case 3 (from the same household as case 2) remained asymptomatic. SARS-CoV-2 detection by RT-qPCR showed that the cat case 1 and the dog case 2 exhibited the lowest cycle threshold (Ct) (Ct < 30) when they presented clinical signs. Viral detection failed in successive samplings. Serological analyses revealed a positive IgG response in cat case 1 and dog cases 3 and 4 shortly after or simultaneously to virus shedding. Dog case 2 was seronegative, but seroconverted 21 days after SARS-CoV-2 detection. SARS-CoV-2 genome sequencing was attempted, and genomes were classified as belonging to the B.1.1.7 lineage.

Ecotoxicogenomic analysis of stress induced on Caenorhabditis elegans in heavy metal contaminated soil after nZVI treatment.

Soil contamination by heavy metals (HMs) is an environmental problem, and nanoremediation by using zero-valent iron nanoparticles (nZVI) has attracted increasing interest. We used ecotoxicological test and global transcriptome analysis with DNA microarrays to assess the suitability of C. elegans as a useful bioindicator to evaluate such strategy of nanoremediation in a highly polluted soil with Pb, Cd and Zn. The HMs produced devastating effect on C. elegans. nZVI treatment reversed this deleterious effect up to day 30 after application, but the reduction in the relative toxicity of HMs was lower at day 120. We stablished gene expression profile in C. elegans exposed to the polluted soil, treated and untreated with nZVI. The percentage of differentially expressed genes after treatment decreases with exposure time. After application of nZVI we found decreased toxicity, but increased biosynthesis of defensive enzymes responsive to oxidative stress. At day 14, when a decrease in toxicity has occurred, genes related to specific heavy metal detoxification mechanisms or to response to metal stress, were down regulated: gst-genes, encoding for glutathione-S-transferase, htm-1 (heavy metal tolerance factor), and pgp-5 and pgp-7, related to stress response to metals. At day 120, we found increased HMs toxicity compared to day 14, whereas the transcriptional oxidative and metal-induced responses were attenuated. These findings indicate that the profiled gene expression in C. elegans may be considered as an indicator of stress response that allows a reliable evaluation of the nanoremediation strategy.

Evaluation of nanoremediation strategy in a Pb, Zn and Cd contaminated soil.

We addressed the efficiency of a nanoremediation strategy using zero-valent iron nanoparticles (nZVI), in a case of co-mingled heavy metals (HM) pollution (Pb, Cd and Zn). We applied a combined set of physical-chemical, toxicological and molecular analyses to assess the effectiveness and ecosafety of nZVI (5% w/w) for environmental restoration. After 120 days, nZVI showed immobilization capacity for Pb (20%), it was scarcely effective for Zn (8%) and negligibly effective for Cd. The HMs immobilization in the nZVI treated soils (compared to control soil), reaches its maximum after 15 days (T3) as reflected in the decrease of HM toxicity towards V. fischeri. The overall abundance of the microbial community was similar in both sets of samples during all experiment, although an increase in the number of metabolically active bacteria was recorded 15 days post treatment. We studied the induced impact of nanoremediation on the soil microbial community structure by Next Generation Sequencing (NGS). Even when higher HM immobilization was recorded, no significant recovery of the microbial community structure was found in nZVI-treated soil. The most marked nZVI-induced structural shifts were observed at T3 (increase in the Firmicutes population with a decrease in Gram-negative bacteria). Predictive metagenomic analysis using PICRUSt showed differences among the predicted metagenomes of nZVI-treated and control soils. At T3 we found decrease in detoxification-related proteins or over-representation of germination-related proteins; after 120 days of nZVI exposure, higher abundance of proteins involved in regulation of cellular processes or sporulation-related proteins was detected. This study highlights the partial effectiveness of nanoremediation in multiple-metal contaminated soil in the short term. The apparent lack of recovery of biodiversity after application of nZVI and the decreased effectiveness of nanoremediation over time must be carefully considered to validate this technology when assurance of medium- to long-term immobilization of HMs is required.

New insights into the impact of nZVI on soil microbial biodiversity and functionality.

Nanoscale zero-valent iron (nZVI) is a strong reducing agent used for in situ remediation of soil. The impacts of nZVI (5-10% w/w) on the soil microbial biodiversity and functionality of two soils (Lufa 2.2 and 2.4) were assessed. Illumina MiSeq technology was used to evaluate the structure of soil microbiomes after 21 days of exposure. Proteobacteria, Verrucomicrobia, Firmicutes and Actinobacteria were the most abundant phyla in both soils. However, the dynamics of bacterial community composition following nZVI addition differed. nZVI exposure induced pronounced shifts in the microbial composition of soil 2.4, but not in soil 2.2; an increase in Verrucomicrobia abundance was the unique common taxonomic pattern observed in both soils. The PICRUSt approach was applied to predict the functional composition of each metagenome. Environmental information processing function (membrane transport) was decreased in both nZVI-spiked soils, although soil 2.4 samples were enriched in functions involved in cellular processes and metabolism. The effects of nZVI on autochthonous bacterial communities clearly varied with the soil type assessed; changes at the phylogenetic level appeared to be more abundant than those observed at the functional level, and thus, the overall effort of the soil ecosystem might involve the maintenance of functionality following nZVI exposure.

Outlining Core Pathways of Amyloid Toxicity in Bacteria with the RepA-WH1 Prionoid.

The synthetic bacterial prionoid RepA-WH1 causes a vertically transmissible amyloid proteinopathy in Escherichia coli that inhibits growth and eventually kills the cells. Recent in vitro studies show that RepA-WH1 builds pores through model lipid membranes, suggesting a possible mechanism for bacterial cell death. By comparing acutely (A31V) and mildly (ΔN37) cytotoxic mutant variants of the protein, we report here that RepA-WH1(A31V) expression decreases the intracellular osmotic pressure and compromise bacterial viability under either aerobic or anaerobic conditions. Both are effects expected from threatening membrane integrity and are in agreement with findings on the impairment by RepA-WH1(A31V) of the proton motive force (PMF)-dependent transport of ions (Fe3+) and ATP synthesis. Systems approaches reveal that, in aerobiosis, the PMF-independent respiratory dehydrogenase NdhII is induced in response to the reduction in intracellular levels of iron. While NdhII is known to generate H2O2 as a by-product of the autoxidation of its FAD cofactor, key proteins in the defense against oxidative stress (OxyR, KatE), together with other stress-resistance factors, are sequestered by co-aggregation with the RepA-WH1(A31V) amyloid. Our findings suggest a route for RepA-WH1 toxicity in bacteria: a primary hit of damage to the membrane, compromising bionergetics, triggers a stroke of oxidative stress, which is exacerbated due to the aggregation-dependent inactivation of enzymes and transcription factors that enable the cellular response to such injury. The proteinopathy caused by the prion-like protein RepA-WH1 in bacteria recapitulates some of the core hallmarks of human amyloid diseases.

Genomic reprograming analysis of the Mesothelial to Mesenchymal Transition identifies biomarkers in peritoneal dialysis patients.

Peritoneal dialysis (PD) is an effective renal replacement therapy, but a significant proportion of patients suffer PD-related complications, which limit the treatment duration. Mesothelial-to-mesenchymal transition (MMT) contributes to the PD-related peritoneal dysfunction. We analyzed the genetic reprograming of MMT to identify new biomarkers that may be tested in PD-patients. Microarray analysis revealed a partial overlapping between MMT induced in vitro and ex vivo in effluent-derived mesothelial cells, and that MMT is mainly a repression process being higher the number of genes that are down-regulated than those that are induced. Cellular morphology and number of altered genes showed that MMT ex vivo could be subdivided into two stages: early/epithelioid and advanced/non-epithelioid. RT-PCR array analysis demonstrated that a number of genes differentially expressed in effluent-derived non-epithelioid cells also showed significant differential expression when comparing standard versus low-GDP PD fluids. Thrombospondin-1 (TSP1), collagen-13 (COL13), vascular endothelial growth factor A (VEGFA), and gremlin-1 (GREM1) were measured in PD effluents, and except GREM1, showed significant differences between early and advanced stages of MMT, and their expression was associated with a high peritoneal transport status. The results establish a proof of concept about the feasibility of measuring MMT-associated secreted protein levels as potential biomarkers in PD.

Genomic and functional analyses unveil the response to hyphal wall stress in Candida albicans cells lacking ß(1,3)-glucan remodeling.

BACKGROUND: The cell wall is essential for the yeast to hypha (Y-H) transition that enables Candida albicans to invade human tissues and evade the immune system. The main constituent, ß(1,3)-glucan, is remodeled by glucanosyltransferases of the GH72 family. Phr1p is responsible of glucan remodeling at neutral-alkaline pH and is essential for morphogenesis and virulence. Due to the pH-regulated expression of PHR1, the phr1Δ phenotype is manifested at pH > 6 and its severity increases with the rise in pH. We exploited the pH-conditional nature of a PHR1 null mutant to analyze the impact of glucan remodeling on the hyphal transcriptional program and the role of chitin synthases in the hyphal wall stress (HWS) response. RESULTS: In hyphal growth inducing conditions, phr1Δ germ tubes are defective in elongation, accumulate chitin, and constitutively activate the signaling pathways mediated by the MAP kinases Mkc1p, Cek1p and Hog1p. The transcriptional profiles revealed an increase of transcript levels for genes involved in cell wall formation (CHS2 and CHS8, CRH11, PGA23, orf19.750, RBR1, RBT4, ECM331, PGA6, PGA13), protein N-glycosylation and sorting in the ER (CWH8 and CHS7), signaling (CPP1, SSK2), ion transport (FLC2, YVC1), stress response and metabolism and a reduced expression of adhesins. A transient up-regulation of DNA replication genes associated with entry into S-phase occurred whereas cell-cycle regulating genes (PCL1, PCL2, CCN1, GIN4, DUN1, CDC28) were persistently up-regulated. To test the physiological relevance of altered CHS gene expression, phr1Δ chsxΔ (x = 2,3,8) mutant phenotypes were analyzed during the Y-H transition. PHR1 deletion was synthetic lethal with CHS3 loss on solid M199 medium-pH 7.5 and with CHS8 deletion on solid M199-pH 8. On Spider medium, PHR1 was synthetic lethal with CHS3 or CHS8 at pH 8. CONCLUSIONS: The absence of Phr1p triggers an adaptive response aimed to reinforce the hyphal cell wall and restore homeostasis. Chs3p is essential in preserving phr1Δ cell integrity during the Y-H transition. Our findings also unveiled an unanticipated essential role of Chs8p during filamentation on solid media. These results highlight the flexibility of fungal cells in maintaining cell wall integrity and contribute to assessments of glucan remodeling as a target for therapy.

Functional and genomic analyses of blocked protein O-mannosylation in baker's yeast.

O-mannosylation is a crucial protein modification in eukaryotes that is initiated by the essential family of protein O-mannosyltransferases (PMTs). Here we demonstrate that in the model yeast Saccharomyces cerevisiae rhodanine-3-acetic acid derivatives affect members of all PMT subfamilies. Specifically, we used OGT2468 to analyse genome-wide transcriptional changes in response to general inhibition of O-mannosylation in baker's yeast. PMT inhibition results in the activation of the cell wall integrity (CWI) pathway. Coinciding, the mitogen-activated kinase Slt2p is activated in vivo and CWI pathway mutants are hypersensitive towards OGT2468. Further, induction of many target genes of the unfolded protein response (UPR) and ER-associated protein degradation (ERAD) is observed. The interdependence of O-mannosylation and UPR/ERAD is confirmed by genetic interactions between HAC1 and PMTs, and increased degradation of the ERAD substrate Pdr5p* in pmtΔ mutants. Transcriptome analyses further suggested that mating and filamentous growth are repressed upon PMT inhibition. Accordingly, in vivo mating efficiency and invasive growth are considerably decreased upon OGT2468 treatment. Quantitative PCR and ChIP analyses suggest that downregulation of mating genes is dependent on the transcription factor Ste12p. Finally, inhibitor studies identified a role of the Ste12p-dependent vegetative signalling cascade in the adaptive response to inhibition of O-mannosylation.

MARQ: an online tool to mine GEO for experiments with similar or opposite gene expression signatures.

The enormous amount of data available in public gene expression repositories such as Gene Expression Omnibus (GEO) offers an inestimable resource to explore gene expression programs across several organisms and conditions. This information can be used to discover experiments that induce similar or opposite gene expression patterns to a given query, which in turn may lead to the discovery of new relationships among diseases, drugs or pathways, as well as the generation of new hypotheses. In this work, we present MARQ, a web-based application that allows researchers to compare a query set of genes, e.g. a set of over- and under-expressed genes, against a signature database built from GEO datasets for different organisms and platforms. MARQ offers an easy-to-use and integrated environment to mine GEO, in order to identify conditions that induce similar or opposite gene expression patterns to a given experimental condition. MARQ also includes additional functionalities for the exploration of the results, including a meta-analysis pipeline to find genes that are differentially expressed across different experiments. The application is freely available at http://marq.dacya.ucm.es.