Microorganisms

BackgroundThe composition and function of the gut microbiome have been shown to contribute to both health and disease. One of the most powerful modulators of microbial composition and function is diet. Materials & MethodsUsing the E{micro}-TCL1 murine model of B-cell chronic lymphocytic leukemia (CLL), we assigned male and female mice to a high-fat, high-carbohydrate Western diet (HF) or standard chow (CH) diet. ResultsMice consuming a HF diet had significantly shorter survival than those consuming a CH diet, irrespective of sex, with female mice exhibiting particularly poor outcomes. We also observed a significant increase in splenic involvement by CLL in the HF diet-fed mice at time of sacrifice. Mice receiving the HF diet demonstrated immediate and profound effects on the gut microbiome, marked by reduced alpha diversity and significantly different community composition as measured by beta diversity. Notably, there was a sustained increase in Akkermansia muciniphila and Bacteroidetes thetaiotaomicron in HF diet-fed mice, coupled with a corresponding increase in microbiome functional pathways related to arginine and histidine biosynthesis, chitin degradation, and nucleotide biosynthesis. DiscussionCollectively our data provides evidence of the profound and sustained impact of a high-fat Western diet upon the gut microbiome community and CLL pathogenesis in the E{micro}-TCL1 murine model of CLL.

The growth of cultures and formation of mucilage blooms in reaction to salt stress of cyanobacterial cultures are investigated with a focus on the influence of pH. In non-buffered medium, cultures show their pH increasing from 6.5 just after inoculation, up to 11 during the exponential phase. We record the time-evolution of concentration and pH, with different initial OD0. In a second set of experiments, we extract the doubling time of the unbuffered cultures in comparison with those inoculated in pH-buffered BG11 media at four different pH from 6.3 to 10.5 : in the most acid media, all cultures die or grow very slowly. At pH = 10.5, we obtain the fastest growth for all four strains, allowing to qualify these cyanobacteria as being alkaliphiles, though for all strains with comparable initial OD0, the doubling time is shorter for unbuffered cultures. Following a previous study [31]), we finally investigate the influence of pH on mucilage formation and biomass uplift induced by salt stress, involving EPS floculation by cations. Our results show that operating in buffered media significantly influences the mucilage formation, though the observed regimes cannot be simply correlated to the pH value.

The biological conservation between fungi and mammals due to a common ancestor has made development of selective antifungal drugs a difficult challenge. Further complicating this situation is the selection of antifungal drug-resistant organisms during drug treatment. The pathogenic yeast Nakaseomyces glabratus (called here Candida glabrata) presents an especially challenging organism due to its tendency to frequently lose susceptibility to the major antifungal drug class the azoles. Additionally, C. glabrata develops resistance to echinocandin drugs, a second, more recently described antifungal agent at 10 times the rate of other organisms. Previous work has established that the sterol responsive transcriptional regulator Upc2A is a key determinant of azole susceptibility in C. glabrata and plays a role in echinocandin resistance. We used a biochemical approach to identify proteins that co-purified with Upc2A and identified the Ypk2 AGC kinase as an interacting protein. Strains lacking YPK2 exhibited increased susceptibility to fluconazole and the echinocandin caspofungin. A ypk2{Delta} strain failed to normally induce transcription of several ERG genes but exhibited normal induction of the CDR1 ATP-binding cassette transporter gene. Isogenic ypk2{Delta} strains were also highly susceptible to the three major classes of antifungal drugs, indicating that this kinase behaves as a multidrug susceptibility factor. RNA-seq analyses indicated that the transcriptional response to exposure is different for each drug and each response is differentially altered upon loss of Ypk2. Our data indicate that Ypk2 plays an important role in coordinating gene expression that impacts susceptibility to all major antifungal drug classes.

In the next years, several space missions will search for evidence of life on exoplanets, focusing on robust biosignatures associated with oxygenic photosynthesis, including atmospheric oxygen accumulation and the Vegetation Red-Edge in surface reflectance spectra. Many potentially habitable rocky exoplanets orbit M-dwarf stars, whose spectral energy distribution may challenge oxygenic photosynthesis. Differently from the Sun, M-dwarf stars emit predominantly far-red (700- 750 nm) and infrared (750-1000 nm) light, and relatively little visible (400-700 nm) radiation, which constitutes photosynthetically active radiation. Some organisms have been found to photosynthesize under such spectrum but less efficiently than under solar light, as their photosynthetic apparatus evolved to harvest visible light emitted by the Sun. Around M-dwarfs, such different irradiation might have selected adaptations optimized for harvesting far-red / infra-red light. On Earth, similar selection can be found in Acaryochloris marina strains, constitutively presenting high chlorophyll d content in photosystem II & I, with in vivo absorption peaks beyond 700 nm. Here we tested the Moss Beach strain under a simulated M-dwarf spectrum and a simulated primeval atmosphere - anoxic and enriched in carbon dioxide. Results underline how this permanently red-shifted photosynthetic apparatus does not require acclimation to the stellar spectrum and enables for a strong growth and oxygen production, higher than under simulated solar light. Moreover, cells reflectance spectrum highlights a shift of the canonical red-edge toward longer wavelengths, resulting in a Chl d-near-infrared edge, suggesting a similar metabolism on exoplanets orbiting M-dwarfs could successfully produce both a gaseous biosignature and a characteristic surface biosignature. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/719884v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@7f91bdorg.highwire.dtl.DTLVardef@1391bdborg.highwire.dtl.DTLVardef@53f7b4org.highwire.dtl.DTLVardef@ab59fa_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Liistro, E. (2026) https://BioRender.com/j2de4ay

BackgroundRespiratory tract infections range from asymptomatic colonisation to an invasive disease. Recent studies suggest that nasopharyngeal microbiota may influence this variability. Emerging evidence points to Dolosigranulum pigrum, a nasopharyngeal commensal, as a potentially protective bacterium. This study aimed to identify variables associated with the presence of D. pigrum in the nasopharynx of children with varying respiratory health statuses. MethodsNasopharyngeal aspirates were collected from children <18 years who were asymptomatic (n=65), had banal viral infection (n=48), or Invasive Pneumococcal Disease (IPD) (n=27). The presence of D. pigrum was defined as >0.1% of total sequences obtained by 16S rRNA gene sequencing. Variables included sex, breastfeeding, delivery mode, S. pneumoniae carriage, respiratory viruses and clinical features. ResultsAmong 140 children (73 males, 67 females), D. pigrum was detected in 79 (56.4%): 44/65 in the healthy group; 26/48 of viral and 9/27 IPD cases. Multivariate analysis revealed significant associations with health status and sex. Healthy children were more likely to carry D. pigrum than IPD cases (44/79 vs. 26/79; p= 0.028). Males were more frequently D. pigrum carriers than females (48/79 vs. 31/79; p= 0.033). ConclusionD. Pigrum was associated with respiratory health, being more prevalent in healthy children, and showed potential sex-related differences.

Beneficial interactions between plants and microorganisms strongly influence plant health and productivity, and root exudates play a central role in shaping these associations. This study analyzed the transcriptional responses of the bacterial endophytes Enterobacter asburiae RCA24 and Kosakonia sacchari RCA25 to root exudates from two commercial Italian rice accessions (Oryza sativa Baldo and Vialone Nano) and from an accession of the wild progenitor of tropical rice, Oryza rufipogon. Bacterial transcriptome analyses revealed that RCA24 responds differently to O. sativa varieties and that RCA25 was more stimulated by O. rufipogon. Changes in bacterial gene expression were mainly related to central metabolism, stress response, and signal transduction, highlighting a precise pattern of interaction. On the other hand, transcriptome analysis of inoculated rice revealed that RCA24 triggered broader transcriptional changes in plants than RCA25. Differentially expressed genes were related, especially in shoots, to defense responses, hormone-mediated signaling, and ribosome biogenesis, revealing that plants discriminate bacterial strains in a genotype-specific manner at the transcriptional level. Our findings suggest that traits beneficial to plant-soil microbiota interactions present in O. rufipogon and lost during domestication and diversification could be identified and reintroduced into modern rice varieties to improve sustainable field performance through beneficial microbial associations.

Since the initial distribution of the SARS-CoV-19 vaccine, its widespread use has been hypothesized to act as a selective pressure that drives the COVID-19 virus to mutate. This study aims to investigate the correlation between global vaccination rates and the mutation rate of the SARS-CoV-2 Beta variant (B.1.351). From January to July 2021, nucleotide diversity increased in tandem with vaccination rates, demonstrating that the virus evolved more rapidly in response to selective pressure from mass vaccination. Statistical analysis revealed statistically significant positive correlations between both vaccination rates and vaccine doses administered with nucleotide diversity. Thus, our findings indicate a positive correlation between rising vaccination rates and nucleotide diversity, suggesting that increased vaccination coverage acted as a selective pressure that accelerated viral evolution of SARS CoV2.

Labyrinthulomycetes are a class of fungus-like heterotrophic protists from the Stramenopiles lineage, recognized for their ecological role as decomposers and contributors to nutrient cycling. They colonize various substrates, from seaweed to terrestrial environments, utilizing ectoplasmic networks for nutrient absorption. This study characterized a novel Labyrinthula strain associated with the marine diatom Biddulphia. Phylogenetic analysis of the full-length 18S rRNA gene positioned this strain as a new species, Labyrinthula merlionensis sp. nov. Scanning electron and light microscopy observations revealed bi-flagellated zoospores and spindle-shaped vegetative cells with ectoplasmic networks. Time-series observations of the interactions between L. merlionensis and Biddulphia were categorised into different phases: establishment, infection, and aggregation. Scanning electron and confocal microscopy observations during the infection phase established the use of ectoplasmic nets to target the marginal ridge regions between diatoms, and the detection of labyrinthulid cells within diatom frustules. These findings enhance the understanding of the diversity, morphology, and ecological roles of Labyrinthulomycetes, particularly their intra- and extra-cellular interactions with diatom hosts.

Plant growth promoting microbes enhance developmental progression of the host by influencing its nutrient availability or by deploying secondary metabolites responsible for manipulating the hormonal crosstalk. Microbacterium bengalense sp. nov. GB16_1_BI (Accession number: SRX9280401), a newly identified ammonium releasing Actinomycetota, could enhance plant growth by manipulating rhizosphere bacteria. Amplicon sequencing of the 16S rRNA V3-V4 region from the rhizosphere of the black rice (Chakhao Poireiton) showed that GB16_1_BI could inhibit most bacteria. However, GB16_1_BI inoculation encouraged the growth of rare bacteria specific to waterlogged rice rhizosphere. Analysis of the OTUs using PICRUSt2 (Phylogenetic investigation of communities by reconstruction of unobserved states) showed increased abundance in the marker genes for nitrogen cycling (nifH, nrfA and nrt) but not for nifD or nifK which was also reflected in the ANOSIM analysis in the OTUs of the N-fixing bacteria. Marker genes for methane metabolism (comA, comB, cofG and cofH) were also more abundant in the inoculated plants than the control; however, ANOSIM studies did not support this observation in the OTUs of methane cycling bacteria. Both Methylosinus and Methylocystis, the two most abundant methanotrophic OTUs, are also known to be nitrogen fixers. Hence, GB16_1_BI could influence plant growth predominantly by manipulating nitrogen cycling microbes. The genome sequence as well as untargeted metabolome analyses of GB16_1_BI showed abundance of secondary metabolites with probable antimicrobial activity. GB16_1_BI could utilize varied carbohydrates and amino acid as energy source and form persister-like cells may help it to survive in the soil in absence of the host plant.

Chia (Salvia hispanica L.) is an emerging crop in Bangladesh valued for its medicinal properties and economic significance. In March 2024, target spot-like symptoms were observed in an experimental chia field (24.75{degrees} N, 90.50{degrees} E) at Bangladesh Agricultural University in Mymensingh, Bangladesh with disease incidence ranging from 23% to 47% across approximately 0.25 ha. Initially appearing as brick-red spots, these symptoms developed into target-shaped concentric rings, affecting leaves, stems, and inflorescences. A total of 24 fungal isolates were recovered from infected tissue; two representative isolates (BGECh-3 and BGECh-4) were randomly selected for details characterization. Pathogen identity was established through morphological traits, multilocus phylogenetic analysis of internal transcribed spacer (ITS) and elongation factor 1-alpha (EF-1) genes sequence, and pathogenicity confirmation through Kochs postulates, collectively identifying the causal agent as Corynespora cassiicola. The isolates demonstrated a broad host range, successfully infecting brinjal, chili, bottle gourd, country bean, tomato, and soybean. In vitro fungicide sensitivity assays with seven commercial fungicides showed that both isolates were highly sensitive to Goldzim (50% carbendazim), which completely inhibited mycelial growth at 10 {micro}g mL-{superscript 1}. Conza (10% Hexaconazole) and Amister top (18.2% azoxystrobin + 11.4% difenoconazole) reduced growth by up to 85% and 67%, respectively at equal concentration. Other fungicides showed comparatively lower efficacy even at higher concentrations. This study represents the first report of target spot disease of chia caused by C. cassiicola in Bangladesh and provides insights for effective disease management strategies.

BackgroundTuberculosis (TB) is the second-leading cause of deaths from infectious agents and remains a global health threat. Ethionamide (ETH) is a prodrug used in regimens for multidrug-resistant TB, and, partly due to side effects that can lead to low treatment adhesion, resistance arises. Changes in EthA, the monooxygenase that activates ETH, are the main mechanism of resistance. Yet, of hundreds of EthA substitutions found in resistant isolates, only a handful have been annotated as resistance determinants. ResultsAn in silico analysis was carried out on a previously described panel of Mycobacterium tuberculosis clinical isolates for which genomes and ETH susceptibility testing results were available. EthA substitutions were mapped, revealing the existence of hotspots in its sequence. Visualization of the hotspots in the EthA structural model shows that they cluster in three regions, including ligand binding pockets. Models were built of twenty-three variants found in resistant isolates and changes in local configuration was mapped to identify investigate impact on ETH activation. Information from these models contributed to establishing five criteria for scoring whether substitutions are most likely to lead to resistance. Using these criteria, EthA D58G was selected and its expression is shown to increase growth in high ETH concentrations. ConclusionFunctionally relevant regions of EthA are revealed and point out priority substitutions for functional studies, enhancing identification and detection of substitutions not been previously associated with resistance.

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection and is increasingly explored for other dysbiosis-related disorders. However, its implementation as a regulated therapeutic strategy still requires robust donor screening, biosafety frameworks, and standardized processing workflows. Here, we describe the establishment of the first fecal microbiota biobank in the south of Brazil and evaluate the incorporation of metagenomic sequencing as a complementary layer of donor safety assessment. A structured donor selection pipeline based on international guidelines was implemented, integrating clinical screening, biochemical and serological testing, and microbiological analyses. Of 100 screened candidates, only four donors met all eligibility criteria and were included in the biobank, highlighting the stringency of the selection process. Shotgun metagenomic sequencing revealed a diverse resistome across all donors, including a shared core set of resistance-related genes alongside marked interindividual variability. Dominant antibiotic resistance genes included tetracycline-associated determinants, as well as ermF, CfxA-type {beta}-lactamases, and aminoglycoside-modifying enzymes, each linked to specific gut taxa. Notably, the relatively high abundance of tetW and ermF in Bacteroides fragilis suggests that this dominant commensal species may act as a reservoir for tetracycline and multidrug resistance determinants within the intestinal microbiota. Rather than serving as exclusion criteria, such determinants highlight the importance of integrating functional genomic profiling into donor characterization. Overall, this study provides a framework for microbiota biobank implementation and supports the use of metagenomics as a complementary strategy to improve biosafety and functional assessment in FMT.

Toxoplasma gondii is a globally prevalent zoonotic parasite with multiple life stages and transmission routes, including ingestion and transplacental transmission. It is a major cause of abortion in sheep, goats and pigs, among other production animals, worldwide. While Type II strains are common in livestock in North America and Europe, non-archetypal, non-clonal genotypes are highly prevalent in South America. This study aimed to determine the molecular epidemiology of T. gondii strains causing sheep abortion in Uruguay. Phylogenomic analyses confirmed significant divergence among typed strains and revealed similarities with genotypes previously detected in the human population. Two novel strains, were isolated and characterized, uncovering the connection between their genetic makeup and phenotypes. Differences in virulence could be correlated to differences in gene copy number of the pseudo kinase ROP5 - further highlighting this virulence factor as relevant in wild strains. Whole-genome sequencing further confirmed the divergence among Uruguayan isolates, uncovering at least three distinct evolutionary origins. Overall, our findings highlight the circulation of virulent non-clonal lineages with links to human infections and underscore the importance of furthering genomic surveillance in South America to better understand Toxoplasmas transmission dynamics, pathogenic potential, and zoonotic risk.

SARS{square}CoV{square}2 infection is associated with marked changes of the upper respiratory tract mycobiome. URT mycobiome Changes in non-hospitalized patients however, remains poorly defined. We performed shotgun metagenomic sequencing of 95 upper respiratory tract swab samples from 48 symptomatic SARS{square}CoV{square}2-positive individuals and 47 healthy controls from central India. Fungal diversity and community structure were compared using alpha- and beta-diversity analyses, while differential taxa were identified using prevalence-based testing and LEfSe. SARS{square}CoV{square}2-positive samples showed significantly higher fungal alpha diversity than controls, with increased Shannon diversity (p = 0.000319) and Simpson diversity (p = 0.017). Beta-diversity analysis showed significant separation between groups for both Bray-Curtis and Jaccard distances (PERMANOVA p = 0.001), with significant dispersion effects as well (PERMDISP p = 0.001). Differential analysis identified more SARS{square}CoV{square}2-enriched than control-enriched taxa, including Candida orthopsilosis, Malassezia furfur, M. sympodialis, M. globosa, Aspergillus niger, A. terreus, and A. nidulans. Aspergillus sydowii was the main control-enriched taxon. LEfSe and concordant multi-test analysis supported these findings, and sensitivity analysis confirmed robustness across thresholds. Certain SARS{square}CoV{square}2-enriched taxa were linked to confirmed or probable COVID{square}19-associated fungal infections, whereas no such pathogens were detected in controls. These findings indicate that SARS{square}CoV{square}2 infection is associated with URT mycobiome dysbiosis and enrichment of clinically relevant opportunistic fungi in community cases.

Ralstonia pseudosolanacearum (Rps) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 109 CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::{Omega} mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::{Omega}. It indicated that the necrotic lesion caused in tomato leaves was hrp-dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

Pathogenic aquatic oomycetes Aphanomyces astaci and Saprolegnia parasitica represent a major threat to biodiversity and aquaculture production, but their interactions with host-associated microbes remain poorly understood. From a collection of bacterial isolates (n = 336) obtained from fish and crayfish hosts, we focused on Pseudomonas spp. (n = 70) and confirmed their previously reported strong inhibitory potential against A. astaci and S. parasitica. However, our results also revealed substantial inter- and intra-species variation in antagonism. To capture this variation, we selected eight isolates belonging to different Pseudomonas species groups (P. fluorescens, P. putida, and P. syringae) and displaying contrasting levels of anti-oomycete activity for further phenotypic assays and comparative genomic analysis. Across these isolates, mycelial inhibition was markedly stronger against A. astaci than against S. parasitica, indicating species-specific differences in susceptibility. Comparative genomic analysis revealed substantial variation in biosynthetic gene cluster (BGC) repertoires among the analysed strains. Strongly inhibitory isolates carried candidate BGCs with similarity to characterised bioactive pathways, including pyoluteorin, rhizoxin, pyrrolnitrin, DAPG, and orfamide, alongside with multiple uncharacterised clusters that were either shared among inhibitory isolates or restricted to individual strains. All analysed genomes also contained clusters related to siderophore and HCN biosynthesis. However, in vitro assays showed that siderophore production was not clearly associated with inhibitory activity and that inhibition was mediated mainly by diffusible rather than volatile compounds. Altogether, our results suggest that Pseudomonas anti-oomycete activity is species- and strain-dependent and likely reflects different combinations of multiple, predominantly diffusible metabolites rather than a single conserved mechanism. In conclusion, this study provides a foundation for future work aimed at resolving mechanisms underlying microbial antagonism toward aquatic oomycete pathogens.

Myxococcus faecalis was recently described from human fecal isolates, although subsequent evidence indicates an environmental distribution for this lineage. Here, we report the isolation and genomic characterization of two M. faecalis strains (BRX-014 and BRX-032) recovered from mangrove ecosystems along the southeastern coast of Brazil, representing the first record of the species in a marine-coastal biome. Phylogenomic reconstruction based on 120 conserved bacterial marker genes, together with Average Nucleotide Identity (ANI >97.6%) and digital DNA-DNA hybridization (dDDH 77.7-90.4%) analyses, confirmed their assignment to M. faecalis and demonstrated high genomic relatedness to strains previously recovered from soil and human feces samples. Pangenome analysis of five available genomes revealed a total repertoire of 9,827 genes, with a large core genome comprising 7,499 genes (76.3%), consistent with a highly conserved and nearly closed pangenome structure. Functional classification based on COG categories showed uniform distributions across all isolates. Comparative analysis of the degradome further revealed strong conservation of proteolytic and carbohydrate-active enzyme repertoires, dominated by serine and metallopeptidases and diverse glycoside hydrolases. The extensive genomic and functional similarity among isolates from geographically distant and ecologically distinct environments supports a broad ecological distribution of M. faecalis and suggests that its large and conserved genomic repertoire underpins its persistence across contrasting habitats. These findings expand the known ecological range of the species and provide a comparative genomic framework for future investigations into its distribution and functional potential across different habitats.

The biotrophic pathogen Puccinia triticina is the causative agent of the most vulnerable foliar disease, namely leaf rust disease of wheat. The pathogen-secreted effectors are essential in modulating fungal virulence and host immune responses. Despite their significance, potential effectors and their underlying mechanisms governing host susceptibility remain elusive. In the present study, we employed an in silico approach to identify and characterise effector proteins from the P. triticina proteome. Later, performed temporal expression profiling to prioritise effector candidates associated with rust disease. Here, a total of 273 high-confidence effector candidates were identified and analysed their physicochemical properties, domains, motifs, and functional annotations, to assess their conservation and dynamics. Although most of the effectors were uncharacterised, the conserved motif virulence-associated [YFW]xC was notably enriched in the effector repertoire. Comparative PHI-base annotation highlighted similarities with known fungal virulence factors involved in host susceptibility. Effectors harbouring CAZyme activity indicate involvement in host cell wall modification. Promoter analysis identified multiple stress- and defence-related transcription factor binding sites, suggesting regulated expression during infection. Transcriptome analysis revealed that 20 effector genes were significantly upregulated during P. triticina infection. qRT-PCR validated the expression of 4 highly induced effector transcripts following P. triticina infection in susceptible wheat variety. Specifically, two of these candidates demonstrated biphasic expression pattern that aligns contrasting PTI- and ETI-mediated defense mechanisms critical for sustained virulence. Overall, this study provides a comprehensive framework for identifying functionally relevant P. triticina effectors and offers insight for future effector-target studies and effector-based leaf rust management strategies.

Cryptosporidium parvum is a protozoan parasite responsible for cryptosporidiosis, significantly threatening immunocompromised individuals, particularly HIV/AIDS patients, by causing severe diarrhea and potential mortality. Current treatments are largely ineffective, prompting investigations into new therapeutic options. This study evaluated two antiparasitic drugs: Mebendazole, used for helminth infections, and Artemisinin, used for malaria. The SKSR gene family encodes virulence factors in C. parvum, and Calcium-dependent protein kinase1 (CpCDPK1) regulates the life cycle of C. parvum; targeting these proteins may reduce growth and infection in hosts. In the current study, molecular docking was conducted taking Mebendazole and Artemisinin drugs as ligands, SKSR gene family and CpCDPK1 proteins as drug targets. Results with SKSR showed binding energy of -4.9 kcal/mol, -6.72 kcal/mol for Mebendazole and Artemisinin, respectively. Whereas, with CpCDPK1, the binding energies were -6.44 kcal/mol, -9.18 kcal/mol for Mebendazole and Artemisinin, respectively. Docking of Nitazoxanide (an in-use drug for C. parvum) with SKSR and CpCDPK1 revealed binding energies -4.2 kcal/mol, -4.81 kcal/mol, respectively. The stability of the proteins (targets) upon binding to the ligands was assessed by performing all-atom MD simulations for 100ns using the GROMACS package. No major variations were observed upon binding of Artemisinin and Mebendazole to SKSR and CpCDPK1. The findings of MD simulations imply that both proteins maintain their stability upon binding of Artemisinin and Mebendazole. Molecular Docking and MD simulation studies suggest that Artemisinin and Mebendazole are potential candidates for repurposing in the treatment of C. parvum infections, with recommendations for in vitro studies to validate these findings.

BackgroundPeriodontitis (Perio) is a progressive oral disease characterized by inflammation and degradation of the periodontal apparatus and is associated with local and systemic morbidity including loss of teeth, cardiovascular disease, and diabetes mellitus, among others. Perio is highly prevalent in domestic canines and exhibits certain parallels in pathogenesis and pathophysiology to Perio in humans, although standard treatments are less effective. In both species, a complex interplay between oral microbiota and host immune response is implicated in the etiology of Perio but is not fully understood. ResultsUsing shotgun metagenomics and RNA-seq on oral samples from companion dogs, we identify features of the oral microbiome and host transcriptional profile that are associated with Perio and its progression. We observe differences in microbiota composition between Perio and non-Perio animals that are largely consistent with what has been described in humans but also identify several species that are distinctly associated with canine Perio. We observe an abrupt shift in host gene expression related to immune response and tissue structure that is associated with disease severity, specifically the progression from mild periodontal disease (PD) to more severe Perio and the initiation of clinical attachment loss. The gingival plaque microbiota exhibits a parallel dynamic, with distinct compositional profiles in mild, moderate, and severe PD. We then examine several of the known mechanistic components of the keystone pathogen hypothesis of PD, identifying specific commonalities between canine and human pathologies, including the involvement of Porphyromonas species and related virulence factors. Additionally, we show infiltration of gingival tissue by Porphyromonas and Tannerella spp. via fluorescence microscopy. Finally, we assess correlations between host gene expression and microbial metabolic pathways which suggest additional potential virulence factors. ConclusionsThis work elucidates the metagenomic and transcriptomic signatures of Perio in companion dogs with the goals of informing veterinary medicine, evaluating the potential of canines as a model organism for the study of Perio, and clarifying the relationship between Perio development and progression, the oral microbiota, and the localized host response. Our findings provide insight into the etiopathogenesis of canine Perio and its relationship to human Perio and suggest novel targets of potential translational interest.