Virulence

Cell-mediated immune responses are crucial for protecting the host against Toxoplasma gondii infection. However, impaired immunity, such as T-cell exhaustion, is a common phenomenon during chronic infection. This may represent a strategy employed by T. gondii to evade host defenses. T-cell immunoglobulin and mucin-domain containing 3 (Tim-3) is an important regulatory molecule involved in cell-mediated immunity. This study examined the expression of Tim-3 and the effects of its blockade in a mouse model of toxoplasmosis. In mice with chronic T. gondii infection, we found that Tim-3 is highly expressed in both cyst-bearing and non-cyst-bearing tissues, and its expression correlates with the parasite burden. Blocking the Tim-3 pathway with an anti-Tim-3 antibody enhances the immune response, resulting in elevated levels of cytokines (IFN-{gamma}, IL-12p70, IL-2, IL-9) and the chemokine CXCL1 in the serum, increased leukocyte infiltration (CD3+, CD14+ cells) in the brain, and downregulation of Tim-3 expression in microglial cells. As a result, the anti-Tim-3 treatment resulted in a 62% reduction in the number of tissue cysts and a trend towards an increase in the homeostatic signature, P2RY12, in microglia. Our study provides proof of concept for an anti-Tim-3 approach in treating chronic T. gondii infection and potentially other brain-residing pathogens.

The tobacco hornworm moth (Manduca sexta) is evaluated as a model of bacterial virulence and host-pathogen dynamics. Infections of Pseudomonas aeruginosa were established by injection of 5th-instar larvae, and multiple assays of virulence were evaluated. Infected larvae exhibited dose-dependent mortality, reduced growth, melanization, behavioral changes, and altered frass constitution. Even low-dose infections that were not fatal exhibited impaired growth, but individual growth trajectories revealed considerable heterogeneity among worms given the same dose. Twice-daily antibiotic treatment with gentamicin or cefepime improved survival four- to five-fold but did not rescue 100%. Heat-killed cells and filtered culture supernatant alone induced significant morbidity and mortality, suggesting secreted bacterial products are important to pathogenesis. Bacterial burden analysis revealed a shifting bacterial distribution over time, with decreasing hemolymph titers and increasing localization in fat body, gut, and carcass. Hornworms thus offer a more sensitive analysis of bacterial infection dynamics and consequences than do larvae of the more commonly used wax moth.

Bilophila wadsworthia is a gut pathobiont implicated in dysbiosis-driven inflammation, yet its pathogenic mechanisms remain poorly investigated. Here, we evaluated the suitability of Galleria mellonella larvae as an in vivo model to study B. wadsworthia infection. Two infection routes were compared: oral inoculation to mimic gastrointestinal colonization and hemolymph injection to model systemic infection. Oral challenge had minimal impact on larval health, whereas hemolymph injection caused marked morbidity, including reduced mobility, impaired cocoon formation, and progressive melanization, indicating that access to the circulatory system is required for overt disease. Infection required live bacteria, with B. wadsworthia capable of intracellular replication within hemocytes, leading to transient depletion of circulating immune cells followed by compensatory hemocyte proliferation. These findings reveal tight coupling between bacterial proliferation and host immune dynamics. Comparison with other sulfidogenic bacteria suggests that Bilophila pathogenicity is likely to involve host-specific interactions. Overall, our results establish G. mellonella as a practical and ethically favorable model to investigate B. wadsworthia virulence, host-pathogen interactions, and mechanisms relevant to gut-associated infection.

Mycoplasma synoviae is an avian pathogen that causes respiratory disease and synovitis, and its hemagglutinin plays a critical role in host cell adhesion. However, the key residues and structural mechanisms underlying hemagglutination remain unclear. In this study, domain analysis of the hemagglutinin family of Mycoplasma synoviae revealed that it contains long-chain and short-chain types, among which LAM HA (VY93_RS01465) was selected as the bait protein due to its complete C-terminal conserved domain. Through yeast two-hybrid screening, 18 host proteins interacting with LAM HA were identified. Furthermore, five key amino acid residues S83, R85, Y88, N124, and K192 were found to mediate hemagglutination activity. Deletion of these residues reduced the hemagglutination titer of LAM HA under acidic conditions. Secondary structure analysis showed that the deletion mutation decreased the -helix content while increasing the proportions of {beta}-sheet and random coil. Molecular dynamics simulations revealed that the mutant exhibited generally higher root mean square deviation and root mean square fluctuation values than the wild-type under different pH conditions, with a marked decrease in structural stability particularly at pH 5.0 and 6.0. These findings indicate that LAM HA, as a critical adhesin, exerts its hemagglutination function dependent on specific key residues and pH-sensitive conformational stability. IMPORTANCEMycoplasma synoviae (M. synoviae) causes significant economic losses to the poultry industry worldwide. Lipid-related membrane protein hemagglutinin (LAM HA) is a surface adhesin essential for host cell attachment, but its precise amino acid residues and structural features have not been defined. In this study, five key residues (S83, R85, Y88, N124, and K192) were identified as critical for LAM HA-mediated hemagglutination activity. Deletion of these residues altered the secondary structure composition, reduced conformational stability under acidic pH conditions, and decreased hemagglutination activity. These findings reveal a previously unknown structure-function relationship of M. synoviae LAM HA, demonstrating that its hemagglutination activity depends on specific residues and pH-sensitive structural integrity. This provides new insights into the molecular mechanisms of M. synoviae adhesion and offers potential targets for the development of novel intervention strategies against avian mycoplasmosis.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, an efficient endoplasmic reticulum (ER) quality control, such as the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in the filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-{beta}-N-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-{beta}-N-acetylhexosaminidase under ER stress. These findings identify cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded animals, progressing through acute and chronic stages. The Akt/mTOR signaling axis plays critical roles in cell survival, proliferation, and metabolism, making it a key target for intracellular pathogens. This study investigated how T. gondii infection modulates this pathway during both infections. Outbred CD-1 mice were infected intraperitoneally with the virulent GT1 strain of T. gondii. Mice for acute studies were sacrificed five days post-infection, while those for chronic studies were treated with sulfadiazine and sacrificed five months post-infection. Phosphoprotein expression of eight Akt/mTOR pathway components was measured in liver tissues using a multiplexed bead-based immunoassay. Acute T. gondii infection caused broad suppression of Akt/mTOR signaling, with 6 of 8 markers significantly downregulated, including pS6RPSer235/236, pAKTS473, pBADSer136, pIRS1S636/639, pPTENSer380, and pGSK-3/{beta}Ser21/9. In contrast, chronic infection selectively activates specific nodes of the pathway in a cyst burden-dependent manner, including pBADSer136, pmTORSer2448, and pGSK-3/{beta}Ser21/9. There are strong correlations in signaling changes between inter-components, which reflect coherent and coordinated pathway-level reprogramming rather than random perturbation. These findings show that acute and chronic T. gondii infections have opposing effects on host Akt/mTOR signaling for their own benefit, which may present new therapeutic targets. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/716682v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8c5021org.highwire.dtl.DTLVardef@1e0cdcaorg.highwire.dtl.DTLVardef@1e690eaorg.highwire.dtl.DTLVardef@342c0b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAcute T. gondii infection broadly suppresses hepatic Akt/mTOR signaling C_LIO_LIChronic infection exerts cyst burden-dependent activation of specific Akt/mTOR nodes C_LIO_LIT. gondii has distinct strategies to manipulate host survival based on its life stages. C_LIO_LIThe Akt/mTOR pathway may serve as a therapeutic target for the treatment of T. gondii. C_LI

Leishmaniases remain a significant global public health threat, with Leishmania amazonensis and Leishmania infantum representing the etiological agents of the cutaneous and visceral forms in the Americas, respectively. Building on our previous identification of Phospholipase A1 (PLA1) in Leishmania braziliensis, this study provides a comprehensive molecular, immunological, and biochemical characterization of PLA1 in L. amazonensis and L. infantum promastigotes. We analyzed PLA1 activity and expression, purified the recombinant enzyme from L. amazonensis, and validated protein expression using a specific anti-PLA1 serum. The major contribution of this research is the first description of the subcellular localization of a PLA1 within the Leishmania genus. Moreover, our results reveal an unprecedented association between PLA1 and lipid droplets within the parasites. This discovery is of particular interest as it provides the first evidence linking this enzyme to lipid storage organelles in Leishmania. Given that PLA1 is an established virulence factor in other trypanosomatids, these findings suggest a specialized role for the enzyme in parasite lipid metabolism and potentially in its pathogenic mechanisms, opening new perspectives for understanding Leishmania biology.

Helicobacter pylori is a prevalent bacterial pathogen that chronically colonizes the human gastric epithelium, but the bacteriums physiological mechanisms that promote this are understudied. Dormancy and low growth are known to facilitate other microbial chronic infections. A critical feature of low growth states is the down regulation of ribosome translational activity via regulation factors. The H. pylori genome is predicted to encode only one ribosome regulation factor, called RsfS (Ribosomal Silencing Factor S). In other bacterial species, RsfS prevents ribosome assembly by binding to a protein called L14 on the 50S large ribosomal subunit. Although H. pylori RsfS has not been experimentally investigated prior to this work, it conserves key residues, suggesting it is a bona fide RsfS homolog. To investigate phenotypes associated with rsfS, the gene was deleted and mutant phenotypes characterized. H. pylori rsfS null mutants had no defects during exponential phase but had viability defects in stationary phase and low growth factor conditions. Additionally, rsfS null mutants could not form biofilms, and instead were only able to form monolayers of multicellular aggregates. These defects were corrected by the re-introduction of rsfS in a second site on the chromosome. To explore whether rsfS is required in vivo, a mouse model was employed. rsfS mutants initially colonized in low numbers in both the glands and total stomach but were unable to develop robust long-term colonization. This work supports that H. pylori requires RsfS for survival in low growth states and to maintain chronic infections in the host. ImportanceH. pylori chronic infections are difficult to cure in part because H. pylori is proposed to adopt low-growth states known to render bacteria tolerant to antibiotics. One key signature of a low growth state includes low translation via ribosome regulation factors. Unlike other bacterial species, H. pylori contain only one known ribosome regulation factor called Ribosomal Silencing Factor S (RsfS). This gene was previously found to be transcriptionally upregulated in at least one low growth state, biofilms. In this work, we found that H. pylori rsfS is required for this microbe to thrive in low growth states and during infection. This study is one of only two studies that investigates the phenotypes of rsfS knockout mutants in any bacterial species and the first to address knowledge gaps in ribosomal regulation by H. pylori in vivo.

Spotty Liver Disease (SLD) is an acute bacterial infection of layer chickens in production, caused by Campylobacter hepaticus, and occurs most frequently in barn-housed and free-range systems. The disease is characterized by a sharp decline in egg production and increased mortality. The hallmark pathological feature is 1-2 mm white to grey necrotic foci distributed across the liver surface. Despite its growing economic impact on commercial poultry, the molecular mechanisms underlying host responses to C. hepaticus infection remain poorly understood. To address this gap, we performed a comprehensive transcriptome analysis of liver tissue from chickens naturally infected with SLD compared to uninfected controls. High-throughput transcriptome sequencing, yielding 9,277 differentially expressed genes (DEG), of which 3,063 were upregulated and 6,214 were downregulated. Functional pathway enrichment analysis revealed significant alterations in immune and metabolic processes associated with SLD pathophysiology. Infected chickens exhibited significant activation of immune response pathways, particularly cytokine-cytokine receptor interactions involving interleukins IL-22, IL-21, and IL-6, along with enhanced cell signaling, and cell adhesion. Among the individual genes, C1QTNF1 and the adhesion molecule gene ADGRD1 were notably overexpressed, indicating enhanced inflammatory activity. In contrast, core hepatic metabolic functions were profoundly reduced, as evidenced by downregulation of oxidative phosphorylation, fatty acid metabolism, iron ion binding, and heme binding pathways. A marked increase in serum amyloid A gene (SAA) expression further confirmed robust acute-phase responses and compromised liver function during infection. Together, these findings demonstrate a complex interplay between inflammatory activation and metabolic dysregulation during SLD. The strong upregulation of acute-phase proteins and pro-inflammatory cytokines demonstrates the hosts vigorous attempt to combat bacterial infection, whereas the concurrent suppression of essential metabolic pathways reflects the pathological consequences of SLD. This study provides a transcriptomic characterization of host responses to C. hepaticus infection, offering insights into SLD pathogenesis and potential avenues for targeted intervention.

The lack of a reliable chronic murine model limits drugs evaluation against Mycobacterium abscessus. Models show discrepancies, especially regarding host factors (mouse strain, sex and age). Using beads-model, we compared BALB/cJRJ and C57BL/6NCrl across sexes and ages. BALB/cJRJ showed more sustained infection and lower variability, with no significant sex- or age-related differences. Considering these results and the higher prevalence of NTM pulmonary infections in female patients, 5-6 weeks-old female BALB/cJRJ are appropriate for M. abscessus beads-model.

Autophagy is a catabolic process used for the degradation of organelles and proteins. Macroautophagy involves the formation of autophagosomes and subsequent fusion with lysosomes to mediate cargo degradation. It also functions as a cellular defence mechanism, known as xenophagy, during infection. Previous studies show that different viruses manipulate the autophagy pathway of the host cell to assure successful replication and/or virion assembly. Vaccinia virus (VACV), the prototypic poxvirus, replicates exclusively in the cytoplasm of host cells. It is known that VACV infection causes LC3 lipidation and prevents autophagosome formation, yet the double membrane vesicles formed during autophagy do not serve as the source of the mature VACV membrane. To date the viral protein(s) causing increased LC3 lipidation have not been identified. Here we developed an image-based screening approach based on LC3 granularity to identify candidate VACV genes affecting its lipidation. We identify several candidate viral membrane proteins as effectors of LC3 lipidation, suggesting that the interplay between VACV and autophagy is more directed than previously thought.

Toxoplasma gondii is a single-celled parasite belonging to the Apicomplexa phylum. Toxoplasmas single mitochondrion is highly dynamic, changing its morphology as the parasite undergoes egress and invasion. Recently, we have demonstrated that mitochondrial morphology is driven by a protein named Lasso Maintenance Factor 1 (LMF1). This protein interacts with IMC10, a protein present at the parasites inner membrane complex (IMC), mediating a unique membrane contact site between the IMC and mitochondrion. Interestingly, parasites lacking either LMF1 or IMC10 have abnormal mitochondrial morphology, cell division defects, and delayed propagation in tissue culture. Although both components of the tether were identified, the functions of this contact site remain unknown. In this work, we show that {Delta}lmf1 parasites exhibit upregulation of egress signaling and downregulation in folate metabolism and pantothenate biosynthesis. {Delta}lmf1 parasites exhibit increased intracellular calcium levels, leading to greater sensitivity to ionophore-induced egress and microneme secretion. We have confirmed that parasites have decreased levels of tetrahydrofolate and coenzyme A, showing a limitation in cofactor production. Interestingly, the {Delta}lmf1 parasites prefer glutamine instead of glucose as a catabolic substrate. Accordingly, we demonstrate for the first time that proper mitochondrial positioning is crucial for folate and Coenzyme A metabolism as well as egress signaling. IMPORTANCEToxoplasma gondii is the causative agent of Toxoplasmosis, a disease that affects a third of the worlds population. This parasite has a single, highly dynamic mitochondrion. The parasites mitochondrion changes shape depending on environmental conditions (inside or outside the host cell) or on stressors, such as drugs. Our laboratory characterized the proteins involved in regulating mitochondrial dynamics in the parasite, but the functional importance of these mitochondrial changes has not yet been described. Here, we show that the shape of Toxoplasmas mitochondrion is important for the synthesis of key cofactors, such as folates and coenzyme A. We show that mitochondrial shape in this parasite is important for signaling the parasites exit from the host cell, a critical process in its life cycle. These findings review a previously unknown function of a parasite-specific organelle contact site, providing new insights into the importance of mitochondria for these parasites.

African swine fever (ASF) is a highly pathogenic disease caused by the African swine fever virus (ASFV) infection, which can affect pigs of all ages and breeds, posing significant threat to the global pig farming industry. The ASFV p30 protein is an early-expressed viral structural protein; however, its function is not fully understood. In this study, the interaction of viral p30 with host TRIM21 was identified. The ectopic TRIM21 inhibited ASFV replication, while knockdown or knockout of TRIM21 promoted ASFV replication. Further, p30 was found to interact with RIG-I-like receptor (RLR) signaling adaptor MAVS, and during ASFV infection, p30-TRIM21-MAVS interacted with each other. Mechanistically, TRIM21 activated the K27 polyubiquitination of MAVS to induce IRF3 mediated type I interferon (IFN) production, whereas p30 counteracted TRIM21 activated MAVS K27 polyubiquitination to evade RLR signaling mediated antiviral IFN induction. In summary, our study revealed a novel function of ASFV p30, and provided new insights into the immune evasion of ASFV.

In Toxoplasma gondii, microneme proteins (MICs) are secreted components of the apical complex that play central roles in motility, host cell attachment, and invasion. Because proteins at the host-parasite interface are often predicted to evolve rapidly, MICs have been suggested as candidates for adaptive diversification. We tested this expectation using comparative analyses of three relatively understudied microneme proteins, MIC13, MIC12, and MIC16. Coding sequences were assembled from GenBank and ToxoDB, aligned by translation, and analyzed using maximum-likelihood phylogenetics, codon-based tests of selection, and predicted protein structure. MIC13 was represented by 51 sequences, MIC12 by 30, and MIC16 by 34, spanning multiple T. gondii haplogroups and including Hammondia hammondi and Neospora caninum as outgroups. All three genes were highly conserved among T. gondii strains, but their phylogenetic trees were topologically incongruent, indicating that individual MICs do not recover a single shared strain history. Contrary to expectation, no positively selected codons were detected in any gene. Instead, purifying selection was detected at one site in MIC13 and 15 sites in MIC12, while no significant codon-specific selection was detected in MIC16. Several constrained MIC12 sites overlapped annotated EGF and calcium-binding EGF-like domains, consistent with structural conservation of extracellular adhesion modules. AlphaFold prediction of MIC13 supported two sialic acid-binding micronemal adhesive repeat regions, but the single constrained MIC13 site did not overlap these motifs. Together, these results indicate that MIC13, MIC12, and MIC16 are shaped more by sequence conservation and heterogeneous gene histories than by strong recurrent positive selection. These findings refine expectations for microneme evolution in T. gondii and highlight conserved domains that may be important for parasite invasion and future functional study.

Endosymbiotic bacteria such as Wolbachia pose significant challenges to genetic and molecular investigation due to their obligate intracellular lifestyle and complex growth requirements.Current understanding of their protein biology relies heavily on functional assignments inferred by homology, which may not reflect the specific roles endosymbiont proteins play within the host. This work addresses the need for robust genetic perturbation by demonstrating the successful application and detection of chemical mutagenesis in the genome of the wMel strain of Wolbachia grown within a stably infected Drosophila melanogaster JW18 cell line. To accurately detect EMS-induced mutations in a large, unsorted cell culture population, in which mutations remain at very low allele frequency, we implemented an ultra-low error rate sequencing strategy, circle sequencing. This technique enables confident detection of EMS-induced single nucleotide polymorphisms (SNPs) that would be swamped by the inherent error rates of standard next-generation sequencing. Circle sequencing library preparations successfully revealed a clear EMS mutation signal in treated cells, characterized by a significant enrichment of canonical C/G>T/A transitions. Furthermore we present a model explaining observed EMS mutation rates across the genome for different sequence contexts. These findings show that EMS-treatment can successfully leave detectable mutation signals in intracellular genomes, and offer promise for the future development of protocols to make targeted edits in Wolbachia genomes. ImportanceAs the use of intracellular symbionts for bioengineering projects grows, so does the need for foundational protocols for the genetic manipulation of intracellular genomes. Ethyl methanesulfonate (EMS), a chemical mutagen, has been a research tool for initial genomic analysis of gene function in plant and animal systems for decades and represents an established way of generating mutations for future functional testing.

Cryptosporidium spp. are protozoan parasites responsible for diarrheal diseases. In humans, cryptosporidiosis is predominantly caused by the human-specific Cryptosporidium hominis and by Cryptosporidium parvum. This second species has been classically reported as zoonotic, with a host preference for ruminants. However, the recently described subspecies C. parvum anthroponosum has been found to be restricted to humans. Here, we generated novel whole genome sequences from West African samples of C. p. anthroponosum, and analyzed them together with all those already available, originating from East Africa, Europe, North America and Asia. Phylogenomics showed that all C. p. anthroponosum isolates are strongly clustered together, forming the sister clade of the zoonotic C. parvum representatives. The phylogenetic variations within C. p. anthroponosum did not present a clear geographic structure, consistent with C. hominis, primarily transmitted in humans. To elucidate the evolution of host species adaptation in C. p. anthroponosum, we then investigated genetic exchanges with C. hominis, detecting an ancestral introgression present in all C. p. anthroponosum isolates. This introgression involved a single gene, encoding for an extracellular galectin-like protein, which we predicted with high confidence to form a protein complex with the human insulin-degrading enzyme, a key metabolic regulator. Considering the role of host insulin metabolism in the proliferation of parasites as well as its known intrinsic differences between humans and ruminants, this molecular interaction could represent a plausible mechanism for an important role of the galectin-like protein in host-parasite interactions and in the host specificity of C. p. anthroponosum.

Bovine viral diarrhea virus (BVDV, genus Pestivirus, family Flaviviridae) is a notifiable pathogen of cattle which significantly impacts animal health, welfare, and the economy. Several cellular factors important for BVDV infection, such as Jiv, CD46 and ADAM17, have already been identified providing new targets development of effective defense strategies. However, our knowledge about BVDV host factor requirements remains limited, as no genome-wide studies of BVDV host resistance factors were performed to date, in part due to lack of accessible whole genome libraries. To close this gap, we have designed a novel bovine whole genome knockout library and successfully used it to identify a set of BVDV host resistance factors. The validity of our approach is highlighted by the strong selection of cells with inactivated ADAM17 and TMEM41B, which have both been described to be of pivotal importance for BVDV infection. In addition, guides targeting VMP1, recently identified as an important factor for flavivirus infection, were also significantly enriched in our screen. Furthermore, we found differential selection of several proteins essential for triggering autophagy, providing additional strong evidence of this process underlying key cellular functions involved in resistance to BVDV.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important zoonotic pathogen that disrupts intestinal epithelial barrier integrity and induces excessive inflammatory responses, thereby leading to impaired growth performance and intestinal injury. EHEC is also an important cause Hemolytic Uremic Syndrome (HUS) in children and older adults. In pig production, chitosan is considered a promising alternative to antibiotics due to its bioadhesive and antimicrobial properties, but the effects and underlying mechanisms of chitosan (COS) under pathogenic challenge remain to be elucidated. One hundred and eight pigs were randomly divided into three treatments: an unchallenged control group (CON), an EHEC-challenged control group (ECON), and an EHEC-challenged group supplemented with 100 mg/kg COS (ECOS). Results show that EHEC challenge increased the feed conversion ratio (FCR), increased inflammatory cytokine levels, disrupted intestinal morphology, and downregulated tight junction and nutrient transporter gene expression (P<0.05). Dietary COS supplementation significantly improved average daily gain (ADG) and FCR during day 6-14 (P<0.05). Moreover, COS reduced fecal shedding of total E. coli (P = 0.085) and EHEC, attenuated systemic inflammation by decreasing serum TNF- and IL-6 levels, and enhanced humoral immunity as indicated by increased IgA and IgM concentrations (P<0.05). Importantly, COS alleviated EHEC-induced intestinal injury by restoring villus height and villus-to-crypt ratio, with enhanced mucosal digestive enzyme activities, and upregulated expression of tight junction proteins (ZO-1 and occludin) and nutrient transporters (SGLT-1 and PEPT1) (P<0.05). In conclusion, these findings indicate that dietary COS improves growth performance in EHEC-challenged weaned pigs, with enhanced intestinal barrier integrity and nutrient transport capacity.