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Toxoplasma gondii is a successful parasite capable of infecting a wide range of warm-blooded animals, including people, livestock, and wildlife. In individuals with intact immune function, T. gondii can invade the host brain tissue by altering the blood-brain barrier permeability, leading to chronic infection. Proteins play crucial regulatory roles in disease progression. By monitoring changes in proteins, a deeper understanding of the molecular mechanisms underlying host resistance to infection and the potential pathogenic mechanisms of pathogens can be gained. This study analyzed differential protein expression and associated signaling pathways in mouse brain tissues during acute and chronic T. gondii infection using proteomic and bioinformatics methods. The results showed that during acute and chronic T. gondii infection stages, 74 and 498 differentially expressed proteins (DEPs) were identified in mouse brain tissue, respectively. Among them, 45 and 309 were up-regulated, while 29 and 189 were down-regulated. GO and KEGG analyses revealed that some of these DEPs were implicated in host immunity, pathogen immune evasion, and T. gondii invasion of the central nervous system, particularly interleukin production and secretion, complement system activation, and alterations in tight junction pathways. Notably, the upregulation of Rab13 was identified as a potential molecular mechanism for T. gondii to regulate blood-brain barrier permeability and facilitate central nervous system invasion. Our findings provided fundamental data for understanding host control of Toxoplasmosis infection and offered new insights into parasite immune evasion and invasion mechanisms within the central nervous system. These insights are crucial for developing strategies to prevent the establishment of chronic T. gondii infection.

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Recent advancements in stem cell biology and tissue engineering have revolutionized the field of neurodegeneration research by enabling the development of sophisticated in vitro human brain models. These models, including 2D monolayer cultures, 3D organoids, organ-on-chips, and bioengineered 3D tissue models, aim to recapitulate the cellular diversity, structural organization, and functional properties of the native human brain. This review highlights how these in vitro brain models have been used to investigate the effects of various pathogens, including viruses, bacteria, fungi, and parasites infection, particularly in the human brain cand their subsequent impacts on neurodegenerative diseases. Traditional studies have demonstrated the susceptibility of different 2D brain cell types to infection, elucidated the mechanisms underlying pathogen-induced neuroinflammation, and identified potential therapeutic targets. Therefore, current methodological improvement brought the technology of 3D models to overcome the challenges of 2D cells, such as the limited cellular diversity, incomplete microenvironment, and lack of morphological structures by highlighting the need for further technological advancements. This review underscored the significance of in vitro human brain cell from 2D monolayer to bioengineered 3D tissue model for elucidating the intricate dynamics for pathogen infection modeling. These in vitro human brain cell enabled researchers to unravel human specific mechanisms underlying various pathogen infections such as SARS-CoV-2 to alter blood-brain-barrier function and Toxoplasma gondii impacting neural cell morphology and its function. Ultimately, these in vitro human brain models hold promise as personalized platforms for development of drug compound, gene therapy, and vaccine. Overall, we discussed the recent progress in in vitro human brain models, their applications in studying pathogen infection-related neurodegeneration, and future directions.

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The fading efficacy of antibiotics is a growing global health concern due to its life-threatening consequences and increased healthcare costs. Non-genetic mechanisms of antimicrobial resistance, such as those employed by Chlamydia pneumoniae and Chlamydia trachomatis, complicate treatment as these bacteria can enter a non-replicative, persistent state under stress, evading antibiotics and linking to inflammatory conditions. Understanding chlamydial persistence at the molecular level is challenging, and new models for studying Chlamydia-host interactions in vivo are urgently needed. Caenorhabditis elegans offers an alternative given its immune system and numerous orthologues of human genes. This study established C. elegans as an in vivo model for chlamydial infection. Both Chlamydia species reduced the worm's lifespan, their DNA being detectable at three- and six-days post-infection. Azithromycin at its MIC (25 nM) failed to prevent the infection-induced lifespan reduction, indicating a persister phenotype. In contrast, the methanolic extract of Schisandra chinensis berries showed anti-chlamydial activity both in vitro (in THP-1 macrophages) and in vivo, significantly extending the lifespan of infected C. elegans and reducing the bacterial load. Moreover, S. chinensis increased the transcriptional activity of SKN-1 in the worms, but was unable to impact the bacterial load or lifespan in a sek-1 defective C. elegans strain. In summary, this study validated C. elegans as a chlamydial infection model and showcased S. chinensis berries' in vivo anti-chlamydial potential, possibly through SEK/SKN-1 signaling modulation.

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Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.

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Hematophagous female mosquitoes are important vectors of numerous devastating human diseases, posing a major public health threat. Effective prevention and control of mosquito-borne diseases rely considerably on progress in understanding the molecular mechanisms of various life activities, and accordingly, the molecules that regulate the various life activities of mosquitoes are potential targets for implementing future vector control strategies. Many long non-coding RNAs (lncRNAs) have been identified in mosquitoes and significant progress has been made in determining their functions. Here, we present a comprehensive overview of the research advances on mosquito lncRNAs, including their molecular identification, function, and interaction with other non-coding RNAs, as well as their synergistic regulatory roles in mosquito life activities. We also highlight the potential roles of competitive endogenous RNAs in mosquito growth and development, as well as in insecticide resistance and virus-host interactions. Insights into the biological functions and mechanisms of lncRNAs in mosquito life activities, viral replication, pathogenesis, and transmission will contribute to the development of novel drugs and safe vaccines.

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Trichophyton rubrum is the leading causative agent of dermatophytosis worldwide. Keratinocytes are the first line of defense that drives an immune response against fungal invasion. Host-specific pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) to trigger immunological pathways. Fungal cell wall components are the primary sources of fungal PAMPs, and some pathogens increase cell wall rearrangement to evade the immune system. Glycolysis and enhanced lactate levels are critical for improving host immune responses to fungal infections. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), we evaluated the transcriptional responses of human genes involved in fungal recognition and glycolytic metabolism and fungal cell-wall-related genes in a co-culture model of human keratinocytes with T. rubrum. We observed the upregulation of several Toll-like receptors (TLRs), NOD-like receptors (NLRs), and glycolytic genes. Complementarily, we measured intra- and extracellular glucose levels and the increase in lactate production in the co-culture supernatant. We noted a distinct transcriptional regulation pattern of fungal cell-wall-related genes from fungal growth on keratin as the primary carbon source compared to co-culture with human keratinocytes. Our results showed new insights into the transcriptional adaptation of keratinocytes, particularly in regulating genes involved in sensing and metabolic processes, during the interaction with T. rubrum.

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Streptococcus canis is a zoonotic agent that causes severe invasive diseases in domestic animals and humans, but little is known about its pathogenesis and virulence mechanisms so far. SCM, the M-like protein expressed by S. canis, is considered one of the major virulence determinants. Here, we report on the two distinct groups of SCM. SCM-1 proteins were already described to interact with its ligands IgG and plasminogen as well as with itself and confer antiphagocytic capability of SCM-1 expressing bacterial isolates. In contrast, the function of SCM-2 type remained unclear to date. Using whole-genome sequencing and subsequent bioinformatics, FACS analysis, fluorescence microscopy and surface plasmon resonance spectrometry, we demonstrate that, although different in amino acid sequence, a selection of diverse SCM-2-type S. canis isolates, phylogenetically representing the full breadth of SCM-2 sequences, were able to bind fibrinogen. Using targeted mutagenesis of an SCM-2 isolate, we further demonstrated that this strain was significantly less able to survive in canine blood. With respect to similar studies showing a correlation between fibrinogen binding and survival in whole blood, we hypothesize that SCM-2 has an important contribution to the pathogenesis of S. canis in the host.

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Bacteria frequently interfere with the post-translational modifications of host cells to facilitate their survival and growth after invasion. SUMOylation, a reversible post-translational modification process, plays an important role in biological life activities. In addition to being critical to host cell metabolism and survival, SUMOylation also regulates gene expression and cell signal transmission. Moreover, SUMOylation in eukaryotic cells can be used by a variety of bacterial pathogens to advance bacterial invasion. In this minireview, we focused on the role and mechanism of host SUMOylation in the pathogenesis of six important clinical bacterial pathogens (Listeria monocytogenes, Shigella flexneri, Salmonella Typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli). Taken together, this review provided new insights for understanding the unique pathogen-host interaction based on host SUMOylation and provided a novel perspective on the development of new strategies to combat bacterial infections in the future.

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Legionella are natural inhabitants of building plumbing biofilms, where interactions with other microorganisms influence their survival, proliferation, and death. Here, we investigated the associations of Legionella with bacterial and eukaryotic microbiomes in biofilm samples extracted from 85 shower hoses of a multiunit residential building. Legionella spp. relative abundance in the biofilms ranged between 0-7.8%, of which only 0-0.46% was L. pneumophila. Our data suggest that some microbiome members were associated with high (e.g. Chthonomonas, Vrihiamoeba) or low (e.g. Aquabacterium, Vannella) Legionella relative abundance. The correlations of the different Legionella variants (30 Zero-Radius OTUs detected) showed distinct patterns, suggesting separate ecological niches occupied by different Legionella species. This study provides insights into the ecology of Legionella with respect to: (i) the colonization of a high number of real shower hoses biofilm samples; (ii) the ecological meaning of associations between Legionella and co-occurring bacterial/eukaryotic organisms; (iii) critical points and future directions of microbial-interaction-based-ecological-investigations.

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How pathogens manipulate host UPRER to mediate immune evasion is largely unknown. Here, we identify the host zinc finger protein ZPR1 as an interacting partner of the enteropathogenic E. coli (EPEC) effector NleE using proximity-enabled protein crosslinking. We show that ZPR1 assembles via liquid-liquid phase separation (LLPS) in vitro and regulates CHOP-mediated UPRER at the transcriptional level. Interestingly, in vitro studies show that the ZPR1 binding ability with K63-ubiquitin chains, which promotes LLPS of ZPR1, is disrupted by NleE. Further analyses indicate that EPEC restricts host UPRER pathways at the transcription level in a NleE-ZPR1 cascade-dependent manner. Together, our study reveals the mechanism by which EPEC interferes with CHOP-UPRER via regulating ZPR1 to help pathogens escape host defense.

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Plants can be infected by multiple pathogens concurrently in natural systems. However, pathogen-pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae-soybean-Fusarium combinations, more than 80% aggravated disease. Different Fusarium species all enhanced P. sojae infection when co-inoculated on soybean. Treatment with Fusarium secreted non-proteinaceous metabolites had an effect equal to the direct pathogen co-inoculation. By screening a Fusarium graminearum mutant library, we identified Fusarium promoting factor of Phytophthora sojae infection 1 (Fpp1), encoding a zinc alcohol dehydrogenase. Fpp1 is functionally conserved in Fusarium and contributes to metabolite-mediated infection promotion, in which vitamin B6 (VB6) produced by Fusarium is key. Transcriptional and functional analyses revealed that Fpp1 regulates two VB6 metabolism genes, and VB6 suppresses expression of soybean disease resistance-related genes. These results reveal that co-infection with Fusarium promotes loss of P. sojae resistance in soybean, information that will inform the sustainable use of disease-resistant crop varieties and provide new strategies to control soybean root rot.

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Plasmodium ookinetes use an invasive apparatus to invade mosquito midguts, and tubulins are the major structural proteins of this apical complex. We examined the role of tubulins in malaria transmission to mosquitoes. Our results demonstrate that the rabbit polyclonal antibodies (pAb) against human α-tubulin significantly reduced the number of P. falciparum oocysts in Anopheles gambiae midguts, while rabbit pAb against human ß-tubulin did not. Further studies showed that pAb, specifically against P. falciparum α-tubulin-1, also significantly limited P. falciparum transmission to mosquitoes. We also generated mouse monoclonal antibodies (mAb) using recombinant P. falciparum α-tubulin-1. Out of 16 mAb, two mAb, A3 and A16, blocked P. falciparum transmission with EC50 of 12 µg/ml and 2.8 µg/ml. The epitopes of A3 and A16 were determined to be a conformational and linear sequence of EAREDLAALEKDYEE, respectively. To understand the mechanism of the antibody-blocking activity, we studied the accessibility of live ookinete α-tubulin-1 to antibodies and its interaction with mosquito midgut proteins. Immunofluorescent assays showed that pAb could bind to the apical complex of live ookinetes. Moreover, both ELISA and pull-down assays demonstrated that insect cell-expressed mosquito midgut protein, fibrinogen-related protein 1 (FREP1), interacts with P. falciparum α-tubulin-1. Since ookinete invasion is directional, we conclude that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 anchors and orients the ookinete invasive apparatus towards the midgut PM and promotes the efficient parasite infection in the mosquito.

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Anthracnose disease of cruciferous plants caused by Colletotrichum higginsianum is a serious fungal disease that affects cruciferous crops such as Chinese cabbage, Chinese flowering cabbage, broccoli, mustard plant, as well as the model plant Arabidopsis thaliana. Dual transcriptome analysis is commonly used to identify the potential mechanisms of interaction between host and pathogen. In order to identify differentially expressed genes (DEGs) in both the pathogen and host, the conidia of wild-type (ChWT) and Chatg8 mutant (Chatg8Δ) strains were inoculated onto leaves of A. thaliana, and the infected leaves of A. thaliana at 8, 22, 40, and 60 h post-inoculation (hpi) were subjected to dual RNA-seq analysis. The results showed that comparison of gene expression between the 'ChWT' and 'Chatg8Δ' samples detected 900 DEGs (306 upregulated and 594 down-regulated) at 8 hpi, 692 DEGs (283 upregulated and 409 down-regulated) at 22 hpi, 496 DEGs (220 upregulated and 276 down-regulated) at 40 hpi, and 3159 DEGs (1544 upregulated and 1615 down-regulated) at 60 hpi. GO and KEGG analyses found that the DEGs were mainly involved in fungal development, biosynthesis of secondary metabolites, plant-fungal interactions, and phytohormone signaling. The regulatory network of key genes annotated in the Pathogen-Host Interactions database (PHI-base) and Plant Resistance Genes database (PRGdb), as well as a number of key genes highly correlated with the 8, 22, 40, and 60 hpi, were identified during the infection. Among the key genes, the most significant enrichment was in the gene encoding the trihydroxynaphthalene reductase (THR1) in the melanin biosynthesis pathway. Both Chatg8Δ and Chthr1Δ strains showed varying degrees of reduction of melanin in appressoria and colonies. The pathogenicity of the Chthr1Δ strain was lost. In addition, six DEGs from C. higginsianum and six DEGs from A. thaliana were selected for real-time quantitative PCR (RT-qPCR) to confirm the RNA-seq results. The information gathered from this study enriches the resources available for research into the role of the gene ChATG8 during the infection of A. thaliana by C. higginsianum, such as potential links between melanin biosynthesis and autophagy, and the response of A. thaliana to different fungal strains, thereby providing a theoretical basis for the breeding of cruciferous green leaf vegetable cultivars with resistance to anthracnose disease.

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Phytoplasmas are uncultivable, phloem-limited, phytopathogenic bacteria that represent a major threat to agriculture worldwide. Phytoplasma membrane proteins are in direct contact with hosts and presumably play a crucial role in phytoplasma spread within the plant as well as by the insect vector. Three highly abundant types of immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). Although recent results indicate that Amp is involved in host specificity by interacting with host proteins such as actin, little is known about the pathogenicity of IDP in plants. In this study, we identified an antigenic membrane protein (Amp) of rice orange leaf phytoplasma (ROLP), which interacts with the actin of its vector. In addition, we generated Amp-transgenic lines of rice and expressed Amp in tobacco leaves by the potato virus X (PVX) expression system. Our results showed that the Amp of ROLP can induce the accumulation of ROLP and PVX in rice and tobacco plants, respectively. Although several studies have reported interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins, this example demonstrates that Amp protein can not only interact with the actin protein of its insect vector but can also directly inhibit host defense responses to promote the infection. The function of ROLP Amp provides new insights into the phytoplasma-host interaction.

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Motivation: The understanding of pathogen-host interactions (PHIs) is essential and challenging research because this potentially provides the mechanism of molecular interactions between different organisms. The experimental exploration of PHI is time-consuming and labor-intensive, and computational approaches are playing a crucial role in discovering new unknown PHIs between different organisms. Although it has been proposed that most machine learning (ML)-based methods predict PHI, these methods are all based on the structure-based information extracted from the sequence for prediction. The selection of feature values is critical to improving the performance of predicting PHI using ML. Results: This work proposed a new method to extract features from phylogenetic profiles as evolutionary information for predicting PHI. The performance of our approach is better than that of structure-based and ML-based PHI prediction methods. The five different extract models proposed by our approach combined with structure-based information significantly improved the performance of PHI, suggesting that combining phylogenetic profile features and structure-based methods could be applied to the exploration of PHI and discover new unknown biological relativity. Availability and implementation: The KPP method is implemented in the Java language and is available at https://github.com/yangfangs/KPP.

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COVID-19 is a highly transmissible disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), affects 226 countries and continents, and has resulted in >6.2 million deaths worldwide. Despite the efforts of all scientific institutions worldwide to identify potential therapeutics, no specific drug has been approved by the FDA to treat the COVID-19 patient. SARS-CoV-2 variants of concerns make the potential of publicly known therapeutics to respond to and detect disease onset highly improbable. The quest for universal therapeutics pointed to the ability of RNA-based molecules to shield and detect the adverse effects of the COVID-19 illness. One such candidate, miRNA (microRNA), works on regulating the differential expression of the target gene post-transcriptionally. The prime focus of this review is to report the critical miRNA molecule and their regular expression in patients with COVID-19 infection and associated comorbidities. Viral and host miRNAs control the etiology of COVID-19 infection throughout the life cycle and host inflammatory response, where host miRNAs are identified as a double-edged showing as a proviral and antiviral response. The review also covered the role of viral miRNAs in mediating host cell signaling expression during disease pathology. Studying molecular interactions between the host and the SARS-CoV-2 virus during COVID-19 pathogenesis offers the chance to use miRNA-based therapeutics to reduce the severity of the illness. By utilizing an appropriate delivery vehicle, these small non-coding RNA could be envisioned as a promising biomarker in designing a practical RNAi-based treatment approach of clinical significance.

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Biotrophic plant pathogenic fungi are widely distributed and are among the most damaging pathogenic organisms of agriculturally important crops responsible for significant losses in quality and yield. However, the pathogenesis of obligate parasitic pathogenic microorganisms is still under investigation because they cannot reproduce and complete their life cycle on an artificial medium. The successful lifestyle of biotrophic fungal pathogens depends on their ability to secrete effector proteins to manipulate or evade plant defense response. By integrating genomics, transcriptomics, and effectoromics, insights into how the adaptation of biotrophic plant fungal pathogens adapt to their host populations can be gained. Efficient tools to decipher the precise molecular mechanisms of rust-plant interactions, and standardized routines in genomics and functional pipelines have been established and will pave the way for comparative studies. Deciphering fungal pathogenesis not only allows us to better understand how fungal pathogens infect host plants but also provides valuable information for plant diseases control, including new strategies to prevent, delay, or inhibit fungal development. Our review provides a comprehensive overview of the efforts that have been made to decipher the effector proteins of biotrophic fungal pathogens and demonstrates how rapidly research in the field of obligate biotrophy has progressed.

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Mycobacterium tuberculosis (Mtb) bacilli are the causative agent of tuberculosis (TB), a major killer of mankind. Although it is widely accepted that local interactions between Mtb and the immune system in the tuberculous granuloma determine whether the outcome of infection is controlled or disseminated, these have been poorly studied due to methodological constraints. We have recently used a spatial transcriptomic technique, in situ sequencing (ISS), to define the spatial distribution of immune transcripts in TB mouse lungs. To further contribute to the understanding of the immune microenvironments of Mtb and their local diversity, we here present two complementary automated bacteria-guided analysis pipelines. These position 33 ISS-identified immune transcripts in relation to single bacteria and bacteria clusters. The analysis was applied on new ISS data from lung sections of Mtb-infected C57BL/6 and C3HeB/FeJ mice. In lungs from C57BL/6 mice early and late post infection, transcripts that define inflammatory macrophages were enriched at subcellular distances to bacteria, indicating the activation of infected macrophages. In contrast, expression patterns associated to antigen presentation were enriched in non-infected cells at 12 weeks post infection. T-cell transcripts were evenly distributed in the tissue. In Mtb-infected C3HeB/FeJ mice, transcripts characterizing activated macrophages localized in apposition to small bacteria clusters, but not in organized granulomas. Despite differences in the susceptibility to Mtb, the transcript patterns found around small bacteria clusters of C3HeB/FeJ and C57BL/6 mice were similar. Altogether, the presented tools allow us to characterize in depth the immune cell populations and their activation that interact with Mtb in the infected lung.

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Opportunistic pathogens belonging to the genus Legionella are among the most reported waterborne-associated pathogens in industrialized countries. Legionella colonize a variety of engineered aquatic ecosystems and persist in biofilms where they interact with a multitude of other resident microorganisms. In this review, we assess how some of these interactions could be used to develop a biological-driven "probiotic" control approach against Legionella. We focus on: (i) mechanisms limiting the ability of Legionella to establish and replicate within some of their natural protozoan hosts; (ii) exploitative and interference competitive interactions between Legionella and other microorganisms; and (iii) the potential of predatory bacteria and phages against Legionella. This field is still emergent, and we therefore specifically highlight research for future investigations, and propose perspectives on the feasibility and public acceptance of a potential probiotic approach.

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