Flow Cytometry and Automated Microscopy Integration for Phagocytic Analysis of Hypervirulent Klebsiella pneumoniae in Dictyostelium discoideum.

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

Helicobacter pylori PldA modulates TNFR1-mediated p38 signaling pathways to regulate macrophage responses for its survival.

Helicobacter pylori, a dominant member of the gastric microbiota was associated with various gastrointestinal diseases and presents a significant challenge due to increasing antibiotic resistance. This study identifies H. pylori's phospholipase A (PldA) as a critical factor in modulating host macrophage responses, facilitating H. pylori 's evasion of the immune system and persistence. PldA alters membrane lipids through reversible acylation and deacylation, affecting their structure and function. We found that PldA incorporates lysophosphatidylethanolamine into macrophage membranes, disrupting their bilayer structure and impairing TNFR1-mediated p38-MK2 signaling. This disruption results in reduced macrophage autophagy and elevated RIP1-dependent apoptosis, thereby enhancing H. pylori survival, a mechanism also observed in multidrug-resistant strains. Pharmacological inhibition of PldA significantly decreases H. pylori viability and increases macrophage survival. In vivo studies corroborate PldA's essential role in H. pylori persistence and immune cell recruitment. Our findings position PldA as a pivotal element in H. pylori pathogenesis through TNFR1-mediated membrane modulation, offering a promising therapeutic target to counteract bacterial resistance.

Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213.

Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213's dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.

Neisseria meningitidis activates pyroptotic pathways in a mouse model of meningitis: role of a two-partner secretion system.

There is evidence that in infected cells in vitro the meningococcal HrpA/HrpB two-partner secretion system (TPS) mediates the exit of bacteria from the internalization vacuole and the docking of bacteria to the dynein motor resulting in the induction of pyroptosis. In this study we set out to study the role of the HrpA/HrpB TPS in establishing meningitis and activating pyroptotic pathways in an animal model of meningitis using a reference serogroup C meningococcal strain, 93/4286, and an isogenic hrpB knockout mutant, 93/4286ΩhrpB. Survival experiments confirmed the role of HrpA/HrpB TPS in the invasive meningococcal disease. In fact, the ability of the hrpB mutant to replicate in brain and spread systemically was impaired in mice infected with hrpB mutant. Furthermore, western blot analysis of brain samples during the infection demonstrated that: i. N. meningitidis activated canonical and non-canonical inflammasome pyroptosis pathways in the mouse brain; ii. the activation of caspase-11, caspase-1, and gasdermin-D was markedly reduced in the hrpB mutant; iii. the increase in the amount of IL-1ß and IL-18, which are an important end point of pyroptosis, occurs in the brains of mice infected with the wild-type strain 93/4286 and is strongly reduced in those infected with 93/4286ΩhrpB. In particular, the activation of caspase 11, which is triggered by cytosolic lipopolysaccharide, indicates that during meningococcal infection pyroptosis is induced by intracellular infection after the exit of the bacteria from the internalizing vacuole, a process that is hindered in the hrpB mutant. Overall, these results confirm, in an animal model, that the HrpA/HrpB TPS plays a role in the induction of pyroptosis and suggest a pivotal involvement of pyroptosis in invasive meningococcal disease, paving the way for the use of pyroptosis inhibitors in the adjuvant therapy of the disease.

Variability of the Microbiota in Chronic Rhinosinusitis: A Scoping Review.

Objective: Chronic rhinosinusitis (CRS) is characterized by a persistent inflammation of the nasal and paranasal sinus mucosa that could be potentially linked to a dysregulation between the microbiota and the immune system. We aim to explore general, methodological, and microbiological aspects of microbiota research in CRS compared to disease-free individuals. Data Sources: Embase, Ovid MEDLINE, PubMed, Scopus, and Web of Science. Review Methods: All studies comparing the composition of the resident microbiota of the sinonasal cavities in 2 groups: CRS and normal participants. We conducted systematic study selection, data extraction, and analysis first using the title and abstract, and then the full texts based on predefined inclusion and exclusion criteria. Compiled and presented findings include sampling site and technique, and microbiological results such as the relative abundance and the variability of the composition of the microbiota in both groups. Results: Twenty-seven studies, using genomic identification with 16s RNA were analyzed. Case definitions primarily followed EPOS or AAO-HNS guidelines, with endoscopic swabs (82%), and middle meatus sampling (74%) being prevalent techniques. Despite relative abundance variability, patterns emerged across studies, indicating an increase in Haemophilus (19%) and Pseudomonas (11%), and decrease in Propionibacterium (15%) and Anaerococcus (11%). Another pattern was observed, showing a decreased alpha diversity (6/19; 22%) in CRS compared to normal individuals. Conclusion: While variations exist among studies, analysis of CRS microbiota suggests an association with dysbiosis, potentially contributing to chronic inflammation. Future research must prioritize standardized criteria for diagnostics and patient selection, fostering a more comprehensive understanding of CRS microbiota.

Transcriptional reprogramming in the entomopathogenic fungus Metarhizium brunneum and its aphid host Myzus persicae during the switch between saprophytic and parasitic lifestyles.

BACKGROUND: The fungus Metarhizium brunneum has evolved a remarkable ability to switch between different lifestyles. It develops as a saprophyte, an endophyte establishing mutualistic relationships with plants, or a parasite, enabling its use for the control of insect pests such as the aphid Myzus persicae. We tested our hypothesis that switches between lifestyles must be accompanied by fundamental transcriptional reprogramming, reflecting adaptations to different environmental settings. RESULTS: We combined high throughput RNA sequencing of M. brunneum in vitro and at different stages of pathogenesis to validate the modulation of genes in the fungus and its host during the course of infection. In agreement with our hypothesis, we observed transcriptional reprogramming in M. brunneum following conidial attachment, germination on the cuticle, and early-stage growth within the host. This involved the upregulation of genes encoding degrading enzymes and gene clusters involved in synthesis of secondary metabolites that act as virulence factors. The transcriptional response of the aphid host included the upregulation of genes potentially involved in antifungal activity, but antifungal peptides were not induced. We also observed the induction of a host flightin gene, which may be involved in wing formation and flight muscle development. CONCLUSIONS: The switch from saprophytic to parasitic development in M. brunneum is accompanied by fundamental transcriptional reprogramming during the course of the infection. The aphid host responds to fungal infection with its own transcriptional reprogramming, reflecting its inability to express antifungal peptides but featuring the induction of genes involved in winged morphs that may enable offspring to avoid the contaminated environment.

Viral- and fungal-mediated behavioral manipulation of hosts: summit disease.

Summit disease, in which infected hosts seek heights (gravitropism), first noted in modern times by nineteenth-century naturalists, has been shown to be induced by disparate pathogens ranging from viruses to fungi. Infection results in dramatic changes in normal activity patterns, and such parasite manipulation of host behaviors suggests a strong selection for convergent outcomes albeit evolved via widely divergent mechanisms. The two best-studied examples involve a subset of viral and fungal pathogens of insects that induce "summiting" in infected hosts. Summiting presumably functions as a means for increasing the dispersal of the pathogen, thus significantly increasing fitness. Here, we review current advances in our understanding of viral- and fungal-induced summit disease and the host behavioral manipulation involved. Viral genes implicated in this process include a host hormone-targeting ecdysteroid UDP-glucosyltransferase (apparently essential for mediating summit disease induced by some viruses but not all) and a protein tyrosine phosphatase, with light dependance implicated. For summit disease-causing fungi, though much remains obscure, targeting of molting, circadian rhythms, sleep, and responses to light patterns appear involved. Targeting of host neuronal pathways by summit-inducing fungi also appears to involve the production of effector molecules and secondary metabolites that affect host muscular, immune, and/or neurological processes. It is hypothesized that host brain structures, particularly Mushroom Bodies (no relation to the fungus itself), important for olfactory association learning and control of locomotor activity, are critical targets for mediating summiting during infection. This phenomenon expands the diversity of microbial pathogen-interactions and host dynamics. KEY POINTS: • Summit disease or height seeking (gravitropism) results from viral and fungal pathogens manipulating insect host behaviors presumably to increase pathogen dispersal. • Insect baculoviruses and select fungal pathogens exhibit convergent evolution in host behavioral manipulation but use disparate molecular mechanisms. • Targets for affecting host behavior include manipulation of host hormones, feeding, locomotion, and immune, circadian, and neurological pathways.

Parasites and Microbiota: Dual Interactions and Therapeutic Perspectives.

The human gut hosts a diverse and active community of bacteria that symbiotically support the physiology, metabolism, and immunity of the intestinal lining. Nevertheless, a dynamic community of parasites (helminths and protozoa) may share a habitat with gut-dwelling microbiota. Both microbiota and parasites can significantly change the physical and immunological environment of the gut, thus generating several mechanisms of interaction. Studying this field is crucial for understanding the pathogenesis of parasitic diseases. Additionally, intestinal microbiota and gut-dwelling parasites may interact with each other and with the host immunity to alleviate or exacerbate the disease. These interactions can alter the pathogenicity of both parasites and microbiota, thereby changing the infection outcomes and the overall disease profile. Parasites and microbiota interactions occur via several mechanisms, including physical alteration in both the gastrointestinal microenvironment and the adaptive and innate immune responses. By modulating the microbiota, treating parasitic infections and microbiota dysbiosis may be improved through knowing the mechanisms and consequences of the interactions between intestinal parasites and the microbiota. Thus, new biological tools of treatment including probiotics can be introduced, particularly with the emergence of drug resistance and adverse effects.

A predictive approach for host-pathogen interactions using deep learning and protein sequences.

Research on host-pathogen interactions (HPIs) has evolved rapidly during the past decades. The more humans discover new pathogens, the more challenging it gets to find a cure and prevent infections caused by those pathogens. Many experimental techniques have been proposed to predict the interactions but most of them are highly costly and time-consuming. Fortunately, computational methods have been proven to be efficient in overcoming such limitations. In this study, we propose utilizing Deep Learning methods to predict HPIs using protein sequences. We use the monoMonoKGap (mMKGap) algorithm with K = 2 to extract features from the sequences. We also used the Negatome Database to generate negative interactions. The proposed method was performed on three separate balanced human-pathogen datasets with 10-fold cross-validation. Our method yielded very high accuracies of 99.65%, 99.52%, and 99.66% (mean accuracy of 99.61%). To further evaluate the performance of the deep Network, we compared it with other classification methods, which were the Random Forest (RF) as multiple Decision Tree, the Support Vector Machine (SVM), and Convolutional Neural Network (CNN). We also tested the Dipeptide Composition algorithm as another feature extraction method to compare the results with the mMKGap method. The experimental results prove that the proposed method is very accurate, robust, and practical and could be used as a reliable framework in HPI research.

A human lectin array for characterizing host-pathogen interactions.

A human lectin array has been developed to probe the interactions of innate immune receptors with pathogenic and commensal micro-organisms. Following the successful introduction of a lectin array containing all of the cow C-type carbohydrate-recognition domains (CRDs), a human array described here contains the C-type CRDs as well as CRDs from other classes of sugar-binding receptors, including galectins, siglecs, R-type CRDs, ficolins, intelectins and chitinase-like lectins. The array is constructed with CRDs modified with single-site biotin tags, ensuring that the sugar-binding sites in CRDs are displayed on a streptavidin-coated surface in a defined orientation and are accessible to the surfaces of microbes. A common approach used for expression and display of CRDs from all of the different structural categories of glycan-binding receptors allows comparisons across lectin families. In addition to previously documented protocols for binding of fluorescently-labeled bacteria, methods have been developed for detecting unlabeled bacteria bound to the array by counter-staining with DNA-binding dye. Screening has also been undertaken with viral glycoproteins and bacterial and fungal polysaccharides. The array provides an unbiased screen for sugar ligands that interact with receptors and many show binding not anticipated from earlier studies. For example, some of the galectins bind with high affinity to bacterial glycans that lack lactose or N-acetyllactosamine. The results demonstrate the utility of the human lectin array for providing a unique overview of the interactions of multiple classes of glycan-binding proteins in the innate immune system with different types of micro-organisms.

The battle within: how Pseudomonas aeruginosa uses host-pathogen interactions to infect the human lung.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen known for its ability to invade the respiratory tract, particularly in cystic fibrosis patients. This review provides a comprehensive analysis of the multifaceted strategies for colonization, virulence, and immune evasion used by P. aeruginosa to infect the host. We explore the extensive protein arsenal of P. aeruginosa, including adhesins, exotoxins, secreted proteases, and type III and VI secretion effectors, detailing their roles in the infective process. We also address the unique challenge of treating diverse lung conditions that provide a natural niche for P. aeruginosa on the airway surface, with a particular focus in cystic fibrosis. The review also discusses the current limitations in treatment options due to antibiotic resistance and highlights promising future approaches that target host-pathogen protein-protein interactions. These approaches include the development of new antimicrobials, anti-attachment therapies, and quorum-sensing inhibition molecules. In summary, this review aims to provide a holistic understanding of the pathogenesis of P. aeruginosa in the respiratory system, offering insights into the underlying molecular mechanisms and potential therapeutic interventions.

Coevolution promotes the coexistence of Tasmanian devils and a fatal, transmissible cancer.

Emerging infectious diseases threaten natural populations, and data-driven modeling is critical for predicting population dynamics. Despite the importance of integrating ecology and evolution in models of host-pathogen dynamics, there are few wild populations for which long-term ecological datasets have been coupled with genome-scale data. Tasmanian devil (Sarcophilus harrisii ) populations have declined range-wide due to devil facial tumor disease (DFTD), a fatal transmissible cancer. Although early ecological models predicted imminent devil extinction, diseased devil populations persist at low densities, and recent ecological models predict long-term devil persistence. Substantial evidence supports evolution of both devils and DFTD, suggesting coevolution may also influence continued devil persistence. Thus, we developed an individual-based, eco-evolutionary model of devil-DFTD coevolution parameterized with nearly two decades of devil demography, DFTD epidemiology, and genome-wide association studies. We characterized potential devil-DFTD coevolutionary outcomes and predicted the effects of coevolution on devil persistence and devil-DFTD coexistence. We found a high probability of devil persistence over 50 devil generations (100 years) and a higher likelihood of devil-DFTD coexistence, with greater devil recovery, than predicted by previous ecological models. These novel results add to growing evidence for long-term devil persistence and highlight the importance of eco-evolutionary modeling for emerging infectious diseases.

mSphere of Influence: Revisiting the central dogma, again!

Dr. Parimal Samir works in the field of host-pathogen interactions. In this mSphere of Influence article, he reflects on how the manuscript entitled "De novo gene synthesis by an antiviral reverse transcriptase" by Samuel Sternberg and colleagues made an impact by reminding him that there is still so much to discover in life sciences.

Extracellular vesicles in parasitic protozoa: Impact of Leishmania exosomes containing Leishmania RNA virus 1 (LRV1) on Leishmania infectivity and disease progression.

This chapter focuses on the interplay between Leishmania parasites and their host, particularly on Leishmania RNA virus (LRVs) and extracellular vesicles (EVs) in modulating host-pathogen interactions. Leishmania EVs have been shown to facilitate gene transfer, including drug-resistance genes, enhancing the parasites' survival and resistance to antileishmanial therapeutics. These EVs also play a significant role in host immune modulation by altering cytokine production in macrophages and promoting an anti-inflammatory environment that favours parasitic persistence. The presence of virulence factors such as GP63 within these EVs further underscores their role in the parasite's immunopathogenesis. Over the last few decades, LRVs have been established as drivers of the severity and persistence of leishmaniasis by exacerbating inflammatory responses and potentially influencing treatment outcomes. This chapter discusses the evolutionary origins and classification of these viruses, and explores their role in parasitic pathogenicity, highlighting their ubiquity across protozoan parasites and their impact on disease progression.

Diverse molecular mechanisms underpinning Staphylococcus aureus small colony variants.

Small colony variants (SCVs) of Staphylococcus aureus are a relatively rare but clinically significant growth morphotype. Infections with SCVs are frequently difficult to treat, inherently antibiotic-resistant, and can lead to persistent infections. Despite a long history of research, the molecular underpinnings of this morphotype and their impact on the clinical trajectory of infections remain unclear. However, a growing body of literature indicates that SCVs are caused by a diverse range of molecular factors. These recent findings suggest that SCVs should be thought of as an ensemble collection of loosely related phenotypes, and not as a single phenomenon. This review describes the diverse mechanisms currently known to contribute to SCVs and proposes an ensemble model for conceptualizing this morphotype.

Juvenile agile frogs spatially avoid ranavirus-infected conspecifics.

Exposure to contagious pathogens can result in behavioural changes, which can alter the spread of infectious diseases. Healthy individuals can express generalized social distancing or avoid the sources of infection, while infected individuals can show passive or active self-isolation. Amphibians are globally threatened by contagious diseases, yet their behavioural responses to infections are scarcely known. We studied behavioural changes in agile frog (Rana dalmatina) juveniles upon exposure to a Ranavirus (Rv) using classic choice tests. We found that both non-infected and Rv-infected focal individuals spatially avoided infected conspecifics, while there were no signs of generalized social distancing, nor self-isolation. Avoidance of infected conspecifics may effectively hinder disease transmission, protecting non-infected individuals as well as preventing secondary infections in already infected individuals. On the other hand, the absence of self-isolation by infected individuals may facilitate it. Since infection status did not affect the time spent near conspecifics, it is unlikely that the pathogen manipulated host behaviour. More research is urgently needed to understand under what circumstances behavioural responses can help amphibians cope with infections, and how that affects disease dynamics in natural populations.

Pangolin scales as adaptations for innate immunity against pathogens.

BACKGROUND: Pangolins are the only mammals that have overlapping scales covering most of their bodies, and they play a crucial role in the ecosystem, biological research, and human health and disease. Previous studies indicated pangolin scale might provide an important mechanical defense to themselves. The origin and exact functions of this unique trait remain a mystery. Using a multi-omics analysis approach, we report a novel functional explanation for how mammalian scales can provide host-pathogen defense. RESULTS: Our data suggest that pangolin scales have a sophisticated structure that could potentially trap pathogens. We identified numerous proteins and metabolites exhibiting antimicrobial activity, which could suggest a role for scales in pathogen defense. Notably, we found evidence suggesting the presence of exosomes derived from diverse cellular origins, including mesenchymal stem cells, immune cells, and keratinocytes. This observation suggests a complex interplay where various cell types may contribute to the release of exosomes and antimicrobial compounds at the interface between scales and viable tissue. These findings indicate that pangolin scales may serve as a multifaceted defense system, potentially contributing to innate immunity. Comparisons with human nail and hair revealed pangolin-specific proteins that were enriched in functions relating to sensing, immune responses, neutrophil degranulation, and stress responses. We demonstrated the antimicrobial activity of key pangolin scale components on pathogenic bacteria by antimicrobial assays. CONCLUSIONS: This study identifies a potential role of pangolin scales and implicates scales, as possible determinants of pathogen defense due to their structure and contents. We indicate for the first time the presence of exosomes in pangolin scales and propose the new functions of scales and their mechanisms. This new mechanism could have implications for multiple fields, including providing interesting new research directions and important insights that can be useful for synthesizing and implementing new biomimetic antimicrobial approaches.

Insights into ubiquitinome dynamics in the host‒pathogen interplay during Francisella novicida infection.

Ubiquitination functions as an important posttranslational modification for orchestrating inflammatory immune responses and cell death during pathogenic infection. The ubiquitination machinery is a major target hijacked by pathogenic bacteria to promote their survival and proliferation. Type I interferon (IFN-I) plays detrimental roles in host defense against Francisella novicida (F. novicida) infection. The effects of IFN-I on the ubiquitination of host proteins during F. novicida infection remain unclear. Herein, we delineate the dynamic ubiquitinome alterations in both wild-type (WT) and interferon-alpha receptor-deficient (Ifnar-/-) primary bone marrow-derived macrophages (BMDMs) during F. novicida infection. Using diGly proteomics and stable isotope labeling (SILAC), we quantified ubiquitination sites in proteins from primary WT and Ifnar-/- BMDMs with and without F. novicida infection. Our mass spectrometry analysis identified 2,491 ubiquitination sites in 1,077 endogenous proteins. Our study revealed that F. novicida infection induces dynamic changes in the ubiquitination of proteins involved in the cell death, phagocytosis, and inflammatory response pathways. IFN-I signaling is essential for both the increase and reduction in ubiquitination in response to F. novicida infection. We identified IFN-I-dependent ubiquitination in proteins involved in glycolysis and vesicle transport processes and highlighted key hub proteins modified by ubiquitination within cell death pathways. These findings underscore the significant influence of IFN-I signaling on modulating ubiquitination during F. novicida infection and provide valuable insights into the complex interplay between the host and F. novicida.

From fat storage to immune hubs: the emerging role of adipocytes in coordinating the immune response to infection.

Adipose tissue is a rich source of diverse cell populations, including immune cells, adipocytes and stromal cells. Interactions between these different cell types are now appreciated to be critical for maintaining tissue structure and function, by governing processes such as adipogenesis, lipolysis and differentiation of white to beige adipocytes. Interactions between these cells also drive inflammation in obesity, leading to an expansion of adipose tissue immune cells, and the secretion of proinflammatory cytokines from immune cells and from adipocytes themselves. However, in evolutionary terms, obesity is a recent phenomenon, raising the question of why adipocytes evolved to express factors that influence the immune response. Studies of various pathogens indicate that adipocytes are highly responsive to infection, altering their metabolic profiles in a way that can be used to release nutrients and fuel the immune response. In the case of infection with the extracellular parasite Trypanosoma brucei, attenuating the ability of adipocytes to sense the cytokine IL-17 results in a loss of control of the local immune response and an increased pathogen load. Intriguingly, comparisons of the adipocyte response to infection suggest that the immune responses of these cells occur in a pathogen-dependent manner, further confirming their complexity. Here, with a focus on murine adipose tissue, we discuss the emerging concept that, in addition to their canonical function, adipocytes are immune signalling hubs that integrate and disseminate signals from the immune system to generate a local environment conducive to pathogen clearance.