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.

Human breast milk isolated lactic acid bacteria: antimicrobial and immunomodulatory activity on the Galleria mellonella burn wound model.

Introduction: Managing burn injuries is a challenge in healthcare. Due to the alarming increase in antibiotic resistance, new prophylactic and therapeutic strategies are being sought. This study aimed to evaluate the potential of live Lactic Acid Bacteria for managing burn infections, using Galleria mellonella larvae as an alternative preclinical animal model and comparing the outcomes with a common antibiotic. Methods: The antimicrobial activity of LAB isolated from human breast milk was assessed in vitro against Pseudomonas aeruginosa ATCC 27853. Additionally, the immunomodulatory effects of LAB were evaluated in vivo using the G. mellonella burn wound infection model. Results and discussion: In vitro results demonstrated the antimicrobial activity of Lactic Acid Bacteria against P. aeruginosa. In vivo results show that their prophylactic treatment improves, statistically significant, larval survival and modulates the expression of immunity-related genes, Gallerimycin and Relish/NF-κB, strain-dependently. These findings lay the foundation and suggest a promising alternative for burn wound prevention and management, reducing the risk of antibiotic resistance, enhancing immune modulation, and validating the potential G. mellonella as a skin burn wound model.

Mechanisms conferring bacterial cell wall variability and adaptivity.

The bacterial cell wall, a sophisticated and dynamic structure predominantly composed of peptidoglycan (PG), plays a pivotal role in bacterial survival and adaptation. Bacteria actively modify their cell walls by editing PG components in response to environmental challenges. Diverse variations in peptide composition, cross-linking patterns, and glycan strand structures empower bacteria to resist antibiotics, evade host immune detection, and adapt to dynamic environments. This review comprehensively summarizes the most common modifications reported to date and their associated adaptive role and further highlights how regulation of PG synthesis and turnover provides resilience to cell lysis.

Human rhinovirus 16 induces an ICAM-1-PKR-ATF2 axis to modulate macrophage functions.

Human rhinovirus (HRV) infections are the leading cause of disease exacerbations in individuals with chronic pulmonary diseases, primarily due to impaired macrophage functions, resulting in defective bacterial elimination. We previously demonstrated that HRV16 impairs macrophages' functions in an ARL5b-dependent manner. In permissive cells, ARL5b acted as an HRV16 restriction factor and was repressed. Here, we delve into the dual regulation of ARL5b by HRV16 in both cell types. We analyzed the effect of HRV16 on primary human macrophages using neutralizing antibodies, specific inhibitors, siRNA, and chromatin immune precipitation. Our study reveals that, while the virus does not replicate in macrophages, it induces interferon and pro-inflammatory responses. We identify the ICAM-1-PKR-ATF2 signaling axis as crucial for ARL5b induction in macrophages, whereas only ICAM-1 plays a role in ARL5b repression in permissive cells. Furthermore, HRV16 triggers epigenetic reprogramming in both cell types at the ARL5b promoter. In macrophages, epigenetic changes are ATF2 dependent. In conclusion, our findings highlight previously unknown signaling pathways activated by HRV16 in macrophages. Targeting these pathways could offer novel strategies to improve outcomes for individuals with respiratory conditions. IMPORTANCE: Human rhinovirus (HRV) infections are the leading cause of disease exacerbations in individuals with chronic pulmonary conditions and are frequently associated with bacterial superinfections due to defective bacterial elimination by macrophages. We previously identified ARL5b-induction by HRV16 to be responsible for the impairment of bacteria elimination. In contrast, in permissive cells, ARL5b is repressed and acts as a restriction factor for HRV16. Here, we investigated the dual regulation of ARL5b by HRV16 in these cells. Our study reveals that the ICAM-1-PKR-ATF2 signaling axis is crucial for ARL5b induction in macrophages. In permissive cells, only ICAM-1 plays a role in ARL5b repression. Moreover, HRV16 triggered epigenetic reprogramming in macrophages. ARL5b promoter was repressed in an ATF2-dependent manner. Collectively, our findings reveal previously unknown signaling pathways activated by HRV16 in macrophages. Targeting these pathways provides novel strategies to target ARL5b expression specifically in macrophages and improve outcomes for individuals with respiratory pathologies.

Microbiome transition mediated plant immune response to Alternaria solani (Ellis & Martin) Jones & Grout infection in tomato (Solanum lycopersicum L.).

Alternaria solani (Ellis & Martin) Jones & Grout, causing early blight infection in solanaceous crops, is a growing threat influencing sustainable crop production. Understanding the variation in the foliar microbiome, particularly the bacterial community during pathogenesis, can provide critical information on host-pathogen interactions, highlighting the host immune response during pathogen invasion. In the present study, early blight (EB) infection was artificially induced in tomato leaves, and the transition in the foliar bacterial community from healthy leaf tissue to infected leaves was analyzed. The 16s sequencing data revealed a significant shift in alpha and beta diversity, with infected leaf tissue exhibiting considerably lower bacterial abundance and diversity. Further interpretation at the genus level highlighted the possible role of the host immune system in recruiting higher nitrogen-fixing bacteria to resist the pathogen. The study, in addition to analyzing the foliar bacterial community transition during pathogenesis, has also shed light on the possible strategy employed by the host in recruiting selective nutrient-enriching microbes. Further application of this research in developing biocontrol agents with higher microbial host colonizing ability will be of tremendous benefit in achieving sustainable EB control measures.

Sensory Dysfunction, Microbial Infections, and Host Responses in Alzheimer's Disease.

Sensory functions of organs of the head and neck allow humans to interact with the environment and establish social bonds. With aging, smell, taste, vision, and hearing decline. Evidence suggests that accelerated impairment in sensory abilities can reflect a shift from healthy to pathological aging, including the development of Alzheimer's disease (AD) and other neurological disorders. While the drivers of early sensory alteration in AD are not elucidated, insults such as trauma and infections can affect sensory function. Herein, we review the involvement of the major head and neck sensory systems in AD, with emphasis on microbes exploiting sensory pathways to enter the brain (the "gateway" hypothesis) and the potential feedback loop by which sensory function may be impacted by central nervous system infection. We emphasize detection of sensory changes as first-line surveillance in senior adults to identify and remove potential insults, like microbial infections, that could precipitate brain pathology.

Subversion from Within and Without: Effector Molecule Transfer from Obligate Intracellular Apicomplexan Parasites to Human Host Cells.

Intracellular protozoan pathogens have to negotiate the internal environment of the host cell they find themselves in, as well as manipulate the host cell to ensure their own survival, replication, and dissemination. The transfer of key effector molecules from the pathogen to the host cell is crucial to this interaction and is technically more demanding to study as compared to an extracellular pathogen. While several effector molecules have been identified, the mechanisms and conditions underlying their transfer to the host cell remain partly or entirely unknown. Improvements in experimental systems have revealed tantalizing details of such intercellular transfer, which form the subject of this chapter.

In silico analysis and functional characterization of a leucine-rich repeat protein of Leptospira interrogans.

Pathogenic spirochetes of the genus Leptospira are the causative agent of leptospirosis, a widely disseminated zoonosis that affects humans and animals. The ability of leptospires to quickly cross host barriers causing infection is not yet fully understood. Thus, understanding the mechanisms of pathogenicity is important to combat leptospiral infection. Outer membrane proteins are interesting targets to study as they are able to interact with host molecules. Proteins containing leucine-rich repeat (LRR) domains are characterized by the presence of multiple regions containing leucine residues and they have putative functions related to host-pathogen interactions. Hence, the present study aimed to clone and express the recombinant protein encoded by the LIC11098 gene, an LRR protein of L. interrogans serovar Copenhageni. In silico analyses predicted that the target protein is conserved among pathogenic strains of Leptospira, having a signal peptide and multiple LRR domains. The DNA sequence encoding the LRR protein was cloned in frame into the pAE vector, expressed without mutations in Escherichia coli and purified by His-tag chromatography. Circular dichroism (CD) spectrum showed that the recombinant protein was predominantly composed of ß-sheets. A dose-dependent interaction was observed with cellular and plasma fibronectins, laminin and the complement system component C9, suggesting a possible role of the protein encoded by LIC11098 gene at the initial stages of infection.

Pathogenicity and virulence of chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-transmitted, RNA virus that causes an often-severe musculoskeletal illness characterized by fever, joint pain, and a range of debilitating symptoms. The virus has re-emerged as a global health threat in recent decades, spreading from its origin in Africa across Asia and the Americas, leading to widespread outbreaks impacting millions of people. Despite more than 50 years of research into the pathogenesis of CHIKV, there is still no curative treatment available. Current management of CHIKV infections primarily involves providing supportive care to alleviate symptoms and improve the patient's quality of life. Given the ongoing threat of CHIKV, there is an urgent need to better understand its pathogenesis. This understanding is crucial for deciphering the mechanisms underlying the disease and for developing effective strategies for both prevention and management. This review aims to provide a comprehensive overview of CHIKV and its pathogenesis, shedding light on the complex interactions of viral genetics, host factors, immune responses, and vector-related factors. By exploring these intricate connections, the review seeks to contribute to the knowledge base surrounding CHIKV, offering insights that may ultimately lead to more effective prevention and management strategies for this re-emerging global health threat.

Interplay between DOT1L and HDAC1 regulates Leishmania donovani infection in human THP-1 cells.

Leishmania donovani, a protozoan parasite, causes visceral leishmaniasis. The parasite modifies the global gene expressions of the host genome, facilitating its survival within the host. Thus, the host epigenetic modulators play important roles in host-pathogen interaction and host epigenetic modification in response to infection. Previously, we had reported that the host epigenetic modulator, histone deacetylase 1 (HDAC1) expression was upregulated on Leishmania donovani infection. This upregulation led to the repression of host defensin genes in response to the infection. In this paper, we have investigated the interplay between the host DOT1L, a histone methyltransferase, and HDAC1 in response to Leishmania donovani infection. We show that the expression of DOT1L is upregulated both at transcript and protein level following infection leading to increase in H3K79me, H3K79me2, and H3K79me3 levels. ChIP experiments showed that DOT1L regulated the expression of HDAC1. Downregulation of DOT1L using siRNA resulted in decreased expression of HDAC1 and increased transcription of defensin genes and thereby, lower parasite load. In turn, HDAC1 regulates the expression of DOT1L on Leishmania donovani infection as downregulation of HDAC1 using siRNA led to reduced expression of DOT1L. Thus, during Leishmania donovani infection, an interplay between DOT1L and HDAC1 regulates the expression of these two histone modifiers leading to downregulation of defensin gene expression.

Epstein-Barr virus nuclear antigen EBNA3A modulates IRF3-dependent IFNß expression.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined. Here, we reveal a mechanism by which the EBV nuclear antigen 3A (EBNA3A) may inhibit IFNß induction. Using proximity biotinylation we identify the histone acetyltransferase P300, a member of the IFNß transcriptional complex, as a binding partner of EBNA3A. We further show that EBNA3A also interacts with the activated IFN-inducing transcription factor interferon regulatory factor 3 that collaborates with P300 in the nucleus. Both events are mediated by the N-terminal domain of EBNA3A. We propose that EBNA3A limits the binding of interferon regulatory factor 3 to the IFNß promoter, thereby hampering downstream IFN I signaling. Collectively, our findings suggest a new mechanism of immune evasion by EBV, affected by its latency gene EBNA3A.

Tad pili contribute to the virulence and biofilm formation of virulent Aeromonas hydrophila.

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170 A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.

The diversity of clinical Mycobacterium abscessus isolates in morphology, glycopeptidolipids and infection rates in a macrophage model.

Introduction. Mycobacterium abscessus (MABS) is a pathogenic bacterium that can cause severe lung infections, particularly in individuals with cystic fibrosis. MABS colonies can exhibit either a smooth (S) or rough (R) morphotype, influenced by the presence or absence of glycopeptidolipids (GPLs) on their surface, respectively. Despite the clinical significance of these morphotypes, the relationship between GPL levels, morphotype and the pathogenesis of MABS infections remains poorly understood.Gap statement. The mechanisms and implications of GPL production and morphotypes in clinical MABS infections are unclear. There is a gap in understanding their correlation with infectivity and pathogenicity, particularly in patients with underlying lung disease.Aim. This study aimed to investigate the correlation between MABS morphology, GPL and infectivity by analysing strains from cystic fibrosis patients' sputum samples.Methodology. MABS was isolated from patient sputum samples and categorized by morphotype, GPL profile and replication rate in macrophages. A high-content ex vivo infection model using THP-1 cells assessed the infectivity of both clinical and laboratory strains.Results. Our findings revealed that around 50 % of isolates displayed mixed morphologies. GPL analysis confirmed a consistent relationship between GPL content and morphotype that was only found in smooth isolates. Across morphotype groups, no differences were observed in vitro, yet clinical R strains were observed to replicate at higher levels in the THP-1 infection model. Moreover, the proportion of infected macrophages was notably higher among clinical R strains compared to their S counterparts at 72 h post-infection. Clinical variants also infected THP-1 cells at significantly higher rates compared to laboratory strains, highlighting the limited translatability of lab strain infection data to clinical contexts.Conclusion. Our study confirmed the general correlation between morphotype and GPL levels in smooth strains yet unveiled more variability within morphotype groups than previously recognized, particularly during intracellular infection. As the R morphotype is the highest clinical concern, these findings contribute to the expanding knowledge base surrounding MABS infections, offering insights that can steer diagnostic methodologies and treatment approaches.

The widespread vulnerability of Hydra oligactis to tumourigenesis confirms its value as a model for studying the effects of tumoural processes on the ecology and evolution of species.

Tumoural processes, ubiquitous phenomena in multicellular organisms, influence evolutionary trajectories of all species. To gain a holistic understanding of their impact on species' biology, suitable laboratory models are required. Such models are characterised by a widespread availability, ease of cultivation, and reproducible tumour induction. It is especially important to explore, through experimental approaches, how tumoural processes alter ecosystem functioning. The cnidarian Hydra oligactis is currently emerging as a promising model due to its development of both transmissible and non-transmissible tumours and the wide breadth of experiments that can be conducted with this species (at the individual, population, mechanistic, and evolutionary levels). However, tumoural hydras are, so far, only documented in Europe, and it is not clear if the phenomenon is local or widespread. In this study we demonstrate that Australian hydras from two independent river networks develop tumours in the laboratory consisting of interstitial stem cells and display phenotypic alterations (supernumerary tentacles) akin to European counterparts. This finding confirms the value of this model for ecological and evolutionary research on host-tumour interactions.

From crisis to cure: harnessing the potential of mycobacteriophages in the battle against tuberculosis.

Tuberculosis (TB) is a serious and fatal disease caused by Mycobacterium tuberculosis (Mtb). The World Health Organization reported an estimated 1.30 million TB-related deaths in 2022. The escalating prevalence of Mtb strains classified as being multi-, extensively, extremely, or totally drug resistant, coupled with the decreasing efficacies of conventional therapies, necessitates the development of novel treatments. As viruses that infect Mycobacterium spp., mycobacteriophages may represent a strategy to combat and eradicate drug-resistant TB. More exploration is needed to provide a comprehensive understanding of mycobacteriophages and their genome structure, which could pave the way toward a definitive treatment for TB. This review focuses on the properties of mycobacteriophages, their potential in diagnosing and treating TB, the benefits and drawbacks of their application, and their use in human health. Specifically, we summarize recent research on mycobacteriophages targeted against Mtb infection and newly developed mycobacteriophage-based tools to diagnose and treat diseases caused by Mycobacterium spp. We underscore the urgent need for innovative approaches and highlight the potential of mycobacteriophages as a promising avenue for developing effective diagnosis and treatment to combat drug-resistant Mycobacterium strains.

Myeloid C-type lectin receptors in host-pathogen interactions and glycan-based targeting.

Lectin-glycan interactions play a crucial role in the immune system. An important class of lectins in the innate immune system is myeloid C-type lectin receptors (CLRs). Myeloid CLRs act as pattern recognition receptors and are predominantly expressed by myeloid cells, such as macrophages, dendritic cells, and neutrophils. In innate immunity, CLRs contribute to self/non-self discrimination. While the recognition of pathogen-associated molecular patterns (PAMPs) by CLRs may contribute to a protective immune response, CLR engagement can also be exploited by pathogens for immune evasion. Since various CLRs act as endocytic receptors and trigger distinct signaling pathways in myeloid cells, CLR targeting has proven useful for drug/antigen delivery into antigen-presenting cells and the modulation of immune responses. This review covers recent discoveries of pathogen/CLR interactions and novel approaches for CLR targeting within the period of the past two years.

Molecular Investigations to Improve Fusarium Head Blight Resistance in Wheat: An Update Focusing on Multi-Omics Approaches.

Fusarium head blight (FHB) is mainly caused by Fusarium graminearum (Fg) and is a very widespread disease throughout the world, leading to severe damage to wheat with losses in both grain yield and quality. FHB also leads to mycotoxin contamination in the infected grains, being toxic to humans and animals. In spite of the continuous advancements to elucidate more and more aspects of FHB host resistance, to date, our knowledge about the molecular mechanisms underlying wheat defense response to this pathogen is not comprehensive, most likely due to the complex wheat-Fg interaction. Recently, due to climate changes, such as high temperature and heavy rainfall, FHB has become more frequent and severe worldwide, making it even more urgent to completely understand wheat defense mechanisms. In this review, after a brief description of the first wheat immune response to Fg, we discuss, for each FHB resistance type, from Type I to Type V resistances, the main molecular mechanisms involved, the major quantitative trait loci (QTLs) and candidate genes found. The focus is on multi-omics research helping discover crucial molecular pathways for each resistance type. Finally, according to the emerging examined studies and results, a wheat response model to Fg attack, showing the major interactions in the different FHB resistance types, is proposed. The aim is to establish a useful reference point for the researchers in the field interested to adopt an interdisciplinary omics approach.

Metabolite profiling of chickpea (Cicer arietinum) in response to necrotrophic fungus Ascochyta rabiei.

Introduction: Ascochyta blight (AB) caused by the necrotrophic fungus Ascochyta rabiei is one of the most significant diseases that limit the production of chickpea. Understanding the metabolic mechanisms underlying chickpea-A.rabiei interactions will provide important clues to develop novel approaches to manage this disease. Methods: We performed metabolite profiling of the aerial tissue (leaf and stem) of two chickpea accessions comprising a moderately resistant breeding line (CICA1841) and a highly susceptible cultivar (Kyabra) in response to one of the highly aggressive Australian A. rabiei isolates TR9571 via non-targeted metabolomics analysis using liquid chromatography-mass spectrometry. Results: The results revealed resistance and susceptibility-associated constitutive metabolites for example the moderately resistant breeding line had a higher mass abundance of ferulic acid while the levels of catechins, phthalic acid, and nicotinic acid were high in the susceptible cultivar. Further, the host-pathogen interaction resulted in the altered levels of various metabolites (induced and suppressed), especially in the susceptible cultivar revealing a possible reason for susceptibility against A.r abiei. Noticeably, the mass abundance of salicylic acid was induced in the aerial tissue of the susceptible cultivar after fungus colonization, while methyl jasmonate (MeJA) was suppressed, elucidating the key role of phytohormones in chickpea-A. rabiei interaction. Many differential metabolites in flavonoid biosynthesis, phenylalanine, Aminoacyl-tRNA biosynthesis, pentose and glucuronate interconversions, arginine biosynthesis, valine, leucine, and isoleucine biosynthesis, and alanine, aspartate, and glutamate metabolism pathways were up- and down-regulated showing the involvement of these metabolic pathways in chickpea-A. rabiei interaction. Discussion: Taken together, this study highlights the chickpea - A. rabiei interaction at a metabolite level and shows how A. rabiei differentially alters the metabolite profile of moderately resistant and susceptible chickpea accessions and is probably exploiting the chickpea defense pathways in its favour.

Using RNAseq to Uncover Transcriptional and Splicing Differences in Host Cells During Rift Valley Fever Virus Infection.

RNAseq is a valuable tool that can aid researchers in uncovering the transcriptional changes that occur when a viral pathogen infects a host cell. Viral infection will invariably cause differential expression of many genes, from transcription of mRNA to alternative splicing and degradation. This change in gene expression can be a result of immune activation or a direct activity of the virus to alter the host cell's environment to make it more favorable for viral replication. Studying the innate immune response to a pathogen can reveal which cellular pathways are active, indicating the steps that the host takes to halt viral infection, and detecting virus-mediated mRNA expression changes can help with identifying the pathways which may be exploited by the virus. Gene expression changes-both cell-caused and virus-caused-can be studied through RNAseq, helping to provide a clearer picture of the cellular changes that occur during viral infection. In this protocol, we outline methods to carry out mRNA sequencing in Rift Valley fever virus-infected cell cultures, from infection to library prep and analysis.

Viral mimicry and endocrine system: Divulging the importance in host-microbial crosstalk.

Host-pathogen interactions are complex associations which evolve over long co-evolutionary histories. Pathogens exhibit different mechanisms to gain advantage over their host. Mimicry of host factors is an influential tool in subverting host mechanisms to ensure pathogenesis. This chapter discusses such molecular mimicry exhibited during viral infections. Understanding the evolutionary relationships, shared identity and functional impact of the virus encoded mimics is critical. With a particular emphasis on viral mimics and their association with cancer and autoimmune diseases, this chapter highlights the importance of molecular mimicry in virus biology.