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The genus Brucella is a group of intracellular bacterial pathogens documented to cause brucellosis, a zoonotic disease of worldwide concern. In the past decade, reported cases of atypical Brucella species in amphibians have increased. These species are considered atypical based on their tropism, phenotypic characteristics, and phylogeny differences. Atypical Brucella species can be misdiagnosed because of their similarity to classical Brucella species and closely related genera. Recent literature has provided evidence for the zoonotic potential of atypical Brucella linked to amphibians. Therefore, understanding the prevalence of these atypical Brucella species in captive amphibian populations is significant for amphibian health, conservation, and public health. This pilot study evaluated the prevalence of atypical Brucella species in a population of marine toads (Rhinella marina) using a validated pan-Brucella qualitative PCR. Skin swab samples from 56 asymptomatic wild-caught marine toads from Miami, FL, were analyzed. Brucella DNA was not detected in any of the skin swab samples analyzed. This pilot study presents a viable non-invasive sampling technique and PCR assay for rapidly screening populations of amphibians for the presence of atypical Brucella species. [ABSTRACT FROM AUTHOR]

ABSTRACT Antigen cross‐presentation is the process whereby small peptides derived from exogenous antigens are attached to MHC‐I molecules triggering CD8+ T lymphocyte activation. The endocytic route of dendritic cells (DCs) is highly specialised for cross‐presentation to initiate cytotoxic immune responses against numerous intracellular pathogens and tumours. In this study, we identify the endosomal protein sorting nexin (SNX) 17 as a key regulator of antigen internalisation and cross‐presentation by DCs. SNX17 expression in DCs guarantees optimal cross‐presentation of soluble, particulate, and Toxoplasma gondii‐associated antigens. The silencing of SNX17 expression in DCs significantly affected the internalisation of exogenous antigens by fluid‐phase endocytosis, phagocytosis, and more strikingly, T. gondii invasion. We show that SNX17 controls proper integrin recycling, actin cytoskeleton organisation, and phagosomal maturation. Altogether, our findings provide compelling evidence that SNX17 plays a central role in the modulation of the DC endocytic network, which is essential for competent antigen cross‐presentation. [ABSTRACT FROM AUTHOR]

Chlamydia pneumoniae (C. pneumoniae) is a specialized intracellular parasitic pathogen capable of causing pneumonia, sinusitis, bronchitis, and other respiratory diseases, which pose significant public health challenges. Therefore, rapid, accurate, and sensitive diagnosis is crucial for the prevention and treatment of respiratory diseases caused by C. pneumoniae. In this study, we combined enzymatic recombination amplification (ERA) with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a system (CRISPR/Cas12a) to develop a dual detection platform termed the Cpn-ERA-CRISPR/Cas12a dual system. This system integrates both the ERA-CRISPR/Cas12a fluorescence system and the ERA-CRISPR/Cas12a lateral flow system. Detection results can be measured using a fluorescence detector or observed with the naked eye on lateral flow strips. The fluorescence system and the lateral flow system detect C. pneumoniae in 30 minutes and 15 minutes, respectively. This dual system exhibits no cross-reactivity with the other seven pathogens, demonstrating high specificity, and achieves a sensitivity of 100 copies/µL. Additionally, the Cpn-ERA-CRISPR/Cas12a dual system was employed to analyze 39 clinical samples, comprising 19 positive and 20 negative samples. The detection rate for positive samples was 100%, with no positive results in the negative samples, indicating a high level of concordance with qPCR results. In summary, the Cpn-ERA-CRISPR/Cas12a dual system represents a novel tool for diagnosing C. pneumoniae and holds promising application potential in grassroots community hospitals. [ABSTRACT FROM AUTHOR]

Autophagy is an important cellular response against intracellular pathogens. However, some viruses have evolved mechanisms to hijack this defensive process to provide favorable conditions for virus replication in host cells. The porcine epidemic diarrhea virus (PEDV) has been shown to alter autophagy pathways; however, it is still unknown through which receptors PEDV induces autophagy in IPEC-J2 cells, whether autophagy facilitates PEDV replication, and which functional domains of PEDV proteins are primarily responsible for inducing autophagy. Here, we found that PEDV infection induces autophagy in host cells via distinct and uncoupled molecular pathways. RNA-seq technology was used to analyze the expression patterns of intracellular genes in PEDV-infected IPEC-J2 cells using transcriptomics. The results demonstrate that PEDV triggers autophagy via the cellular pathogen receptor TLR4 and the AKT-mTOR pathway. As evidenced by autophagosome detection, PEDV infection increases autophagosomes and light chain 3 (LC3)-II as well as downregulated AKT-mTOR phosphorylation. Our study revealed that the binding of the viral protein NSP61-2C (56-151aa) to TLR4 triggers autophagy and inactivates the AKT-mTOR pathway, both of which are critical for facilitating PEDV infection. Through screening and analysis, TLR4 was found to be a key gene involved in PEDV-induced autophagy. The screening of the key functional domains of NSP6 (56-151aa) for their ability to induce autophagy in IPEC-J2 cells provided a basis for the in-depth analysis of the pathogenic mechanism of PEDV infection-induced autophagy and promotion of self-replication and also provided an important target for the study of PEDV antiviral drugs. In conclusion, we elucidated that the PEDV infection of IPEC-J2 cells could induce autophagy and found that PEDV could use autophagy to promote its own replication. [ABSTRACT FROM AUTHOR]

Tuberculosis caused by the obligate intracellular pathogen, Mycobacterium tuberculosis, is one among the prime causes of death worldwide. An urgent remedy against tuberculosis is of paramount importance in the current scenario. However, the complex nature of this appalling disease contributes to the limitations of existing medications. The quest for better treatment approaches is driving the research in the field of host epigenomics forward in context with tuberculosis. The interplay between various host epigenetic factors and the pathogen is under investigation. A comprehensive understanding of how Mycobacterium tuberculosis orchestrates such epigenetic factors and favors its survival within the host is in increasing demand. The modifications beneficial to the pathogen are reversible and possess the potential to be better targets for various therapeutic approaches. The mechanisms, including histone modifications, DNA methylation, and miRNA modification, are being explored for their impact on pathogenesis. In this article, we are deciphering the role of mycobacterial epigenetic regulators on various strategies like cytokine expression, macrophage polarization, autophagy, and apoptosis, along with a glimpse of the potential of host-directed therapies. [ABSTRACT FROM AUTHOR]

In the competition between the pathogen infection and the host defense, infectious microorganisms may enter the host cells by evading host defense mechanisms and use the intracellular biomolecules as replication nutrient. Among them, intracellular Staphylococcus aureus relies on the host cells to protect itself from the attacks by antibiotics or immune system to achieve long-term colonization in the host, and the consequent clinical therapeutic failures and relapses after antibiotic treatment. Here, we demonstrate that intracellular S. aureus surviving well even in the presence of vancomycin can be effectively eliminated using an emerging cell-mimicking therapeutic strategy. These cell mimics with natural killer cell-like activity (NKMs) are composed of a redox-responsive degradable carrier, and perforin and granzyme B within the carrier. NKMs perform far more effectivly than clinical antibiotics in treating intracellular bacterial infections, providing a direct evidence of the NK cell-mimicking immune mechanism in the treatment of intracellular S. aureus. [ABSTRACT FROM AUTHOR]

As cytoplasmic protein complexes that are pivotal for innate immunity, inflammasomes act primarily through the detection of pathogen‐ or danger‐associated molecular patterns. Nucleotide oligomerisation domain‐like receptor family and caspase activation recruitment domain‐containing protein 4 (NLRC4) inflammasomes identify and eliminate intracellular pathogens, a process contingent on the ligand‐recognition capabilities of neuronal apoptosis inhibitory proteins (NAIPs). Upon detection of specific molecules indicative of intracellular infection, NAIPs discern distinct pathogenic components and subsequently transmit signals to NLRC4, thus initiating their activation and triggering an inflammatory response. However, the mechanisms underlying NLRC4 inflammasome remain unclear. In this study, we elucidated the critical role of ATG16L2 in activating the NLRC4 inflammasome. ATG16L2‐deficient macrophages exhibited reduced NLRC4 inflammasome activation, characterised by decreased oligomerisation of apoptosis‐associated speck‐like protein containing a CARD and attenuated cleavage of Pro‐caspase‐1, Pro‐IL‐1β and gasdermin D. Co‐immunoprecipitation assays revealed an interaction between ATG16L2 and NAIPs. Furthermore, ATG16L2 enhanced the association between NAIPs and NLRC4 by binding to NAIPs. For ATG16L2‐knockout mice infected with Salmonella typhimurium, pathogen clearance and survival rates markedly decreased. Collectively, our findings suggest that ATG16L2 is a significant modulator of the innate immune system, influencing the activity of the NLRC4 inflammasome and the host's defensive response to intracellular pathogens. [ABSTRACT FROM AUTHOR]

Phages are gaining attention for their ability to target drug-resistant bacteria, disrupt biofilms, and reach intracellular pathogens, offering promising alternatives to traditional antibiotics. The Collection discusses advances in phage therapy, including their application in vaccine development, cancer immunotherapy, and gene delivery systems. Key research gaps are identified, such as challenges related to phage stability, immune response, and regulatory hurdles. Despite the progress, phage therapy faces obstacles in maintaining phage viability, evading immune detection, and navigating complex regulatory frameworks. The articles collectively address these challenges and propose potential solutions to enhance the effectiveness and acceptance of phage-based treatments. By overcoming these barriers, bacteriophage research has the potential to revolutionize medical therapies, providing innovative approaches to some of the most pressing healthcare challenges today. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei (Bpm) is the causative agent of the disease melioidosis. As a facultative intracellular pathogen, Bpm has a complex lifestyle that culminates in cell-to-cell fusion and multinucleated giant cells (MNGCs) formation. The virulence factor responsible for MNGC formation is the type 6 secretion system (T6SS), a contractile nanomachine. MNGC formation is a cell-to-cell spread strategy that allows the bacteria to avoid the extracellular immune system and our previous data highlighted cell death, apoptosis, and inflammation as pathways significantly impacted by T6SS activity. Thusly, we investigated how the T6SS influences these phenotypes within the macrophage and pulmonary models of infection. Here we report that the T6SS is responsible for exacerbating apoptotic cell death during infection in both macrophages and the lungs of infected mice. We also demonstrate that although the T6SS does not influence differential macrophage polarization, the M2 polarization observed is potentially beneficial for Bpm pathogenesis and replication. Finally, we show that the T6SS contributes to the severity of inflammatory nodule formation in the lungs, which might be potentially connected to the amount of apoptosis that is triggered by the bacteria. Author summary: Burkholderia pseudomallei (Bpm) is an intracellular pathogen and is the etiological agent of melioidosis. This neglected tropical disease results in an estimated 89,000 fatalities a year, however, this number is believed to be severely underreported. The complex intracellular lifestyle and host response to infection is poorly understood, basic cell interactions and responses are critical to defense against infection. Bpm utilizes an array of virulence factors to successfully replicate and disseminate cell-to-cell, one of these is the T6SS which is responsible for MNGC formation and cell-to-cell spread. In this work, we characterized how macrophages respond to infection in the presence or absence of the critical T6SS virulence factor. Macrophages are a primary replicative niche for Bpm but how intracellular replication disrupts macrophages response to infection is poorly understood. By understanding macrophage cell death patterns and polarization, we can dissect responses that are triggered by T6SS activity with the expectation of finding exploitable responses for host directed therapies. [ABSTRACT FROM AUTHOR]

Leishmaniasis is considered one of the most critical health concerns in the world. Unfortunately, no protective vaccines exist and conventional treatments are relatively ineffective. Therefore, new strategies are necessary against leishmaniasis. In recent years, exosomes have shown promising therapeutic outcomes in various diseases, including infectious diseases. In this regard, we aimed to explore the effect of the exosome, pyrimethamine and their combination on the anti‐parasitic function of RAW264.7 cells against Leishmania major. Exosomes were isolated from the C57BL/6 peritoneal macrophages. L. major infected and non‐infected RAW264.7 cells treated with exosomes, pyrimethamine (PM), and exosomes along with PM. The effect of the treatments was analysed on phagocytosis, efferocytosis, the intracellular parasite count, arginase activity, nitric oxide (NO) and reactive oxygen species (ROS) production. Exosomes could significantly elevate the phagocytosis, efferocytosis, NO and ROS in both infected and non‐infected groups (Pv < 0.05). The exosomes reduced the arginase activity in both groups (Pv < 0.05). The intracellular parasite count was significantly lower after treatment with exosomes (Pv < 0.05). These results demonstrate that MQ‐derived exosomes can enhance in vitro anti‐parasitic responses against L. major. This provides a potential pathway for more effective treatments and underscores the importance of further research in this area. [ABSTRACT FROM AUTHOR]

Autophagy is the process by which damaged regions of the cytoplasm and intracellular pathogens are degraded. This mechanism often serves an adaptive role in cells, enhancing their survival. It plays a direct or indirect role in the development of various pathological conditions within the body. This phenomenon is common in various malignant diseases, where autophagy is associated with the resistance of transformed cells to chemotherapy. Conversely, abnormal activation of autophagy can trigger cell death, a process often seen in neurodegenerative conditions. Given that dysregulation of autophagy is associated with the progression of numerous pathological conditions, this is of significant interest to the developers of drugs that can effectively modulate autophagy for both basic research and clinical applications. Here, we provide a brief description of the mechanism of macroautophagy, the most prevalent form of autophagy identified in humans. We also discuss the clinical potential of drugs that can modulate autophagy, highlighting their use in combating diseases associated with direct or indirect dysregulation of this essential process. [ABSTRACT FROM AUTHOR]

Intracellular parasites of the genus Glugea Thélohan, 1891 (Microsporidia) comprise about 34 putative species capable of causing high morbidity and mortality in freshwater and marine teleost fishes. In this study, we report on the first mass mortality event associated with Glugea sp. infecting free‐ranging round sardinella Sardinella aurita in the southern Tyrrhenian Sea (Italy). Here, we describe the ultrastructure of mature spores of this microsporidian and characterize it molecularly, as well as report its phylogenetic position. Most of the affected fish showed an irregular swelling of its abdomen. At necropsy, a variable number of xenomas, spherical to ellipsoidal in shape, were found in the peritoneal cavity strongly attached to the viscera of all fish. Histological analysis revealed varying severity of chronic inflammation along with occasional necrosis in visceral organs associated with multiple xenoma proliferation. These pathological findings were considered the main cause of this mass mortality event. Morphologically, the present material was closely related to G. sardinellesis and G. thunni. The phylogenetically closest taxa to the newly SSU rDNA sequence were G. thunni and an erroneusly identified G. plecoglossi, which were very closely related to each other, also suggesting that all these sequences might belong to the same species. [ABSTRACT FROM AUTHOR]

Simple Summary: Microsporidia are a group of fungus-related eukaryotes with worldwide distribution. The microsporidian species Encephalitozoon cuniculi and E. hellem occur in mammals, birds and even humans. Knowledge of their relevance in wild rabbits is very limited so far. Thus, the aim of the present study was to investigate the occurrence of E. cuniculi and E. hellem in wild rabbit populations in southern Germany (Bavaria). Therefore, blood and organ samples of 158 wild rabbits were investigated by serological and PCR-based assays. Antibodies to E. cuniculi were detected in 24 of the 158 (15.2%) wild rabbits, while DNA of E. cuniculi was found in 7 (4.4%) and DNA of E. hellem was found in 3 (1.9%). Sequencing identified E. cuniculi genotype 1. This study provides the first E. cuniculi genotype determination in free-living wild rabbits worldwide and the first evidence of E. hellem in rabbits worldwide. Wild rabbits should, therefore, be regarded as a reservoir for both pathogens and as a source of infection for domestic rabbits, other animals and humans. Encephalitozoon cuniculi and Encephalitozoon hellem are fungus-related, obligate intracellular pathogens belonging to the microsporidia. Both microorganisms occur in mammals, birds and even humans, thus revealing a zoonotic potential. Knowledge of their relevance in wild rabbits is very limited so far. Thus, the aim of the present study was to investigate the occurrence of E. cuniculi and E. hellem in wild rabbit populations in southern Germany (Bavaria). Therefore, blood and organ samples (brain/kidney) of 158 wild rabbits were investigated by immunofluorescence and PCR-based assays. Antibodies to E. cuniculi were detected in 24 of the 158 (15.2%) wild rabbits, while DNA of E. cuniculi was found in 7 (4.4%) and DNA of E. hellem was found in 3 (1.9%). Sequencing identified E. cuniculi genotype 1. This study provides the first E. cuniculi genotype determination in free-living wild rabbits worldwide and the first evidence of E. hellem in rabbits worldwide. Wild rabbits should, therefore, be regarded as a reservoir for both pathogens and, on the basis of molecular evidence from kidney tissue and presumed urine excretion, also as a source of infection for E. cuniculi for animals and humans. [ABSTRACT FROM AUTHOR]

The intracellular bacterial pathogen Legionella pneumophila utilizes the Dot/Icm system to translocate over 330 effectors into the host cytosol. These virulence factors modify a variety of cell processes, including pathways involved in cell death and survival, to promote bacterial proliferation. Here, we show that the effector LegK3 is a eukaryotic-like Ser/Thr kinase that functions to suppress host apoptosis. Mechanistically, LegK3 directly phosphorylates multiple caspases involved in apoptosis signaling, including Caspase-3, Caspase-7, and Caspase-9. LegK3-induced phosphorylation of these caspases occurs at serine (Ser29 in Caspase-3 and Ser199 in Caspase-7) or threonine (Thr102 in Caspase-9) residues located in the prodomain or interdomain linkers. These modifications interfere with the suitability of the caspases as the substrates of initiator caspases or upstream regulators without impacting their proteolytic activity. Collectively, our study reveals a novel strategy used by L. pneumophila to maintain the integrity of infected cells for its intracellular growth. The bacterial pathogen Legionella pneumophila delivers hundreds of effector proteins to promote infection. Here, Ge et al show that effector LegK3 suppresses host apoptosis via direct phosphorylation of caspases. [ABSTRACT FROM AUTHOR]

Treating bone infections with common antibiotics is challenging, since pathogens like Staphylococcus aureus can reside inside macrophages. To target these intracellular bacteria, we have proposed nanoparticles (NPs) as drug carriers. This study aims to investigate the efficacy of hydroxyapatite and gelatin NPs, selected in view of their bone mimicry and potential for targeted delivery, as carriers for the antibacterial agents zinc and vancomycin. Therefore, two distinct NPs are fabricated: zinc-doped hydroxyapatite (ZnHA) and vancomycin-loaded gelatin (VGel) NPs. The NPs are characterized based on morphology, size, chemical composition, cellular internalization, and intracellular bactericidal efficacy. Specifically, the intracellular bactericidal efficacy is tested using a validated coculture model of human THP-1 derived macrophages and phagocytosed S. aureus bacteria. Scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FTIR) results show that the spherical NPs are synthesized successfully. These NPs are internalized by THP-1 cells and show >75% colocalization with lysosomes without compromising the viability of the THP-1 cells. Both ZnHA and VGel NPs substantially reduce the intracellular survival of S. aureus compared to the direct addition of dissolved zinc and vancomycin. Concluding, our NPs are highly effective drug delivery vehicles to kill intracellular S. aureus , which stress the potential of these NPs for future clinical translation. [ABSTRACT FROM AUTHOR]

Edwardsiella piscicida is an emerging bacterial pathogen and the aetiological agent of edwardsiellosis among cultured and wild fish species globally. The increased frequency of outbreaks of this Gram‐negative, facultative intracellular pathogen pose not only a threat to the aquaculture industry but also a possible foodborne/waterborne public health risk due to the ill‐defined zoonotic potential. Thus, understanding the role of temperature on the virulence of this emerging pathogen is essential for comprehending the pathogenesis of piscine edwardsiellosis in the context of current warming trends associated with climate change, as well as providing insight into its zoonotic potential. In this study, significant temperature‐dependent alterations in bacterial growth patterns were observed, with bacterial isolates grown at 17°C displaying higher peak growth sizes, extended lag times, and slower maximal growth rates than isolates grown at 27or 37°C. When E. piscicida isolates were grown at 37°C compared to 27 and 17°C, mass spectrometry analysis of the E. piscicida proteome revealed significant downregulation of crucial virulence proteins, such as Type VI secretion system proteins and flagellar proteins. Although in vivo models of infection are warranted, this in vitro data suggests possible temperature‐associated alterations in the virulence and pathogenic potential of E. piscicida in poikilotherms and homeotherms. [ABSTRACT FROM AUTHOR]

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens that infect eukaryotic cells and reside within a host-derived vacuole known as the inclusion. To facilitate intracellular replication, these bacteria must engage in host-pathogen interactions to obtain nutrients and membranes required for the growth of the inclusion, thereby sustaining prolonged bacterial colonization. Autophagy is a highly conserved process that delivers cytoplasmic substrates to the lysosome for degradation. Pathogens have developed strategies to manipulate and/or exploit autophagy to promote their replication and persistence. This review delineates recent advances in elucidating the interplay between Chlamydia trachomatis infection and autophagy in recent years, emphasizing the intricate strategies employed by both the Chlamydia pathogens and host cells. Gaining a deeper understanding of these interactions could unveil novel strategies for the prevention and treatment of Chlamydia infection. [ABSTRACT FROM AUTHOR]

Microsporidia are intracellular parasites that all possess a unique infection apparatus involving a polar tube. Upon contact with a host cell, this tube forms the conduit through which the parasite enters the host. Infecting mostly animals, microsporidian species can be transmitted vertically or horizontally, and exert various effects on their hosts: infections range from being relatively benign to lethal. Microsporidian genomes possess highly divergent sequences and are often substantially reduced in size. Their divergent sequences and unique morphology created early challenges to our understanding of their phylogenetic position within the tree of eukaryotes. Over the last couple of decades, advances in both sequencing technology and phylogenetic methodology supported a clear relationship between microsporidia and fungi. However, the specifics of this relationship were muddied by the lack of known microsporidian relatives. With increased taxon discovery and the morphological and molecular characterization of microsporidia‐like taxa, rozellids and aphelids, a better resolved picture is emerging. Here we review the history of microsporidian taxonomy and current status of genomics of microsporidia and their nearest relatives, with an aim to understand their morphological and metabolic differences, along with their evolutionary relationships. [ABSTRACT FROM AUTHOR]

Since the advent of sequencing techniques and due to their continuous evolution, it has become easier and less expensive to obtain the complete genome sequence of any organism. Nevertheless, to elucidate all biological processes governing organism development, quality annotation is essential. In genome annotation, predicting gene structure is one of the most important and captivating challenges for computational biology. This aspect of annotation requires continual optimization, particularly for genomes as unusual as those of microsporidia. Indeed, this group of fungal‐related parasites exhibits specific features (highly reduced gene sizes, sequences with high rate of evolution) linked to their evolution as intracellular parasites, requiring the implementation of specific annotation approaches to consider all these features. This review aimed to outline these characteristics and to assess the increasingly efficient approaches and tools that have enhanced the accuracy of gene prediction for microsporidia, both in terms of sensitivity and specificity. Subsequently, a final part will be dedicated to postgenomic approaches aimed at reinforcing the annotation data generated by prediction software. These approaches include the characterization of other understudied genes, such as those encoding regulatory noncoding RNAs or very small proteins, which also play crucial roles in the life cycle of these microorganisms. [ABSTRACT FROM AUTHOR]

Microsporidia and Apicomplexa are eukaryotic, single‐celled, intracellular parasites with huge public health and economic importance. Typically, these parasites are studied separately, emphasizing their uniqueness and diversity. In this review, we explore the huge amount of genomic data that has recently become available for the two groups. We compare and contrast their genome evolution and discuss how their transitions to intracellular life may have shaped it. In particular, we explore genome reduction and compaction, genome expansion and ploidy, gene shuffling and rearrangements, and the evolution of centromeres and telomeres. [ABSTRACT FROM AUTHOR]

Microsporidia are prolific producers of effector molecules, encompassing both proteins and nonproteinaceous effectors, such as toxins, small RNAs, and small peptides. These secreted effectors play a pivotal role in the pathogenicity of microsporidia, enabling them to subvert the host's innate immunity and co‐opt metabolic pathways to fuel their own growth and proliferation. However, the genomes of microsporidia, despite falling within the size range of bacteria, exhibit significant reductions in both structural and physiological features, thereby affecting the repertoire of secretory effectors to varying extents. This review focuses on recent advances in understanding how microsporidia modulate host cells through the secretion of effectors, highlighting current challenges and proposed solutions in deciphering the complexities of microsporidial secretory effectors. [ABSTRACT FROM AUTHOR]

The nematode Caenorhabditis elegans is an invaluable host model for studying infections caused by various pathogens, including microsporidia. Microsporidia represent the first natural pathogens identified in C. elegans, revealing the previously unknown Nematocida genus of microsporidia. Following this discovery, the utilization of nematodes as a model host has rapidly expanded our understanding of microsporidia biology and has provided key insights into the cell and molecular mechanisms of antimicrosporidia defenses. Here, we first review the isolation history, morphological characteristics, life cycles, tissue tropism, genetics, and host immune responses for the four most well‐characterized Nematocida species that infect C. elegans. We then highlight additional examples of microsporidia that infect related terrestrial and aquatic nematodes, including parasitic nematodes. To conclude, we assess exciting potential applications of the nematode‐microsporidia system while addressing the technical advances necessary to facilitate future growth in this field. [ABSTRACT FROM AUTHOR]

Microsporidia are obligate intracellular parasites of the Fungal Kingdom that cause widespread infections in nature, with important effects on invertebrates involved in food production systems. The two microsporidian species Vairimorpha (Nosema) ceranae (and the less common Vairimorpha (Nosema) apis) can cause individual disease in honey bees and contribute to colony collapse. The efficacy, safety, and availability of fumagillin, the only drug currently approved to treat microsporidia infection in bees, is uncertain. In this review, we will discuss some of the most promising alternative strategies for the mitigation of Vairimorpha spp. with an emphasis on infection by V. ceranae, now the dominant species infecting bees. We will focus on pharmacologic interventions where the mechanism of action is known and examine both pathogen‐directed and host‐directed approaches. As limiting toxicity to host cells has been especially emphasized in treating bees that are already facing numerous stressors, strategies that disrupt pathogen‐specific targets may be especially advantageous. Therefore, efforts to increase the knowledge and tools for facilitating the discovery of such targets and pharmacologic agents directed against them should be prioritized. [ABSTRACT FROM AUTHOR]

The microbiome is the collection of microbes that are associated with a host. Microsporidia are intracellular eukaryotic parasites that can infect most types of animals. In the last decade, there has been much progress to define the relationship between microsporidia and the microbiome. In this review, we cover an increasing number of reports suggesting that microsporidia are common components of the microbiome in both invertebrates and vertebrates. These microsporidia infections can range from mutualistic to pathogenic, causing several physiological phenotypes, including death. Infection with microsporidia often causes a disruption in the normal microbiome, with both increases and decreases of bacterial, fungal, viral, and protozoan species being observed. This impact on the microbiome can occur through upregulation and downregulation of innate immunity as well as morphological changes to tissues that impact interactions with these microbes. Other microbes, particularly bacteria, can inhibit microsporidia and have been exploited to control microsporidia infections. These bacteria can function through regulating immunity, secreting anti‐microsporidia compounds, and, in engineered versions, expressing double‐stranded RNA targeting microsporidia genes. We end this review by discussing potential future directions to further understand the complex interactions between microsporidia and the other members of the microbiome. [ABSTRACT FROM AUTHOR]

Mammalian autophagy is a highly regulated and conserved cellular homeostatic process. Its existence allows the degradation of self‐components to mediate cell survival in different stress conditions. Autophagy is involved in the regulation of cellular metabolic needs, protecting the cell or tissue from starvation through the degradation and recycling of cytoplasmic materials and organelles to basic molecular building blocks. It also plays a critical role in eliminating damaged or harmful proteins, organelles, and intracellular pathogens. Thus, a deterioration of the process may result in pathological conditions, such as aging‐associated disorders and cancer. Understanding the crucial role of autophagy in maintaining the normal physiological function of cells, tissue, or organs has led to copious and expansive research regarding the regulation of this process. So far, most of the research has revolved around transcriptional and post‐translational regulation. Here, we discuss the regulation of autophagy‐related (ATG) mRNA transcripts by RNA‐binding proteins (RBPs). This analysis focuses on how RBPs modulate autophagy in disease. A deeper understanding of the involvement of RBPs in autophagy can facilitate further research and treatment of a variety of human diseases. [ABSTRACT FROM AUTHOR]

IFN-γ-inducible immunity-related GTPases (IRG proteins) and guanylate binding proteins (GBP proteins) confer resistance against T. gondii by accumulating at the parasite´s parasitophorous vacuolar membrane, a necessary prerequisite for its destruction. The IRG system represents one of the most polymorphic genetic systems described in mammals so far and likely explains the existence of IRG-specific polymorphic effectors of the parasite, enabling evasion of IRG-mediated immunity. A central element of T. gondii virulence is the pseudokinase ROP5 which provides a scaffold for the assembly of multiprotein complexes that inactivate individual IRG proteins. The reciprocal polymorphism between parasite effectors and host IRG proteins strongly implies ongoing or recent co-evolution. Studying these co-evolutionary dynamics can provide insights into the molecular choreography of T. gondii infection. Toxoplasmosis is a common parasitic zoonosis that can be life-threatening in immunocompromised patients. About one-third of the human population is infected with Toxoplasma gondii. Primary infection triggers an innate immune response wherein IFN-γ-induced host cell GTPases, namely IRG and GBP proteins, serve as a vital component for host cell resistance. In the past decades, interest in elucidating the function of these GTPase families in controlling various intracellular pathogens has emerged. Numerous T. gondii effectors were identified to inactivate particular IRG proteins. T. gondii is re-optimizing its effectors to combat IRG function and in this way secures transmission. We discuss the IRG-specific effectors employed by the parasite in murine infections, contributing to a better understanding of T. gondii virulence. [ABSTRACT FROM AUTHOR]

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila , an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function. [ABSTRACT FROM AUTHOR]

Phage therapy, the use of bacteriophages (phages) to treat bacterial infections, is regaining momentum as a promising weapon against the rising threat of multidrug-resistant (MDR) bacteria. This comprehensive review explores the historical context, the modern resurgence of phage therapy, and phage-facilitated advancements in medical and technological fields. It details the mechanisms of action and applications of phages in treating MDR bacterial infections, particularly those associated with biofilms and intracellular pathogens. The review further highlights innovative uses of phages in vaccine development, cancer therapy, and as gene delivery vectors. Despite its targeted and efficient approach, phage therapy faces challenges related to phage stability, immune response, and regulatory approval. By examining these areas in detail, this review underscores the immense potential and remaining hurdles in integrating phage-based therapies into modern medical practices. [ABSTRACT FROM AUTHOR]

Intracellular bacterial pathogens pose significant public health challenges due to their ability to evade immune defenses and conventional antibiotics. Drug repurposing has recently been explored as a strategy to discover new therapeutic uses for established drugs to combat these infections. Utilizing high-throughput screening, bioinformatics, and systems biology, several existing drugs have been identified with potential efficacy against intracellular bacteria. For instance, neuroleptic agents like thioridazine and antipsychotic drugs such as chlorpromazine have shown effectiveness against Staphylococcus aureus and Listeria monocytogenes. Furthermore, anticancer drugs including tamoxifen and imatinib have been repurposed to induce autophagy and inhibit bacterial growth within host cells. Statins and anti-inflammatory drugs have also demonstrated the ability to enhance host immune responses against Mycobacterium tuberculosis. The review highlights the complex mechanisms these pathogens use to resist conventional treatments, showcases successful examples of drug repurposing, and discusses the methodologies used to identify and validate these drugs. Overall, drug repurposing offers a promising approach for developing new treatments for bacterial infections, addressing the urgent need for effective antimicrobial therapies. [ABSTRACT FROM AUTHOR]

Purpose: Visceral leishmaniasis (VL) is caused by an intracellular parasite that is transmitted to humans by sandfly bites. It is prevalent throughout Asia, Africa, the Americas, and the Mediterranean area, where 147 million people are at risk of contracting the illness. The manifestation of heterotrophic illness relies on both Leishmania implicated and the host's immunological response, ranging from asymptomatic to severe leishmaniasis with potentially lethal effects. Method: We reviewed the literature (published till 31st December 2023) on the worldwide situation of leishmaniasis, standard and novel detection techniques, and traditional and modern treatment strategies and endeavors to eliminate VL. Moreover, epidemiological data was collected from the World Health Organization's publicly available databases. GraphPad Prism Version 8 was used to analyze and produce figures based on the epidemiological data. Results: Diagnosis of parasites in tissues or serology is commonly employed. Diagnosis by identifying parasite DNA using molecular techniques is becoming more popular. Despite recent findings of L. donovani resistance to pentavalent antimoniate medications, it continues to be the cornerstone in the medical management of VL. Amphotericin B and its lipid formulations, injectable paromomycin, and oral miltefosine are among the new therapy options being researched. The number of reported VL cases has reduced remarkably over the last decade due to human interventions made to eliminate VL. Particularly countries from the South East Asian region have experienced momentous progress in reducing VL cases and eliminating this disease from this region. Owing to the robust elimination programs, countries such as Bangladesh has eliminated VL as a public health concern. India and Nepal are on the verge of its elimination. Conclusion: Rapid diagnosis, effective and inexpensive treatment, simple access to newly discovered medications, appropriate vector control, and a well-designed vaccine are all required for the elimination of this disease burden in impoverished areas of the globe. [ABSTRACT FROM AUTHOR]

Ubiquitination is a posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. Here, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on phosphoribosyl-Ub conjugated to host targets by Sde. Remarkably, Ub moieties within poly-Ub chains are either modified with a phosphoribosyl group by PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated phosphoribosyl-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors and therefore exclude host autophagy adaptors from the LCV. In this work, we shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV. Ubiquitination plays an essential role in intracellular pathogen infections. Here, Wan et al. report how Legionella pneumophila hijacks the host ubiquitin system by building modified poly-ubiquitin chains at the Legionella-containing vacuole, preventing the recognition by host autophagy adaptors. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei (Bp) causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. Bp is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing Bp infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as Bp protrudes toward neighboring cells. We have characterized BP1026B_ II1561 and determined that it has a role in pathogenesis. A deletional mutant of BP1026B_II1561 is attenuated in RAW264.7 macrophage and BALB/c mouse models of infection. Using RNA-seq, we found that BP1026B_II1561 controls secondary metabolite biosynthesis, fatty acid degradation, and propanoate metabolism. In addition, we identified that BP1026B_II1561 directly controls expression of an outer membrane porin and genes in the shikimate biosynthetic pathway using ChIP-seq. Transposon mutants of genes within the BP1026B_ II1561 regulon show defects during intracellular replication in RAW264.7 cells confirming the role of this transcriptional regulator and the pathways it controls in pathogenesis. BP1026B_II1561 also up-regulates the majority of the enzymes in shikimate and tryptophan biosynthetic pathways, suggesting their importance for Bp physiology. To investigate this, we tested fluorinated analogs of anthranilate and tryptophan, intermediates and products of the shikimate and tryptophan biosynthetic pathways, respectively, and showed inhibition of Bp growth at nanomolar concentrations. The expression of these pathways by BP1026b_II1561 and during intracellular infection combined with the inhibition of Bp growth by fluorotryptophan/anthranilate highlights these pathways as potential targets for therapeutic intervention against melioidosis. In the present study, we have identified BP1026B_II1561 as a critical transcriptional regulator for Bp pathogenesis and partially characterized its role during host cell infection. [ABSTRACT FROM AUTHOR]

The host ESCRT-machinery repairs damaged endolysosomal membranes. If damage persists, selective macroautophagy/autophagy clears the damaged compartment. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that damages the phagosomal membrane and targets ESCRT-mediated repair as part of its virulence program. The E3 ubiquitin ligases PRKN and SMURF1 promote autophagic capture of damaged, Mtb-containing phagosomes. Because ubiquitination is a reversible process, we anticipated that host deubiquitinases (DUBs) would also be involved. Here, we screened all predicted mouse DUBs for their role in ubiquitin targeting and control of intracellular Mtb. We show that USP8 (ubiquitin specific peptidase 8) colocalizes with intracellular Mtb, recognizes phagosomal membrane damage, and is required for ESCRT-dependent membrane repair. Furthermore, we show that USP8 regulates the NFE2L2/NRF2-dependent antioxidant signature. Taken together, our study demonstrates a central role of USP8 in promoting Mtb intracellular growth by promoting phagosomal membrane repair, limiting ubiquitin-driven selective autophagy, and reducing oxidative stress.Abbreviation: BMDMs: bone marrow-derived macrophages; CFUs: colony-forming units; DUB: deubiquitinase; ESCRT: endosomal sorting complexes required for transport; LLOMe: L-leucyl-L-leucine methyl ester; MFI: mean fluorescence intensity; MOI: multiplicity of infection; Mtb: Mycobacterium tuberculosis; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; PMA: phorbol 12-myristate 13-acetate; ROS: reactive oxygen species; USP8: ubiquitin specific peptidase 8 [ABSTRACT FROM AUTHOR]

To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization. The Chlamydia pneumoniae effector SemD binds and activates the actin regulator N-WASP, structurally and functionally mimicking the endogenous N-WASP activator and small GTPase Cdc42GTP, hijacking the host cell endocytic machinery via this interaction. [ABSTRACT FROM AUTHOR]

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival. [ABSTRACT FROM AUTHOR]

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture. [ABSTRACT FROM AUTHOR]

In Chile, Piscirickettsia salmonis contains two genetically isolated genogroups, LF-89 and EM-90. However, the impact of a potential co-infection with these two variants on Salmonid Rickettsial Septicemia (SRS) in Atlantic salmon (Salmo salar) remains largely unexplored. In our study, we evaluated the effect of P. salmonis LF-89-like and EM-90-like co-infection on post-smolt Atlantic salmon after an intraperitoneal challenge to compare changes in disease dynamics and host immune response. Co-infected fish had a significantly lower survival rate (24.1%) at 21 days post-challenge (dpc), compared with EM-90-like single-infected fish (40.3%). In contrast, all the LF-89-like single-infected fish survived. In addition, co-infected fish presented a higher presence of clinical lesions than any of the single-infected fish. The gene expression of salmon immune-related biomarkers evaluated in the head kidney, spleen, and liver showed that the EM-90-like isolate and the co-infection induced the up-regulation of cytokines (e.g., il-1β, ifnγ, il8, il10), antimicrobial peptides (hepdicin) and pattern recognition receptors (PRRs), such as TLR5s. Furthermore, in serum samples from EM-90-like and co-infected fish, an increase in the total IgM level was observed. Interestingly, specific IgM against P. salmonis showed greater detection of EM-90-like antigens in LF-89-like infected fish serum (cross-reaction). These data provide evidence that P. salmonis LF-89-like and EM-90-like interactions can modulate SRS disease dynamics in Atlantic salmon, causing a synergistic effect that increases the severity of the disease and the mortality rate of the fish. Overall, this study contributes to achieving a better understanding of P. salmonis population dynamics. [ABSTRACT FROM AUTHOR]

Brucella spp. are facultative intracellular pathogens that cause zoonosis- brucellosis worldwide. There has been a trend of the re-emergence of brucellosis worldwide in recent years. The epidemic situation of brucellosis is serious in Xinjiang. To analyze the epidemic situation of Brucella spp. in Xinjiang among humans and animals, this study identified 144 Brucella isolates from Xinjiang using classical identification and 16 S rRNA sequencing. MLVA, drug resistance testing, and wgSNP detection were also performed. At the same time, analysis was conducted based on the published data of Brucella isolates worldwide. The results showed that the dominant species was B. melitensis biovar 3, which belonged to GT42 (MLVA-8 typing) and the East Mediterranean lineage. The correlation among isolates was high both in humans or animals. The isolates in Xinjiang exhibited higher polymorphism compared to other locations in China, with polymorphism increasing each year since 2010. No amikacin/kanamycin-resistant strains were detected, but six rifampicin-intermediate isolates were identified without rpoB gene variation. The NJ tree of the wgSNP results indicated that there were three main complexes of the B. melitensis epidemic in Xinjiang. Based on the results of this study, the prevention and control of brucellosis in Xinjiang should focus on B. melitensis, particularly strains belonging to B. melitensis bv.3 GT42 (MLVA-8 typing) and East Mediterranean lineage. Additionally, the rifampicin- and trimethoprim-sulfamethoxazole- resistance of isolates in Xinjiang should be closely monitored to avoid compromising the therapeutic efficacy and causing greater losses. These results provide essential data for the prevention and control of brucellosis in Xinjiang and China. Although the isolates from Xinjiang have significant characteristics among Chinese isolates and can reflect the epidemiological situation of brucellosis in China to some extent, this study cannot represent the characteristics of isolates from other regions. [ABSTRACT FROM AUTHOR]

Obligate intracellular protozoan parasite, Leishmania donovani, causative agent of visceral leishmaniasis, led to impaired macrophage functions. It is well documented that many of these changes were induced by parasite‐mediated reduction in macrophage cholesterol content. Leishmania‐mediated alteration in the other lipids has not been explored in detail yet. Here, we found that the expression of key cholesterol biosynthetic genes and total cellular cholesterol were reduced during L. donovani infection. Further, we have also identified that this reduction in the cholesterol led to increased membrane fluidity and inhibition of antigen‐presenting potential of macrophages. In addition to this, we studied the relative changes in different lipids in THP‐1‐derived macrophages during L. donovani infection through liquid chromatography‐mass spectrometry. We found that Sphingomyelin (16:0) and ceramide (20:1, 26:0 and 26:1) were significantly reduced in infected macrophages. We further observed that the majority of different sub‐classes of phospholipids were downregulated significantly. Overall ratio of phosphatidylcholine versus phosphotidylethanolamine was decreased which indicated the compensatory mechanism of cell in response to cholesterol reduction. The observed Leishmania‐mediated alteration in macrophage‐lipidome provided the novel insights into mechanism of host‐pathogen interactions. [ABSTRACT FROM AUTHOR]

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy. [ABSTRACT FROM AUTHOR]

The antioxidant defense is critical for the survival of intracellular pathogens such as Mycobacterium tuberculosis complex (MTBC) species, including Mycobacterium bovis, which are often exposed to an oxidative environment caused by reactive oxygen species (ROS) in hosts. However, the signaling pathway in mycobacteria for sensing and responding to oxidative stress remains largely unclear. In this study, we characterize a TetR-type transcription regulator BCG_3893c, designated AotM, as a novel redox sensor in Mycobacterium bovis that increases mycobacterial tolerance to oxidative stress. AotM is required for the growth of M. bovis in the presence of 1 mM hydrogen peroxide. Loss of the aotM gene leads to altered transcriptional profiles with 352 genes significantly up-regulated and 25 genes significantly down-regulated. AotM recognizes a 14-bp palindrome sequence motif and negatively regulates the expression of a FAD-dependent oxidoreductase encoded by bcg_3892c. Overexpression of BCG_3892c increases intracellular ROS production and reduces the growth of M. bovis. In summary, we propose that AotM enhances the mycobacterial resistance against oxidative stress probably by inhibiting intracellular ROS production. Our findings reveal a novel underlying regulatory mechanism behind mycobacterial oxidative stress adaptation. [ABSTRACT FROM AUTHOR]

Autophagy is a unique catabolic process that degrades irrelevant or damaged components in eukaryotic cells to maintain homeostasis and eliminate infections from pathogenesis. Pathogenic bacteria have developed many autophagy manipulation techniques that affect host immune responses and intracellular bacterial pathogens have evolved to avoid xenophagy. However, reducing its effectiveness as an innate immune response has not yet been elucidated. Bacterial pathogens cause autophagy in infected cells as a cell-autonomous defense mechanism to eliminate the pathogen. However, harmful bacteria have learned to control autophagy and defeat host defenses. Intracellular bacteria can stimulate and control autophagy, while others inhibit it to prevent xenophagy and lysosomal breakdown. This review evaluates the putative functions for xenophagy in regulating bacterial infection, emphasizing that successful pathogens have evolved strategies to disrupt or exploit this defense, reducing its efficiency in innate immunity. Instead, animal models show that autophagy-associated proteins influence bacterial pathogenicity outside of xenophagy. We also examine the consequences of the complex interaction between autophagy and bacterial pathogens in light of current efforts to modify autophagy and develop host-directed therapeutics to fight bacterial infections. Therefore, effective pathogens have evolved to subvert or exploit xenophagy, although autophagy-associated proteins can influence bacterial pathogenicity outside of xenophagy. Finally, this review implies how the complex interaction between autophagy and bacterial pathogens affects host-directed therapy for bacterial pathogenesis. [ABSTRACT FROM AUTHOR]

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen posing a significant global public health threat. There is a pressing need for dependable and effective treatments, alongside an urgency for further research into the molecular characterization of its genome. Within the genomic landscape of Coxiella burnetii, numerous hypothetical proteins remain unidentified, underscoring the necessity for in-depth study. In this study, we conducted comprehensive in silico analyses to identify and prioritize potential hypothetical protein of Coxiella burnetii, aiming to elucidate the structure and function of uncharacterized protein. Furthermore, we delved into the physicochemical properties, localization, and molecular dynamics and simulations, and assessed the primary, secondary, and tertiary structures employing a variety of bioinformatics tools. The in-silico analysis revealed that the uncharacterized protein contains a conserved Mth938-like domain, suggesting a role in preadipocyte differentiation and adipogenesis. Subcellular localization predictions indicated its presence in the cytoplasm, implicating a significant role in cellular processes. Virtual screening identified ligands with high binding affinities, suggesting the protein's potential as a drug target against Q fever. Molecular dynamics simulations confirmed the stability of these complexes, indicating their therapeutic relevance. The findings provide a structural and functional overview of an uncharacterized protein from C. burnetii, implicating it in adipogenesis. This study underscores the power of in-silico approaches in uncovering the biological roles of uncharacterized proteins and facilitating the discovery of new therapeutic strategies. The findings provide valuable preliminary data for further investigation into the protein's role in adipogenesis. [ABSTRACT FROM AUTHOR]

Mechanosensitive ion channels play an essential role in reacting to environmental signals and sustaining cell integrity by facilitating ion flux across membranes. For obligate intracellular pathogens like microsporidia, adapting to changes in the host environment is crucial for survival and propagation. Despite representing a eukaryote of extreme genome reduction, microsporidia have expanded the gene family of mechanosensitive ion channels of small conductance (mscS) through repeated gene duplication and horizontal gene transfer. All microsporidian genomes characterized to date contain mscS genes of both eukaryotic and bacterial origin. Here, we investigated the cryo-electron microscopy structure of the bacterially derived mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, an intracellular pathogen of Caenorhabditis elegans. MscS2 is the most compact MscS-like channel known and assembles into a unique superstructure in vitro with six heptameric MscS2 channels. Individual MscS2 channels are oriented in a heterogeneous manner to one another, resembling an asymmetric, flexible six-way cross joint. Finally, we show that microsporidian MscS2 still forms a heptameric membrane channel, however the extreme compaction suggests a potential new function of this MscS-like protein. [ABSTRACT FROM AUTHOR]

In the decades since the discovery, Type I interferon (IFN-I) has been intensively studied for their antiviral activity. However, increasing evidences suggest that it may also play an important role in the infection of Toxoplasma gondii, a model organism for intracellular parasites. Recent studies demonstrated that the induction of IFN-I by the parasite depends on cell type, strain genotype, and mouse strain. IFN-I can inhibit the proliferation of T. gondii, but few studies showed that it is beneficial to the growth of the parasite. Meanwhile, T. gondii also can secrete proteins that impact the pathway of IFN-I production and downstream induced interferon-stimulated genes (ISGs) regulation, thereby escaping immune destruction by the host. This article reviews the major findings and progress in the production, function, and regulation of IFN-I during T. gondii infection, to thoroughly understand the innate immune mechanism of T. gondii infection, which provides a new target for subsequent intervention and treatment. [ABSTRACT FROM AUTHOR]

Apoptosis, a major form of programmed cell death, is an essential component of host defense against invading intracellular pathogens. Viruses encode inhibitors of apoptosis to evade host responses during infection, and to support their own replication and survival. Therefore, hosts and their viruses are entangled in a constant evolutionary arms race to control apoptosis. Until now, apoptosis in the context of the antiviral immune system has been almost exclusively studied in vertebrates. This limited phyletic sampling makes it impossible to determine whether a similar mechanism existed in the last common ancestor of animals. Here, we established assays to probe apoptosis in the sea anemone Nematostella vectensis, a model species of Cnidaria, a phylum that diverged approximately 600 million years ago from the rest of animals. We show that polyinosinic:polycytidylic acid (poly I:C), a synthetic long double-stranded RNA mimicking viral RNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), is sufficient to induce apoptosis in N. vectensis. Furthermore, at the transcriptomic level, apoptosis related genes are significantly enriched upon poly(I:C) exposure in N. vectensis as well as bilaterian invertebrates. Our phylogenetic analysis of caspase family genes in N. vectensis reveals conservation of all four caspase genes involved in apoptosis in mammals and revealed a cnidarian-specific caspase gene which was strongly upregulated. Altogether, our findings suggest that apoptosis in response to a viral challenge is a functionally conserved mechanism that can be traced back to the last common ancestor of Bilateria and Cnidaria. Author summary: Apoptosis is a programmed cell death mechanism used by vertebrates to efficiently block viral infection. The presence of long double-stranded RNA (dsRNA) in the cytosol is a key feature of DNA and RNA virus replication and is absent from uninfected mammalian host cells. Therefore, the ability to sense and respond to viral dsRNAs is crucial for organismal survival. Indeed, numerous studies in mammals have shown that dsRNA has the capacity to trigger a robust apoptosis as antiviral response. However, such studies were limited to vertebrates, and it remained largely unclear how such systems have evolved. Here, we used the sea anemone Nematostella vectensis, member of phylum Cnidaria (jellyfish, corals, hydroids and sea anemones), to address this topic. We demonstrated that indeed dsRNA is sufficient to induce apoptosis in Nematostella and uncovered a conserved network of genes involved in this process. Further, by comparing the results of gene expression analyses in sea anemones and other diverse animal groups such as oysters and lancelets, we show that apoptosis is prevalent in many animal groups and was already part of the response to dsRNA in the last common ancestor of Cnidaria and most other animals that lived 600 million years ago. [ABSTRACT FROM AUTHOR]

Pathogenic bacteria secrete protein effectors to hijack host machinery and remodel their infectious niche. Rickettsia spp. are obligate intracellular bacteria that can cause life-threatening disease, but their absolute dependence on the host cell has impeded discovery of rickettsial effectors and their host targets. We implemented bioorthogonal non-canonical amino acid tagging (BONCAT) during R. parkeri infection to selectively label, isolate, and identify effectors delivered into the host cell. As the first use of BONCAT in an obligate intracellular bacterium, our screen more than doubles the number of experimentally validated effectors for the genus. The seven novel secreted rickettsial factors (Srfs) we identified include Rickettsia-specific proteins of unknown function that localize to the host cytoplasm, mitochondria, and ER. We further show that one such effector, SrfD, interacts with the host Sec61 translocon. Altogether, our work uncovers a diverse set of previously uncharacterized rickettsial effectors and lays the foundation for a deeper exploration of the host-pathogen interface. Rickettsia species secrete effector proteins into host cells to promote infection, but few effectors have been identified. Here, Sanderlin et al use a protein labeling approach to identify an expanded repertoire of effectors acting at the host-pathogen interface. [ABSTRACT FROM AUTHOR]

The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation. The bacterial pathogen Legionella pneumophila secretes effectors that affect protein ubiquitination within host cells, involving production of phosphoribosyl ubiquitin (PR-Ub). Here, the authors identify additional effectors that convert PR-Ub back into functional ubiquitin, thus maintaining ubiquitin homeostasis in infected cells. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a re-emerging food-borne zoonosis of warm-blooded animals caused by Toxoplasma gondii , an obligate intracellular parasite with a complex biological cycle. Until now, the genotypic approach for discovering putative resistance immuno-genetic markers has never been adopted for T. gondii infection. Thus, this study aimed, for the first time, to deepen these novel insights. In particular, pigs, reared in different farms in Central Italy, were phenotypically characterized by serological assays performed on diaphragm meat juice. Out of 179 tested animals, 98 resulted seropositive (54.74%), 57 seronegative (31.84%), and 24 doubtful (13.40%), underlining a possible re-emerging diffusion of this protozoan in the investigated areas. Contextually, an RT-PCR assay, followed by Sanger sequencing for IL-18 , a pro-inflammatory cytokine with a key role upstream of the infection, was developed. At this stage, for this interleukin, significant polymorphic variations were not detected compared to the reference sequence, except in a seropositive animal. However, the starting outcomes of this novel and preliminary study will be investigated in depth with different approaches also on other target genes, with a crucial activity in the immunity pathway towards T. gondii , to unveil the possible presence of resistance genetic mechanisms and, finally, to make pork derived food safer. [ABSTRACT FROM AUTHOR]

IFN‐γ plays a critical role in host defense against intracellular pathogens. IFN‐γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN‐γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN‐γ has beneficial effects on eradication of Pneumocystis, endogenous IFN‐γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell‐depleted IFN‐γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin‐1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN‐γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis‐phagocytic activity. These results indicate that endogenous IFN‐γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin‐1. [ABSTRACT FROM AUTHOR]