The varieties of immunological experience: of pathogens, stress, and dendritic cells.

In the 40 years since their discovery, dendritic cells (DCs) have been recognized as central players in immune regulation. DCs sense microbial stimuli through pathogen-recognition receptors (PRRs) and decode, integrate, and present information derived from such stimuli to T cells, thus stimulating immune responses. DCs can also regulate the quality of immune responses. Several functionally specialized subsets of DCs exist, but DCs also display functional plasticity in response to diverse stimuli. In addition to sensing pathogens via PRRs, emerging evidence suggests that DCs can also sense stress signals, such as amino acid starvation, through ancient stress and nutrient sensing pathways, to stimulate adaptive immunity. Here, I discuss these exciting advances in the context of a historic perspective on the discovery of DCs and their role in immune regulation. I conclude with a discussion of emerging areas in DC biology in the systems immunology era and suggest that the impact of DCs on immunity can be usefully contextualized in a hierarchy-of-organization model in which DCs, their receptors and signaling networks, cell-cell interactions, tissue microenvironment, and the host macroenvironment represent different levels of the hierarchy. Immunity or tolerance can then be represented as a complex function of each of these hierarchies.

Virome characterization of game animals in China reveals a spectrum of emerging pathogens.

Game animals are wildlife species traded and consumed as food and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1,941 game animals, representing 18 species and five mammalian orders, sampled across China. From this, we identified 102 mammalian-infecting viruses, with 65 described for the first time. Twenty-one viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high-risk viruses. We inferred the transmission of bat-associated coronavirus from bats to civets, as well as cross-species jumps of coronaviruses from bats to hedgehogs, from birds to porcupines, and from dogs to raccoon dogs. Of note, we identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

The Genomics and Cell Biology of Host-Beneficial Intracellular Infections.

Microbes gain access to eukaryotic cells as food for bacteria-grazing protists, for host protection by microbe-killing immune cells, or for microbial benefit when pathogens enter host cells to replicate. But microbes can also gain access to a host cell and become an important-often required-beneficial partner. The oldest beneficial microbial infections are the ancient eukaryotic organelles now called the mitochondrion and plastid. But numerous other host-beneficial intracellular infections occur throughout eukaryotes. Here I review the genomics and cell biology of these interactions with a focus on intracellular bacteria. The genomes of host-beneficial intracellular bacteria have features that span a previously unfilled gap between pathogens and organelles. Host cell adaptations to allow the intracellular persistence of beneficial bacteria are found along with evidence for the microbial manipulation of host cells, but the cellular mechanisms of beneficial bacterial infections are not well understood.

Mucosal plasma cells are required to protect the upper airway and brain from infection.

While blood antibodies mediate protective immunity in most organs, whether they protect nasal surfaces in the upper airway is unclear. Using multiple viral infection models in mice, we found that blood-borne antibodies could not defend the olfactory epithelium. Despite high serum antibody titers, pathogens infected nasal turbinates, and neurotropic microbes invaded the brain. Using passive antibody transfers and parabiosis, we identified a restrictive blood-endothelial barrier that excluded circulating antibodies from the olfactory mucosa. Plasma cell depletions demonstrated that plasma cells must reside within olfactory tissue to achieve sterilizing immunity. Antibody blockade and genetically deficient models revealed that this local immunity required CD4+ T cells and CXCR3. Many vaccine adjuvants failed to generate olfactory plasma cells, but mucosal immunizations established humoral protection of the olfactory surface. Our identification of a blood-olfactory barrier and the requirement for tissue-derived antibody has implications for vaccinology, respiratory and CNS pathogen transmission, and B cell fate decisions.

Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination.

Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.

The mechanosensor Piezo1 rings the alarm on epithelial intruders.

Recognition of invasive pathogens by the epithelium that is constantly exposed to microbial products remains incompletely understood. In a recent study, Tadala et al. demonstrated that the entry process of intracellular bacteria is itself a mechanical signal that is detected by the stretch-activated channel Piezo1, which triggers innate immune signaling.

Genomic Approaches to Antifungal Drug Target Identification and Validation.

The last several decades have witnessed a surge in drug-resistant fungal infections that pose a serious threat to human health. While there is a limited arsenal of drugs that can be used to treat systemic infections, scientific advances have provided renewed optimism for the discovery of novel antifungals. The development of chemical-genomic assays using Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of molecules in a living cell. Advances in molecular biology techniques have enabled complementary assays to be developed in fungal pathogens, including Candida albicans and Cryptococcus neoformans. These approaches enable the identification of target genes for drug candidates, as well as genes involved in buffering drug target pathways. Here, we examine yeast chemical-genomic assays and highlight how such resources can be utilized to predict the mechanisms of action of compounds, to study virulence attributes of diverse fungal pathogens, and to bolster the antifungal pipeline.

Modulation of host immunity by sensory neurons.

Recent studies have uncovered a new role for sensory neurons in influencing mammalian host immunity, challenging conventional notions of the nervous and immune systems as separate entities. In this review we delve into this groundbreaking paradigm of neuroimmunology and discuss recent scientific evidence for the impact of sensory neurons on host responses against a wide range of pathogens and diseases, encompassing microbial infections and cancers. These valuable insights enhance our understanding of the interactions between the nervous and immune systems, and also pave the way for developing candidate innovative therapeutic interventions in immune-mediated diseases highlighting the importance of this interdisciplinary research field.

"Under Pressure" - How fungi evade, exploit, and modulate cells of the innate immune system.

The human immune system uses an arsenal of effector mechanisms to prevent and counteract infections. Yet, some fungal species are extremely successful as human pathogens, which can be attributed to a wide variety of strategies by which these fungi evade, exploit, and modulate the immune system. These fungal pathogens normally are either harmless commensals or environmental fungi. In this review we discuss how commensalism, but also life in an environmental niche without human contact, can drive the evolution of diverse and specialized immune evasion mechanisms. Correspondingly, we discuss the mechanisms contributing to the ability of these fungi to cause superficial to life-threatening infections.

Identification of an active RNAi pathway in Candida albicans.

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Glucuronic acid confers colonization advantage to enteric pathogens.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

Ancient pathogens provide a window into health and well-being.

This perspective draws on the record of ancient pathogen genomes and microbiomes illuminating patterns of infectious disease over the course of the Holocene in order to address the following question. How did major changes in living circumstances involving the transition to and intensification of farming alter pathogens and their distributions? Answers to this question via ancient DNA research provide a rapidly expanding picture of pathogen evolution and in concert with archaeological and historical data, give a temporal and behavioral context for heath in the past that is relevant for challenges facing the world today, including the rise of novel pathogens.

Sialic acid plays a pivotal role in licensing Citrobacter rodentium's transition from the intestinal lumen to a mucosal adherent niche.

Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses and an established microbiota remain poorly defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy likely involves metabolic adaptation to the host's intestinal luminal environment, as a necessary precursor to reach and infect the mucosal surface. Suspecting this adaptation involved the intestinal mucus layer, we found that C. rodentium was able to catabolize sialic acid, a monosaccharide derived from mucins, and utilize it as its sole carbon source for growth. Moreover, C. rodentium also sensed and displayed chemotactic activity toward sialic acid. These activities were abolished when the nanT gene, encoding a sialic acid transporter, was deleted (ΔnanT). Correspondingly, the ΔnanT C. rodentium strain was significantly impaired in its ability to colonize the murine intestine. Intriguingly, sialic acid was also found to induce the secretion of two autotransporter proteins, Pic and EspC, which possess mucinolytic and host-adherent properties. As a result, sialic acid enhanced the ability of C. rodentium to degrade intestinal mucus (through Pic), as well as to adhere to intestinal epithelial cells (through EspC). We thus demonstrate that sialic acid, a monosaccharide constituent of the intestinal mucus layer, functions as an important nutrient and a key signal for an A/E bacterial pathogen to escape the colonic lumen and directly infect its host's intestinal mucosa.

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses.

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructural protein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells in vitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.

Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Translocation expands the scope of the large clostridial toxin family.

Large clostridial toxins (LCTs) are a family of six homologous disease-causing proteins characterised by their large size (>200 kDa) and conserved multidomain architectures. Using their central translocation and receptor-binding domain (T domain), LCTs bind host cell receptors and translocate their upstream glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol. The recent discovery of hundreds of LCT-like T domains in diverse genomic contexts and domain architectures from bacteria other than clostridia has provided significant new insights into the enigmatic process of LCT translocation, but also has put the definition of what constitutes an LCT into question. In this opinion article, we discuss how these findings have expanded our understanding of LCT translocation and reshaped the scope of the LCT family.

Fatal attraction: How Phytophthora zoospores find their host.

Oomycete plant pathogens, such as Phytophthora and Pythium species produce motile dispersal agents called zoospores that actively target host plants. Zoospores are exceptional in their ability to display taxis to chemical, electrical and physical cues to navigate the phyllosphere and reach stomata, wound sites and roots. Many components of root exudates have been shown attractive or repulsive to zoospores. Although some components possess very strong attractiveness, it seems that especially the mix of components exuded by the primary host is most attractive to zoospores. Zoospores actively approach attractants with swimming behaviour reminiscent of other microswimmers. To achieve a unified description of zoospore behaviour when sensing an attractant, we propose the following terms for the successive stages of the homing response: reorientation, approaching, retention and settling. How zoospores sense and process attractants is poorly understood but likely involves signal perception via cell surface receptors. Since zoospores are important for infection, undermining their activity by luring attractants or blocking receptors seem promising strategies for disease control.

Recent developments in plant-downy mildew interactions.

Downy mildews are obligate oomycete pathogens that attack a wide range of plants and can cause significant economic impacts on commercial crops and ornamental plants. Traditionally, downy mildew disease control relied on an integrated strategies, that incorporate cultural practices, deployment of resistant cultivars, crop rotation, application of contact and systemic pesticides, and biopesticides. Recent advances in genomics provided data that significantly advanced understanding of downy mildew evolution, taxonomy and classification. In addition, downy mildew genomics also revealed that these obligate oomycetes have reduced numbers of virulence factor genes in comparison to hemibiotrophic and necrotrophic oomycetes. However, downy mildews do deploy significant arrays of virulence proteins, including so-called RXLR proteins that promote virulence or are recognized as avirulence factors. Pathogenomics are being applied to downy mildew population studies to determine the genetic diversity within the downy mildew populations and manage disease by selection of appropriate varieties and management strategies. Genome editing technologies have been used to manipulate host disease susceptibility genes in different plants including grapevine and sweet basil and thereby provide new soucres of resistance genes against downy mildews. Previously, it has proved difficult to transform and manipulate downy mildews because of their obligate lifestyle. However, recent exploitation of RNA interference machinery through Host-Induced Gene Silencing (HIGS) and Spray-Induced Gene Silencing (SIGS) indicate that functional genomics in downy mildews is now possible. Altogether, these breakthrough technologies and attendant fundamental understanding will advance our ability to mitigate downy mildew diseases.