MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria.

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Identification of type VI secretion system effector-immunity pairs using structural bioinformatics.

The type VI secretion system (T6SS) is an important mediator of microbe-microbe and microbe-host interactions. Gram-negative bacteria use the T6SS to inject T6SS effectors (T6Es), which are usually proteins with toxic activity, into neighboring cells. Antibacterial effectors have cognate immunity proteins that neutralize self-intoxication. Here, we applied novel structural bioinformatic tools to perform systematic discovery and functional annotation of T6Es and their cognate immunity proteins from a dataset of 17,920 T6SS-encoding bacterial genomes. Using structural clustering, we identified 517 putative T6E families, outperforming sequence-based clustering. We developed a logistic regression model to reliably quantify protein-protein interaction of new T6E-immunity pairs, yielding candidate immunity proteins for 231 out of the 517 T6E families. We used sensitive structure-based annotation which yielded functional annotations for 51% of the T6E families, again outperforming sequence-based annotation. Next, we validated four novel T6E-immunity pairs using basic experiments in E. coli. In particular, we showed that the Pfam domain DUF3289 is a homolog of Colicin M and that DUF943 acts as its cognate immunity protein. Furthermore, we discovered a novel T6E that is a structural homolog of SleB, a lytic transglycosylase, and identified a specific glutamate that acts as its putative catalytic residue. Overall, this study applies novel structural bioinformatic tools to T6E-immunity pair discovery, and provides an extensive database of annotated T6E-immunity pairs.

Immunological functional differentiation of two transmembrane variants of Dscam in Chinese mitten crab.

Down syndrome cell adhesion molecule (Dscam), also called hypervariable Dscam (Dscam-hv), is an important player in arthropod alternative splicing that connects neurons and immune regulation, acting as a pathogen-specific recognition molecule. Dscam-hv has two forms: transmembrane (TM) Dscam (mDscam) and soluble Dscam (sDscam). Herein, we investigated two transmembrane variants of mDscam resulting from alternative splicing of the transmembrane domain, focusing on differences in their immune regulation. We characterized the Dscam[TM1] and Dscam[TM2] genes of Chinese mitten crab (Eriocheir sinensis) through bioinformatics analysis. Both genes are expressed in the gill, intestine, and other immune tissues. Following gram-positive and gram-negative bacteria stimulation, EsDscam[TM1] and EsDscam[TM2] mRNA expression levels increased significantly in hemocytes. Sequencing showed that EsDscam[TM1] was more abundant in hemocytes than EsDscam[TM2]. Additionally, the two subtypes differ in their regulation of antimicrobial peptides, the proportion of exon 33 carried, and bacterial phagocytosis.

Insights from targeting transferrin receptors to develop vaccines for pathogens of humans and food production animals.

While developing vaccines targeting surface transferrin receptor proteins in Gram-negative pathogens of humans and food production animals, the common features derived from their evolutionary origins has provided us with insights on how improvements could be implemented in the various stages of research and vaccine development. These pathogens are adapted to live exclusively on the mucosal surfaces of the upper respiratory or genitourinary tract of their host and rely on their receptors to acquire iron from transferrin for survival, indicating that there likely are common mechanisms for delivering transferrin to the mucosal surfaces that should be explored. The modern-day receptors are derived from those present in bacteria that lived over 320 million years ago. The pathogens represent the most host adapted members of their bacterial lineages and may possess factors that enable them to have strong association with the mucosal epithelial cells, thus likely reside in a different niche than the commensal members of the bacterial lineage. The bacterial pathogens normally lead a commensal lifestyle which presents challenges for development of relevant infection models as most infection models either exclude the early stages of colonization or subsequent disease development, and the immune mechanisms at the mucosal surface that would prevent disease are not evident. Development of infection models emulating natural horizontal disease transmission are also lacking. Our aim is to share our insights from the study of pathogens of humans and food production animals with individuals involved in vaccine development, maintaining health or regulation of products in the human and animal health sectors.

JAK2-STAT Epigenetically Regulates Tolerized Genes in Monocytes in the First Encounter With Gram-Negative Bacterial Endotoxins in Sepsis.

Microbial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.

Bacterial Outer Membrane Vesicles as a Versatile Tool in Vaccine Research and the Fight against Antimicrobial Resistance.

Gram-negative bacteria include a number of pathogens that cause disease in humans and animals. Although antibiotics are still effective in treating a considerable range of infections caused by Gram-negative bacteria, the alarming increase of antimicrobial resistance (AMR) induced by excessive use of antibiotics has raised global concerns. Therefore, alternative strategies must be developed to prevent and treat bacterial infections and prevent the advent of a postantibiotic era. Vaccines, one of the greatest achievements in the history of medical science, hold extraordinary potential to prevent bacterial infections and thereby reduce the need for antibiotics. Novel bacterial vaccines are urgently needed, however, and outer membrane vesicles (OMVs), naturally produced by Gram-negative bacteria, represent a promising and versatile tool that can be employed as adjuvants, antigens, and delivery platforms in the development of vaccines against Gram-negative bacteria. Here, we provide an overview of the many roles OMVs can play in vaccine development and the mechanisms behind these applications. Methods to improve OMV yields and a comparison of different strategies for OMV isolation aiming at cost-effective production of OMV-based vaccines are also reviewed.

Microbiota Signals during the Neonatal Period Forge Life-Long Immune Responses.

The microbiota regulates immunological development during early human life, with long-term effects on health and disease. Microbial products include short-chain fatty acids (SCFAs), formyl peptides (FPs), polysaccharide A (PSA), polyamines (PAs), sphingolipids (SLPs) and aryl hydrocarbon receptor (AhR) ligands. Anti-inflammatory SCFAs are produced by Actinobacteria, Bacteroidetes, Firmicutes, Spirochaetes and Verrucomicrobia by undigested-carbohydrate fermentation. Thus, fiber amount and type determine their occurrence. FPs bind receptors from the pattern recognition family, those from commensal bacteria induce a different response than those from pathogens. PSA is a capsular polysaccharide from B. fragilis stimulating immunoregulatory protein expression, promoting IL-2, STAT1 and STAT4 gene expression, affecting cytokine production and response modulation. PAs interact with neonatal immunity, contribute to gut maturation, modulate the gut-brain axis and regulate host immunity. SLPs are composed of a sphingoid attached to a fatty acid. Prokaryotic SLPs are mostly found in anaerobes. SLPs are involved in proliferation, apoptosis and immune regulation as signaling molecules. The AhR is a transcription factor regulating development, reproduction and metabolism. AhR binds many ligands due to its promiscuous binding site. It participates in immune tolerance, involving lymphocytes and antigen-presenting cells during early development in exposed humans.

GMMA-Based Vaccines: The Known and The Unknown.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria engineered to provide an over-vesiculating phenotype, which represent an attractive platform for the design of affordable vaccines. GMMA can be further genetically manipulated to modulate the risk of systemic reactogenicity and to act as delivery system for heterologous polysaccharide or protein antigens. GMMA are able to induce strong immunogenicity and protection in animal challenge models, and to be well-tolerated and immunogenic in clinical studies. The high immunogenicity could be ascribed to their particulate size, to their ability to present to the immune system multiple antigens in a natural conformation which mimics the bacterial environment, as well as to their intrinsic self-adjuvanticity. However, GMMA mechanism of action and the role in adjuvanticity are still unclear and need further investigation. In this review, we discuss progresses in the development of the GMMA vaccine platform, highlighting successful applications and identifying knowledge gaps and potential challenges.

A human apolipoprotein L with detergent-like activity kills intracellular pathogens.

Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.

Staphylococcus aureus Vaccine Research and Development: The Past, Present and Future, Including Novel Therapeutic Strategies.

Staphylococcus aureus is one of the most important human pathogens worldwide. Its high antibiotic resistance profile reinforces the need for new interventions like vaccines in addition to new antibiotics. Vaccine development efforts against S. aureus have failed so far however, the findings from these human clinical and non-clinical studies provide potential insight for such failures. Currently, research is focusing on identifying novel vaccine formulations able to elicit potent humoral and cellular immune responses. Translational science studies are attempting to discover correlates of protection using animal models as well as in vitro and ex vivo models assessing efficacy of vaccine candidates. Several new vaccine candidates are being tested in human clinical trials in a variety of target populations. In addition to vaccines, bacteriophages, monoclonal antibodies, centyrins and new classes of antibiotics are being developed. Some of these have been tested in humans with encouraging results. The complexity of the diseases and the range of the target populations affected by this pathogen will require a multipronged approach using different interventions, which will be discussed in this review.

Two atypical gram-negative bacteria-binding proteins are involved in the antibacterial response in the pea aphid (Acyrthosiphon pisum).

The activation of immune pathways is triggered by the recognition of pathogens by pattern recognition receptors (PRRs). Gram-negative bacteria-binding proteins (GNBPs)/ß-1,3-glucan recognition proteins (ßGRPs) are a conserved family of PRRs in insects. Two GNBPs are predicted in the genome database of pea aphids; however, little is known about their functions in the aphid immune system. Here, we show that pea aphid GNBPs possess domain architectures and sequence features distinct from those of typical GNBPs/ßGRPs and that their expression is induced by bacterial infection. Knockdown of their expression by dsRNA resulted in lower phenoloxidase activity, higher bacterial loads and higher mortality in aphids after infection. Our data suggest that these two atypical GNBPs are involved in the antibacterial response in the pea aphid, likely acting as PRRs in the prophenoloxidase pathway.

Outer Membrane Vesicles (OMVs) Produced by Gram-Negative Bacteria: Structure, Functions, Biogenesis, and Vaccine Application.

Gram-negative bacteria produce outer membrane vesicles (OMVs) with 10 to 300 nm of diameter. The contribution of OMVs to bacterial pathogenesis is a topic of great interest, and their capacity to be combined with antigens impact in the future to the development of vaccines.

Breaching the Bacterial Envelope: The Pivotal Role of Perforin-2 (MPEG1) Within Phagocytes.

The membrane attack complex (MAC) of the complement system and Perforin-1 are well characterized innate immune effectors. MAC is composed of C9 and other complement proteins that target the envelope of gram-negative bacteria. Perforin-1 is deployed when killer lymphocytes degranulate to destroy virally infected or cancerous cells. These molecules polymerize with MAC-perforin/cholesterol-dependent cytolysin (MACPF/CDC) domains of each monomer deploying amphipathic ß-strands to form pores through target lipid bilayers. In this review we discuss one of the most recently discovered members of this family; Perforin-2, the product of the Mpeg1 gene. Since their initial description more than 100 years ago, innumerable studies have made macrophages and other phagocytes some of the best understood cells of the immune system. Yet remarkably it was only recently revealed that Perforin-2 underpins a pivotal function of phagocytes; the destruction of phagocytosed microbes. Several studies have established that phagocytosed bacteria persist and in some cases flourish within phagocytes that lack Perforin-2. When challenged with either gram-negative or gram-positive pathogens Mpeg1 knockout mice succumb to infectious doses that the majority of wild-type mice survive. As expected by their immunocompromised phenotype, bacterial pathogens replicate and disseminate to deeper tissues of Mpeg1 knockout mice. Thus, this evolutionarily ancient gene endows phagocytes with potent bactericidal capability across taxa spanning sponges to humans. The recently elucidated structures of mammalian Perforin-2 reveal it to be a homopolymer that depends upon low pH, such as within phagosomes, to transition to its membrane-spanning pore conformation. Clinical manifestations of Mpeg1 missense mutations further highlight the pivotal role of Perforin-2 within phagocytes. Controversies and gaps within the field of Perforin-2 research are also discussed as well as animal models that may be used to resolve the outstanding issues. Our review concludes with a discussion of bacterial counter measures against Perforin-2.

Ex vivo tumor necrosis factor-alpha response of blood leukocytes in Danish Holstein-Friesian cows stimulated by Gram-positive and Gram-negative bacteria isolated from mastitic milk.

A whole blood stimulation assay was used to investigate the effects of parity, number of weeks after calving and Gram-positive and Gram-negative bacteria on the ex vivo TNF-α responsiveness of Danish Holstein-Friesian cows of first to third lactation (n = 28). Blood samples were collected in weeks 2, 3, 5 and 8 after parturition and stimulated with Escherichia coli LPS (10 µg/mL), Staphylococcus aureus peptidoglycan (PGN, 10 µg/mL) and dead Escherichia coli, Streptococcus uberis, Staphylococcus aureus, and Streptococcus dysgalactiae at a concentration of 2.5 × 106/mL. The antibiotic polymyxin-B (100 µg/mL) was added to the Gram-positive bacteria to avoid the influence of environmental endotoxin by ELISA test. Overall, parity had no effect, whereas number of weeks after calving altered the TNF-α responsiveness of the majority of the stimulants. Ex vivo, Gram-positive bacteria always resulted in a higher TNF-α response than Gram-negative bacteria with large differences within the individual cows. High correlations were found within the Gram-negative stimulants panel (r = 0.83) and within the Gram-positive (r = 0.81 to 0.86) stimulants panel except PGN. The higher TNF-α responsiveness by Gram-positive bacteria is in agreement with in vitro studies in human but in contrast to the in vivo TNF-α responsiveness in bovine udder.

ELAVL1a is an immunocompetent protein that protects zebrafish embryos from bacterial infection.

Previous studies have shown that ELAVL1 plays multiple roles, but its overall biological function remains ill-defined. Here we clearly demonstrated that zebrafish ELAVL1a was a lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs/embryos of zebrafish. ELAVL1a acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, as well as bacteria, but also as an effector molecule, capable of inhibiting the growth of Gram-positive and -negative bacteria. Furthermore, we reveal that the C-terminal 62 residues of ELAVL1a positioned at 181-242 were indispensable for ELAVL1a antibacterial activity. Additionally, site-directed mutagenesis revealed that the hydrophobic residues Val192/Ile193, as well as the positively charged residues Arg203/Arg204, were the functional determinants contributing to the antimicrobial activity of rELAVL1a. Importantly, microinjection of rELAVL1a into embryos markedly promoted their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was considerably reduced by co-injection of anti-ELAVL1a antibody or by knockdown with morpholino for elavl1a. Collectively, our results indicate that ELAVL1a is a maternal immune factor that can protect zebrafish embryos from bacterial infection. This work also provides another angle for understanding the biological roles of ELAVL1a.

More than a Pore: Nonlytic Antimicrobial Functions of Complement and Bacterial Strategies for Evasion.

The complement system is an evolutionarily ancient defense mechanism against foreign substances. Consisting of three proteolytic activation pathways, complement converges on a common effector cascade terminating in the formation of a lytic pore on the target surface. The classical and lectin pathways are initiated by pattern recognition molecules binding to specific ligands, while the alternative pathway is constitutively active at low levels in circulation. Complement-mediated killing is essential for defense against many Gram-negative bacterial pathogens, and genetic deficiencies in complement can render individuals highly susceptible to infection, for example, invasive meningococcal disease. In contrast, Gram-positive bacteria are inherently resistant to the direct bactericidal activity of complement due to their thick layer of cell wall peptidoglycan. However, complement also serves diverse roles in immune defense against all bacteria by flagging them for opsonization and killing by professional phagocytes, synergizing with neutrophils, modulating inflammatory responses, regulating T cell development, and cross talk with coagulation cascades. In this review, we discuss newly appreciated roles for complement beyond direct membrane lysis, incorporate nonlytic roles of complement into immunological paradigms of host-pathogen interactions, and identify bacterial strategies for complement evasion.

Compensatory intestinal immunoglobulin response after vancomycin treatment in humans.

Intestinal immunoglobulins (Ig) are abundantly secreted antibodies that bind bacteria and bacterial components in the gut. This binding is considered to accelerate bacterial transit time and prevent the interaction of potentially immunogenic compounds with intestinal immune cells. Ig secretion is regulated by alterations in gut microbiome composition, an event rarely mapped in an intervention setting in humans. Here, we determined the intestinal and systemic Ig response to a major intervention in gut microbiome composition. Healthy humans and humans with metabolic syndrome received oral vancomycin 500 mg four times per day for 7 days. Coinciding with a vancomycin-induced increase in Gram-negative bacteria, fecal levels of the immunogenic bacterial components lipopolysaccharide (LPS) and flagellin drastically increased. Intestinal antibodies (IgA and IgM) significantly increased, whereas peripheral antibodies (IgG, IgA, and IgM) were mostly unaffected by vancomycin treatment. Bacterial cell sorting followed by 16S rRNA sequencing revealed that the majority of Gram-negative bacteria, including opportunistic pathogens, were IgA-coated after the intervention. We suggest that the intestinal Ig response after vancomycin treatment prevents the intrusion of pathogens and bacterial components into systemic sites.

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes.

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.

Planarian gamma-interferon-inducible lysosomal thiol reductase (GILT) is required for gram-negative bacterial clearance.

The powerful regenerative ability of planarians has long been a concern of scientists, but recently, their efficient immune system has attracted more and more attention from researchers. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is related not only to antigen presentation but also to bacteria invasions. But the systematic studies are not yet to be conducted on the relationship between bacterial infection. Our study reveals for the first time that GILT of planarian (DjGILT) plays an essential role in the clearance of Gram-negative bacteria by conducting H2O2 concentration in planarians. In animals that DjGILT was silenced, it persisted for up to 9 days before all bacteria were cleared, compared with 6 days of the control group. When infected with E. coli and V. anguillarum, the level of H2O2 was significantly increased in DjGILT-silenced planarians, and concomitantly, mRNA level of C-type lectin DjCTL, which modulates agglutination and clearance efficiency of invading bacteria, was decreased. Further study showed that the decrease of H2O2 level led to a significant increase in DjCTL transcripts. Collectively, we proposed a mechanism model for the involvement of GILT gene in bacterial elimination. We have for the first time revealed the specific mechanism of GILT in innate immune response against bacterial infection.

ADP-heptose: a bacterial PAMP detected by the host sensor ALPK1.

The innate immune response constitutes the first line of defense against pathogens. It involves the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), the production of inflammatory cytokines and the recruitment of immune cells to infection sites. Recently, ADP-heptose, a soluble intermediate of the lipopolysaccharide biosynthetic pathway in Gram-negative bacteria, has been identified by several research groups as a PAMP. Here, we recapitulate the evidence that led to this identification and discuss the controversy over the immunogenic properties of heptose 1,7-bisphosphate (HBP), another bacterial heptose previously defined as an activator of innate immunity. Then, we describe the mechanism of ADP-heptose sensing by alpha-protein kinase 1 (ALPK1) and its downstream signaling pathway that involves the proteins TIFA and TRAF6 and induces the activation of NF-κB and the secretion of inflammatory cytokines. Finally, we discuss possible delivery mechanisms of ADP-heptose in cells during infection, and propose new lines of thinking to further explore the roles of the ADP-heptose/ALPK1/TIFA axis in infections and its potential implication in the control of intestinal homeostasis.