Toxigenic Clostridium perfringens isolated from at-risk pediatric inflammatory bowel disease patients.

BACKGROUND AND AIMS: The goal was to identify microbial drivers of IBD, by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from pediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying C. perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in 8 of 9 patients' mucosal biopsies, correlating with hemolytic activity, while not in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults (18.7-27.1%) versus healthy (5.1%). In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial, neuroblasts, and neutrophils, while impact on epithelial cells was less pronounced, suggesting C. perfringens may be damaging particularly when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed PFO toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Alternative therapeutic strategies to treat antibiotic-resistant pathogens.

Resistance threatens to render antibiotics - which are essential for modern medicine - ineffective, thus posing a threat to human health. The discovery of novel classes of antibiotics able to overcome resistance has been stalled for decades, with the developmental pipeline relying almost entirely on variations of existing chemical scaffolds. Unfortunately, this approach has been unable to keep pace with resistance evolution, necessitating new therapeutic strategies. In this Review, we highlight recent efforts to discover non-traditional antimicrobials, specifically describing the advantages and limitations of antimicrobial peptides and macrocycles, antibodies, bacteriophages and antisense oligonucleotides. These approaches have the potential to stem the tide of resistance by expanding the physicochemical property space and target spectrum occupied by currently approved antibiotics.

Bacterial biofilms in the human body: prevalence and impacts on health and disease.

Bacterial biofilms can be found in most environments on our planet, and the human body is no exception. Consisting of microbial cells encased in a matrix of extracellular polymers, biofilms enable bacteria to sequester themselves in favorable niches, while also increasing their ability to resist numerous stresses and survive under hostile circumstances. In recent decades, biofilms have increasingly been recognized as a major contributor to the pathogenesis of chronic infections. However, biofilms also occur in or on certain tissues in healthy individuals, and their constituent species are not restricted to canonical pathogens. In this review, we discuss the evidence for where, when, and what types of biofilms occur in the human body, as well as the diverse ways in which they can impact host health under homeostatic and dysbiotic states.

Returning to Nature for the Next Generation of Antimicrobial Therapeutics.

Antibiotics found in and inspired by nature are life-saving cures for bacterial infections and have enabled modern medicine. However, the rise in resistance necessitates the discovery and development of novel antibiotics and alternative treatment strategies to prevent the return to a pre-antibiotic era. Once again, nature can serve as a source for new therapies in the form of natural product antibiotics and microbiota-based therapies. Screening of soil bacteria, particularly actinomycetes, identified most of the antibiotics used in the clinic today, but the rediscovery of existing molecules prompted a shift away from natural product discovery. Next-generation sequencing technologies and bioinformatics advances have revealed the untapped metabolic potential harbored within the genomes of environmental microbes. In this review, we first highlight current strategies for mining this untapped chemical space, including approaches to activate silent biosynthetic gene clusters and in situ culturing methods. Next, we describe how using live microbes in microbiota-based therapies can simultaneously leverage many of the diverse antimicrobial mechanisms found in nature to treat disease and the impressive efficacy of fecal microbiome transplantation and bacterial consortia on infection. Nature-provided antibiotics are some of the most important drugs in human history, and new technologies and approaches show that nature will continue to offer valuable inspiration for the next generation of antibacterial therapeutics.

IL-22 alters gut microbiota composition and function to increase aryl hydrocarbon receptor activity in mice and humans.

BACKGROUND: IL-22 is induced by aryl hydrocarbon receptor (AhR) signaling and plays a critical role in gastrointestinal barrier function through effects on antimicrobial protein production, mucus secretion, and epithelial cell differentiation and proliferation, giving it the potential to modulate the microbiome through these direct and indirect effects. Furthermore, the microbiome can in turn influence IL-22 production through the synthesis of L-tryptophan (L-Trp)-derived AhR ligands, creating the prospect of a host-microbiome feedback loop. We evaluated the impact IL-22 may have on the gut microbiome and its ability to activate host AhR signaling by observing changes in gut microbiome composition, function, and AhR ligand production following exogenous IL-22 treatment in both mice and humans. RESULTS: Microbiome alterations were observed across the gastrointestinal tract of IL-22-treated mice, accompanied by an increased microbial functional capacity for L-Trp metabolism. Bacterially derived indole derivatives were increased in stool from IL-22-treated mice and correlated with increased fecal AhR activity. In humans, reduced fecal concentrations of indole derivatives in ulcerative colitis (UC) patients compared to healthy volunteers were accompanied by a trend towards reduced fecal AhR activity. Following exogenous IL-22 treatment in UC patients, both fecal AhR activity and concentrations of indole derivatives increased over time compared to placebo-treated UC patients. CONCLUSIONS: Overall, our findings indicate IL-22 shapes gut microbiome composition and function, which leads to increased AhR signaling and suggests exogenous IL-22 modulation of the microbiome may have functional significance in a disease setting. Video Abstract.

The role of bacterial membrane vesicles in antibiotic resistance.

Bacterial survival during antibiotic exposure is a complex and multifaceted phenomenon. On top of antibiotic resistance genes, biofilm formation, and persister tolerance, bacterial membrane vesicles (MVs) provide a layer of protection that has been largely overlooked. MVs are spherical nanoparticles composed of lipid membranes and are common to Gram-positive and Gram-negative bacteria. Although the importance of MVs in bacterial pathogenesis and virulence factor transport has been firmly established, a growing body of work now identifies MVs as key contributors to bacterial survival during antibiotic exposure. Herein, we highlight the ability of MVs to reduce antibiotic efficacy and transmit resistance elements. We also discuss the potential of targeting MV production as an unconventional therapeutic approach.

Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

Monitoring Mitochondrial Perturbations During Infection.

Mitochondria are pivotal organelles in the cell that regulate a myriad of cellular functions, which eventually govern cellular physiology and homeostasis. Intriguingly, microbial infection is known to trigger morphological and functional alterations of mitochondria. In fact, a number of bacteria and viruses have been reported to hijack mitochondrial functions including cell death induction and regulation of immune signaling to evade detection, promote their intracellular growth and subsequent dissemination. Here we describe methodologies that can be applied to assess mitochondrial functions upon infection. More specifically, we outline experimental procedures used to evaluate different parameters including mitochondrial morphology, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS) levels, and mitophagy. Together these parameters can help gauge the overall health of mitochondria upon infection.

Sialic acid diversity in the human gut: Molecular impacts and tools for future discovery.

Sialic acids are a family of structurally related sugars that are prevalent in mucosal surfaces, including the human intestine. In the gut, sialic acids have diverse biological roles at the interface of the host epithelium and the microbiota. N-acetylneuraminic acid (Neu5Ac), the best studied sialic acid, is a nutrient source for bacteria and, when displayed on the cell surface, a binding site for host immune factors, viruses, and bacterial toxins. Neu5Ac is extensively modified by host and microbial enzymes, and the impacts of Neu5Ac derivatives on host-microbe interactions, and generally on human and microbial biology, remain underexplored. In this mini-review, we highlight recent reports describing how host and microbial proteins differentiate Neu5Ac and its derivatives, draw attention to gaps in knowledge related to sialic acid biology, and suggest cutting-edge methodologies that may expand our appreciation and understanding of Neu5Ac in health and disease.

Acinetobacter baumannii Secretes a Bioactive Lipid That Triggers Inflammatory Signaling and Cell Death.

Acinetobacter baumannii is a highly pathogenic Gram-negative bacterium that causes severe infections with very high fatality rates. A. baumannii infection triggers innate as well as adaptive immunity, however, our understanding of the inflammatory factors secreted by A. baumannii that alarm the immune system remains limited. In this study, we report that the lab adapted and clinical strains of A. baumannii secrete an inflammatory bioactive factor which activates TLR2, leading to canonical IRAK4-dependent NF-κB signaling and production of pro-inflammatory cytokines interleukin (IL)-6 and IL-8 and activation of the inflammasome pathway causing pyroptotic cell death. Biochemical fractionation of the A. baumannii culture filtrate revealed the hydrophobic nature of the inflammatory factor. Concordantly, lipase treatment of the culture filtrate or TLR2 inhibition in macrophages abrogated NF-κB activation and cell death induction. Culture filtrates from the LPS- and lipoprotein-deficient A. baumannii mutants retain immuno-stimulatory properties suggesting that a lipid other than these known stimulatory molecules can trigger inflammation during A. baumannii infection. Our results reveal that A. baumannii secretes a previously unappreciated inflammatory bioactive lipid that activates multiple pro-inflammatory signaling pathways and induces cell death in human and murine macrophages.

Host immunity and cellular responses to bacterial outer membrane vesicles.

All Gram-negative bacteria produce outer membrane vesicles (OMVs) which are minute spherical structures emanating from the bacterial outer membrane. OMVs are primarily enriched in lipopolysaccharide (LPS) and phospholipids, as well as outer membrane and periplasmic proteins. Recent research has provided convincing evidence for their role in multiple aspects of bacterial physiology and their interaction with vertebrate host cells. OMVs play vital roles in bacterial colonization, delivery of virulence factors, and disease pathogenesis. Here, we discuss the interactions of OMVs with mammalian host cells with a focus on how bacteria use OMVs to modulate host immune responses that eventually enable bacteria to evade host immunity.

Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection.

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.

Outer membrane vesicles containing OmpA induce mitochondrial fragmentation to promote pathogenesis of Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic resistant Gram-negative bacterium that causes life-threatening infections in humans with a very high mortality rate. A. baumannii is an extracellular pathogen with poorly understood virulence mechanisms. Here we report that A. baumannii employs the release of outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) to promote bacterial pathogenesis and dissemination. OMVs containing OmpAAb are taken up by mammalian cells where they activate the host GTPase dynamin-related protein 1 (DRP1). OmpAAb mediated activation of DRP1 enhances its accumulation on mitochondria that causes mitochondrial fragmentation, elevation in reactive oxygen species (ROS) production and cell death. Loss of DRP1 rescues these phenotypes. Our data show that OmpAAb is sufficient to induce mitochondrial fragmentation and cytotoxicity since its expression in E. coli transfers its pathogenic properties to E. coli. A. baumannii infection in mice also induces mitochondrial damage in alveolar macrophages in an OmpAAb dependent manner. We finally show that OmpAAb is also required for systemic dissemination in the mouse lung infection model. In this study we uncover the mechanism of OmpAAb as a virulence factor in A. baumannii infections and further establish the host cell factor required for its pathogenic effects.

Mitochondrial Functions in Infection and Immunity.

Mitochondria have a central role in regulating a range of cellular activities and host responses upon bacterial infection. Multiple pathogens affect mitochondria dynamics and functions to influence their intracellular survival or evade host immunity. On the other side, major host responses elicited against infections are directly dependent on mitochondrial functions, thus placing mitochondria centrally in maintaining homeostasis upon infection. In this review, we summarize how different bacteria and viruses impact morphological and functional changes in host mitochondria and how this manipulation can influence microbial pathogenesis as well as the host cell metabolism and immune responses.

Publisher Correction: Definitions and guidelines for research on antibiotic persistence.

In Figure 2b, the minimal duration for killing (MDK) 99% of tolerant cells was erroneously labelled as MDK99.99 instead of MDK99. This has now been corrected in all versions of the Review. The publisher apologizes to the authors and to readers for this error.

Definitions and guidelines for research on antibiotic persistence.

Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.

Optimized arylomycins are a new class of Gram-negative antibiotics.

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.

Structural basis for dual-mode inhibition of the ABC transporter MsbA.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

A Tet-Off gene expression system for validation of antifungal drug targets in a murine invasive pulmonary aspergillosis model.

Aspergillus fumigatus is one of the major causes of invasive pulmonary aspergillosis in immunocompromised patients. Novel antifungal therapy is in urgent need due to emerging resistance and adverse toxicity of current antifungal drugs. Gene products that are essential for Aspergillus viability during infection are attractive drug targets. To characterize these genes in vivo we developed a Tet-Off gene expression system in A. fumigatus, whereby the administration of doxycycline resulted in down regulation of the gene whose expression is under the control of the Tet-Off promoter. We tested the system on two potential drug targets, inosine 5'-monophosphate dehydrogenase (IMPDH) and L-ornithine N5-oxygenase (sidA) in a murine invasive pulmonary aspergillosis model. We show that depletion of IMPDH attenuated but did not completely abolish virulence in vivo whereas turning off the expression of sidA, which is required for iron acquisition, resulted in avirulence. We also investigated whether sidA expression could be controlled in a time-dependent manner in mice. Our results demonstrated that timing of doxycycline administration dramatically affects survival rate, suggesting that this genetic system can be used for testing whether an antifungal drug target is critical for fungal growth post-infection.