Microbiome and infectious disease: diagnostics to therapeutics.

Over 300 years of research on the microbial world has revealed their importance in human health and disease. This review explores the impact and potential of microbial-based detection methods and therapeutic interventions, integrating research of early microbiologists, current findings, and future perspectives.

Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota.

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.

Gut microbial signatures are associated with Lynch syndrome (LS) and cancer history in Druze communities in Israel.

Lynch syndrome (LS) is a hereditary cancer syndrome caused by autosomal dominant mutations, with high probability of early onset for several cancers, mainly colorectal cancer (CRC). The gut microbiome was shown to be influenced by host genetics and to be altered during cancer development. Therefore, we aimed to determine alterations in gut microbiome compositions of LS patients with and without cancer. We performed fecal microbiome analyses on samples of LS and non-LS members from the Druze ethnoreligious community in Israel, based on both their LS mutation and their cancer history. Our analysis revealed specific bacterial operational taxonomic units (OTUs) overrepresented in LS individuals as well as bacterial OTUs differentiating between the LS individuals with a history of cancer. The identified OTUs align with previous studies either correlating them to pro-inflammatory functions, which can predispose to cancer, or to the cancer itself, and as such, these bacteria can be considered as future therapeutic targets.

Combinatorial fluorescent labeling of live anaerobic bacteria via the incorporation of azide-modified sugars into newly synthesized macromolecules.

The human gut microbiome modulates physiological functions and pathologies; however, a mechanistic understanding of microbe-host and microbe-microbe interactions remains elusive owing to a lack of suitable approaches to monitor obligate anaerobic bacterial populations. Common genetically encoded fluorescent protein reporters, derived from the green fluorescent protein, require an oxidation step for fluorescent light emission and therefore are not suitable for use in anaerobic microbes residing in the intestine. Fluorescence in situ hybridization is a useful alternative to visualize bacterial communities in their natural niche; however, it requires tissue fixation. We therefore developed an approach for the real-time detection and monitoring of live communities of anaerobic gut commensals in their natural environment. We leverage the bacterial cells' reliance on sugars for macromolecule synthesis in combinatorial click chemistry labeling, where the addition of azide-modified sugars to the culturing media enables the fluorescence labeling of newly synthesized molecules via the addition of combinations of exogenous fluorophores conjugated to cyclooctynes. This process is suitable for labeling communities of live anaerobic gut bacteria with combinations of fluorophores that do not require oxygen to mature and fluoresce, and that can be detected over time in their natural environments. The labeling procedure requires 4-9 d, depending on the varying growth rates of different bacterial strains, and an additional 1-2 d for the detection and monitoring steps. The protocol can be completed by users with basic expertise in bacterial culturing.

A sound mind in a sound body: Stress-induced glucocorticoids exacerbate gut inflammation.

The notion that psychological stress can deteriorate our health is widely accepted. However, the mechanisms at play are poorly understood. In this issue of Cell, Schneider et al. identify the impact of glucocorticoids on enteric glia and neurons and elucidate the underlying mechanisms that link psychological stress to the exacerbation of gut inflammation.

Extracellular vesicles of the Gram-positive gut symbiont Bifidobacterium longum induce immune-modulatory, anti-inflammatory effects.

The gut microbiota is now well known to affect the host's immune system. One way of bacterial communication with host cells is via the secretion of vesicles, small membrane structures containing various cargo. Research on vesicles secreted by Gram-positive gut bacteria, their mechanisms of interaction with the host and their immune-modulatory effects are still relatively scarce. Here we characterized the size, protein content, and immune-modulatory effects of extracellular vesicles (EVs) secreted by a newly sequenced Gram-positive human gut symbiont strain - Bifidobacterium longum AO44. We found that B. longum EVs exert anti-inflammatory effects, inducing IL-10 secretion from both splenocytes and dendritic cells (DC)-CD4+ T cells co-cultures. Furthermore, the EVs protein content showed enrichment in ABC transporters, quorum sensing proteins, and extracellular solute-binding proteins, which were previously shown to have a prominent function in the anti-inflammatory effect of other strains of B. longum. This study underlines the importance of bacterial vesicles in facilitating the gut bacterial immune-modulatory effects on the host and sheds light on bacterial vesicles as future therapeutics.

Global Thiol Proteome Analysis Provides Novel Insights into the Macrophage Inflammatory Response and Its Regulation by the Thioredoxin System.

Aims: Oxidative modifications of cysteine (Cys) thiols regulate various physiological processes, including inflammatory responses. The thioredoxin (Trx) system plays a key role in thiol redox control. The aim of this study was to characterize the dynamic cysteine proteome of human macrophages upon activation by the prototypical proinflammatory agent, bacterial lipopolysaccharide (LPS), and/or perturbation of the Trx system. Results: In this study, we profiled the cellular and redox proteome of human THP-1-derived macrophages during the early phase of LPS activation and/or inhibition of Trx system activity by auranofin (AF) by employing a peptide-centric, resin-assisted capture, redox proteomic workflow. Among 4200 identified cysteines, oxidation of nearly 10% was selectively affected by LPS or AF treatments. Notably, the proteomic analysis uncovered a subset of ∼100 thiols, mapped to proteins involved in diverse processes, whose oxidation is antagonistically regulated by LPS and Trx. Compared with the redox proteome, the cellular proteome was largely unchanged, highlighting the importance of redox modification as a mechanism that allows for rapid modulation of macrophage activities in response to a proinflammatory or pro-oxidant insult. Structural-functional analyses provided mechanistic insights into redox regulation of selected proteins, including the glutathione-synthesizing enzyme, glutamate-cysteine ligase, and the autophagy adaptor, SQSTM1/p62, suggesting mechanisms by which macrophages adapt and fine-tune their responses according to a changing inflammatory and redox environment. Innovation: This study provides a rich resource for further characterization of redox mechanisms that regulate macrophage inflammatory activities. Conclusion: The dynamic thiol redox proteome allows macrophages to efficiently respond and adapt to redox and inflammatory challenges. Antioxid. Redox Signal. 38, 388-402.

Automated device for multi-stage paper-based assays enabled by an electroosmotic pumping valve.

We leverage electroosmotic-flow generation in porous media in combination with a hydrophobic air gap to create a controllable valve capable of operating in either finite dosing or continuous flow mode, enabling the implementation of multi-step assays on paper-based devices. The hydrophobic air gap between two paper pads creates a barrier keeping the valve nominally closed. Electroosmotic actuation, implemented using a pair of electrodes under the upstream pad, generates sufficient pressure to overcome the barrier and connect the two pads. We present a model describing the flow and governing parameters, including the electric potentials required to open and close the valve and the threshold potential for switching between the modes of operation. We construct the air gap using a hierarchical superhydrophobic surface and study the stability of the closed valve under strenuous conditions and find good agreement between our model and experimental results, as well as stable working conditions for practical applications. We present a straightforward design for a compact and automated device based on paper pads placed on top of printed circuit boards (PCB), equipped with heating and actuation electrodes and additional power and logic capabilities. Finally, we demonstrate the use of the device for amplification of SARS-CoV-2 sequences directly from raw saliva samples, using a loop-mediated isothermal amplification (LAMP) protocol requiring sample lysis followed by enzymatic deactivation and delivery to multiple amplification sites. Since PCB costs scale favorably with mass-production, we believe that this approach could lead to a low-cost diagnostic device that offers the sensitivity of amplification methods.

Microbiome Characterization of Infected Diabetic Foot Ulcers in Association With Clinical Outcomes: Traditional Cultures Versus Molecular Sequencing Methods.

Background: Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. These potentially limb-threatening ulcers are challenging to treat due to impaired wound healing characterizing diabetic patients and the complex microbial environment of these ulcers. Aim: To analyze the microbiome of IDFU in association with clinical outcomes. Methods: Wound biopsies from IDFU were obtained from hospitalized patients and were analyzed using traditional microbiology cultures, 16S rRNA sequencing and metagenomic sequencing. Patients' characteristics, culture-based results and sequencing data were analyzed in association with clinical outcomes. Results: A total of 31 patients were enrolled. Gram-negative bacteria dominated the IDFU samples (79%, 59% and 54% of metagenomics, 16S rRNA and cultures results, respectively, p<0.001). 16S rRNA and metagenomic sequencing detected significantly more anaerobic bacteria, as compared to conventional cultures (59% and 76%, respectively vs. 26% in cultures, p=0.001). Culture-based results showed that Staphylococcus aureus was more prevalent among patients who were treated conservatively (p=0.048). In metagenomic analysis, the Bacteroides genus was more prevalent among patients who underwent amputation (p<0.001). Analysis of metagenomic-based functional data showed that antibiotic resistance genes and genes related to biofilm production and to bacterial virulent factors were more prevalent in IDFU that resulted in amputation (p<0.001). Conclusion: Sequencing tools uncover the complex biodiversity of IDFU and emphasize the high prevalence of anaerobes and Gram-negative bacteria in these ulcers. Furthermore, sequencing results highlight possible associations among certain genera, species, and bacterial functional genes to clinical outcomes.

Combinatorial Click Chemistry Labeling to Study Live Human Gut-Derived Microbiota Communities.

Gut bacteria were shown to exert pivotal effects on health and disease. However, mechanistic studies of gut bacterial communities are limited due to the lack of technologies for real-time studies on live bacteria. Here, we developed COMBInatorial cliCK-chemistry (COMBICK) labeling on human gut-derived bacteria, both aerobic and anaerobic strains, to enable dynamic tracing of live, heterogeneous bacterial communities on the strain level, including clinical isolates of the Enterobacteriaceae family. We further show that COMBICK labeling is applicable on anaerobic bacterial strains directly isolated from stool. In COMBICK, the number of labeled bacteria that can be simultaneously differentiated increases exponentially depending on the availability of fluorophores and machine capabilities. This method allows real-time studies of bacterial communities from a variety of ecosystems, and can significantly advance mechanistic research in the microbiome field.

Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis.

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.

Direct on-the-spot detection of SARS-CoV-2 in patients.

IMPACT STATEMENT: Humanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.

Gut Bacteria-Not for the Faint of Heart.

The role of gut bacteria in complications of heart inflammation has previously not been fully understood. Gil-Cruz et al. (2019) demonstrate a commensal Bacteroides species that triggers a cross-immune response against a bacterial protein and heart epitope, causing cardiomyopathy. This study links commensal bacteria with heart inflammation mechanistically in mice and correlatively in patients.

A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP).

SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown strategy in which many workplaces and activities are completely closed, leading to substantial economy costs. Here, we present a protocol we recently developed that allows rapid and simple detection of SARS-CoV-2 for the large population, eliminating costs and involvement of professional teams and laboratories. This protocol is based on Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP). We tested this protocol directly on patient samples, both nasal and throat clinical swabs as well as saliva. Notably, this protocol is simple, cheap and can be easily applied to other pathogens as well.

Tumor-targeted silencing of the peptide transporter TAP induces potent antitumor immunity.

Neoantigen burden is a major determinant of tumor immunogenicity, underscored by recent clinical experience with checkpoint blockade therapy. Yet the majority of patients do not express, or express too few, neoantigens, and hence are less responsive to immune therapy. Here we describe an approach whereby a common set of new antigens are induced in tumor cells in situ by transient downregulation of the transporter associated with antigen processing (TAP). Administration of TAP siRNA conjugated to a broad-range tumor-targeting nucleolin aptamer inhibited tumor growth in multiple tumor models without measurable toxicity, was comparatively effective to vaccination against prototypic mutation-generated neoantigens, potentiated the antitumor effect of PD-1 antibody or Flt3 ligand, and induced the presentation of a TAP-independent peptide in human tumor cells. Treatment with the chemically-synthesized nucleolin aptamer-TAP siRNA conjugate represents a broadly-applicable approach to increase the antigenicity of tumor lesions and thereby enhance the effectiveness of immune potentiating therapies.

What Came First: The Microbiota or the Tr(egg) Cells?

Studies have shown that gut commensals facilitate the differentiation of peripheral regulatory T cells (pTregs) via their metabolic products. In this issue of Immunity, Campbell et al. (2018) demonstrate a reciprocal effect of pTregs on the metabolic functions of specific gut commensals that affects their overall energy harvest capacity.

Potentiating tumor immunity using aptamer-targeted RNAi to render CD8+ T cells resistant to TGFß inhibition.

TGFß secreted by tumor cells and/or tumor infiltrating stromal cells is a key mediator of tumor growth and immune suppression at the tumor site. Nonetheless, clinical trials in cancer patients targeting the TGFß pathway exhibited at best a modest therapeutic benefit. A likely reason, a common limitation of many cancer drugs, is that the physiologic roles of TGFß in tissue homeostasis, angiogenesis, and immune regulation precluded the dose escalation necessary to achieve a profound clinical response. Murine studies have suggested that countering immune suppressive effects of TGFß may be sufficient to inhibit tumor growth. Here we describe an approach to render vaccine-activated CD8+ T cells transiently resistant to TGFß inhibition using an siRNA against Smad4 to inhibit a key step in the canonical TGFß signaling pathway. The siRNA was targeted to vaccine activated CD8+ T cells in the mouse by conjugation to a 4-1BB binding oligonucleotide (ODN) aptamer ligand (4-1BB-Smad4 conjugate). In vitro the 4-1BB-Smad4 conjugate rendered T cells partially resistant to TGFß inhibition, and treatment of tumor bearing mice with systemically administered 4-1BB-Smad4 conjugate enhanced vaccine- and irradiation-induced antitumor immunity. Limiting the inhibitory effects of TGFß to tumor-specific T cells will not interfere with its multiple physiologic roles and hence reduce the risk of toxicity.

A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice.

T cell immunoglobulin-3 (TIM-3) is a negative regulator of interferon-γ (IFN-γ) secreting CD4+ T cells and CD8+ T cytotoxic cells. Recent studies have highlighted the role of TIM-3 as an important mediator of CD8+ T cell exhaustion in the setting of chronic viral infections and cancer. In murine tumor models, antibody blockade of TIM-3 with anti-TIM-3 antibodies as monotherapy has no or minimal antitumor activity, suggesting that TIM-3 signaling exerts an accessory or amplifying effect in keeping immune responses in check. Using a combined bead and cell-based systemic evolution of ligands by exponential enrichment (SELEX) protocol, we have isolated nuclease-resistant oligonucleotide aptamer ligands that bind to cell-associated TIM-3 with high affinity and specificity. A trimeric form of the TIM-3 aptamer blocked the interaction of TIM-3 with Galectin-9, reduced cell death, and enhanced survival, proliferation, and cytokine secretion in vitro. In tumor-bearing mice, the aptamer delayed tumor growth as monotherapy and synergized with PD-1 antibody in prolonging the survival of the tumor-bearing mice. Both in vitro and in vivo, the trimeric aptamer displayed superior activity compared to the currently used RMT3-23 monoclonal antibody. This study suggests that multi-valent aptamers could represent an alternative platform to generate potent ligands to manipulate the function of TIM-3 and other immune modulatory receptors.

Formation of multimeric antibodies for self-delivery of active monomers.

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

The effect of haptens on protein-carrier immunogenicity.

The immune response against hapten is T-cell-dependent, and so requires the uptake, processing and presentation of peptides on MHC class II molecules by antigen-presenting cells to the specific T cell. Some haptens, following conjugation to the available free amines on the surface of the carrier protein, can reduce its immunogenicity. The purpose of this study was to explore the mechanism by which this occurs. Four proteins were tested as carriers and six molecules were used as haptens. The immune response to the carrier proteins was reduced > 100-fold by some of the haptens (termed carrier immunogenicity reducing haptens--CIRH), whereas other haptens did not influence the protein immunogenicity (carrier immunogenicity non-reducing haptens--nCIRH). Conjugation of the protein to a CIRH affected protein degradation by lysosomal cathepsins, leading to the generation of peptides that differ in length and sequence from those derived from the same native protein or that protein modified with nCIRH. Injection of CIRH-conjugated protein into mice induced an increase in the population of regulatory T cells. The results of this study provide a putative mechanism of action for the reduction of immune response to haptenated proteins.