Marcos Pires

Nature Communications, Published online: 26 June 2020; doi:10.1038/s41467-020-16967-2

High levels of Fusobacterium nucleatum have been associated with poor overall survival in patients with colorectal and esophageal cancer. Here, the authors show that F. nucleatum is abundant in breast cancer samples and that the colonization by F. nucleatum accelerates tumor growth and metastasis in preclinical breast cancer models.

A co‐assembled vaccine, composed of lipidated antigens and lipophilic adjuvants, is reported. This vaccine design possesses both antigen multivalency and antigen‐specific immunostimulation properties, and induces a robust immune response. This simple vaccine initiated a potent immune response without requiring complex synthesis, allowing efficient and practical development of self‐adjuvanting vaccines.

Abstract

Co‐assembling vaccines composed of a lipidated HER2‐derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam₃CSK₄, α‐GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen‐specific immunostimulation properties, observed in reported self‐adjuvanting vaccine candidates, by using self‐assembly and adjuvant‐conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co‐assembly with lipidated CH401, which boosted the anti‐CH401 IgG and IgM production. In particular, α‐GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co‐assembling vaccine design opens the door for efficient and practical self‐adjuvanting vaccine development.

Many bacteria exist in a state of metabolic quiescence where energy consumption must be minimized so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for down-regulation...

by Andrea Sánchez-Vallet, Hui Tian, Luis Rodriguez-Moreno, Dirk-Jan Valkenburg, Raspudin Saleem-Batcha, Stephan Wawra, Anja Kombrink, Leonie Verhage, Ronnie de Jonge, H. Peter van Esse, Alga Zuccaro, Daniel Croll, Jeroen R. Mesters, Bart P. H. J. Thomma

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.

Li et al. present a general synthetic biotechnology platform to create covalent protein biologics from various protein-protein interactions by incorporating an unnatural amino acid with latent bioreactivity into one of the proteins, which can then target the other in vitro, on cell surfaces, and in mice. As a proof of concept, the authors create a synthetic form of PD-1 that enhances the activation of T cells and CAR-T cells more than wildtype PD-1 and can inhibit tumor growth more potently in immune-humanized mice by binding covalently to PD-L1.

Nature Communications, Published online: 23 June 2020; doi:10.1038/s41467-020-16950-x

Peptide antibiotics often display a very narrow therapeutic index. Here, the authors present an optimized peptide antibiotic with broad-spectrum in vitro activities, in vivo efficacy in multiple disease models against multidrug-resistant Gram-negative infections, and reduced toxicity.

by Dennis J. Doorduijn, Bart W. Bardoel, Dani A. C. Heesterbeek, Maartje Ruyken, Georgina Benn, Edward S. Parsons, Bart W. Hoogenboom, Suzan H. M. Rooijakkers

An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

ABSTRACT

We propose the concept that administration of an unrelated live attenuated vaccine, such as MMR (measles, mumps, rubella), could serve as a preventive measure against the worst sequelae of coronavirus disease 2019 (COVID-19). There is mounting evidence that live attenuated vaccines provide nonspecific protection against lethal infections unrelated to the target pathogen of the vaccine by inducing "trained" nonspecific innate immune cells for improved host responses against subsequent infections. Mortality in COVID-19 cases is strongly associated with progressive lung inflammation and eventual sepsis. Vaccination with MMR in immunocompetent individuals has no contraindications and may be especially effective for health care workers who can easily be exposed to COVID-19. Following the lead of other countries conducting clinical trials with the live attenuated Mycobacterium bovis BCG (BCG) vaccine under a similar concept, a clinical trial with MMR in high-risk populations may provide a "low-risk–high-reward" preventive measure in saving lives during this unprecedented COVID-19 pandemic.

ABSTRACT

Staphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When S. aureus was grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus require a living or nondenatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed that CEs and TGs were the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host.

IMPORTANCE Comprehensive lipidomics of S. aureus grown in the presence of human serum suggests that human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo. Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.

Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4)...

Nature, Published online: 17 June 2020; doi:10.1038/s41586-020-2403-9

Chimeric antigen receptor (CAR) T cells targeting uPAR, a cell-surface protein that is upregulated on senescent cells, eliminate senescent cells in vitro and in vivo and reduce liver fibrosis in mice.

Pretarget then activate : Functionalization of single‐walled carbon nanotubes (SWCNTs) with tetrazines led to a stable and biocompatible material that undergoes a rapid inverse‐electron‐demand Diels–Alder reaction with trans‐cyclooctene caged molecules. This approach enables bioorthogonal activation of responsive drugs or probes in solid tumours in a strictly controlled manner.

Abstract

The bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated...

The consequences of cholesterol metabolism by the gut microbiome were largely unknown. Kenny, Plichta et al. identified and characterized microbial cholesterol dehydrogenase enzymes encoded by uncultured metagenomic species of bacteria. The presence of these cholesterol-metabolizing bacteria is associated with decreased stool and serum total cholesterol in human cohorts.

Cirovic and de Bree et al. investigate the effects of BCG vaccination in humans and reveal the induction of a transcriptomic rewiring of human stem and progenitor cells toward the myeloid cell lineage, instructed by hepatic nuclear factors, resulting in epigenetic and functional changes within CD14+ peripheral monocytes.

ABSTRACT

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 alr1 alr2 dat strain (SE) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SE colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SE showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SE as a live biotherapeutic strain whose growth can be controlled by d-alanine.

IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans.

Related Articles

Differential Peptidoglycan Recognition Assay Using Varied Surface Presentations.

J Am Chem Soc. 2020 Jun 10;:

Authors: D'Ambrosio EA, Bersch KL, Lauro ML, Grimes CL

Abstract
Bacterial peptidoglycan (PG) is recognized by the human innate immune system to generate an appropriate downstream response. In order to gain an appreciation of how this essential polymer is sensed, a surface plasmon resonance (SPR) assay using varied PG surface presentation was developed. PG derivatives were synthesized and immobilized on the surface at different positions on the molecule to assess effects of ligand orientation on the binding affinities of NOD-like receptors (NLRs). NLRP1 and NOD2 are cytosolic innate immune proteins known to generate an immune response to PG. Both possess conserved leucine rich repeat domains (LRR) as proposed site of molecular recognition, though limited biochemical evidence exists regarding the mechanisms of PG recognition. Here we show direct biochemical evidence for the association of PG fragments to NOD2 and NLRP1 with nanomolar affinity. The orientations in which the fragments were presented on the SPR surface greatly influenced the strength of PG recognition by both NLRs. This assay displays fundamental differences in binding preferences for PG by innate immune receptors and reveals unique recognition mechanisms between the LRRs. Each receptor uses specific ligand structural features to achieve optimal binding, which will be critical information to manipulate these responses and combat diseases. This assay will be valuable in teasing apart subtle but critical structural features of a variety of receptors with therapeutic potential.

PMID: 32520538 [PubMed - as supplied by publisher]

Clostridioides difficile encounters high concentrations of heme at the host-pathogen interface. Knippel et al. discover that the HsmRA system sequesters heme and utilizes the heme bound by the membrane protein HsmA to protect against oxidative stress. The HsmRA system decreases sensitivity to vancomycin in the presence of heme during infection.

Nature Microbiology, Published online: 08 June 2020; doi:10.1038/s41564-020-0737-6

In this Article, using a nutrient-limited, media-based compound screening, the authors discover that the old antibiotic rifabutin is highly active against extensively drug-resistant Acinetobacter baumannii.

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the...

Nature Communications, Published online: 05 June 2020; doi:10.1038/s41467-020-16620-y

Bacterial colonization of surfaces has a profound environmental, technological and medical impact. Here, Secchi et al. show how fluid flow affects the magnitude and location of bacterial colonization on curved surfaces through its coupling with cell morphology and motility.