Brianna Dalesandro

Elife. 2022 Apr 27;11:e73055. doi: 10.7554/eLife.73055.

ABSTRACT

L,D-transpeptidase function predominates in atypical 3 → 3 transpeptide networking of peptidoglycan (PG) layer in Mycobacterium tuberculosis. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or β-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by β-lactams. Here, we report identification of an allosteric site at a distance of 21 Å from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second β-lactam molecule and influences binding at the catalytic site. We provide evidence that two β-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-pocket and one covalently at the catalytic site. This dual β-lactam-binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new drugs against M. tuberculosis.

PMID:35475970 | PMC:PMC9094749 | DOI:10.7554/eLife.73055

Mol Ther Methods Clin Dev. 2022 Mar 22;25:250-263. doi: 10.1016/j.omtm.2022.03.012. eCollection 2022 Jun 9.

ABSTRACT

Chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in the treatment of B cell malignancies. Current CAR T cell manufacturing protocols are complex and costly due to their reliance on viral vectors. Non-viral systems of genetic modification, such as with transposase and transposon systems, offer a potential streamlined alternative for CAR T cell manufacture and are currently being evaluated in clinical trials. In this study, we utilized the previously described transposase from the little brown bat, designated piggyBat, for production of CD19-specific CAR T cells. PiggyBat demonstrates efficient CAR transgene delivery, with a relatively low variability in integration copy number across a range of manufacturing conditions as well as a similar integration site profile to super-piggyBac transposon and viral vectors. PiggyBat-generated CAR T cells demonstrate CD19-specific cytotoxic efficacy in vitro and in vivo. These data demonstrate that alternative, naturally occurring DNA transposons can be efficiently re-tooled to be exploited in real-world applications.

PMID:35474955 | PMC:PMC9018555 | DOI:10.1016/j.omtm.2022.03.012

Cancer Immunol Immunother. 2022 Apr 19. doi: 10.1007/s00262-022-03195-4. Online ahead of print.

ABSTRACT

Chimeric antigen receptor (CAR) T-cell therapy achieves great success for hematological malignancies. However, clinical trials have revealed some limitations in both improving the efficacy and reducing the relapse, which calls for innovative strategies to engineer more powerful CAR-T cells. Promoting the formation of CAR clusters provides an alternative approach and potentially improves current CAR T-cell therapy against cancers. Here, we generated CAR_Cys-T cells using a 4-1BB-derived hinge region including 11 cysteines residues. The cysteines in the hinge were found to facilitate CAR_Cys clustering upon antigen stimulation and promote the antitumor activity of CAR-T cells. Compared with most conventionally used CAR-T cells with CD8α-derived hinge (CAR_conv-T cells), CAR_Cys-T cells exhibited larger diameter of CAR clusters and enhanced antigen-specific tumor lysis both in vitro and in vivo. In addition, the CAR_Cys-mediated enhancement could be applied to HER2, CD19 as well as GPC3-targeted CAR-T cells. More importantly, CAR_Cys-T cells showed potent antitumor efficacy in clinically relevant patient-derived primary tumor cells and organoids. Thus, the novel hinge containing 11 cysteines provides a promising strategy to facilitate CAR clustering and maximize anti-tumor activity of CAR-T cells, which emphasizes the importance of CAR clustering to improve CAR T-cell therapy in the clinic.

PMID:35441325 | DOI:10.1007/s00262-022-03195-4

RSC Adv. 2021 Apr 15;11(23):14161-14168. doi: 10.1039/d1ra01316e. eCollection 2021 Apr 13.

ABSTRACT

The development of quick and efficient methods for the detection of pathogenic bacteria is urgently needed for the diagnosis of infectious diseases and the control of microbiological contamination in global waterways, potable water sources and the food industry. This contribution will describe the synthesis of gold nanoparticles and their conjugation to broad spectrum, polypeptide and β-lactam antibiotics that function as both reducing agents and surface protectants (ATB@AuNP). Nanoparticle colloids examined using transmission electron microscopy are generally spherical in shape and range from 2-50 nm in size. Dynamic light scattering and infrared spectroscopy were also used to confirm encapsulation of the AuNP surface by antibiotic molecules. ATB@AuNP were then used to detect 3 common pathogenic bacterial species: Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The colour of the AuNP colloid was monitored visually and using UV-visible spectroscopy. A red shift of the UV visible absorbance and a visible colour change following introduction of each pathogen is indicative of ATB binding to the bacteria surface, ascribed to AuNP agglomeration. This work demonstrates that ATB@AuNP may be an efficient and high throughput tool for the rapid detection of common bacterial contaminants.

PMID:35423905 | PMC:PMC8697705 | DOI:10.1039/d1ra01316e

Chemistry. 2022 Apr 19. doi: 10.1002/chem.202200547. Online ahead of print.

ABSTRACT

TLR4 is a key pattern recognition receptor that can sense pathogen- and danger- associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro-inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species-specific, and barely predictable structure-activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan-based lipid A-mimicking molecules that can activate human and murine TLR4-mediated signaling with picomolar affinity. Exploiting crystal structure - based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)-linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non-reducing βGlcN(1↔1´)βGlcN scaffold. Excellent stereoselectivity in a challenging β,β -1,1´ glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β-1,1´-linked diglucosamine-derived glycolipids entailing multiple long-chain ( R )-3- O -acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D-molecular shape and conformational rigidity of unnatural β,β -1,1´-linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4-mediated signaling in a species-independent manner.

PMID:35439332 | DOI:10.1002/chem.202200547

Science. 2022 Apr 15;376(6590):248-249. doi: 10.1126/science.abo7933. Epub 2022 Apr 14.

ABSTRACT

Peptidoglycans from gut microbiota modulate appetite through hypothalamic circuits.

PMID:35420955 | DOI:10.1126/science.abo7933

Adv Drug Deliv Rev. 2022 Jun;185:114295. doi: 10.1016/j.addr.2022.114295. Epub 2022 Apr 13.

ABSTRACT

The clinical application of bacteria-mediated immune therapy dates back over a century ago. In recent years, these strategies have advanced greatly with the rapid development of synthetic biology and nanotechnology. Several bacterial therapies have been developed allowing for more effective treatments for cancers, and Salmonella is one of the most studied bacterial species. Here, we review the advances in the bioengineered and functionalized Salmonella Typhimurium strains as drug delivery carries, including the various genetic circuits for programing these bacteria, the surface modification strategies using nanoparticles or other therapeutic agents for richer and broader features, and the bacterial component-based vehicles for cancer immunotherapy. This review will include the promises and challenges of these optimized Salmonella-based delivery systems and their related clinical trials. Ultimately, we hope to provide a spark of thought in the field of drug delivery and find important crosstalk between bacteria-mediated therapy and other different forms of treatments.

PMID:35429576 | DOI:10.1016/j.addr.2022.114295

J Biol Chem. 2022 Apr 14:101940. doi: 10.1016/j.jbc.2022.101940. Online ahead of print.

ABSTRACT

Trafficking of M-protein (Mprt) from the cytosol of Streptococcus pyogenes (GAS) occurs via Sec translocase membrane channels that associate with Sortase A (SrtA), an enzyme that catalyzes cleavage of Mprt at the proximal C-terminal [-LPST³⁵⁵*GEAA-] motif and subsequent transpeptidation of the Mprt-containing product to the cell wall (CW). This facilitates stable exposure of the N-terminus of Mprt to the extracellular milieu where it interacts with ligands. Previously, we found that inactivation of SrtA in GAS cells eliminated Mprt CW transpeptidation but effected little reduction in its cell surface exposure, indicating that the C-terminus of M-protein retained in the cell membrane (CM) extends its N-terminus to the cell surface. Herein, we assessed the effects of mutating the Thr³⁵⁵ residue in the wild-type SrtA consensus sequence, [-LPST³⁵⁵*GEAA-] SrtA-cleavage site in a specific Mprt, PAM. In vitro, we found that synthetic peptides with mutations (LPSX³⁵⁵GEAA) in the SrtA cleavage site displayed slower cleavage activities with rSrtA than the wild-type peptide. Aromatic residues at X had the lowest activities. Nonetheless, PAM/[Y³⁵⁵G] still transpeptidated the CW in vivo. However, when using isolated CMs from srtA-inactivated GAS cells, rapid cleavage of PAM/[LPSY³⁵⁵GEAA] occurred at E³⁵⁷* but transpeptidation did not take place. These results show that another CM-resident enzyme nonproductively cleaved PAM/[LPSYGE³⁵⁷*AA]. However, SrtA associated with the translocon channel in vivo cleaved and transpeptidated PAM/[LPSX³⁵⁵*GEAA] variants. These CM features allow diverse cleavage site variants to covalently attach to the CW despite the presence of other potent nonproductive CM proteases.

PMID:35430253 | DOI:10.1016/j.jbc.2022.101940

Biomacromolecules. 2022 Jul 11;23(7):2793-2802. doi: 10.1021/acs.biomac.2c00166. Epub 2022 Apr 15.

ABSTRACT

In recent years, much research has focused on assemblies formed by charged polyelectrolytes via electrostatic interactions in aqueous solutions as they have the potential to be used in a variety of biomedical applications. In this study, we analyzed supramolecular architectures fabricated from poly(N-allyl glycine) modified with cysteamine (PNAG-NH₂) and folic acid (FA) via electrostatic interactions. The PNAG-NH₂/FA complex exhibits a reversible pH-responsive morphological transformation from vesicles (pH = 7.0) to nanofibers (pH = 5.0). Besides, we demonstrated that homopolypeptoids electrostatically interact with FA, thereby facilitating ribbon- and disk-like H-bonded FA patterns and inducing the formation of vesicle nanostructures and fiber arrays, respectively. Additionally, we systematically studied the influence of the degree of polymerization of the polymers, concentration, charge mixing ratio, and type of the polymer and the small-molecule acid on the assemblies. We show the superior stability of the polypeptoid/FA complex as compared to those based on other polymers. We established that the polypeptoid/FA complex exhibits a superior stability than those based on other polymers. By applying these beneficial properties, we encapsulated the anticancer drug doxorubicin (DOX) in the complex vesicle to obtain a pH-induced drug carrier. Cytotoxicity studies and internalization assays revealed that the DOX-loaded PNAG-NH₂/FA complex vesicles display an enhanced therapeutic efficacy via typical FA-folate receptor-mediated endocytosis in vitro.

PMID:35427120 | DOI:10.1021/acs.biomac.2c00166

Carbohydr Polym. 2022 Jul 1;287:119325. doi: 10.1016/j.carbpol.2022.119325. Epub 2022 Mar 15.

ABSTRACT

Intermediate-state bacteria produced in the process of bacteriostasis have always been a potential threat to public health, but they are very easy to be overlooked. As a natural and non-toxic biological antibacterial agent, chitooligosaccharide (COS) has attracted the public's attention. However, little is known about the microbial stress response during the antibacterial process of COS. In this study, the antibacterial mechanisms of COS were expounded, and the formation of sublethal and viable but nonculturable (VBNC) state were further investigated. The COS was shown to bind to the cell envelopes, and the permeability and integrity of bacterial cell membrane were damaged severely, accompanied by the increase of intracellular reactive oxygen species and decrease of adenosine triphosphate content. Flow cytometry analysis indicated that COS finally inactivated Escherichia coli through the sublethal injury process. While for Staphylococcus aureus, some cells were induced into VBNC state by COS, causing incomplete inactivation.

PMID:35422291 | DOI:10.1016/j.carbpol.2022.119325

Cell Host Microbe. 2022 Apr 13;30(4):556-569.e5. doi: 10.1016/j.chom.2022.03.014.

ABSTRACT

Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.

PMID:35421351 | DOI:10.1016/j.chom.2022.03.014

Org Biomol Chem. 2022 May 4;20(17):3619-3628. doi: 10.1039/d1ob02394b.

ABSTRACT

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths to design a palette of fluorogens with emissions spanning the green to red range, opening new possibilities for multicolor imaging. The probes were studied in glycerol and in the presence of HaloTag and exhibited good fluorogenic properties thanks to a viscosity-sensitive emission. In live-cell confocal imaging, the fluorogens yielded only a very low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast. Combining experimental studies and theoretical investigation of the protein/fluorogen complexes by molecular dynamics, these results offer new insight into the design of molecular rotor-based fluorogenic HaloTag probes in order to improve reaction rates and the imaging contrast.

PMID:35420083 | DOI:10.1039/d1ob02394b

Int J Mol Sci. 2022 Mar 22;23(7):3424. doi: 10.3390/ijms23073424.

ABSTRACT

T cell activation plays a central role in supporting and shaping the immune response. The induction of a functional adaptive immune response requires the control of signaling processes downstream of the T cell receptor (TCR). In this regard, protein phosphorylation and dephosphorylation have been extensively studied. In the past decades, further checkpoints of activation have been identified. These are E3 ligases catalyzing the transfer of ubiquitin or ubiquitin-like proteins to protein substrates, as well as specific peptidases to counteract this reaction, such as deubiquitinating enzymes (DUBs). These posttranslational modifications can critically influence protein interactions by targeting proteins for degradation by proteasomes or mediating the complex formation required for active TCR signaling. Thus, the basic aspects of T cell development and differentiation are controlled by defining, e.g., the threshold of activation in positive and negative selection in the thymus. Furthermore, an emerging role of ubiquitination in peripheral T cell tolerance has been described. Changes in the function and abundance of certain E3 ligases or DUBs involved in T cell homeostasis are associated with the development of autoimmune diseases. This review summarizes the current knowledge of E3 enzymes and their target proteins regulating T cell signaling processes and discusses new approaches for therapeutic intervention.

PMID:35408787 | PMC:PMC8998186 | DOI:10.3390/ijms23073424

Microbiology (Reading). 2022 Apr;168(4). doi: 10.1099/mic.0.001159.

ABSTRACT

The lipopolysaccharide (LPS) is a characteristic molecule of the outer leaflet of the Gram-negative bacterial outer membrane, which consists of lipid A, core oligosaccharide, and O antigen. The lipid A is embedded in outer membrane and provides an efficient permeability barrier, which is particularly important to reduce the permeability of antibiotics, toxic cationic metals, and antimicrobial peptides. LPS, an important modulator of innate immune responses ranging from localized inflammation to disseminated sepsis, displays a high level of structural and functional heterogeneity, which arise due to regulated differences in the acylation of the lipid A and the incorporation of non-stoichiometric modifications in lipid A and the core oligosaccharide. This review focuses on the current mechanistic understanding of the synthesis and assembly of the lipid A molecule and its most salient non-stoichiometric modifications.

PMID:35394417 | DOI:10.1099/mic.0.001159

J Biol Chem. 2022 Apr 7:101915. doi: 10.1016/j.jbc.2022.101915. Online ahead of print.

ABSTRACT

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.6 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved β-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.

PMID:35398351 | DOI:10.1016/j.jbc.2022.101915

Methods Enzymol. 2022;665:233-258. doi: 10.1016/bs.mie.2021.12.006. Epub 2022 Jan 20.

ABSTRACT

Teixobactin is a promising new antibiotic that kills a spectrum of Gram-positive pathogens that are considered to be urgent threats by the CDC and the WHO. Better understanding of the novel mechanism of action of teixobactin may assist in developing new antibiotics and furthering our understanding of antibiotic resistance. This chapter describes the synthesis and application of fluorescent teixobactin analogs in fluorescence microscopy to study the mode of action of teixobactin. The first part of this chapter describes the synthesis and purification of fluorescent teixobactin analogs using two synthetic approaches. The second part of this chapter describes the application of the fluorescent teixobactin analogs to visualize their interactions with molecular targets in B. subtilis using fluorescence microscopy. The methods described herein provide synthetic access to chemical probes that may help further the understanding of antibiotic resistance.

PMID:35379436 | DOI:10.1016/bs.mie.2021.12.006

Methods Enzymol. 2022;665:259-279. doi: 10.1016/bs.mie.2021.11.019. Epub 2022 Feb 2.

ABSTRACT

The bacterial cell wall, whose main component is peptidoglycan (PG), provides cellular rigidity and prevents lysis from osmotic pressure. Moreover, the cell wall is the main interface between the external environment and internal cellular components. Given its essentiality, many antibiotics target enzymes related to the biosynthesis of cell wall. Of these enzymes, transpeptidases (TPs) are central to proper cell wall assembly and their inactivation is the mechanism of action of many antibiotics including β-lactams. TPs are responsible for stitching together strands of PG to make the crosslinked meshwork of the cell wall. This chapter focuses on the use of solid-phase peptide synthesis to build PG analogs that become site-selectively incorporated into the cell wall of live bacterial cells. This method allows for the design of fluorescent handles on PG probes that will enable the interrogation of substrate preferences of TPs (e.g., amidation at the glutamic acid residue, crossbridge presence) by analyzing the level of probe incorporation within the native cell wall of live bacterial cells.

PMID:35379437 | DOI:10.1016/bs.mie.2021.11.019