Brianna Dalesandro

Microbiol Spectr. 2022 Jul 7:e0101122. doi: 10.1128/spectrum.01011-22. Online ahead of print.

ABSTRACT

A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures; however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilizing a genome-wide association approach, we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA are required for the release of several cytolytic toxins from the bacterial cells. We show that this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to the process of toxin release by a major human pathogen while also demonstrating that clinical isolates can utilize WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities. IMPORTANCE The production and release of cytolytic toxins is a critical aspect for the pathogenicity of many bacterial pathogens. In this study, we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aureus cells into the extracellular environment. Our findings suggest that this effect is mediated by a gradient of electrostatic charge which the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.

PMID:35863033 | DOI:10.1128/spectrum.01011-22

Bioact Mater. 2022 Jun 29;20:548-560. doi: 10.1016/j.bioactmat.2022.05.037. eCollection 2023 Feb.

ABSTRACT

Bacterial outer membrane vesicles (OMVs) are potent immuno-stimulating agents and have the potentials to be bioengineered as platforms for antitumor nanomedicine. In this study, OMVs are demonstrated as promising antitumor therapeutics. OMVs can lead to beneficial M2-to-M1 polarization of macrophages and induce pyroptosis to enhance antitumor immunity, but the therapeutic window of OMVs is narrow for its toxicity. We propose a bioengineering strategy to enhance the tumor-targeting ability of OMVs by macrophage-mediated delivery and improve the antitumor efficacy by co-loading of photosensitizer chlorin e6 (Ce6) and chemotherapeutic drug doxorubicin (DOX) into OMVs as a therapeutic platform. We demonstrate that systemic injection of the DOX/Ce6-OMVs@M therapeutic platform, providing combinational photodynamic/chemo-/immunotherapy, eradicates triple-negative breast tumors in mice without side effects. Importantly, this strategy also effectively prevents tumor metastasis to the lung. This OMVs-based strategy with bioengineering may serve as a powerful therapeutic platform for a synergic antitumor therapy.

PMID:35846843 | PMC:PMC9253654 | DOI:10.1016/j.bioactmat.2022.05.037

BioTech (Basel). 2021 Oct 11;10(4):21. doi: 10.3390/biotech10040021.

ABSTRACT

Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs (fluorescently labeled) probes directed against DNA or (m)RNA, for instance targeting a gene or microorganism of interest and provides information on a single-cell level. Furthermore, by combining Flow-FISH with antibody-based protein detection, proteins of interest can be measured simultaneously with genetic material. Additionally, depending on the type of Flow-FISH assay, Flow-FISH can also be multiplexed, allowing for the simultaneous measurement of multiple gene targets and/or microorganisms. Together, this allows for, e.g., single-cell gene expression analysis or identification of (sub)strains in mixed cultures. Flow-FISH has been used in mammalian cells but has also been extensively employed to study diverse microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Specifically, the detection of (intracellular) pathogens, studying microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, with a particular focus on the viruses EBV, HIV-1, and SARS-CoV-2.

PMID:35822795 | DOI:10.3390/biotech10040021

Nat Biomed Eng. 2022 Jul 7. doi: 10.1038/s41551-022-00903-4. Online ahead of print.

ABSTRACT

Orally delivered antibiotics can reach the caecum and colon, and induce gut dysbiosis. Here we show that the encapsulation of antibiotics in orally administered positively charged polymeric nanoparticles with a glucosylated surface enhances absorption by the proximal small intestine through specific interactions of glucose and the abundantly expressed sodium-dependent glucose transporter 1. This improves bioavailability of the antibiotics, and limits their exposure to flora in the large intestine and their accumulation in caecal and faecal contents. Compared with the standard administration of the same antibiotics, the oral administration of nanoparticle-encapsulated ampicillin, chloramphenicol or vancomycin in mice with bacterial infections in the lungs effectively eliminated the infections, decreased adverse effects on the intestinal microbiota by protecting the animals from dysbiosis-associated metabolic syndromes and from opportunistic pathogen infections, and reduced the accumulation of known antibiotic-resistance genes in commensal bacteria. Glucosylated nanocarriers may be suitable for the oral delivery of other drugs causing gut dysbiosis.

PMID:35798834 | DOI:10.1038/s41551-022-00903-4

Nat Commun. 2022 Jul 7;13(1):3905. doi: 10.1038/s41467-022-31570-3.

ABSTRACT

Whole-cell screening for Mycobacterium tuberculosis (Mtb) inhibitors is complicated by the pathogen's slow growth and biocontainment requirements. Here we present a synthetic biology framework for assaying Mtb drug targets in engineered E. coli. We construct Target Essential Surrogate E. coli (TESEC) in which an essential metabolic enzyme is deleted and replaced with an Mtb-derived functional analog, linking bacterial growth to the activity of the target enzyme. High throughput screening of a TESEC model for Mtb alanine racemase (Alr) revealed benazepril as a targeted inhibitor, a result validated in whole-cell Mtb. In vitro biochemical assays indicated a noncompetitive mechanism unlike that of clinical Alr inhibitors. We establish the scalability of TESEC for drug discovery by characterizing TESEC strains for four additional targets.

PMID:35798732 | DOI:10.1038/s41467-022-31570-3

JACS Au. 2022 May 18;2(6):1324-1337. doi: 10.1021/jacsau.2c00002. eCollection 2022 Jun 27.

ABSTRACT

HaloTag labeling technology has introduced unrivaled potential in protein chemistry and molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, a tetramethylrhodamine- and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We showed that highly efficient labeling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from the wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates the need for expensive and laborious protein engineering.

PMID:35783171 | PMC:PMC9241015 | DOI:10.1021/jacsau.2c00002

Anticancer Res. 2022 Jul;42(7):3453-3461. doi: 10.21873/anticanres.15832.

ABSTRACT

BACKGROUND/AIM: Cytotoxic payload conjugation to antibodies efficiently suppresses tumors and contributes to the improvement of cancer survival. In our previous study, c-Kit targeting antibody-drug conjugate (2G4-DM1) with DM1, a microtubule inhibitor, efficiently suppressed tumor growth. However, slow-growing c-Kit-positive tumors, such as GIST-48, did not efficiently respond to DM1. In this study, we aimed to treat tumors using 2G4 immunotoxin with Pseudomonas exotoxin A (PE) as a payload.

MATERIALS AND METHODS: Modified FcBP-PE24 containing p-benzoyl-L-phenylalanine, unnatural amino acid, was expressed in E. coli and purified. Then, photoconjugation of 2G4 antibody and FcBP-PE24 at 365 nm was carried out and 2G4 immunotoxin was purified using anion exchange chromatography. In vitro cytotoxicity of 2G4 immunotoxins was assessed in HMC-1.2, GIST-48, and MDA-MB-453 cells. Then, in vivo efficacy analysis was performed using C.B-17 SCID mice.

RESULTS: 2G4 immunotoxin efficiently induced cytotoxicity in 2G4-DM1-resistant HMC-1.2 and GIST-48 cells by inhibiting protein synthesis but not in c-Kit-negative MDA-MB-453 cells. The results showed ~200-fold or more increase in cytotoxicity against c-Kit-positive cells compared to IC50 of 2G4-DM1. In addition, 2G4 immunotoxin suppressed tumor growth in the in vivo xenograft mouse model.

CONCLUSION: 2G4 immunotoxins could be an alternative therapeutic strategy for microtubule inhibitor- resistant cancer cells.

PMID:35790260 | DOI:10.21873/anticanres.15832

ACS Infect Dis. 2022 Jul 8;8(7):1241-1252. doi: 10.1021/acsinfecdis.2c00004. Epub 2022 Jun 28.

ABSTRACT

Penicillin-binding proteins (PBPs) make up an essential class of bacterial enzymes that carry out the final steps of peptidoglycan synthesis and regulate the recycling of this polymeric structure. PBPs are an excellent drug target and have been the most clinically relevant antibacterial target since the 1940s with the introduction of β-lactams. Despite this, a large gap in knowledge remains regarding the individual function and regulation of each PBP homologue in most bacteria. This can be attributed to a lack of chemical tools and methods that enable the study of individual PBPs in an activity-dependent manner and in their native environment. The development of such methods in Gram-negative bacteria has been particularly challenging due to the presence of an outer membrane and numerous resistance mechanisms. To address this, we have developed an optimized live-cell assay for screening inhibitors of the PBPs in Escherichia coli MG1655. We utilized EDTA to permeabilize Gram-negative cells, enabling increased penetration of our readout probe, Bocillin-FL, and subsequent analysis of PBP-inhibition profiles. To identify scaffolds for future development of PBP-selective activity-based probes, we screened ten β-lactams, one diazabicyclooctane, and one monobactam for their PBP-selectivity profiles in E. coli MG1655. These results demonstrate the utility of our assay for the screening of inhibitors in live, non-hypersusceptible Gram-negative organisms.

PMID:35763562 | DOI:10.1021/acsinfecdis.2c00004

Methods Mol Biol. 2022;2523:151-160. doi: 10.1007/978-1-0716-2449-4_10.

ABSTRACT

Nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NOD2 have been identified as intracellular receptors for bacterial peptidoglycan for almost two decades; however, the direct binding with their respective ligands has only been recently demonstrated due to the difficulty of achieving large quantity of proteins with high purity. Here we describe a strategy combining immunoprecipitation of GFP-tagged proteins and microscale thermophoresis (MST) for efficient one-step purification of NOD1-GFP and NOD2-GFP and easy measurement of the binding affinities of NOD1 or NOD2 with sphingosine-1-phosphate (S1P) using small amount of proteins (nM range). This method will allow the identification of novel agonists/antagonists for NOD1/2.

PMID:35759196 | DOI:10.1007/978-1-0716-2449-4_10

Int J Mol Sci. 2022 Jun 8;23(12):6415. doi: 10.3390/ijms23126415.

ABSTRACT

Staphylococcus aureus is a notorious biofilm-producing pathogen that is frequently isolated from implantable medical device infections. As biofilm ages, it becomes more tolerant to antimicrobial treatment leading to treatment failure and necessitating the costly removal of infected devices. In this study, we performed in-solution digestion followed by TMT-based high-throughput mass spectrometry and investigated what changes occur in the proteome of S. aureus biofilm grown for 3-days and 12-days in comparison with 24 h planktonic. It showed that proteins associated with biosynthetic processes, ABC transporter pathway, virulence proteins, and shikimate kinase pathway were significantly upregulated in a 3-day biofilm, while proteins associated with sugar transporter, degradation, and stress response were downregulated. Interestingly, in a 3-day biofilm, we observed numerous proteins involved in the central metabolism pathways which could lead to biofilm growth under diverse environments by providing an alternative metabolic route to utilize energy. In 12-day biofilms, proteins associated with peptidoglycan biosynthesis, sugar transporters, and stress responses were upregulated, whereas proteins associated with ABC transporters, DNA replication, and adhesion proteins were downregulated. Gene Ontology analysis revealed that more proteins are involved in metabolic processes in 3dwb compared with 12dwb. Furthermore, we observed significant variations in the formation of biofilms resulting from changes in the level of metabolic activity in the different growth modes of biofilms that could be a significant factor in S. aureus biofilm maturation and persistence. Collectively, potential marker proteins were identified and further characterized to understand their exact role in S. aureus biofilm development, which may shed light on possible new therapeutic regimes in the treatment of biofilm-related implant-associated infections.

PMID:35742863 | PMC:PMC9223533 | DOI:10.3390/ijms23126415

PLoS Pathog. 2022 Jun 22;18(6):e1010516. doi: 10.1371/journal.ppat.1010516. eCollection 2022 Jun.

ABSTRACT

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

PMID:35731836 | PMC:PMC9216600 | DOI:10.1371/journal.ppat.1010516

Annu Rev Biochem. 2022 Jun 21;91:221-243. doi: 10.1146/annurev-biochem-040320-103817.

ABSTRACT

Genetic code reprogramming has enabled us to ribosomally incorporate various nonproteinogenic amino acids (npAAs) into peptides in vitro. The repertoire of usable npAAs has been expanded to include not only l-α-amino acids with noncanonical sidechains but also those with noncanonical backbones. Despite successful single incorporation of npAAs, multiple and consecutive incorporations often suffer from low efficiency or are even unsuccessful. To overcome this stumbling block, engineering approaches have been used to modify ribosomes, EF-Tu, and tRNAs. Here, we provide an overview of these in vitro methods that are aimed at optimal expansion of the npAA repertoire and their applications for the development of de novo bioactive peptides containing various npAAs.

PMID:35729073 | DOI:10.1146/annurev-biochem-040320-103817

Talanta. 2022 May 27:123564. doi: 10.1016/j.talanta.2022.123564. Online ahead of print.

ABSTRACT

Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the ubiquitin and ubiquitin chains. The preferences of UBDs for ubiquitin chains of specific length and linkage are central to their functions. We demonstrated that an artificial tandem hybrid UBD (ThUBD) exhibits an unbiased high affinity to all ubiquitin chains and is a promising tool for global ubiquitination profiling research. In this study, we labeled fluorescein on the four cysteine residues in the N-terminal glutathione S-transferase (GST) tag of ThUBD, generating a fluorescein-labeled ThUBD (ThUBD-Flu) probe for direct polyubiquitination signal imaging and visualization. Compared to the canonical ubiquitin antibody method, the ThUBD-Flu is hyper-sensitive and accurate to detect ubiquitination signal. More importantly, the ThUBD-Flu probe provided, for the first time, a widely applicable, super-sensitive and unbiased technique for in situ detection of intracellular polyubiquitination signal through immunofluorescence staining, which was only achievable with recombinant fluorescence tag fused ubiquitin gene previously. We propose that ThUBD-Flu, combined with evolving microscopy technology, could serve as prototypes to track and trace cellular polyubiquitination signal in vivo.

PMID:35710467 | DOI:10.1016/j.talanta.2022.123564