Biology subjects

Rosenberg, O.

Publications and source records attributed to Rosenberg, O..

9 recordsLinked to original sources

bioRxiv2025

Archaeal SegAB forms a bipolar structure that promotes chromosome segregation in spherical cells

Archaeal segAB operons are thought to promote chromosome segregation, but their mechanism remains unknown. We employ comparative genomics, structural biology, genetic knockouts, and quantitative cell biology to investigate how SegA and SegB proteins work together to segregate chromosomes in the thermophilic archaeon Sulfolobus acidocaldarius. In vitro, SegB binds a centromeric DNA sequence adjacent to the segAB operon, and in vivo forms a distinct focus on each segregating chromosome. SegA, a ParA-like ATPase, binds DNA non-specifically in vitro and promotes chromosome compaction and segregation in vivo. During division, SegA shifts from chromosome-associated puncta to form a single, elongated figure that runs between separating SegB foci. Late in division, SegA retreats to regions surrounding separated SegB foci. Elongated SegA figures appear in segB knockout cells but no longer lie perpendicular to the division plane. We propose that SegA and SegB interact to form a bipolar, DNA-segregating structure radically different from bacterial ParABS systems.

cell biology↗

bioRxiv2024

How Mycobacterium tuberculosis builds a home: Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs.

Mycobacterium tuberculosis (MTB) infects and replicates in lung mononuclear phagocytes (MNPs) with astounding ability to evade elimination. ESX-1, a type VII secretion system, acts as a virulence determinant that contributes to MTBs ability to survive within MNPs, but its effect on MNP recruitment and/or differentiation remains unknown. Here, using single-cell RNA sequencing, we studied the role of ESX-1 in MNP heterogeneity and response in mice and murine bone marrow-derived macrophages (BMDM). We found that ESX-1 is required for MTB to recruit diverse MNP subsets with high MTB burden. Further, MTB induces an anti-inflammatory signature in MNPs and BMDM in an ESX-1 dependent manner. Similarly, spatial transcriptomics revealed an upregulation of anti-inflammatory signals in MTB lesions, where monocyte-derived macrophages concentrate near MTB-infected cells. Together, our findings suggest that MTB ESX-1 mediates the recruitment and differentiation of anti-inflammatory MNPs, which MTB can infect and manipulate for survival.

microbiology↗

bioRxiv2023

Chemoenzymatic syntheses of fluorine-18-labeled disaccharides from FDG yield potent sensors of living bacteria in vivo

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach, that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-D-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with {beta}-D-glucose-1-phosphate in the presence of maltose phosphorylase, both the -1,4 and -1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (,-1,1), laminaribiose ({beta}-1,3), and cellobiose ({beta}-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. The lead sakebiose-derived tracer [18F]FSK was stable in human serum and showed high uptake in preclinical models of myositis and vertebral discitis-osteomyelitis. Both the synthetic ease, and high sensitivity of [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

microbiology↗

bioRxiv2020

Cyclic gallium-68 labeled peptides for specific detection of human angiotensin-converting enzyme 2

In this study, we developed ACE2-specific, peptide-derived 68Ga-labeled radiotracers, motivated by the hypotheses that (1) ACE2 is an important determinant of SARS-CoV-2 susceptibility, and (2) that modulation of ACE2 in COVID-19 drives severe organ injury. MethodsA series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears two cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with 68Ga to generate peptide radiotracers ([68Ga]NOTA-PEP). The aminocaproate-derived radiotracer [68Ga]NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model. ResultsCyclic DX-600 derived peptides had markedly lower IC50s than their linear counterparts. The three cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated IC50s similar to, or lower than the parent DX600 molecule. Peptides were readily labeled with 68Ga, and the biodistribution of [68Ga]NOTA-PEP4 was determined in a hACE2 transgenic murine cohort. Pharmacologic concentrations of co-administered NOTA-PEP ("blocking") showed significant reduction of [68Ga]NOTA-PEP4 signals in the in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in vivo results. ConclusionsNOTA-conjugated, cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for 68Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer [68Ga]NOTA-PEP4 showed specific binding in the heart, liver, lungs and intestine - organs known to be affected in SARS-CoV-2 infection. These results suggest that [68Ga]NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2 infected murine models and COVID-19 patients. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/412809v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@f3047org.highwire.dtl.DTLVardef@12ab9a9org.highwire.dtl.DTLVardef@33f43org.highwire.dtl.DTLVardef@12e77ed_HPS_FORMAT_FIGEXP M_FIG For Table of Contents use only C_FIG

biochemistry↗

bioRxiv2020

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

A serious public health crisis is currently unfolding due to the SARS-CoV-2 pandemic. SARS-CoV-2 viral entry depends on an interaction between the receptor binding domain of the trimeric viral Spike protein (Spike-RBD) and the dimeric human angiotensin converting enzyme 2 (ACE2) receptor. While it is clear that strategies to block the Spike/ACE2 interaction are promising as anti-SARS-CoV-2 therapeutics, our current understanding is insufficient for the rational design of maximally effective therapeutic molecules. Here, we investigated the mechanism of Spike/ACE2 interaction by characterizing the binding affinity and kinetics of different multimeric forms of recombinant ACE2 and Spike-RBD domain. We also engineered ACE2 into a split Nanoluciferase-based reporter system to probe the conformational landscape of Spike-RBDs in the context of the Spike trimer. Interestingly, a dimeric form of ACE2, but not monomeric ACE2, binds with high affinity to Spike and blocks viral entry in pseudotyped virus and live SARS-CoV-2 virus neutralization assays. We show that dimeric ACE2 interacts with an RBD on Spike with limited intra-Spike avidity, which nonetheless contributes to the affinity of this interaction. Additionally, we demonstrate that a proportion of Spike can simultaneously interact with multiple ACE2 dimers, indicating that more than one RBD domain in a Spike trimer can adopt an ACE2-accessible "up" conformation. Our findings have significant implications on the design strategies of therapeutic molecules that block the Spike/ACE2 interaction. The constructs we describe are freely available to the research community as molecular tools to further our understanding of SARS-CoV-2 biology.

biochemistry↗

bioRxiv2019

A large inner membrane pore defines the ESX translocon

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane; however, the architecture and mechanism of translocation has remained elusive. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the model organism Mycobacterium smegmatis. The structure reveals two large -helical membrane pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric cytoplasmic domain is poised to gate and regulate the pores function. Our study provides mechanistic insights into the ESX systems and will guide structure-based design of drugs targeting this unique bacterial translocon.\n\nOne Sentence SummaryThe structure of the ESX-3 secretion system reveals a pore of sufficient size for the transit of folded substrates and a complex, cytoplasmic regulatory apparatus.

microbiology↗

bioRxiv2019

Modulating pathogenesis with Mobile-CRISPRi

Pathogens express a set of proteins required for establishing and maintaining an infection, termed virulence life-style genes (VLGs). Due to their outsized importance in pathogenesis, VLG products are attractive targets for the next generation of antimicrobials. However, precise manipulation of VLG expression in the context of infection is technically challenging, limiting our ability to understand the roles of VLGs in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of gene knockdown tools that are transferred by conjugation and stably integrate into pathogen genomes that we call \"Mobile-CRISPRi\". Here we show the efficacy of Mobile-CRISPRi in controlling VLG expression in a murine infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid non-specific toxicity. As a proof of principle, we demonstrate that knockdown of a VLG encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of VLGs across many bacterial species and pathogenesis models.\n\nImportanceAntibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically these genetic drivers have been difficult to manipulate, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study virulence life-style genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

microbiology↗

bioRxiv2019

Two accessory proteins govern MmpL3 mycolic acid transport in mycobacteria

Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterial specific antibiotics, and a mediator of M. tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of M. smegmatis and M. tuberculosis, is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate transport to the cell wall. In light of these findings we propose Msmeg_0736/Rv0383c be named \"TMM transport factor A\", TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex, but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3 mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it stabilizes the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis.

microbiology↗

bioRxiv2018

Determination of biophysical parameters for enhancement of human osteoblast proliferation by mechanical vibration in the infrasonic frequency range

Experimental methods for studying an enhancement of osteoblast proliferation in vitro provide tools for the research of biochemical processes involved in bone turnover in vivo. Some of the current methods used for this purpose are based on the ability of the osteoblasts to enhance proliferation by mechanical stimulation. We describe an experimental approach of biomechanical stimulation of cultured human osteoblast-like cells by vibration. This method is based on the specially designed controlled vibration setup that consists of an electric actuator, with horizontally mounted well plate containing cell cultures. Previously this method found to be effective to enhance cell proliferation, but the exact mechanical parameters of effective vibration were elusive. The current low friction system for mechanical stimulation of osteoblast-like cells in vitro provides recording of narrow range mechanical parameters in the infrasonic spectrum.\n\nWe exposed human osteoblast-like cells in explant monolayer culture to mechanical vibration in the 10-70Hz range of frequencies and found that 50-70 Hz of vibration frequency is optimal for inducing osteoblast proliferation that was deduced from interrelation between unchanged cell number in culture samples with significant decrease in cell death rate (decreased LDH activity in culture media, p<.05) and with parallel decrease of their maturation level (p<.01).\n\nIn this report we determined the optimal mechanical parameters and excitation protocol for induction of osteoblast proliferation in vitro by using a tunable and versatile mechanical platform, which can be used in the research of cell mechanotransduction.

cell biology↗