Biology subjects

Caffrey, M.

Publications and source records attributed to Caffrey, M..

5 recordsLinked to original sources

bioRxiv2024

Structural insights into Pseudomonas aeruginosa lysine-specific uptake mechanism for extremely low pH regulation

Under conditions of extremely low pH, in addition to transporting lysine, bacterial lysine-specific permease (LysP) interacts with the transcriptional regulator CadC to upregulate cadBA operon expression. cadBA encodes CadA, which decarboxylates lysine to cadaverine, and CadB, which exports cadaverine to the environment to reduce acidity. This process is crucial for survival of pathogenic bacteria in their hosts. Here, we report the inward-occluded (3.2 - 5.3 [A]) cryo-EM structure of Pseudomonas aeruginosa LysP bound to L-lysine and in complex with a nanobody. L-lysine is coordinated by hydrophobic stacking, cation-{pi} interactions and hydrogen bonding mostly with polar uncharged LysP residues. LysP reconstituted into liposomes showed robust and specific transport of L-lysine with the transport being inhibited by L-4-thialysine (S-2-aminoethyl-L-cysteine). These findings inform our understanding of the specific recognition, inhibition, and transport mechanism of L-lysine by LysP, which will have important ramifications for the design of antibiotics to target bacterial LysP.

biochemistry↗

bioRxiv2024

Towards Real-Time Airborne Pathogen Sensing: Electrostatic Capture and On-Chip LAMP Based Detection of Airborne Viral Pathogens

Considerable loss of life, economic slowdown, and public health risk associated with the transmission of airborne respiratory pathogens was underscored by the recent COVID-19 pandemic. Airborne transmission of zoonotic diseases such as the highly pathogenic avian influenza (HPAI) and porcine reproductive and respiratory syndrome virus (PRRSV) has caused major disruptions to domestic and global food security. Current ambient air pathogen monitoring systems involves the collection of air samples from indoor settings suspected of viral contamination, followed by subsequent processing of capture samples to determine the presence and species of airborne viral matter. Nucleic acid amplification techniques are considered the gold standard for pathogen diagnostics. Currently, the necessary extraction and purification of viral RNA from air collector systems prior to sample analysis is both time consuming and performed manually. A monitoring system with separate air sampling and biochemical detection procedures is prone to delay the response to emergent viral threats. In this paper, we present a pathogen monitoring system that overcomes these limitations related to extraction and purification of viral samples and lays the groundwork for a real-time monitor for airborne viral pathogens. We demonstrate a high flow electrostatic precipitator system, that uses small collection wells as counter electrodes for pathogen collection. Integrated reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is used for detection of captured viral matter within wells. On-chip heating of collection wells is enabled by integrated planar heaters and small volumes of reagent (30 L) directly to the collection wells. We present the design of such a system and show experimental results that demonstrate the use of this device for detection of aerosolized SARS-CoV-2 virus like particles (VLPs), a model pathogen for SARV-CoV-2.

bioengineering↗

bioRxiv2024

VLP-Based Model for Study of Airborne Viral Pathogens

The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution, as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus like particles (VLP). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling study of BSL3 and BSL4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins including those of newly discovered pathogens and mutant variants; (v) the ability to introduce viral sequences to develop nucleic acid amplification assays. ImportanceStudy and detection of airborne pathogens is hampered by the lack of appropriate model systems. In this work we demonstrate that noninfectious Virus Like Particles (VLP) represent attractive models to study airborne viral pathogens. Specifically, VLP are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

molecular biology↗

bioRxiv2022

The SARS-CoV-2 envelope (E) protein forms a calcium- and voltage-activated calcium channel

The function of ion channels is essential in the infectious cycle of many viruses. To facilitate viral uptake, maturation and export, viruses must modify the ionic balance of their host cells, in particular of calcium ions (Ca2+). Viroporins encoded in the viral genome play a key part in altering the cells ionic homeostasis. In SARS-Coronavirus-2 (SARS-CoV-2) - the causative agent of Covid-19 - the envelope (E) protein is considered to form ion channels in ERGIC organellar membranes, whose function is closely linked to disease progression and lethality. Deletion, blockade, or loss-of-function mutation of coronaviral E proteins results in propagation-deficient or attenuated virus variants. The exact physiological function of the E protein, however, is not sufficiently understood. Since one of the key features of the ER is its function as a Ca2+ storage compartment, we investigated the activity of E in the context of this cation. Molecular dynamics simulations and voltage-clamp electrophysiological measurements show that E exhibits ion channel activity that is regulated by increased luminal Ca2+ concentration, membrane voltage, post-translational protein modification, and negatively charged ERGIC lipids. Particularly, calcium ions bind to a distinct region at the ER-luminal channel entrance, where they activate the channel and maintain the pore in an open state. Also, alongside monovalent ions, the E protein is highly permeable to Ca2+. Our results suggest that the physiological role of the E protein is the release of Ca2+ from the ER, and that the distinct Ca2+ activation site may serve as a promising target for channel blockers, potentially inhibiting the infectious cycle of coronaviruses.

biophysics↗

bioRxiv2021

Reversing the miRNA -5p/-3p stoichiometry reveals physiological roles and targets of miR-140 miRNAs

The chondrocyte specific miR-140 miRNAs are necessary for normal endochondral bone growth in mice. miR-140 deficiency causes dwarfism and craniofacial deformity. However, the physiologically important targets of miR-140 miRNAs are still unclear. The miR-140 gene (Mir140) encodes three chondrocyte-specific microRNAs, miR-140-5p, derived from the 5 strand of primary miR-140, and miR140-3p.1 and -3p.2, derived from the 3 strand of primary miR-140. miR-140-3p miRNAs are ten times more abundant than miR-140-5p likely due to the non-preferential loading of miR-140-5p to Argonaute proteins. To differentiate the role of miR-140-5p and -3p miRNAs in endochondral bone development, two distinct mouse models, miR140-C>T, in which the first nucleotide of miR-140-5p was altered from cytosine to uridine, and miR140-CG, where the first two nucleotides of miR-140-3p were changed to cytosine and guanine, were created. These changes are expected to alter Argonaute protein loading preference of -5p and -3p to increase -5p loading and decrease -3p loading without changing the function of miR140-5p. These models presented a mild delay in epiphyseal development with delayed chondrocyte maturation. Using RNA-sequencing analysis of the two models, direct targets of miR140-5p, including Wnt11, were identified. Disruption of the predicted miR140-5p binding site in the 3 untranslated region of Wnt11 was shown to increase Wnt11 mRNA expression and caused a modest acceleration of epiphyseal development. These results show that the relative abundance of miRNA-5p and -3p can be altered by changing the first nucleotide of miRNAs in vivo, and this method can be useful to identify physiologically important miRNA targets.

genetics↗