Biology subjects

Sabnis, A.

Publications and source records attributed to Sabnis, A..

9 recordsLinked to original sources

bioRxiv2025

Immune-deficient bacteria serve as gateways to genetic exchange and microbial evolution

Horizontal gene transfer plays a key role in bacterial evolution, yet its efficiency under natural conditions, especially between genetically distinct strains, remains unclear. Using Staphylococcus aureus as a model, we found that gene transfer via various mechanisms is significantly restricted between strains from different clonal complexes (CCs), with the notable exception of lateral transduction, which occurs at high frequency. Interestingly, some strains exhibited a "promiscuous" ability to accept diverse mobile genetic elements. These strains were defective in the key immune defences of S. aureus, specifically the Type I restriction-modification systems that normally protect against foreign DNA. A broader analysis revealed that such immune-deficient mutants are widespread within S. aureus populations. Our study uncovered a trade-off that explains their persistence in nature: although these mutants are more susceptible to phage attack, they gain an evolutionary advantage by acquiring beneficial genes - such as those conferring antibiotic resistance - which enhance survival under selective pressure. These immune-deficient cells act as gateways for foreign DNA, which, once integrated and advantageous, can spread within the same CC. Our findings highlight an unexpected role for immune-deficient bacteria in facilitating the emergence of novel virulence factors and antibiotic resistance, emphasising their importance in shaping bacterial evolution.

microbiology↗

bioRxiv2024

The retina's neurovascular unit: Mueller glial sheaths and neuronal contacts

The neurovascular unit (NVU), comprising vascular, glial and neural elements, supports the energetic demands of neural computation, but this aspect of the retinas trilaminar vessel network is poorly understood. Only the innermost vessel layer - the superficial vascular plexus (SVP) - is ensheathed by astrocytes, like brain capillaries, whereas glial ensheathment in other layers derives from radial Muller glia. Using serial electron microscopy reconstructions from mouse and primate retina, we find that Muller processes cover capillaries in a tessellating pattern, mirroring the tiled astrocytic endfeet wrapping brain capillaries. However, gaps in the Muller sheath, found mainly in the intermediate vascular plexus (IVP), permit different neuron types to contact pericytes and the endothelial cells directly. Pericyte somata are a favored target, often at spine-like structures with a reduced or absent vascular basement lamina. Focal application of adenosine triphosphate (ATP) to the vitreal surface evoked Ca2+ signals in Muller sheaths in all three vascular layers. Pharmacological experiments confirmed that Muller sheaths express purinergic receptors that, when activated, trigger intracellular Ca2+ signals that are amplified by IP3-controlled intracellular Ca2+ stores. When rod photoreceptors die in a mouse model of retinitis pigmentosa (rd10), Muller sheaths dissociate from the deep vascular plexus (DVP) but are largely unchanged within the IVP or SVP. Thus, Muller glia interact with retinal vessels in a laminar, compartmentalized manner: glial sheathes are virtually complete in the SVP but fenestrated in the IVP, permitting direct neural-to-vascular contacts. In the DVP, the glial sheath is only modestly fenestrated and is vulnerable to photoreceptor degeneration.

neuroscience↗

bioRxiv2023

IS1-related large-scale deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli

Nitrofurantoin is a broad-spectrum first-line antimicrobial used for managing uncomplicated urinary tract infection. Loss-of-function mutations in chromosomal genes nfsA, nfsB, and ribE of Escherichia coli are known to reduce nitrofurantoin susceptibility. Here, we report monoclonal nitrofurantoin heteroresistance in E. coli and a novel genetic mechanism associated with this phenomenon. Subpopulations with reduced nitrofurantoin susceptibility in cultures of two E. coli blood strains were identified using population analysis profiling. Four colonies of each strain growing on agar with 0.5xMIC nitrofurantoin were sub-cultured in broth with 0.5xMIC nitrofurantoin (n=2) or without nitrofurantoin (n=2). Moreover, one colony of each strain growing without nitrofurantoin exposure was selected as a reference for genomic comparison. Whole-genome sequencing of all isolates were conducted on Illumina and Nanopore MinION systems. Both strains had a nitrofurantoin MICs of 64 mg/L. The proportion of cells grown at 0.5xMIC was two and 99 per million, respectively, which is distinct to that of a homogeneously susceptible or resistant isolate. All isolates grown at 0.5xMIC had 11-66 kbp deletions in chromosomal regions harbouring nfsB, and all these deletions were immediately adjacent to IS1-family insertion sequences. Although this study is limited to E. coli and nitrofurantoin, our findings suggest IS1-associated genetic deletion represents a hitherto unrecognised mechanism of heteroresistance that could compromise infection management and impact conventional antimicrobial susceptibility testing. Impact statementNitrofurantoin is widely used for treating and preventing urinary tract infection. Prevalence of nitrofurantoin resistance generally is low in E. coli. Here, we report nitrofurantoin heteroresistance in two E. coli blood strains and attribute this phenotype to IS1-associated deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase gene nfsB. Our discoveries demonstrate a novel genetic mechanism of heteroresistance and suggest detecting nitrofurantoin heteroresistance in E. coli urinary isolates for improving prescribing. Data summaryWhole-genome sequencing reads and genome assemblies generated in this study have been deposited under BioProject PRJEB58678 in the European Nucleotide Archive (ENA). Accession numbers are listed in Supplementary Table 1. Previously generated Illumina whole-genome sequencing reads of parental isolates EC0026B and EC0880B are available under ENA accessions ERR3142418 and ERR3142524, respectively.

microbiology↗

bioRxiv2023

Neurexin 3 is required for the specific S-cone to S-cone bipolar cell synapse in the mammalian retina

Precise wiring within sensory systems is critical for the accurate transmission of information. In the visual system, S-cone photoreceptors specialize in detecting short-wavelength light, crucial to color perception and environmental cue detection. S-cones form specific synapses with S-cone bipolar cells (SCBCs), a connection that is remarkably consistent across species. Yet, the molecular mechanisms guiding this specificity remain unexplored. To address this, we used the cone-dominant ground squirrel for deep-sequencing of cone subtype transcriptomes and identified Nrxn3 as an essential molecule for the S-cone to SCBC synapse. Using transgenic mouse models, we further examined the role of Nrxn3 in S-cones and discovered a significant reduction of SCBC connections in the absence of Nrxn3. This finding extends the known functions of neurexins, typically associated with synapse regulation, by highlighting their essential role in a specific synaptic connection for the first time. Moreover, the differentially expressed genes identified here pave the way for further investigations into the unique functions of cone subtypes.

neuroscience↗

bioRxiv2023

Investigating the roles of reflexes and central pattern generators in the control and modulation of human locomotion using a physiologically plausible neuromechanical model

Studying the neural components regulating movement in human locomotion is obstructed by the inability to perform invasive experimental recording. Neuromechanical simulations can provide insights by modeling the locomotor circuits. Past neuromechanical models proposed control of locomotion either driven by central pattern generators (CPGs) with simple sensory commands or by a purely reflex-based network regulated by state-machine mechanisms. However, the physiological interpretation of these state-machines remains unclear. Here, we present a physiologically plausible model to investigate spinal control and modulation of human locomotion. We propose a bio-inspired controller composed of two coupled central pattern generators (CPGs) that produce the rhythm and pattern and a reflex-based network simulating low-level reflex pathways and Renshaw cells. This reflex network is based on leaky-integration neurons, and the whole system does not rely on changing reflex gains according to the gait cycle state. The only component of the controller that maintains a state-machine mechanism is the balance controller of the trunk. The musculoskeletal model is composed of a skeletal structure and 9 muscles per leg generating movement in sagittal plane. After optimizing the open parameters, human locomotion replicating kinematics and muscle activation naturally emerged. Furthermore, when CPGs were not activated, no stable and physiologically plausible gaits could be achieved through optimization, suggesting the necessity of this component to generate rhythmic behavior without a state machine mechanism regulating reflex activation. The controller could reproduce a wide range of speeds from 0.3 to 1.9 m/s. The results also showed that the net influence of feedback on motoneurons during perturbed locomotion is predominantly inhibitory and that the CPG network provides the timing of motoneurons activation by exciting or inhibiting muscles in specific gait phases. The proposed bio-inspired controller could contribute to our understanding of locomotor circuits of the intact spinal cord and could be used to study neuromotor disorders.

neuroscience↗

bioRxiv2022

Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin Against Cystic Fibrosis Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonise and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate anti-pseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers; potentiating the activities of antibiotics when given in combination restoring and/or enhancing antibiotic efficacy. Growth, viability, minimum inhibitory concentration (MIC)-determination and synergy analysis showed that GA improved the efficacy of TOB against reference strains of P. aeruginosa, reducing TOB MICs and synergising with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the concentration of TOB required to inhibit 50% (MIC50) of viable cells (from 1.69 [95%CI 0.26-8.97] to 0.62 [95%CI 0.15-3.94] mg/L, p=0.002) and inhibit 90% (MIC90) (from 7.00 [95%CI 1.18-26.50] to 2.20 [95%CI 0.99-15.03] mg/L, p=0.001) compared with TOB-only. Investigating mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarisation of cytoplasmic membranes and permeabilisation of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three.

microbiology↗

bioRxiv2021

Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic

Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to lysis and bacterial death. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilised colistin resistance (mcr) genes. Both these colistin resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilisation of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilisation of the outer membrane of colistin resistant isolates by the polymyxin is in turn sufficient to sensitise bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in E. coli is typically due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

microbiology↗

bioRxiv2020

A high-density narrow-field inhibitory retinal interneuron with direct coupling to Müller glia

Amacrine cells are interneurons comprising the most diverse cell type in the mammalian retina. They help encode visual features such as edges or directed motion by mediating excitatory and inhibitory interactions between input (i.e. bipolar) and output (i.e. ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, neurovascular control and metabolic regulation. Here, we report that a previously poorly characterized, but relatively abundant, inhibitory amacrine cell type in the mouse retina is coupled directly to Muller glia. Electron microscopic reconstructions of this amacrine type revealed extensive associations with Muller glia, whose processes often completely ensheathe the neurites of this amacrine cell type. Microinjections of small tracer molecules into the somas of these amacrine cells led to selective labelling of nearby Muller glia, leading us to suggest the name "Muller glia-coupled amacrine cell" or MAC. Our electrophysiological data also indicate that MACs release glycine at conventional chemical synapses with amacrine, bipolar and retinal ganglion cells (RGCs), and viral transsynaptic tracing showed connections to several known RGC types. Visually-evoked responses revealed a strong preference for light increments; these "ON" responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling to other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function. Significance StatementGap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system, and play a myriad of roles including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells are rare and poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Muller glia.

neuroscience↗

bioRxiv2018

Colistin requires de novo lipopolysaccharide biosynthesis for activity

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

microbiology↗