Biology subjects

Rosch, J.

Publications and source records attributed to Rosch, J..

11 recordsLinked to original sources

bioRxiv2026

Antibiotic Persistence Emerges from Cell-State-Driven Transcriptional Reprogramming

Antibiotic persistence allows a subpopulation of bacterial cells to survive antibiotic treatment without acquiring resistance mutations, often contributing to treatment failure. Although many genes have been linked to persistence, their deletion rarely abolishes the phenotype, highlighting redundancy in persistence mechanisms. Moreover, the transient and heterogeneous nature of persister formation has made it difficult to resolve its molecular basis using bulk analyses. Here, we use bacterial single-cell RNA sequencing and functional assays in Klebsiella pneumoniae to demonstrate how transcriptional heterogeneity and redundancy in stress responses shape persistence outcomes. Using growth phase as a biologically meaningful axis of transcriptional variation, we reveal that even within an isogenic population, distinct transcriptional responses can be induced and co-contribute to survival. These responses are shaped by the cells pre-treatment transcriptional state and the mechanism of antibiotic action. Genetic and environmental perturbations, such as rpoS deletion and nutrient supplementation, shift pre-treatment cell states and alter persistence frequencies. Our findings establish the biological significance of transcriptional heterogeneity shaped by pre-treatment cell states, providing a systems-level framework for understanding persistence and suggesting strategies to enhance antibiotic efficacy by modulating cell states.

microbiology↗

bioRxiv2026

RNA Quality Control Enables Antibiotic Tolerance

The mechanisms underlying adaptation to antibiotic pressure within complex host environments remain incompletely understood. By experimentally evolving Streptococcus pneumoniae subjected to various antibiotics and immune states, we demonstrate populations adopting distinct adaptive strategies depending on specific selective context. General antibiotic stress drives convergent mutations in rny, encoding the RNA degradosome scaffold RNase Y, that exhibit broad-spectrum antibiotic tolerance and accelerated recovery. Single-cell transcriptomics revealed antibiotic-induced death is driven by transcriptional collapse, a catastrophic loss of RNA quantity and integrity. In contrast, rny mutants avert this via a bet-hedging strategy: a resilient minority maintains a baseline transcriptional profile, while a quiescent majority undergoes selective RNA degradation to preserve transcript fidelity. Upon stress removal, these populations execute a prioritized transcriptional ribosomal reboot, facilitating accelerated recovery. These findings identify RNA turnover as a tunable master regulator to survive combined pressures of antibiotics and immunity.

microbiology↗

bioRxiv2023

Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

bioengineering↗

bioRxiv2022

Generalized strategy for engineering mammalian cell-compatible RNA-based biosensors from random sequence libraries

Fluorescent RNA-based biosensors are useful tools for real-time detection of molecules in living cells. These biosensors typically consist of a chromophore-binding aptamer and a target-binding aptamer, whereby the chromophore-binding aptamer is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding. However, known riboswitches only exist for a limited number of molecules, significantly constraining biosensor design. To overcome this challenge, we designed a framework for producing mammalian cell-compatible biosensors using aptamers selected from a large random library by capture-SELEX. As a proof-of-concept, we generated and characterized a fluorescent RNA biosensor against L-dopa, the precursor of several neurotransmitters. Overall, we suggest that this approach will have utility for generating RNA biosensors that can reliably detect custom targets in mammalian cells.

molecular biology↗

bioRxiv2022

Pneumococcal Evasion of Antibiotics via Metabolic Adaptation During Infection

Streptococcus pneumoniae is a major human pathogen of global health concern, causing a range of mild to severe infections, including acute otitis media, pneumonia, sepsis, and meningitis. The rapid emergence of antibiotic resistance among S. pneumoniae isolates poses a serious public health problem worldwide. Resistant pneumococcal strains have rendered the mainstay treatment with beta-lactams, fluoroquinolones, and macrolides, ineffective. Antibiotic resistance in S. pneumoniae has spread globally via the emergence of de novo mutations and horizontal transfer of resistance. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage, as is often the case with other classes of antibiotics. In this study, we demonstrated that deleterious fitness costs constrain the emergence of individual fluoroquinolone resistance mutations in either topoisomerase IV or gyrase A in S. pneumoniae. Generation of double point mutations in the target enzymes in topoisomerase IV and gyrase A conferred high-level fluoroquinolone resistance while restoring fitness comparable to the sensitive wild-type. During an in vivo model of antibiotic resistance evolution, S. pneumoniae was able to circumvent deleterious fitness costs imposed by resistance determinants through development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, an effect that could be recapitulated pharmacologically. The metabolic mutants conferring tolerance resulted in a fitness benefit during infection following antibiotic treatment with fluroquinolones. These data suggest that emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by host fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes may be an important contributor to the evasion of antibiotic mediated killing.

microbiology↗

bioRxiv2021

Albumin-binding Aptamer Chimeras for Improved siRNA Bioavailability

Short interfering RNAs (siRNAs) are potent nucleic acid-based drugs designed to target disease driving genes that may otherwise be undruggable with small molecules. However, the potential of administering therapeutic siRNA in vivo is limited by poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. Nanocarriers have traditionally been explored as means to overcome these challenges, but they have intrinsic downsides such as dose-limiting toxicity and synthetic complexity. Backpacking on natural carriers such as albumin, which is present at high concentration and has a long half-life in serum, is an effective way to modify pharmacokinetics of biologic drugs that otherwise have poor bioavailability. In this work, we sought to develop albumin-binding aptamer-siRNA chimeras to improve the bioavailability of siRNA. We used a Systematic Evolution of Ligands through Exponential Enrichment (SELEX) approach to obtain RNA aptamers with modified bases that bind albumin with high affinity. We then fused the aptamers directly to an siRNA to generate the chimera structure. These aptamer-siRNA chimeras are stable in serum, exhibit potent gene knockdown capabilities in vitro, and display extended circulation time in vivo. We suggest that this albumin-binding aptamersiRNA chimera approach is a promising strategy for drug delivery applications.

bioengineering↗

bioRxiv2021

Immunosuppression broadens evolutionary pathways to treatment failure during Acinetobacter baumannii pneumonia

Acinetobacter baumannii is increasingly refractory to antibiotic treatment in healthcare settings. As is true of most human pathogens, the genetic path to antimicrobial resistance (AMR) and the role that the immune system plays in modulating AMR during disease are poorly understood. Here we reproduced several routes to fluoroquinolone resistance, performing evolution experiments using sequential lung infections in mice that are replete or depleted of neutrophils, providing two key insights into the evolution of drug resistance. First, neutropenic hosts acted as reservoirs for the accumulation of drug resistance during drug treatment. Selection for variants with altered drug sensitivity profiles arose readily in the absence of neutrophils, while immunocompetent animals restricted the appearance of these variants. Secondly, antibiotic treatment failure in the immunocompromised host was shown to occur without clinically defined resistance, an unexpected result that provides a model for how antibiotic failure occurs clinically in the absence of AMR. The genetic mechanism underlying both these results is initiated by mutations activating the drug egress pump regulator AdeL, which drives persistence in the presence of antibiotic. Therefore, antibiotic persistence mutations present a two-pronged risk during disease, causing drug treatment failure in the immunocompromised host while simultaneously increasing the emergence of high-level AMR.

microbiology↗

bioRxiv2020

Identification of colonisation and virulence determinants of Streptococcus pneumoniae via experimental evolution in mouse infection models

Streptococcus pneumoniae is a commensal of the human nasopharynx and a major cause of respiratory and invasive disease. Pneumococcus stimulates upper respiratory tract inflammation that promotes shedding from mucosal surfaces and transmission to new hosts. Colonisation and transmission are partially antagonistic processes. Adhesion to surfaces and evasion of host responses favours the former, whilst detachment, promoted by inflammation, is necessary for the latter. We sought to determine how adaptation and evolution of pneumococcus within its nasopharyngeal niche might progress when selective pressures associated with transmission were removed. This was achieved by serial passage of pneumococci in mouse models of nasopharyngeal carriage, manually transferring bacteria between mice. To assess the role of host environmental factors on pneumococcal evolution, we also performed analogous experimental evolution in a mouse pneumonia model, passaging pneumococci through lungs. Nasopharynx-passaged pneumococci became more effective colonisers, whilst those evolved within lungs showed reduced virulence. We observed selection of mutations in genes associated with cell wall biogenesis and metabolism in both nasopharynx and lung lineages, but identified prominent examples of parallel evolution that were niche specific. We focussed on gpsA, a gene in which the same single nucleotide polymorphism arose in two independently evolved nasopharynx-passaged lineages. We identified a single nucleotide change conferring resistance to oxidative stress and enhanced nasopharyngeal colonisation potential. We show that gpsA is also a frequent target of mutation during human colonisation. These findings highlight the role played by the host environment in determining trajectories of bacterial evolution and the potential of experimental evolution in animal infection models for identification of novel pathogen virulence and colonisation factors.

microbiology↗

bioRxiv2020

Respiratory bacteria stabilize and promote airborne transmission of influenza A virus

Influenza A virus (IAV) is a major pathogen of the human respiratory tract where the virus co-exists and interacts with bacterial populations comprising the respiratory microbiome. Synergies between IAV and respiratory bacterial pathogens promote enhanced inflammation and disease burden that exacerbate morbidity and mortality. We demonstrate that direct interactions between IAV and encapsulated bacteria commonly found in the respiratory tract promote environmental stability and infectivity of IAV. Antibiotic-mediated depletion of the respiratory bacterial flora abrogated IAV transmission in ferret models, indicating that these viral-bacterial interactions are operative for airborne transmission of IAV. Restoring IAV airborne transmission in antibiotic treated ferrets by co-infection with Streptococcus pneumoniae confirmed a role for specific members of the bacterial respiratory community in promoting IAV transmission. These results implicate a role for the bacterial respiratory flora in promoting airborne transmission of IAV.

microbiology↗

bioRxiv2019

Dynamic pneumococcal genetic adaptations support bacterial growth and inflammation during coinfection with influenza

Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These bacterial coinfections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms, but the specific bacterial genes that contribute to post-influenza pathogenicity are not known. Here, we used genome-wide transposon mutagenesis (Tn-Seq) to reveal bacterial genes that confer improved fitness in influenza-infected hosts. The majority of the 32 identified genes are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We generated single-gene deletion (SGD) mutants of five identified genes: SPD1414, SPD2047 (cbiO1), SPD0058 (purD), SPD1098, and SPD0822 (proB), to investigate their effect on in vivo fitness, disease severity, and host immune responses. Growth of SGD mutants was slightly attenuated in vitro and in vivo, but each still grew to high titers in the lungs of mock- and influenza-infected hosts. Despite high bacterial loads, mortality was significantly reduced or delayed with all SGD mutants. Time-dependent reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were also observed. Immunohistochemical staining further revealed that neutrophil phenotype and distribution was altered in the lungs of influenza-SGD coinfected animals. These studies demonstrate a critical role for specific bacterial genes and for bacterial metabolism in driving virulence and modulating immune function during influenza-associated bacterial pneumonia.

microbiology↗

bioRxiv2019

Allergic inflammation hinders synergistic viral-bacterial co-infection in C57BL/6 mice

Asthma is a chronic airways disease that can be exacerbated during respiratory infections. Our previous findings that the inflammatory state of allergic airways at the time of influenza A virus (IAV) infection in combination with epidemiologic findings that asthmatics were less likely to suffer from severe influenza during the 2009 pandemic suggest that additional complications of influenza, such as increased susceptibility to bacterial superinfection, may be mitigated in the allergic host. To test this hypothesis, we developed a murine model of triple-disease in which mice were first rendered allergic to Aspergillus fumigatus and co-infected with IAV and Streptococcus pneumoniae seven days apart. Significant alterations to known synergistic effects of co-infection were noted in the allergic mice including reduced morbidity and mortality, bacterial burden, maintenance of alveolar macrophages, and reduced lung inflammation and damage. The lung microbiome of allergic mice differed from that of non-allergic mice during co-infection. To investigate the impact of the microbiome on the pathogenesis of lung disease, we induced a perturbation with a short course of fluoroquinolone antibiotic that is often prescribed for lung infections. A significant change in the microbiome was complemented with alterations to the inflammatory profile and a drastic increase in pro-inflammatory cytokines in allergic mice which were now susceptible to severe disease from IAV and S. pneumoniae co-infection. Our data suggest that responses to co-infection in allergic hosts likely depends on the immune and microbiome states and that antibiotics should be used with caution in individuals with underlying chronic lung disease. Author Summary Asthma is a condition of the lungs that affects millions worldwide. Traditionally, respiratory infections are considered to have a negative impact on asthmatics. However, epidemiological data surrounding the 2009 influenza pandemic suggest that asthmatics may be better equipped to counter severe influenza including bacterial pneumonia. Herein, we introduce a novel mouse model system designed to recapitulate an influenza virus and Streptococcal co-infection in a host with fungal asthma. We found that underlying allergic asthma protects against severe disease induced by co-infection. Mice with underlying allergic inflammation had reduced damage to the lungs and did not show signs of respiratory distress. Among the differences noted in the allergic mice that were protected from viral and bacterial co-infection, was the lung microbiome. Allergic mice lost their protection from co-infection after we perturbed their lung microbiome with antibiotics suggesting that the lung microbiome plays a role in host immunity against invading pathogens.

pathology↗