Biology subjects

Carey, A. F.

Publications and source records attributed to Carey, A. F..

6 recordsLinked to original sources

bioRxiv2026

FunctionaL Assigning Sequence Homing (FLASH) maps phenotype to sequence with deep and machine learning

Genome-wide association studies (GWAS) map genetic variation to a reference genome and correlate variants to phenotypes. Yet, GWAS and similar procedures have limitations, including an inability to predict phenotype on variants never seen during the discovery phase and difficulty integrating structural variants. Deep and machine learning alternatives have not been successful at consistent prediction of resistance phenotypes (Hu et al. 2024). Here, we introduce FLASH: a new interpretable, statistically-based deep learning framework that operates directly on raw sequencing reads. In over 35,000 isolates of bacteria, fungi and viruses, FLASH achieves uniformly high accuracy on independent test data, including on variation never seen in training, meeting or exceeding bespoke state of the art methods. FLASH identifies canonical drug targets ab initio and new pan-species predictors of virulence, including those lacking annotation and those only partially aligned to NCBI reference databases. Further, FLASH can predict phenotypes beyond the possibility of GWAS, such as bacterial host range of phage, a task that to our knowledge is impossible today. FLASH is simple to run, highly efficient and constitutes a new approach for predicting gene function and phenotype across the tree of life. It is especially valuable when bioethical concerns and the vast genetic complexity of pathogenic microbes limit the feasibility of experimental validation.

bioinformatics↗

bioRxiv2024

Genetic requirements of Mycobacterium tuberculosis for survival under pathogen-specific immunity

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), remains a persistent global health challenge due to the lack of an effective vaccine. The only licensed TB vaccine, Bacille Calmette-Guerin (BCG), is a live attenuated strain of Mycobacterium bovis, that protects young children from severe disease but fails to provide protection through adulthood. It is unclear why BCG-induced immunity provides incomplete protection despite inducing a robust CD4+ T cell response, a known critical correlate of protection against Mtb. We set out to interrogate mycobacterial determinants of vaccine escape using a functional genomics approach, TnSeq, to define mycobacterial genes required for survival in mice vaccinated with BCG, the live attenuated Mtb vaccine, {Delta}LprG, and in mice with Mtb immunity conferred by prior infection. We find that critical virulence genes associated with acute infection and exponential growth are less essential in hosts with adaptive immunity, including genes encoding the Esx-1 and Mce1 systems. Genetic requirements for Mtb growth in vaccinated and previously Mtb-infected hosts mirror the genetic requirements reported for bacteria grown under in vitro conditions that reflect aspects of the adaptive immune response as well as in mouse genetic backgrounds where bacterial growth is restricted. Across distinct immunization conditions, differences in genetic requirements between live attenuated vaccines and vaccination routes are observed, suggesting that different immunization strategies impose distinct bacterial stressors. Collectively, these data support the idea that Mtb requires genes that enable stress adaptation and growth arrest upon encountering the restrictive host environment induced by the adaptive immune response. We demonstrate that TnSeq can be used to understand the bacterial genetic requirements for survival in vaccinated hosts across pre-clinical live attenuated vaccines and therefore may be applied to other vaccine modalities. Understanding how Mtb survives in the setting of vaccine-induced immunity could uncover bacterial vulnerabilities to inform the development and prioritization of new vaccines or adjuvant therapies.

microbiology↗

bioRxiv2024

Description of Bacterial RNA Transcripts Detected in Mycobacterium tuberculosis Infected Cells from Peripheral Human Granulomas using Single Cell RNA Sequencing

Mycobacterium tuberculosis (Mtb) remains a global human health threat and a significant cause of human morbidity and mortality. We document here the capture of Mtb transcripts in libraries designed to amplify eukaryotic mRNA. These reads are often considered spurious or nuisance and are rarely investigated. Because of early literature suggesting the possible presence of polyadenylated transcripts in Mtb RNA, we included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples from patients presenting at the TB clinic, Port Moresby General Hospital, Papua New Guinea. We used 10X Genomics single-cell RNA sequencing transcriptomics pipeline, which initiates mRNA amplification with poly-T primers on [~]30-micron beads designed to capture, in this case, human mRNA associated with individual cells in the clinical samples. Utilizing the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences (rrs and rrl, respectively) and other bacterial gene transcripts in the cell culture and patient samples. We interpret Mtb reads associated with the host cells unique molecular identifier (UMI) and transcriptome to indicate infection of that individual host cell. The Mtb transcripts detected showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least 1 sequence difference compared to the H37Rv reference genome. The data presented includes only bacterial sequences from patients with TB infections that were confirmed by the hospital pathology lab using acid-fast microscopy and/or GeneXpert analysis. The repeated, non-random nature of the sequence variations detected in Mtb rrs and rrl transcripts from multiple patients, suggests that, even though this appears to be a stochastic process, there is possibly some selective pressure that limits the types and locations of sequence variation allowed. The variation does not appear to be entirely artefactual, and it is hypothesized that it could represent an additional mechanism of adaptation to enhance bacterial fitness against host defenses or chemotherapy.

microbiology↗

bioRxiv2024

Single Cell Analysis of Peripheral TB-Associated Granulomatous Lymphadenitis

We successfully employed a single cell RNA sequencing (scRNA-seq) approach to describe the cells and the communication networks characterizing granulomatous lymph nodes of TB patients. When mapping cells from individual patient samples, clustered based on their transcriptome similarities, we uniformly identify several cell types that known to characterize human and non-human primate granulomas. Whether high or low Mtb burden, we find the T cell cluster to be one of the most abundant. Many cells expressing T cell markers are clearly quantifiable within this CD3 expressing cluster. Other cell clusters that are uniformly detected, but that vary dramatically in abundance amongst the individual patient samples, are the B cell, plasma cell and macrophage/dendrocyte and NK cell clusters. When we combine all our scRNA-seq data from our current 23 patients (in order to add power to cell cluster identification in patient samples with fewer cells), we distinguish T, macrophage, dendrocyte and plasma cell subclusters, each with distinct signaling activities. The sizes of these subclusters also varies dramatically amongst the individual patients. In comparing FNA composition we noted trends in which T cell populations and macrophage/dendrocyte populations were negatively correlated with NK cell populations. In addition, we also discovered that the scRNA-seq pipeline, designed for quantification of human cell mRNA, also detects Mtb RNA transcripts and associates them with their host cells transcriptome, thus identifying individual infected cells. We hypothesize that the number of detected bacterial transcript reads provides a measure of Mtb burden, as does the number of Mtb-infected cells. The number of infected cells also varies dramatically in abundance amongst the patient samples. CellChat analysis identified predominating signaling pathways amongst the cells comprising the various granulomas, including many interactions between stromal or endothelial cells and the other component cells, such as Collagen, FN1 and Laminin. In addition, other more selective communications pathways, including MIF, MHC-1, MHC-2, APP, CD 22, CD45, and others, are identified as originating or being received by individual immune cell components. Author SummaryThe research conducted describes the cellular composition and communication networks within granulomatous lymph nodes of tuberculosis (TB) patients, employing a single-cell RNA sequencing (scRNA-seq) approach. By analyzing individual patient samples and clustering cells based on their transcriptome similarities, the study reveals several consistent cell types described to be present in both human and non-human primate granulomas. Notably, T cell clusters emerge as abundant in most samples. Additionally, variations in the abundance of B cells, plasma cells, macrophages/dendrocytes, and NK cells among patient samples are observed. Pooling scRNA-seq data from 23 patients enabled the identification of T, macrophage, dendrocyte, and plasma cell subclusters, each displaying distinct signaling activities. Moreover, the study uncovers a surprising capability of the scRNA-seq pipeline to detect Mtb RNA transcripts within host cells, providing insights into individual infected cells and Mtb burden. CellChat analysis unveils predominant signaling pathways within granulomas, highlighting interactions between stromal/endothelial cells and other immune cell components. Moreover, selective communication pathways involving molecules such as Collagen, FN1, Laminin, CD99, MIF, MHC-1, APP and CD45 are identified, shedding light on the intricate interplay within granulomatous lymph nodes during TB infection.

pathology↗

bioRxiv2022

Multiplexed strain phenotyping defines consequences of genetic diversity in Mycobacterium tuberculosis for infection and vaccination outcomes

There is growing evidence that genetic diversity in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, contributes to the outcomes of infection and public health interventions, such as vaccination. Epidemiological studies suggest that among the phylogeographic lineages of Mtb, strains belonging to Lineage 2 (L2) are associated with concerning clinical features including hypervirulence, treatment failure, and vaccine escape. The global expansion and increasing prevalence of L2 has been attributed to the selective advantage conferred by these characteristics, yet confounding host and environmental factors make it difficult to identify the bacterial determinants driving these associations in human studies. Here, we developed a molecular barcoding strategy to facilitate high-throughput, experimental phenotyping of Mtb clinical isolates. This approach allowed us to characterize growth dynamics for a panel of genetically diverse Mtb strains during infection and after vaccination in the mouse model. We found that L2 strains exhibit distinct growth dynamics in vivo and are resistant to the immune protection conferred by Bacillus Calmette-Guerin (BCG) vaccination. The latter finding corroborates epidemiological observations and demonstrates that mycobacterial features contribute to vaccine efficacy. To investigate the genetic and biological basis of L2 strains distinctive phenotypes, we performed variant analysis, transcriptional studies, and genome-wide transposon sequencing. We identified functional genetic changes across multiple stress- and host-response pathways in a representative L2 strain that are associated with variants in regulatory genes. These adaptive changes may underlie the distinct clinical characteristics and epidemiological success of this lineage.

microbiology↗

bioRxiv2018

Concurrent infection with Mycobacterium tuberculosis confers robust protection against secondary infection in macaques

Introduction Introduction Main text Methods References For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural M. tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted B ...

microbiology↗