Biology subjects

Stamm, C. E.

Publications and source records attributed to Stamm, C. E..

3 recordsLinked to original sources

bioRxiv2026

Targeted Delivery of Nucleic Acid and Protein Cargos into Primary Human Hematopoietic Stem Cells Using Bacteriophage T4

Here, we report, for the first time, delivery of mRNA and/or protein payloads into primary adult human hematopoietic stem cells (HSCs) using a bacteriophage-derived nanoparticle vector. We have been developing a new category of bacteriophage T4-engineered nanoparticles, termed "artificial viral vectors" (AVVs), for delivering therapeutic nucleic acid and protein complexes into human cells. Using a defined in vitro assembly-line platform, we decorated the capsid surface with mRNA and protein complexes through two outer capsid proteins, Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein). First, we displayed a Hoc-protein G fusion protein to which HSC-targeted monoclonal antibodies (mAbs) are attached. Then we decorated the nanoparticle with the Soc-fused HIV-TAT molecule to create a positively charged capsid surface with cell penetrating function. Reporter mRNA molecules are then displayed on the capsid surface and the nanoparticle is coated with lipids. Such T4-AVVs transduced HSCs and delivered GFP and Luciferase reporter mRNAs at levels as high as 20% efficiency and exhibited targeted Ab-dependent phenotypes. Furthermore, we demonstrate simultaneous delivery of both protein and mRNA payloads, by decorating the capsid with mRNA and [~]507 kDa tetrameric {beta}-galactosidase. The AVV-transduced HSCs maintain viability, and once optimized, the T4-AVV platform will provide enormous versatility to target various types of human cells and deliver next generation therapies for cancer and genetic diseases.

bioengineering↗

bioRxiv2024

RECON gene disruption enhances host resistance to enable genome-wide evaluation of intracellular pathogen fitness during infection.

Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq in vivo is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here we have developed a murine model with a disruption in Akr1c13 that renders the resulting RECON-/- mouse resistant to high dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen Listeria monocytogenes in vivo. We identified 139 genes which were required for L. monocytogenes growth in mice including novel genes not previously identified for host survival. We identified organ specific requirements for L. monocytogenes survival and investigated the role of the folate enzyme FolD in L. monocytogenes liver pathogenesis. A mutant lacking folD was impaired for growth in murine livers by 2.5-log10 compared to wildtype and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in alsR, which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log10 and 3-log10, respectively, but showed modest phenotypes in in vitro models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the in vivo growth defect, as deletion of the operon in the {Delta}alsR background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for L. monocytogenes host infection, which highlights the utility of the RECON-/- mouse model for future screening efforts. ImportanceListeria monocytogenes is the gram-positive bacterium responsible for the food-borne disease Listeriosis. Although infections with L. monocytogenes are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for L. monocytogenes in vivo survival will present new opportunities for treatment and prevention of Listeriosis. We developed a murine model of infection using a RECON-/- mouse that is restrictive to systemic L. monocytogenes infection. We utilized this model to screen for L. monocytogenes genes required in vivo via Tn-seq. We identified the liver-specific gene folD and a repressor, alsR that only exhibits an in vivo growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic Listeria. A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.

microbiology↗

bioRxiv2018

Screening Mycobacterium tuberculosis secreted proteins identifies Mpt64 as eukaryotic membrane-binding virulence factor

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb exported proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 exported proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb exported proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and was necessary for Mtb survival in primary human macrophages. These data highlight the importance of exported proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.\n\nImportanceAdvances have been made to identify exported proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis exported proteins for eukaryotic membrane binding, host subcellular localization and interactions with host vesicular trafficking. In addition, we observed the localization of one exported protein, Mpt64, during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the Type VII Secretion System. Finally, we observed that Mpt64 contributes to the virulence of M. tuberculosis during infection of primary human macrophages.

microbiology↗