Biology subjects

Assad-Garcia, N.

Publications and source records attributed to Assad-Garcia, N..

5 recordsLinked to original sources

bioRxiv2026

Selection-free whole genome transplantation revives dead microbes

We present a living, synthetic bacterial cell made by transplanting a complete genome into a dead cell. After killing Mycoplasma capricolum cells by chemically crosslinking their genome with Mitomycin C (MMC), we installed synthetic Mycoplasma mycoides genomes into the resulting dead cells using Whole Genome Transplantation (WGT)1,2. During WGT, a synthetic donor genome is placed into a recipient cell, thereby reprogramming that cell to adopt a new genetic identity3. WGT has only been demonstrated using species within one phylogenetic clade of Mollicutes bacteria4. A major barrier to expanding WGT to diverse bacterial species has been the inability to inactivate the recipient genome, leading to false positive transplants due to homologous recombination of antibiotic resistance markers from the donor genome into the recipient cell genome. Here, we address this key limitation by removing reliance on an antibiotic resistance marker to select for transplants; recipient cells are dead unless revived by the installation of a new genome. Our work demonstrates a general approach to fully inactivate the recipient cell genome, reports the first living synthetic bacterial cell constructed from non-living parts, and advances WGT for building engineered or synthetic cells for diverse applications.

synthetic biology↗

bioRxiv2023

SARS-CoV-2 ORF8 modulates lung inflammation and clinical disease progression

The virus severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of the current COVID-19 pandemic. It possesses a large 30 kilobase (kb) genome that encodes structural, non-structural, and accessory proteins. Although not necessary to cause disease, these accessory proteins are known to influence viral replication and pathogenesis. Through the synthesis of novel infectious clones of SARS-CoV-2 that lack one or more of the accessory proteins of the virus, we have found that one of these accessory proteins, ORF8, is critical for the modulation of the host inflammatory response. Mice infected with a SARS-CoV-2 virus lacking ORF8 exhibit increased weight loss and exacerbated macrophage infiltration into the lungs. Additionally, infection of mice with recombinant SARS-CoV-2 viruses encoding ORF8 mutations found in variants of concern reveal that naturally occurring mutations in this protein influence disease severity. Our studies with a virus lacking this ORF8 protein and viruses possessing naturally occurring point mutations in this protein demonstrate that this protein impacts pathogenesis. SignificanceSince its emergence in 2019, SARS-CoV-2 has accrued mutations throughout its 30kb genome. Of particular interest are the mutations present in the ORF8 protein, which occur in every major variant. The precise function and impact of this protein on disease severity and pathogenesis remains understudies. Our studies reveal that the ORF8 protein modulates the immune response by impacting macrophage infiltration into the lungs. Additionally, we have shown that the ORF8 protein of SARS-CoV-2 has accrued mutations throughout its evolution that lead to a loss of function phenotype in this protein. Our work reveals that the ORF8 protein of SARS-CoV-2 contributes significantly to disease progression through modulation of the inflammatory response.

microbiology↗

bioRxiv2022

Minimal cell JCVI-syn3B as a chassis to reveal the mechanisms behind Mycoplasma host-pathogen interactions

Mycoplasmas are atypical bacteria that are obligate parasites of animals and plants. They are known for their capacity to contaminate and parasitize mammalian cell cultures, thereby offering fortuitous models to examine the host-microbe interaction. We used a near wildtype strain of the goat pathogen Mycoplasma mycoides (Mmc) and several minimized Mmc strains lacking genes not essential for growth in laboratory media to investigate host-mycoplasma interactions. Stains with near minimal genomes were incapable of surviving in co-culture with mammalian HEK-293T and HeLa cells. We identified a cluster of eight non-essential genes that when added back to the minimized strains enabled mycoplasma attachment to mammalian cells. Those genes did not restore the capacity of the minimal cell to grow when co-cultured with mammalian cells indicating processes of attaching to mammalian cells and parasitism involve different genes. Furthermore, minimized Mmc strains were not phagocytized by human myeloid cells unlike the near wildtype Mmc.

microbiology↗

bioRxiv2022

SARS-CoV-2 Variant Spike and accessory gene mutations alter pathogenesis

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against SARS-CoV-2, the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the Spike protein. Although these differences in Spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome which may affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other non-spike mutations on SARS-CoV-2 pathogenesis, we synthesized viruses where the WA1 Spike is replaced by each variant spike genes in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to the full variant viruses. Our work has revealed that non-spike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host and can lead to attenuating phenotypes in circulating variants of concern. This work suggests that while Spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may lead to less clinical disease, extended time toward knowing an infection exists in a person and thus increased time for transmission to occur. SignificanceA hallmark of the COVID19 pandemic has been the emergence of SARS-CoV-2 variants that have increased transmission and immune evasion. Each variant has a set of mutations that can be tracked by sequencing but little is known about their affect on pathogenesis. In this work we first identify accessory genes that are responsible for pathogenesis in vivo as well as identify the role of variant spike genes on replication and disease in mice. Isolating the role of Spike mutations in variants identifies the non-Spike mutations as key drivers of disease for each variant leading to the hypothesis that viral fitness depends on balancing increased Spike binding and immuno-evasion with attenuating phenotypes in other genes in the SARS-CoV-2 genome.

microbiology↗

bioRxiv2020

Genetic requirements for cell division in a genomically minimal cell

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. While this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics resulting in irregular morphologies. The addition of 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents significantly less morphological variation than JCVI-syn3.0. We further identified seven of these 19 genes, including two known cell division genes ftsZ and sepF and five genes of unknown function, required together to restore cell morphology and division similar to JCVI-syn1.0. This surprising result emphasizes the polygenic nature of cell morphology, as well as the importance of a Z-ring and membrane properties in the physiology of genomically minimal cells.

synthetic biology↗