Browsing by Author "Utama, Budi"

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    Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA–MurG interaction
    (Elsevier, 2023) Zhou, Yue; Utama, Budi; Pratap, Shivendra; Supandy, Adeline; Song, Xinhao; Tran, Truc T.; Mehta, Heer H.; Arias, Cesar A.; Shamoo, Yousif
    Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to “resensitize” enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure–function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA–MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.
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    Transcriptional activators YAP/TAZ and AXL orchestrate dedifferentiation, cell fate, and metastasis in human osteosarcoma
    (Springer Nature, 2021) Lamhamedi-Cherradi, Salah-Eddine; Mohiuddin, Sana; Mishra, Dhruva K.; Krishnan, Sandhya; Velasco, Alejandra Ruiz; Vetter, Amelia M.; Pence, Kristi; McCall, David; Truong, Danh D.; Cuglievan, Branko; Menegaz, Brian A.; Utama, Budi; Daw, Najat C.; Molina, Eric R.; Zielinski, Rafal J.; Livingston, John A.; Gorlick, Richard; Mikos, Antonios G.; Kim, Min P.; Ludwig, Joseph A.
    Osteosarcoma (OS) is a molecularly heterogeneous, aggressive, poorly differentiated pediatric bone cancer that frequently spreads to the lung. Relatively little is known about phenotypic and epigenetic changes that promote lung metastases. To identify key drivers of metastasis, we studied human CCH-OS-D OS cells within a previously described rat acellular lung (ACL) model that preserves the native lung architecture, extracellular matrix, and capillary network. This system identified a subset of cells—termed derived circulating tumor cells (dCTCs)—that can migrate, intravasate, and spread within a bioreactor-perfused capillary network. Remarkably, dCTCs highly expressed epithelial-to-mesenchymal transition (EMT)-associated transcription factors (EMT-TFs), such as ZEB1, TWIST, and SOX9, which suggests that they undergo cellular reprogramming toward a less differentiated state by coopting the same epigenetic machinery used by carcinomas. Since YAP/TAZ and AXL tightly regulate the fate and plasticity of normal mesenchymal cells in response to microenvironmental cues, we explored whether these proteins contributed to OS metastatic potential using an isogenic pair of human OS cell lines that differ in AXL expression. We show that AXL inhibition significantly reduced the number of MG63.2 pulmonary metastases in murine models. Collectively, we present a laboratory-based method to detect and characterize a pure population of dCTCs, which provides a unique opportunity to study how OS cell fate and differentiation contributes to metastatic potential. Though the important step of clinical validation remains, our identification of AXL, ZEB1, and TWIST upregulation raises the tantalizing prospect that EMT-TF-directed therapies might expand the arsenal of therapies used to combat advanced-stage OS.
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    Visualization of RNA virus infection in a marine protist with a universal biomarker
    (Springer Nature, 2023) Coy, Samantha R.; Utama, Budi; Spurlin, James W.; Kim, Julia G.; Deshmukh, Harshavardhan; Lwigale, Peter; Nagasaki, Keizo; Correa, Adrienne M. S.
    Half of the marine virosphere is hypothesized to be RNA viruses (kingdom Orthornavirae) that infect abundant micro-eukaryotic hosts (e.g. protists). To test this, quantitative approaches that broadly track infections in situ are needed. Here, we describe a technique—dsRNA-Immunofluorescence (dsRIF)—that uses a double-stranded RNA (dsRNA) targeting monoclonal antibody to assess host infection status based on the presence of dsRNA, a replicative intermediate of all Orthornavirae infections. We show that the dinoflagellate Heterocapsa circularisquama produces dsRIF signal ~ 1000 times above background autofluorescence when infected by the + ssRNA virus HcRNAV. dsRNA-positive virocells were detected across > 50% of the 48-h infection cycle and accumulated to represent at least 63% of the population. Photosynthetic and chromosomal integrity remained intact during peak replication, indicating HcRNAV infection does not interrupt these processes. This work validates the use of dsRIF on marine RNA viruses and their hosts, setting the stage for quantitative environmental applications that will accelerate understanding of virus-driven ecosystem impacts.