Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Genomic and microbiological analyses of iron acquisition pathways among respiratory and environmental nontuberculous mycobacteria from Hawai'i.

As environmental opportunistic pathogens, nontuberculous mycobacteria (NTM) can cause severe and difficult to treat pulmonary disease. In the United States, Hawai'i has the highest prevalence of infection. Rapid growing mycobacteria (RGM) such as Mycobacterium abscessus and M. porcinum and the slow growing mycobacteria (SGM) including M. intracellulare subspecies chimaera are common environmental NTM species and subspecies in Hawai'i. Although iron acquisition is an essential process of many microorganisms, iron acquisition via siderophores among the NTM is not well-characterized. In this study, we apply genomic and microbiological methodologies to better understand iron acquisition via siderophores for environmental and respiratory isolates of M. abscessus, M. porcinum, and M. intracellulare subspecies chimaera from Hawai'i. Siderophore synthesis and transport genes, including mycobactin (mbt), mmpL/S, and esx-3 were compared among 47 reference isolates, 29 respiratory isolates, and 23 environmental Hawai'i isolates. Among all reference isolates examined, respiratory isolates showed significantly more siderophore pertinent genes compared to environmental isolates. Among the Hawai'i isolates, RGM M. abscessus and M. porcinum had significantly less esx-3 and mbt genes compared to SGM M. chimaera when stratified by growth classification. However, no significant differences were observed between the species when grown on low iron culture agar or siderophore production by the chrome azurol S (CAS) assay in vitro. These results indicate the complex mechanisms involved in iron sequestration and siderophore activity among diverse NTM species.

Comparative survival of environmental and clinical Mycobacterium abscessus isolates in a variety of diverse host cells.

AIMS: Mycobacterium abscessus subsp. abscessus (MABS) is an emerging, opportunistic pathogen found globally in freshwater biofilms and soil. Typically, isolates are treated as a uniform group of organisms and very little is known about their comparative survival in healthy host cells. We posit that environmentally- and clinically derived isolates, show differential infectivity in immune cells and resistance to innate defenses. METHODS AND RESULTS: Six MABS isolates were tested including three water biofilm/soil and three sputum-derived isolates. A clinical MABS type strain and an environmental isolate of Arthrobacter were also included. MABS counts were significantly higher compared to Arthrobacter after co-culture with Acanthamoeba lenticulata, BEAS-2B epithelial cells, alveolar macrophages and the THP-1 macrophage cell line. A rough sputum-derived MABS isolate emerged as an isolate with higher virulence compared to others tested, as both a pellicle and cord former, survivor in the human cell models tested, inducer of high and prolonged production of pro-inflammatory cytokines, and the capacity to evade LL-37. CONCLUSIONS: Findings support intraspecies variation between MABS isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate subversion of host immune defenses by environmental and clinical MABS isolates is nuanced and maybe isolate dependent, providing new information regarding the pathogenesis of NTM infections.