Mutation rates and adaptive variation among the clinically dominant clusters of Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is a multidrug-resistant pathogen increasingly responsible for severe pulmonary infections. Analysis of whole-genome sequences (WGS) of Mab demonstrates dense genetic clustering of clinical isolates collected from disparate geographic locations. This has been interpreted as supporting patient-to-patient transmission, but epidemiological studies have contradicted this interpretation. Here, we present evidence for a slowing of the Mab molecular clock rate coincident with the emergence of phylogenetic clusters. We performed phylogenetic inference using publicly available WGS from 483 Mab patient isolates. We implement a subsampling approach in combination with coalescent analysis to estimate the molecular clock rate along the long internal branches of the tree, indicating a faster long-term molecular clock rate compared to branches within phylogenetic clusters. We used ancestry simulation to predict the effects of clock rate variation on phylogenetic clustering and found that the degree of clustering in the observed phylogeny is more easily explained by a clock rate slowdown than by transmission. We also find that phylogenetic clusters are enriched in mutations affecting DNA repair machinery and report that clustered isolates have lower spontaneous mutation rates in vitro. We propose that Mab adaptation to the host environment through variation in DNA repair genes affects the organism's mutation rate and that this manifests as phylogenetic clustering. These results challenge the model that phylogenetic clustering in Mab is explained by person-to-person transmission and inform our understanding of transmission inference in emerging, facultative pathogens.

Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness.

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.