Comparative Genomic and Transcriptomic Analyses of Mycobacterium kansasii Subtypes Provide New Insights Into Their Pathogenicity and Taxonomy

Mycobacterium kansasii is an important opportunistic pathogen of humans and has a close phylogenetic relationship with Mycobacterium tuberculosis. Seven subtypes (I–VII) have been identified using molecular biology approaches, of which subtype I is the most frequent causative agent of human disease. To investigate the genotypes and pathogenic components of M. kansasii, we sequenced and compared the complete base-perfect genomes of different M. kansasii subtypes. Our findings support the proposition that M. kansasii “subtypes” I-VI, whose assemblies are currently available, should be considered as different species. Furthermore, we identified the exclusive presence of the espACD operon in M. kansasii subtype I, and we confirmed its role in the pathogenicity of M. kansasii in a cell infection model. The espACD operon is exclusively present in mycobacterial species that induce phagosomal rupture in host phagocytes and is known to be a major determinant of ESX1-mediated virulence in pathogenic mycobacteria. Comparative transcriptome analysis of the M. kansasii I-V strains identified genes potentially associated with virulence. Using a comparative genomics approach, we designed primers for PCR genotyping of M. kansasii subtypes I-V and tested their efficacy using clinically relevant strains of M. kansasii.Work in AP's laboratory was supported by the KAUST faculty baseline fund (BAS/1/1020-01- 01

Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion

Genomic expression catalogue of a global collection of BCG vaccine strains show evidence for highly diverged metabolic and cell-wall adaptations.

Although Bacillus Calmette-Guérin (BCG) vaccines against tuberculosis have been available for more than 90 years, their effectiveness has been hindered by variable protective efficacy and a lack of lasting memory responses. One factor contributing to this variability may be the diversity of the BCG strains that are used around the world, in part from genomic changes accumulated during vaccine production and their resulting differences in gene expression. We have compared the genomes and transcriptomes of a global collection of fourteen of the most widely used BCG strains at single base-pair resolution. We have also used quantitative proteomics to identify key differences in expression of proteins across five representative BCG strains of the four tandem duplication (DU) groups. We provide a comprehensive map of single nucleotide polymorphisms (SNPs), copy number variation and insertions and deletions (indels) across fourteen BCG strains. Genome-wide SNP characterization allowed the construction of a new and robust phylogenic genealogy of BCG strains. Transcriptional and proteomic profiling revealed a metabolic remodeling in BCG strains that may be reflected by altered immunogenicity and possibly vaccine efficacy. Together, these integrated-omic data represent the most comprehensive catalogue of genetic variation across a global collection of BCG strains

Transdifferentiation of Mouse Embryonic Fibroblasts into Dopaminergic Neurons Reactivates LINE-1 Repetitive Elements

In mammals, LINE-1 (L1) retrotransposons constitute between 15% and 20% of the genome. Although only a few copies have retained the ability to retrotranspose, evidence in brain and differentiating pluripotent cells indicates that L1 retrotransposition occurs and creates mosaics in normal somatic tissues. The function of de novo insertions remains to be understood. The transdifferentiation of mouse embryonic fibroblasts to dopaminergic neuronal fate provides a suitable model for studying L1 dynamics in a defined genomic and unaltered epigenomic background. We found that L1 elements are specifically re-expressed and mobilized during the initial stages of reprogramming and that their insertions into specific acceptor loci coincides with higher chromatin accessibility and creation of new transcribed units. Those events accompany the maturation of neuronal committed cells. We conclude that L1 retrotransposition is a non-random process correlating with chromatin opening and lncRNA production that accompanies direct somatic cell reprogramming

Transdifferentiation of Mouse Embryonic Fibroblasts into Dopaminergic Neurons Reactivates LINE-1 Repetitive Elements

In mammals, LINE-1 (L1) retrotransposons constitute between 15% and 20% of the genome. Although only a few copies have retained the ability to retrotranspose, evidence in brain and differentiating pluripotent cells indicates that L1 retrotransposition occurs and creates mosaics in normal somatic tissues. The function of de novo insertions remains to be understood. The transdifferentiation of mouse embryonic fibroblasts to dopaminergic neuronal fate provides a suitable model for studying L1 dynamics in a defined genomic and unaltered epigenomic background. We found that L1 elements are specifically re-expressed and mobilized during the initial stages of reprogramming and that their insertions into specific acceptor loci coincides with higher chromatin accessibility and creation of new transcribed units. Those events accompany the maturation of neuronal committed cells. We conclude that L1 retrotransposition is a non-random process correlating with chromatin opening and lncRNA production that accompanies direct somatic cell reprogramming

Impact of the SARS-CoV-2 nucleocapsid 203K/204R mutations on the inflammatory immune response in COVID-19 severity

Abstract Background The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the nucleocapsid (N) protein correlate with increased disease severity in COVID-19 patients. However, links with the host immune response remain unclear. Methods Here, we grouped nasopharyngeal swab samples of COVID-19 patients into two cohorts based on the presence and absence of SARS-CoV-2 nucleocapsid KR mutations. We performed nasopharyngeal transcriptome analysis of age, gender, and ethnicity-matched COVID-19 patients infected with either SARS-CoV-2 with KR mutations in the N protein (KR patients n = 39) or with the wild-type N protein (RG patients n = 39) and compared to healthy controls (n = 34). The impact of KR mutation on immune response was further characterized experimentally by transcriptomic and proteomic profiling of virus-like-particle (VLP) incubated cells. Results We observed markedly elevated expression of proinflammatory cytokines, chemokines, and interferon-stimulated (ISGs) genes in the KR patients compared to RG patients. Using nasopharyngeal transcriptome data, we found significantly higher levels of neutrophils and neutrophil-to-lymphocyte (NLR) ratio in KR patients than in the RG patients. Furthermore, transcriptomic and proteomic profiling of VLP incubated cells confirmed a similar hyper-inflammatory response mediated by the KR variant. Conclusions Our data demonstrate an unforeseen connection between nucleocapsid KR mutations and augmented inflammatory immune response in severe COVID-19 patients. These findings provide insights into how mutations in SARS-CoV-2 modulate host immune output and pathogenesis and may contribute to more efficient therapeutics and vaccine development

Genomic expression catalogue of a global collection of BCG vaccine strains show evidence for highly diverged metabolic and cell-wall adaptations

Although Bacillus Calmette-Guérin (BCG) vaccines against tuberculosis have been available for more than 90 years, their effectiveness has been hindered by variable protective efficacy and a lack of lasting memory responses. One factor contributing to this variability may be the diversity of the BCG strains that are used around the world, in part from genomic changes accumulated during vaccine production and their resulting differences in gene expression. We have compared the genomes and transcriptomes of a global collection of fourteen of the most widely used BCG strains at single base-pair resolution. We have also used quantitative proteomics to identify key differences in expression of proteins across five representative BCG strains of the four tandem duplication (DU) groups. We provide a comprehensive map of single nucleotide polymorphisms (SNPs), copy number variation and insertions and deletions (indels) across fourteen BCG strains. Genome-wide SNP characterization allowed the construction of a new and robust phylogenic genealogy of BCG strains. Transcriptional and proteomic profiling revealed a metabolic remodeling in BCG strains that may be reflected by altered immunogenicity and possibly vaccine efficacy. Together, these integrated-omic data represent the most comprehensive catalogue of genetic variation across a global collection of BCG strains