SUMMARY Pathogenic mycobacteria cause chronic and acute diseases ranging from human tuberculosis (TB) to nontubercular infections. Mycobacterium tuberculosis causes both acute and chronic human tuberculosis. Environmentally acquired nontubercular mycobacteria (NTM) cause chronic disease in humans and animals. Not surprisingly, NTM and M. tuberculosis often use shared molecular mechanisms to survive within the host. The ESX-1 system is a specialized secretion system that is essential for virulence and is functionally conserved between M. tuberculosis and Mycobacterium marinum. M. marinum is an NTM found in both salt water and freshwater that is often used to study mycobacterial virulence. Since the discovery of the secretion system in 2003, the use of both M. tuberculosis and M. marinum has defined the conserved molecular mechanisms underlying protein secretion and the lytic and regulatory activities of the ESX-1 system. Here, we review the trajectory of the field, including key discoveries regarding the ESX-1 system. We highlight the contributions of M. marinum studies and the conserved and unique aspects of the ESX-1 secretion system.

Colorectal cancer (CRC) is a common, and often incurable, form of cancer. Gene silencing by CpG island hypermethylation often plays a role in CRC progression. Certain regions of the genome, called high confidence differentially‐methylated regions (DMRs), are consistently hypermethylated across numerous patient samples. In this study, we used bisulfite PCR sequencing to investigate methylation levels at DMRs in the promoter region of CDKN2A, DKK3, EN1, MiR34b, SPG20, and TLX1 in HCT‐116 CRC cells. We observed that for all DMRs except CDKN2A, the demethylating agent decitabine significantly reduced CpG methylation. Using ENCODE project data, we observed that transcriptional activator binding inversely correlates with DNA methylation at all of these sites across diverse cancers and cell types. Our data increase resolution of the methylation status at the above DMRs, show the reversibility of methylation at these sites by decitabine, and the likely role of hypermethylation at these sites in gene silencing. In the future, we plan to test if DMR any specific gene silencing protects HCT116 cells.Support or Funding InformationThis work is supported by the Lake Forest College biochemistry and molecular biology program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

According to the World Health Organization, Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, we seek to develop a genetic prophylaxis for TB. We have determined D-cycloserine to be the optimal target for this approach due to its relatively short six-enzyme biosynthetic pathway. D-CS is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB in human cells, we endeavored to express DcsE in human cells and test its functionality. We overexpressed DcsE tagged with FLAG and GFP in A549 lung cancer cells as determined using fluorescence microscopy. We observed that purified DcsE catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, DcsE synthesized in human cells is a functional enzyme capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells.

Parkinson's disease (PD) is characterized by a‐synuclein misfolding and the death of midbrain neurons. PD can be described as familial, or sporadic, both of which are influenced by a multitude of environmental and genetic factors. Familial PD is directly caused by a mutation in one of at least ten genes, including SNCA, DJ‐1, VPS35, and ATP13A2. SNCA, which encodes a‐synuclein, has six identified missense mutations (A30P, E46K, H50Q, G51D, A53E, and A53T) that each cause autosomal dominant PD. Sporadic PD is linked with several risk genes and loci, including VPS13, the Sac I domain of SYNJ1, and the Swa2 domain of DNAJC6. Using our previously established budding yeast model system for α‐synuclein, we first show that wild‐type (WT), E46K, A53T, H50Q, and A53E a‐synuclein are toxic to yeast and show varying degrees of membrane binding and aggregation, while A30P and G51D a‐synuclein are relatively non‐toxic and shows cytoplasmic diffuse localization. What is still not well understood is whether the other PD‐causing and risk genes mentioned above can influence toxicity and localization properties of WT a‐synuclein and these six familial PD mutants. To test the hypothesis that they do influence a‐synuclein, WT and familial mutant forms of a‐synuclein were studied in haploid yeast strains that were singly deleted for these six PD‐linked genes (all of which are linked to loss‐of‐function in PD). Results show that some gene deletions increase (Δhsp31) or decrease (Δatp13, Δvps35) a‐synuclein toxicity and alter its localization in a highly familial mutant specific way, while others more broadly increase a‐synuclein toxicity or aggregation (Δvps13, Δsac1), while still others no effect (Δswa2). Our findings suggest that WT and each familial mutant of a‐synuclein create cellular toxicity and alter localization in distinct ways and that each is likely regulated by different subsets of genes, opening doors for mutant‐specific mechanistic insight into the varying modes of a‐synuclein toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Parkinson's Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are mainly composed of misfolded α‐synuclein protein. α‐synuclein is a highly post‐translationally modified protein. While phosphorylation and nitration of α‐synuclein are well studied in the context of PD pathology, less is known about sumoylation, which is proposed to be neuroprotective based on limited studies. The majority of sumoylation takes place on the lysine‐96 and lysine‐102 sites of α‐synuclein, and it increases the protein's solubility. The goal of this research was to better understand the role of sumoylation in regulating α‐synuclein toxicity, and we performed four studies towards it. First, we evaluated the effects of blocking sumoylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more aggregated, gains toxicity, and loses localization at the plasma membrane. Second, we evaluated the effects of altering sumoylation pathways by using yeast strains with reduced (ulp1ts) or excessive sumoylation (smt3ts), and found that α‐synuclein aggregates more with reduced sumoylation, but becomes less toxic with increased sumoylation. Third, we asked how altering phosphorylation of α‐synuclein would alter sumoylation's protective role and found that blocking phosphorylation (in α‐synuclein already blocking sumoylation) reduced the protein's toxicity. Finally, we began evaluating whether blocking sumoylation and altering phosphorylation on familial PD mutant versions of α‐synuclein would exacerbate its toxicity. We found preliminary evidence that the toxicity of the A53T mutation increases when sumoylation is blocked. In the future, we will conduct further studies to understand how sumoylation affects other variants and modifications of α‐synuclein.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.