ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the gene. Finally, we found that the loss of WhiB6 resulted in a virulent strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in are fine-tuned by negative feedback control, linking the expression of the gene to the presence, not the functionality, of the ESX-1 membrane complex.

N-terminal acetylation (NTA) is a post-transcriptional modification of proteins that is conserved from bacteria to humans. In bacteria, the enzymes that mediate protein NTA also promote antimicrobial resistance. In pathogenic mycobacteria, which cause human tuberculosis and other chronic infections, NTA has been linked to pathogenesis and stress response, yet the fundamental biology underlying NTA of mycobacterial proteins remains unclear. We enriched, defined, and quantified the NT-acetylated populations of both cell-associated and secreted proteins from both the human pathogen, Mycobacterium tuberculosis, and the nontuberculous opportunistic pathogen, Mycobacterium marinum. We used a parallel N-terminal enrichment strategy from proteolytic digests coupled to charge-based selection and stable isotope ratio mass spectrometry. We show that NTA of the mycobacterial proteome is abundant, diverse, and primarily on Thr residues, which is unique compared with other bacteria. We isolated both the acetylated and unacetylated forms of 256 proteins, indicating that NTA of mycobacterial proteins is homeostatic. We identified 16 mycobacterial proteins with differential levels of NTA on the cytoplasmic and secreted forms, linking protein modification and localization. Our findings reveal novel biology underlying the NTA of mycobacterial proteins, which may provide a basis to understand NTA in mycobacterial physiology, pathogenesis, and antimicrobial resistance.

Objective: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2 V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2 V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl 3 -induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. Conclusions: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

is a nontuberculous pathogen of poikilothermic fish and an opportunistic human pathogen. Like tuberculous mycobacteria, the M strain requires the ESX-1 (ESAT-6 system 1) secretion system for virulence in host cells. EsxB and EsxA, two major virulence factors exported by the ESX-1 system, are encoded by the genes within the ESX-1 locus. Deletion of the genes abrogates ESX-1 export and attenuates in and models of infection. Interestingly, there are several duplications of the and genes (, ,, , and) in the M genome located outside the ESX-1 locus. We sought to understand if this region, known as ESX-6, contributes to ESX-1-mediated virulence. We found that deletion of the_ gene alone or the entire ESX-6 locus did not impact ESX-1 export or function, supporting the idea that the genes present at the ESX-1 locus are the primary contributors to ESX-1-mediated virulence. Nevertheless, overexpression of the locus complemented ESX-1 function in the Δ strain, signifying that the two loci are functionally equivalent. Our findings raise questions about why duplicate versions of the genes are maintained in the M genome and how these proteins, which are functionally equivalent to virulence factors, contribute to mycobacterial biology. is the causative agent of the human disease tuberculosis (TB). There are 10.4 million cases and 1.7 million TB-associated deaths annually, making TB a leading cause of death globally. Nontuberculous mycobacteria (NTM) cause chronic human infections that are acquired from the environment. Despite differences in disease etiology, both tuberculous and NTM pathogens use the ESX-1 secretion system to cause disease. The nontubercular mycobacterial species, , has additional copies of specific ESX-1 genes. Our findings demonstrate that the duplicated genes do not contribute to virulence but can substitute for virulence factors in These findings suggest that the duplicated genes may play a specific role in NTM biology.

Currently, we have insufficient understanding of venous thromboembolism in cancer patients. In this article, the authors reveal a novel mechanism for colon cancer-associated venous thrombosis using a murine model.