Characterization of EccA3, a CbbX family ATPase from the ESX-3 secretion pathway of M. tuberculosis.

EccA family proteins are conserved components of ESX secretion pathways in M. tuberculosis H37Rv. Here, we report the characterization of EccA3 (Rv0282), a CbbX family AAA (ATPases Associated with diverse cellular Activities) protein from the ESX-3 pathway that is required for in vitro growth of mycobacteria, secretion of virulence factors, and acquisition of iron and zinc. EccA3 is a thermostable ATPase with a molecular weight of ~68kDa. It exists as a dodecamer in the apo form and associates as a hexamer in the presence of ATP. Its C-terminal region consists of a CbbX-like AAA-domain while the N-terminal region contains a tetratricopeptide repeat (TPR) domain with lower homology to other EccA-type proteins. Further, the C-terminal domain functions as the oligomerization domain and also exhibits ATPase activity. Mutational analysis, steady state kinetics and molecular docking studies identify R573 as the important 'sensor arginine' and R505 as an 'arginine finger' in EccA3. Dynamic fluorescence quenching experiments suggest that the N-terminal domain moves closer to the C-terminal domain upon ATP-binding. The ATP-dependent 'open-close' relative movements of the two domains might help EccA3 interaction and secretion of essential virulence factors.

Comprehensive essentiality analysis of the Mycobacterium kansasii genome by saturation transposon mutagenesis and deep sequencing.

Mycobacterium kansasii (Mk) is an opportunistic pathogen that is frequently isolated from urban water systems, posing a health risk to susceptible individuals. Despite its ability to cause tuberculosis-like pulmonary disease, very few studies have probed the genetics of this opportunistic pathogen. Here, we report a comprehensive essentiality analysis of the Mk genome. Deep sequencing of a high-density library of Mk Himar1 transposon mutants revealed that 86.8% of the chromosomal thymine-adenine (TA) dinucleotide target sites were permissive to insertion, leaving 13.2% TA sites unoccupied. Our analysis identified 394 of the 5,350 annotated open reading frames (ORFs) as essential. The majority of these essential ORFs (84.8%) share essential mutual orthologs with Mycobacterium tuberculosis (Mtb). A comparative genomics analysis identified 139 Mk essential ORFs that share essential orthologs in four other species of mycobacteria. Thirteen Mk essential ORFs share orthologs in all four species that were identified as being not essential, while only two Mk essential ORFs are absent in all species compared. We used the essentiality data and a comparative genomics analysis reported here to highlight differences in essentiality between candidate Mtb drug targets and the corresponding Mk orthologs. Our findings suggest that the Mk genome encodes redundant or additional pathways that may confound validation of potential Mtb drugs and drug target candidates against the opportunistic pathogen. Additionally, we identified 57 intergenic regions containing four or more consecutive unoccupied TA sites. A disproportionally large number of these regions were located upstream of pe/ppe genes. Finally, we present an essentiality and orthology analysis of the Mk pRAW-like plasmid, pMK1248. IMPORTANCE Mk is one of the most common nontuberculous mycobacterial pathogens associated with tuberculosis-like pulmonary disease. Drug resistance emergence is a threat to the control of Mk infections, which already requires long-term, multidrug courses. A comprehensive understanding of Mk biology is critical to facilitate the development of new and more efficacious therapeutics against Mk. We combined transposon-based mutagenesis with analysis of insertion site identification data to uncover genes and other genomic regions required for Mk growth. We also compared the gene essentiality data set of Mk to those available for several other mycobacteria. This analysis highlighted key similarities and differences in the biology of Mk compared to these other species. Altogether, the genome-wide essentiality information generated and the results of the cross-species comparative genomics analysis represent valuable resources to assist the process of identifying and prioritizing potential Mk drug target candidates and to guide future studies on Mk biology.

ESX secretion system: The gatekeepers of mycobacterial survivability and pathogenesis.

Mycobacterium tuberculosis, the causative agent of Tuberculosis has plagued humankind for ages and has surfaced stronger than ever with the advent of drug resistance. Mycobacteria are adept at evading the host immune system and establishing infection by engaging host factors and secreting several virulence factors. Hence these secretion systems play a key role in mycobacterial pathogenesis. The type VII secretion system or ESX (early secretory antigenic target (ESAT6) secretion) system is one such crucial system that comprises five different pathways having distinct roles in mycobacterial proliferation, pathogenesis, cytosolic escape within macrophages, regulation of macrophage apoptosis, metal ion homeostasis, etc. ESX 1-5 systems are implicated in the secretion of a plethora of proteins, of which only a few are functionally characterized. Here we summarize the current knowledge of ESX secretion systems of mycobacteria with a special focus on ESX-1 and ESX-5 systems that subvert macrophage defenses and help mycobacteria to establish their niche within the macrophage.

BCG-Prime and boost with Esx-5 secretion system deletion mutant leads to better protection against clinical strains of Mycobacterium tuberculosis.

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (TCM), follicular helper T cells (TFH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.