Assembly of the <i>Mycobacterium tuberculosis</i> type VII ESX-1 secretion system in <i>Mycobacterium smegmatis</i> identifies a new transcriptional activator of <i>esx-1</i> genes and a novel TB vaccine

<i>Mycobacterium tuberculosis</i> (<i>M. tb</i>) uses its type VII secretion system (T7SS) ESX-1 to export immunogenic, virulence-mediating protein effectors. In this study, the fast-growing, non-pathogenic model mycobacteria <i>Mycobacterium smegmatis</i> mc²-155 was engineered to express the <i>M. tb</i> T7SS ESX-1 system. We found that <i>M. smegmatis</i> transformed with <i>M. tb esx-1</i> locus genes only, as well as <i>M. smegmatis</i> transformed with <i>M. tb esx-1</i> and <i>espACD</i> operon genes (designated MSX-1), produces and secretes the <i>M. tb</i> ESX-1 protein effectors EsxA, EsxB, and EspB. However, the abundance of these proteins was higher inside the cell and culture filtrate of the MSX-1 strain. Although ESX-1 is critical for <i>M. tb</i> pathogenesis, expression of <i>M. tb</i> ESX-1 did not make the recombinant <i>M. smegmatis</i> strains virulent in macrophages. Serendipitously, transformation of <i>M. smegmatis</i> with a modified <i>esx-1</i> locus in this study revealed <i>rv3860</i>, a gene of previously unknown function, to be required for the transcription of <i>pe35</i>, <i>ppe68</i>, <i>esxB,</i> and <i>esxA</i> genes. Finally, mice vaccinated with MSX-1 were found to be as protected as mice vaccinated with <i>Mycobacterium bovis</i> BCG against <i>M. tb</i> infection, without becoming sensitized to tuberculin. These results show that a functional <i>M. tb</i> ESX-1 system can be assembled in <i>M. smegmatis</i> to uncover novel facets of the secretion machinery and that the modified <i>M. smegmatis</i> strain can function as a tuberculosis (TB) vaccine. Unlike BCG, however, its deployment may be compatible with tests currently used to diagnose TB.IMPORTANCEIn this study, we modified <i>Mycobacterium smegmatis</i>, which is often used as a surrogate model organism in mycobacterial research, to produce and assemble a functional <i>Mycobacterium tuberculosis</i> (<i>M. tb</i>) ESX-1 protein secretion system. One such <i>M. smegmatis</i> strain named MSX-1 was found to make a functional <i>M. tb</i> ESX-1 system without becoming virulent. And in using <i>M. smegmatis</i> as a chassis to study the ESX-1 system, we found that <i>rv3860</i>, an <i>M. tb</i> gene of previously unknown function, is needed for the production of key ESX-1 proteins. Finally, mice vaccinated with MSX-1 were as protected from tuberculosis (TB) as mice given BCG, the only approved TB vaccine. Notably, we found that unlike BCG, MSX-1 does not sensitize mice to the antigens used in existing TB diagnostic tests. These observations, taken together, highlight the utility of <i>M. smegmatis</i> as a chassis to study the <i>M. tb</i> ESX-1 secretion machinery.