Abstract
BackgroundMycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host’s immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive.ResultsParallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs.ConclusionWe conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1569-2) contains supplementary material, which is available to authorized users.
Highlights
Mycobacterium tuberculosis is one of the world’s most successful human pathogens, with almost 10 million new cases every year [1] and more than 2 billion people latently infected
This study shows strikingly that many of these PPE proteins are important for fitness in the human Dendritic cells (DC) with over half of the annotated PPE mutants lost from the library by day 7 (p = 1.5 × 10−6), including many of the PPE genes chromosomally and functionally associated with ESX-5 in M. marinum (PPE28, PPE29, PPE30, PPE31, PPE32 and PPE33) [111]
We find that many of the genes that have been associated with resistance to reactive nitrogen in macrophages are important during DC infection e.g. fbiC, Rv2115c, Rv2097 [123], the nucleotide excision repair system encoded by uvrABCD1D2 [124] and nitrate reductase associated genes
Summary
Mycobacterium tuberculosis is one of the world’s most successful human pathogens, with almost 10 million new cases every year [1] and more than 2 billion people latently infected. People are usually infected with M. tuberculosis by inhaling respired droplets that pass through the upper respiratory tract and deposit the bacilli in the alveoli. They are phagocytosed by a range of cells including neutrophils [2,3], macrophages [3] and dendritic cells (DCs) [3,4], all of which contribute to a co-ordinated immunological response to the infection that is often protective. In some cases M. tuberculosis is able to manipulate both the innate, and the subsequent adaptive immune response, to establish an infection that may persist for a lifetime. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive
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