Lipid metabolism and Type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells

Abstract

Background: Mycobacterium 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.

Results: Parallel 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.

Conclusion: We 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.

Figure 1

Dynamics of intracellular and extracellular M. tuberculosis and their dendritic cell host. The number of (a) intracellular M. tuberculosis per DC (b) the percentage of viable post-infection DCs and (c) the number of extracellular M. tuberculosis per DC. Total numbers of bacteria per well are shown in the plot insets. Error bars are standard errors of the mean from 5 separate experiments each with different PBMC donors.

Figure 2

Overview of mutant ‘fitness’ during dendritic cell infection. (a) A Euler diagram (Venn diagram with areas proportional to the values) showing the numbers of mutants with significant fitness costs (p < 0.05) at different times during M. tuberculosis infection of DCs (Additional file 1), (b) a plot of the ‘fitness’ rank of mutations in individual genes 3 days and 7 days after infection, Spearman’s rank-order correlation ρ = 0.856, p < 1 × 10⁻⁶. (c) a genome plot of the ‘fitness’ of intracellular M. tuberculosis mutants 7 days after infection. Red lines indicate fitness of the gene knockouts 7 days after DC infection. Black lines are genes assigned as essential in vitro [43] and so excluded from the analysis. Relevant regions of co-functionality are indicated around the perimeter.

Figure 3

Violin plots of the ‘fitness’ of mutants ordered by functional groups 7 days after dendritic cell infection. Violin plots describing the distribution of the genes within functional gene groupings ranked by their ‘fitness’ associated p values 7 days after DC infection. Violin plots [139] are similar to box plots, but use a kernel density estimation of the probability density, similar to a smoothed distribution histogram (light grey zone), to better illustrate the overall distribution of the data. The probabilities that mutations of the genes of the functional groups had a decreased intracellular fitness, calculated using the Mann–Whitney U test, are given in brackets. Median values are indicated by the bold black lines, each gene by smaller dark grey lines, the dashed line represents the median rank for all non-essential genes [43] in the data-set.

Figure 4

Summary of the major cellular components and processes that are associated with fitness costs during M. tuberculosis survival within DC.