Rv0100: An essential acyl carrier protein from M. tuberculosis important in dormancy

Abstract

We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen.

Fig 1. Rv0100 operon.

The genomic structure surrounding Rv0100 including all the genes contained in the same operon. This includes all genes contained within the same operon as our target of interest, Rv0100. Here, the nrp gene (encoding the Rv0101 protein) is shown to be only 18 nucleotides apart from Rv0100. The overlapping stop codon of Rv0100 and start codon of Rv0101 make them likely protein partners.

Fig 2. Growth profile of MTB WT, Rv0100 KO, and its respective complement strain in the Wayne model.

The Rv0100 KO strain showed significantly attenuated growth following exposure to anaerobic conditions for 7 days compared to the complement and WT strains. This effect was further pronounced after 14 days of exposure where the difference in growth reached statistical significance compared to both complement and WT strains. n = 1(day0)-2 biological replicates. Data represented as mean ± SEM CFU/ml across 2 technical replicates per timepoint. Repeated measures mixed-effect ANOVA followed by Tukey’s test. *p<0.05:Rv0100 KO vs. WT and complement; †p<0.05:Rv0100 KO vs. complement. CFU = colony forming unit; KO = knock out; WT = wild type.

Fig 3. Effect of Rv0100 KO on PDIM synthesis.

Qualitative analysis of lipid extracts from MTB WT and Rv0100 KO reveals that disruption of the Rv0100 gene is associated with disrupted synthesis PGI and the virulence enhancing PDIM, DIM B, but not DIM A. In a separate repeat experiment, we also compared the complement strain to WT and found that PGI was restored (S2 Fig). Representative image shown from two biological and two technical replicates. MTB = Mycobacterium tuberculosis; WT = wild type; KO = knock out; PDIM = phthiocerol dimycocerosates; PGI = phosphoglucoisomerase: Dim B = dimycocerosates of phthiodiolone; Dim A = dimycocerosates of phthiocerol A.

Fig 4. Effect of Rv0100 KO on growth in the mouse lung.

The growth of MTB WT was compared with mutant Rv0100 KO and complement strains in vivo using a murine aerosol lung infection model. The Rv0100 KO strain exhibited significantly reduced growth compared to WT at post-infection days 29 and 99. In contrast, restoring Rv0100 through complementation resulted in a growth profile that was indistinguishable from WT at the same time points. There was also a small, but significant increase in CFUs found in the complement strain versus WT at post-infection day 1. Data represented as mean ± SEM CFU/ml lung tissue homogenate, n = 6 biological replicates per group for each time point. Two-way repeated measures ANOVA followed by Tukey’s test. *p<0.05:Rv0100 KO vs. WT and complement; #p<0.05:Rv0100 complement vs. WT. MTB = Mycobacterium tuberculosis; CFU = colony forming unit; KO = knock out; WT = wild type.

Fig 5. Effect of Rv0100 KO on intracellular survivability in J774A macrophages.

The ability of the mutant Rv0100 KO strain to survive phagocytosis by macrophages was assessed by comparing its growth with MTB WT and the Rv0100 complement strains at three time points. The results showed a significant reduction of CFUs in Rv0100 KO strain compared to complement at post-infection day 7. The WT strain also exhibited attenuation at this time point but the effect did not reach statistical significance. n = 1(day1;WT)-3 biological replicates per strain for each time point. Data represented as mean ± SEM CFU/ml across n = 1(day1;WT)-3 technical replicates per group for each time point. Repeated measures mixed-effect ANOVA followed by Tukey’s test. #p<0.05:Rv0100 complement vs. WT. MTB = Mycobacterium tuberculosis; CFU = colony forming unit; KO = knock out; WT = wild type.