The Phosphatidyl- myo-Inositol Dimannoside Acyltransferase PatA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

Abstract

Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids considered to be not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane-associated acyltransferase that transfers a palmitoyl moiety from palmitoyl coenzyme A (palmitoyl-CoA) to the 6-position of the mannose ring linked to the 2-position of inositol in PIM₁/PIM₂ We validate that the function of PatA is vital for M. tuberculosisin vitro and in vivo We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significantly reduced levels of Ac₁PIM₂, an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo Altogether, the experimental data highlight the significance of the early steps of the PIM biosynthetic pathway for M. tuberculosis physiology and reveal that PatA is a novel target for drug discovery programs against this major human pathogen.IMPORTANCE Tuberculosis (TB) is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis, the etiologic agent of TB, emphasizes the need to identify new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined that PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis These results highlight the importance of early steps of the PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target.

Keywords: Mycobacterium tuberculosis; acyltransferase; conditional mutant; glycolipid; mycobacterium; tuberculosis.

FIG 1

The PIMs from M. tuberculosis. (A) The chemical structure of PIMs. The major PIM species have two (PIM₂) or six (PIM₆) mannose residues with different degrees of acylation. (B) The current model of PIM biosynthesis. The currently accepted model proposes that PimA and PimB catalyze the two first consecutive mannosylation steps in the cytoplasmic face of the plasma membrane. The identity and location of the enzyme(s) responsible for the third and fourth mannosylation steps, as well as the existence of a flippase, are under debate. The fifth mannosylation step is catalyzed by PimE in the periplasmic face of the plasma membrane. The identity of the enzyme responsible for the sixth mannosylation step is still unknown. PIMs can be also acylated by the action of two acyltransferases: PatA catalyzes the transfer of an acyl chain to the mannose ring transferred by PimA at the cytosolic face of the plasma membrane, whereas an unknown acyltransferase catalyzes the transfer of an acyl chain to the inositol ring of the glycolipid. In the inset, the acylation steps on Ac₂PIM₂ are shown as an example. (C) PatA transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM₁/PIM₂.

FIG 2

patA is an essential gene in M. tuberculosis. (A) Serial dilutions of log-phase cultures of the conditional mutant TB506.1 were spotted on Middlebrook 7H10 plates with or without 500 ng/ml ATc. (B) Growth curves of the conditional knockdown TB506.1 in the presence (triangles) or absence (circles) of 500 ng/ml ATc; 1:10 dilutions of the cultures in fresh medium with or without ATc (dashed arrows) were performed until the growth of the culture with ATc stopped. Gray bars represent the number of CFU per milliliter in the culture with ATc after its growth arrested. The results represent the average of three independent experiments. *, P < 0.5; **, P < 0.05.

FIG 3

Loss of Ac₁PIM₂ in the PatA-depleted strain. (A) Lipids were extracted from M. tuberculosis TB38 and TB506.1 grown in the presence of 500 ng ml⁻¹ ATc at different time points (0, 4, and 12 days), separated by TLC in the solvent CHCl₃-CH₃OH-concentrated NH₄OH-H₂O (65:25:0.5:4) and visualized with α-naphthol. (B) Alternatively, samples from day 12 were separated by 2D-TLC using the same solvent system as in panel A for dimension 1 and chloroform, acetic acid, methanol, and water (50:60:2.3:3) for dimension 2 and visualized as in panel A. Data are representative of two independent experiments with similar results. At the indicated days, cultures were submitted to serial dilutions in fresh medium with ATc in order to visualize a progressive decrease in the Ac₁PIM₂ levels.

FIG 4

Growth curves of TB506.1 and of the parental strain TB38 in THP-1-derived macrophages. The patA conditional mutant TB506.1 and the parental strain TB38 were used to infect THP-1-derived macrophages. While TB506.1 was able to replicate intracellularly only in the absence of ATc (A), TB38 was able to divide regardless of the presence of ATc in the cell culture medium (B). Filled circles, no ATc; open circles, 500 ng/ml ATc added to the culture medium. Data are representative of two independent experiments with similar results. ***, P < 0.001 one-way ANOVA with Tukey posttest.

FIG 5

Validation of patA essentiality in the mouse model of infection. The graph illustrates the bacterial loads in the lungs (A) and spleens (B) of C57BL/6JOlaHsd mice infected with TB506.1. Silencing of patA was achieved by supplying animals with water supplemented with 5% sucrose containing doxycycline. Results represent the mean value and error for 5 mice per group and time point. **, P < 0.01; ***, P < 0.001 one-way ANOVA with Tukey posttest.

FIG 6

Essentiality of the early steps in PIM biosynthesis. Two pathways were originally proposed for the biosynthesis of Ac₁PIM₂ in the cytoplasmic phase of the mycobacterial inner membrane as follows: (i) PI is mannosylated to form PIM₂ by the consecutive action of the GT-B glycosyltransferases PimA and PimB, respectively, and PIM₂ is then acylated by PatA to form Ac₁PIM₂; and (ii) PI is mannosylated by PimA to form PIM₁ and then acylated by PatA to form Ac₁PIM₁, which in turn is mannosylated by PimB to form Ac₁PIM₂. In vitro experimental evidence indicates that although both pathways might coexist in mycobacteria, the sequence of events PI→PIM₁→PIM₂→Ac₁PIM₂ is favored (20, 28). The production and purification of high yields of PimA, PimB, and PatA, along with the determination of their three-dimensional structure, place us in an unprecedented position to initiate a drug discovery program focusing on those enzymes (32, 33, 51–53).