Mycobacterium tuberculosis ftsH expression in response to stress and viability

Abstract

FtsH is an essential membrane-bound protease that degrades integral membrane proteins as well as cytoplasmic proteins. We show that Mycobacterium tuberculosis (Mtb) ftsH expression levels are upregulated upon exposure to agents that produce reactive oxygen and nitrogen intermediates (ROI and RNI) and growth in macrophages. In partial support of this result is our observation that the Mtb merodiploid overexpressing ftsH shows increased resistance to ROI. ftsH transcript levels are downregulated during stationary phase and starvation. ftsH overexpression strain shows delayed growth and reduced viability in vitro and ex vivo. Finally, we show that the intracellular levels of FtsZ, an essential cell-division protein, are reduced in ftsH-overexpressing strain. Together, our results suggest that Mtb FtsH is a stress-response protein that promotes the pathogen's ability to deal with ROI stress and is possibly involved in the regulation of FtsZ levels.

Figure 1

ftsH expression levels under different growth conditions. ftsH expression levels in Mtb grown under various indicated conditions were quantitated by QRT-PCR. cDNA was synthesized for ftsH along with 16S rRNA and QRT-PCR was carried out as described under material and methods. The expression levels of ftsH mRNA were normalized to 16s mRNA levels. Mean ±SD from three independent experiments are shown.

Figure 2

FtsH protein levels and growth of ftsH strains. A. FtsH protein levels in Mtb strains were quantitated by immunoblotting. Protein lysates from ftsH strains were resolved on SDS-PA gels, transferred to nitrocellulose membrane and probed with α-FtsH and α-sigma³² antibodies. Bands were visualized on a BioRad molecular imager and quantitated using the QuantityOne software. FtsH levels were normalized to SigA. Symbols: WT – wild type Mtb, Mtb-6 – ftsH overexpressing strain and Mtb-33 – ftsH antisense strain. Inset: Immunoblot showing FtsH and SigA bands. B. Growth of ftsH strains was monitored by measuring absorbance at 600 nm and plotted. Actively growing cultures were also spread on agar plats for determining CFU (inset).

Figure 2

FtsH protein levels and growth of ftsH strains. A. FtsH protein levels in Mtb strains were quantitated by immunoblotting. Protein lysates from ftsH strains were resolved on SDS-PA gels, transferred to nitrocellulose membrane and probed with α-FtsH and α-sigma³² antibodies. Bands were visualized on a BioRad molecular imager and quantitated using the QuantityOne software. FtsH levels were normalized to SigA. Symbols: WT – wild type Mtb, Mtb-6 – ftsH overexpressing strain and Mtb-33 – ftsH antisense strain. Inset: Immunoblot showing FtsH and SigA bands. B. Growth of ftsH strains was monitored by measuring absorbance at 600 nm and plotted. Actively growing cultures were also spread on agar plats for determining CFU (inset).

Figure 3

FtsH protein levels and resistance to ROI. Exponential cultures of Mtb-6, Mtb-33 and wild type strains were grown in the presence of menadione (70μM) or H₂O₂ (5mM) for 48 hrs and plated on 7H10-OADC plates. CFU were counted and percent survival was calculated. Data presented are normalized to no-treatment control for each strain.

Figure 4

Growth of ftsH strains in THP-I macrophages. Monolayers of human macrophage cell line, THP-1, were infected with wild type (H37Rv, squares) or merodiploids overexpressing ftsH (Mtb-6, diamonds) or ftsH antisense (Mtb-33, triangles) at a moi of 1:1. At indicated periods of time, macrophages were lysed and viable bacterial count was determined by plating for CFU.

Figure 5

FtsZ levels in ftsH strains. Exponential cultures of ftsH strains were grown in the presence of 0.2% acetamide for 3 days and examined for FtsZ levels by immunoblotting as described above in Fig. 2. Symbols: WT – wild type Mtb, Mtb-6 – ftsH overexpressing strain and Mtb-33 – ftsH antisense strain. Inset: Immunoblot showing FtsZ protein in the above strains.