senX3-independent contribution of regX3 to Mycobacterium tuberculosis virulence

Abstract

Background: Mycobacterium tuberculosis (Mtb) must adapt to various stress conditions during host infection. The two-component regulatory system (2CRS) SenX3-RegX3 is required for Mtb virulence. We showed recently that the senX3-regX3 intergenic region contains promoter activity, driving senX3-independent regX3 expression. In the current study, we tested the hypothesis that RegX3 has a SenX3-independent role in Mtb virulence. The gene expression patterns, growth, and survival of mutants containing transposon insertions in senX3 (senX3::Tn) and regX3 (regX3::Tn) were compared to those of their respective complemented strains and the isogenic wild-type parent strain during axenic growth in nutrient-rich broth, phosphate depletion, nutrient starvation, and in the lungs of BALB/c mice.

Results: regX3 expression was reduced in senX3::Tn during phosphate depletion and nutrient starvation, and expression of the phosphate-specific transport gene pstC2 was reduced similarly in senX3::Tn and regX3::Tn during phosphate depletion. Although senX3 and regX3 were each dispensable for Mtb growth in nutrient-rich broth, disruption of senX3 or regX3 caused a similar growth defect during phosphate depletion. Interestingly, senX3::Tn, in which monocistronic regX3 expression is preserved, showed significantly higher survival relative to regX3::Tn after 7 days of nutrient starvation (p <0.01), and in mouse lungs at Day 31 (p < 0.01), Day 62 (p < 0.01), and Day 124 (p = 0.05) after aerosol infection.

Conclusion: Our data demonstrate the specificity of the senX3-regX3 2CRS for sensing and responding to low ambient phosphate, but also raise the possibility that RegX3 may function independently of its cognate sensor histidine kinase.

Figure 1

Genetic confirmation of complementation of senX3 ::Tn and regX3 ::Tn. (A). Diagram of a 2824-bp fragment containing the senX3-regX3 operon from the genome of wild-type CDC1551. (B) FseI digestion of genomic DNA and hybridization with dig-labeled senX3 probe reveals fragments measuring 3.7-kb and 5.7-kb in CDC1551 and senX3::Tn, respectively, as well as 5.7-kb and 7.7-kb fragments in senX3::Tn Comp. (C) SphI digestion of genomic DNA followed by hybridization with dig-labeled regX3 probe reveals 5.5-kb and 7.5-kb fragments in CDC1551 and regX3::Tn, respectively, as well as 7.5-kb and 4.6-kb fragments in regX3::Tn Comp. (D) Southern analysis of senX3 complementation. Lanes 1 and 2: SenX3::Tn Comp candidates with appropriately-sized bands; Lane 3: SenX3::Tn; Lane 4: Wild-type CDC1551. (E) Southern analysis of regX3 complementation. Lane 1: Wild-type CDC551; Lane 2: regX3::Tn; Lanes 3–5: regX3::Tn Comp candidates with appropriately-sized bands.

Figure 2

Gene expression and regulation of senX3 , regX3, and the phosphate-specific transport gene pstC2 in each strain by qRT-PCR. An equal amount of total RNA samples were used for cDNA synthesis with random primers. The cycle threshold value (C_T) of each gene of interest was normalized to that of the housekeeping gene sigA to generate ∆C_T, which was then converted to fold change (1.83e-∆C_T). (A) Expression of the genes senX3 and regX3 in Middlebrook 7H9 broth. (B) Expression of the genes senX3 and regX3 during P_i depletion. (C) Expression of the pstC2 gene in Middlebrook 7H9 broth. (D) Expression of the pstC2 gene during P_i depletion. The samples were prepared as duplicates and the experiment was repeated twice with similar results. *p <0.05; **p <0.01.

Figure 3

Mtb senX3 and regX3 are required for optimal growth during P _i depletion, but not in P _i -replete broth. The wild-type, senX3::Tn, senX3::Tn Comp, regX3::Tn, and regX3::Tn Comp strains were each cultured in reconstituted 7H9 broth containing 25 mM P_i (A) and P_i-depleted broth (50 μM P_i) (B). Each experiment was repeated twice with similar values of optimal density (O.D.600_nm).

Figure 4

The contribution of senX3 and regX3 to bacillary elongation during P _i depletion. Transmission electron microscopy (TEM) was used to compare the morphology of all the strains after growth in reconstituted 7H9 broth containing 50 μM P_i for 21 days. The images are representative of the following strains: A. Wild type; B. senX3::Tn; C. senX3::Tn Comp; D. regX3::Tn; E. regX3::Tn Comp (20,000x magnification).

Figure 5

Both senX3 and regX3 are required for Mtb survival during nutrient starvation, although there appears to be a senX3 -independent contribution of regX3 . The wild-type CDC1551, senX3::Tn, senX3::Tn Comp, regX3::Tn, and regX3::Tn Comp were subcultured in 1xPBS with 0.05% Tween-80 and CFU were counted at different time points after plating the diluted cultures on Middlebrook 7H10 plates (mean ± SD) and incubating for 21 days. Triplicate samples were used in the experiment and the experiment was repeated twice under the same condition. *p <0.05; **p <0.01.

Figure 6

Both senX3 ::Tn and regX3 ::Tn are attenuated, but there appears to be a senX3 -independent contribution of regX3 for long-term Mtb survival in mouse lungs. Separate groups of BalB/c mice (4–5 mice per group) were aerosol-infected with the wild-type CDC1551, senX3::Tn, senX3::Tn Comp, regX3::Tn, and regX3::Tn Comp. *p <0.05; **p <0.01.

Figure 7

Histological evaluation of infected mouse lungs at Day 124 post-infection. Hematoxylin-eosin stains, 200x magnification. A. CDC1551; B. senX3::Tn mutant; C. senX3::Tn Comp; D. regX3::Tn; E. regX3::Tn Comp.