Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc

Abstract

Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

Figure 1. Physical map of the B. mallei T6SS-1 gene cluster.

Genes encoding components of T6SS-1 are shown in green (BMAA0744-0730). The Burkholderia intracellular motility (bim) genes are shown in red (BMAA0751-0747), the VirAG two-component regulatory system is shown in blue (virAG; BMAA0746-0745), the deubiquitinase encoding gene (tssM; BMAA0729) is shown in black, and downstream genes tssN and folE are shown in white. Homologous gene clusters are also present in B. pseudomallei (BPSS1490-1514) and B. thailandensis (BTH_II0877-0854).

Figure 2. Hcp1 is produced during growth of B. mallei in minimal media.

Whole cell lysates were prepared from overnight cultures of (A) B. mallei SR1A (pvirAG) and BM0742 (pvirAG) grown in LB4G, or (B) B. mallei SR1A (pBHR2) and SR1A (pvirAG) grown in LB4G or M9G, and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowhead.

Figure 3. Minimal media supplements influence B. mallei Hcp1 production.

Whole cell lysates were prepared from overnight cultures of B. mallei SR1A, ATCC 23344, NCTC 3708 and NCTC 3709 grown in LB4G, M9G, M9CG or M9TG, and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowhead.

Figure 4. Quantitation of B. mallei hcp1 and virG transcript during growth in minimal media.

Transcript levels of hcp1, virG and dnaK were determined by qRT-PCR using gene specific primers and dual-labeled Taqman probes. RNA was harvested from B. mallei SR1A grown for 8 h in LB4G, M9G, M9CG or M9TG. Relative mRNA levels were calculated using the ΔΔC_T method and represent fold changes in comparison to growth in LB4G. All values have been normalized to the internal control, rpoA. Results represent the means and standard deviations of three independent experiments performed in triplicate.

Figure 5. B. mallei T6SS-1 expression in M9CG media is VirG-dependent.

(A) Whole cell lysates were prepared from overnight cultures of B. mallei SR1A or BM0746 (ΔvirG) grown in M9CG and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowhead. (B) Transcript levels of bimA, tssA, hcp1, virG, tssM and dnaK were determined by qRT-PCR using gene specific primers and dual-labeled Taqman probes. RNA was harvested from B. mallei strains grown for 8 h in M9CG. Relative mRNA levels were calculated using the ΔΔC_T method and represent fold changes in comparison to SR1A. All values have been normalized to the internal control, rpoA. Results represent the means and standard deviations of three independent experiments performed in triplicate. ND, not detected.

Figure 6. Iron and zinc inhibit B. mallei T6SS-1 expression.

Whole cell lysates were prepared from overnight cultures of B. mallei SR1A grown in: (A) M9CG, M9TG or M9TG-C; (B) M9CG alone (none), M9CG supplemented with copper, iron, magnesium, manganese, nickel and zinc (pooled; 10 µM each), M9CG individually supplemented with copper, iron, magnesium, manganese, nickel or zinc (Cu, Fe, Mg, Mn, Ni or Zn; 10 µM each) or; (C) M9CG alone (none), M9CG supplemented with iron and zinc (Fe/Zn, 10 µM each), 2x iron (2x Fe, 20 µM) or 2x zinc (2x Zn, 20 µM); and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowheads. (D) Transcript levels of bimA, tssA, hcp1, virG, tssM and dnaK were determined by qRT-PCR using gene specific primers and dual-labeled Taqman probes. RNA was harvested from B. mallei strains grown for 8 h in M9CG (none) or M9CG plus iron and zinc (Fe/Zn; 10 µM each). Relative mRNA levels were calculated using the ΔΔC_T method and represent fold changes in comparison to M9CG. All values have been normalized to the internal control, rpoA. Results represent the means and standard deviations of three independent experiments performed in triplicate.

Figure 7. B. pseudomallei T6SS-1 expression is suppressed by iron and zinc.

(A) Whole cell lysates were prepared from overnight cultures of B. pseudomallei K96243 and 1026b grown in M9CG alone (none), M9CG supplemented with copper, iron, magnesium, manganese, nickel and zinc (pooled; 10 µM each), or M9CG individually supplemented with copper, iron, magnesium, manganese, nickel or zinc (Cu, Fe, Mg, Mn, Ni or Zn; 10 µM each), and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowheads. (B) Transcript levels of bimA, tssA, hcp1, virG, tssM and dnaK were determined by qRT-PCR using gene specific primers and dual-labeled Taqman probes. RNA was harvested from B. pseudomallei K96243 grown for 8 h in M9CG (none), M9CG plus iron and zinc (Fe/Zn; 10 µM each), M9CG plus iron (Fe; 10 µM), or M9CG plus zinc (Zn; 10 µM). Relative mRNA levels were calculated using the ΔΔC_T method and represent fold changes in comparison to M9CG. All values have been normalized to the internal control, rpoA. Results represent the means and standard deviations of three independent experiments performed in triplicate.

Figure 8. Hcp1 is not produced by B. thailandensis during growth in minimal media.

Whole cell lysates were prepared from overnight cultures of B. thailandensis E264 grown in LB4G, M9G, M9CG or M9TG, and then assayed for Hcp1 production by Western immunoblotting using anti-BtHcp1 polyclonal rabbit serum. A whole cell lysate prepared from B. thailandensis DW503 (pBtvirAG) grown overnight in LB was used as a positive control (+ve) for Hcp1 expression. The protein band corresponding to Hcp1 is indicated by the black arrowhead.