Trehalase plays a role in macrophage colonization and virulence of Burkholderia pseudomallei in insect and mammalian hosts

Abstract

Trehalose is a disaccharide formed from two glucose molecules. This sugar molecule can be isolated from a range of organisms including bacteria, fungi, plants and invertebrates. Trehalose has a variety of functions including a role as an energy storage molecule, a structural component of glycolipids and plays a role in the virulence of some microorganisms. There are many metabolic pathways that control the biosynthesis and degradation of trehalose in different organisms. The enzyme trehalase forms part of a pathway that converts trehalose into glucose. In this study we set out to investigate whether trehalase plays a role in both stress adaptation and virulence of Burkholderia pseudomallei. We show that a trehalase deletion mutant (treA) had increased tolerance to thermal stress and produced less biofilm than the wild type B. pseudomallei K96243 strain. We also show that the ΔtreA mutant has reduced ability to survive in macrophages and that it is attenuated in both Galleria mellonella (wax moth larvae) and a mouse infection model. This is the first report that trehalase is important for bacterial virulence.

Keywords: Burkholderia pseudomallei; Galleria mellonella; biofilm; thermal stress; trehalase; trehalose; virulence.

Figure 1.

Growth of B. pseudomallei wild type, ΔtreA mutant or complemented mutants in M9 medium containing 0.4% glucose with agitation (A) or 0.4% trehalose with agitation (B). The ability of complemented mutants (ΔtreA/pBHR-nattreA and ΔtreA/pDA17-treA) to grow in M9 medium containing 0.4% trehalose, in a static 96 well plate format, was recorded after 48 hours (C). Values represent the mean from one experiment performed in triplicate. Error bars show standard error of the mean (SEM).

Figure 2.

Survival of B. pseudomallei wild type ΔtreA mutant, pSCrhaB2-treA complement, pDA17-treA complement or pBHR-nattreA complement after exposure to heat stress at 65°C (A) or cold stress at 4°C (B) Values represent the mean from 5 independent experiments performed in triplicate or 2 independent experiments with 9 replicates respectively. Error bars show SEM. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001 following one way Anova, Tukey post-test.

Figure 3.

Intracellular survival of B. pseudomallei wild type, ΔtreA mutant or pDA17-treA complemented mutant in J774.A.1 macrophages. Macrophages were infected with bacteria at an MOI of 10 and intracellular bacteria enumerated on LB agar. Values represent the mean from 3 independent experiments, performed in triplicate, with error bars showing the SEM s. * = p < 0.05, *** = p < 0.001 following students t-test.

Figure 4.

Response of B. pseudomallei wild type, ΔtreA mutant or pDA17-treA complemented mutant to 0.04 M tBOOH by disc diffusion assay. Values represent the mean from 3 independent experiments performed in triplicate, with error bars showing SEM. * = p < 0.05 following students t-test.

Figure 5.

Relative biofilm formation by B. pseudomallei wild type, ΔtreA mutant or pDA17-treA complement measured as crystal violet (CV) bound (OD490nm) / turbidity of bacterial culture (OD590nm). Values represent the mean from 3 independent experiments each with 12 replicates. Error bars show SEM. *** = p < 0.001, **** = p < 0.0001 following one-way Anova, Tukey post-test.

Figure 6.

Virulence of B. pseudomallei wild type or ΔtreA mutant in G. mellonella larvae or in mice. (A) Groups of 10 G. mellonella larvae were challenged with 10³ CFU of B. pseudomallei K96243 or the ΔtreA mutant. Values represent the mean from 3 independent experiments. Error bars show SEM. * = p < 0.05 following 2 way Anova, Sidak's multiple comparisons test. (B) Groups of 6 BALB/c mice challenged via i.p. route with 3.4 × 10⁴ CFU of B. pseudomallei K96243 (solid line) or 6.7 × 10⁴ CFU of the ΔtreA mutant (dotted line).