The Type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages

Abstract

SummaryBurkholderia cenocepacia is a Gram-negative opportunistic pathogen of patients with cystic fibrosis and chronic granulomatous disease. The bacterium survives intracellularly in macrophages within a membrane-bound vacuole (BcCV) that precludes the fusion with lysosomes. The underlying cellular mechanisms and bacterial molecules mediating these phenotypes are unknown. Here, we show that intracellular B. cenocepacia expressing a type VI secretion system (T6SS) affects the activation of the Rac1 and Cdc42 RhoGTPase by reducing the cellular pool of GTP-bound Rac1 and Cdc42. The T6SS also increases the cellular pool of GTP-bound RhoA and decreases cofilin activity. These effects lead to abnormal actin polymerization causing collapse of lamellipodia and failure to retract the uropod. The T6SS also prevents the recruitment of soluble subunits of the NADPH oxidase complex including Rac1 to the BcCV membrane, but is not involved in the BcCV maturation arrest. Therefore, T6SS-mediated deregulation of Rho family GTPases is a common mechanism linking disruption of the actin cytoskeleton and delayed NADPH oxidase activation in macrophages infected with B. cenocepacia.

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

“…Previous work in our laboratory demonstrated that the B. cenocepacia T6SS is essential for virulence in vivo (Hunt et al, 2004), and also required for the formation of actin-rich protrusions in infected macrophages (Aubert et al, 2010(Aubert et al, , 2008Rosales-Reyes et al, 2012) through inactivation of Rac1 (Flannagan et al, 2012;Rosales-Reyes et al, 2012). To determine whether the suhB Bc deletion affects T6SS function, we infected macrophages with B. cenocepacia-K56-2, DsuhB Bc and DsuhB Bc -pSM67.…”

“…The ability of B. cenocepacia to infect and persist in macrophages is associated with delayed phagolysosomal fusion (Huynh et al, 2010;Keith et al, 2009;Lamothe et al, 2007;Rosales-Reyes et al, 2012). To identify whether suhB Bc plays a role in the ability of B. cenocepacia to survive intracellularly we deleted the suhB Bc gene in the MH1K strain, an isogenic derivative of strain K56-2 that exhibits high susceptibility to gentamicin (Hamad et al, 2010).…”

“…In macrophages, the maturation of the B. cenocepacia-containing vacuole (BcCV) is altered, as revealed by decreased acidification and delayed fusion with lysosomes (Lamothe et al, 2007) and Rab7 inactivation (Huynh et al, 2010). Also, intracellular infection by B. cenocepacia induces Rac1 inactivation (Flannagan et al, 2012;Rosales-Reyes et al, 2012), delayed assembly of NADPH oxidase complex onto the BcCV membrane (Keith et al, 2009;Rosales-Reyes et al, 2012) and downregulation of the autophagy pathway (Abdulrahman et al, 2011).…”

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

“…B. cepacia adopts an extracellular or intracellular lifestyle (23,24). This bacterium can survive within a variety of eukaryotic cells such as amoebae, epithelial cells, and macrophages (25)(26)(27)(28).…”

Burkholderia cenocepacia J2315-mediated destruction of Staphylococcus aureus NRS77 biofilms.

“…The type IV secretion system has been implicated in the ability of B. cenocepacia to survive macrophage phagocytosis by evasion of lysosomal fusion [89]. The type VI secretion system has been shown to reduce production of reactive oxygen species inside macrophages [90].…”