e Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsBencoding gene within the regulatory gene operon.
Pseudomonas aeruginosa is a Gram-negative bacterium that thrives in a variety of environments (1, 2) and can colonize diverse hosts, from invertebrates to humans (3, 4). It represents a real threat to human health, being responsible for the most frequent hospital-acquired infections along with Escherichia coli and Staphylococcus aureus in France (5). Moreover, it is the first cause of mortality and morbidity for people suffering from cystic fibrosis (6). As most clinical isolates are multiresistant to antibiotics, it is imperative to find new antibacterial strategies against this pathogen (7,8). In this perspective, a better understanding of the virulence mechanisms of P. aeruginosa turned out to be a valuable approach to identify therapeutic targets and eventually develop new and more specific drugs.One major virulence factor of P. aeruginosa is the type III secretion system (T3SS). This system is well conserved among bacterial pathogens, such as Yersinia pestis, the causative agent of plague, and Salmonella species or Shigella flexneri, responsible for intestinal diseases (9). It has been shown that the T3SS of P. aeruginosa is particularly active during acute infections and is associated with poor clinical outcomes (10). Indeed, the presence of active T3SS is correlated with an increased mortality (11).The T3SS allows the bacterium to inject toxins directly into the host cell cytoplasm in order to hijack several cellular pathways, leading to disruption of the actin cytoskeleton and to cell death. This injection machinery consists of more than 20 proteins, assembled in a syringe-like structure and composed of three complexes: the basal body, the needle, and the translocon (12). In the cytoplasm, a proteinaceous ring makes the connection between the basal body and other soluble proteins. The basal body spans the two bacterial memb...
