Date Published: July 23, 2012
Publisher: Public Library of Science
Author(s): Marlies Galle, Shouguang Jin, Pieter Bogaert, Mira Haegman, Peter Vandenabeele, Rudi Beyaert, Dipshikha Chakravortty. http://doi.org/10.1371/journal.pone.0041547
The type III secretion system (T3SS) is a complex nanomachine of many pathogenic Gram-negative bacteria. It forms a proteinaceous channel that is inserted into the host eukaryotic cell membrane for injection of bacterial proteins that manipulate host cell signaling. However, few studies have focused on the effector-independent functions of the T3SS. Using a murine model of acute lung infection with Pseudomonas aeruginosa, an important human opportunistic pathogen, we compared the pathogenicity of mutant bacteria that lack all of the known effector toxins ( ΔSTY), with mutant bacteria that also lack the major translocator protein PopB (ΔSTY/ΔPopB) and so cannot form a functional T3SS channel in the host cell membrane. Mortality was higher among mice challenged with ΔSTY compared to mice challenged with ΔSTY/ΔPopB mutant bacteria. In addition, mice infected with ΔSTY showed decreased bacterial clearance from the lungs compared to those infected with ΔSTY/ΔPopB. Infection was in both cases associated with substantial killing of lung infiltrating macrophages. However, macrophages from ΔSTY-infected mice died by pro-inflammatory necrosis characterized by membrane permeabilization and caspase-1 mediated IL-1β production, whereas macrophages from ΔSTY/ΔPopB infected mice died by apoptosis, which is characterized by annexin V positive staining of the cell membrane and caspase-3 activation. This was confirmed in macrophages infected in vitro. These results demonstrate a T3SS effector toxin independent role for the T3SS, in particular the T3SS translocator protein PopB, in the pathogenicity of P. aeruginosa during acute lung infection.
Pseudomonas aeruginosa is a Gram-negative bacterium found ubiquitously in soil and water habitats. It is an opportunistic pathogen that causes serious, often antibiotic resistant, infections in immunocompromised individuals, burn victims and patients requiring mechanical ventilation , . For example, once P. aeruginosa is established in the airways of cystic fibrosis patients, it is almost impossible to eradicate, and the result is frequently mortality . Most clinical isolates of P. aeruginosa secrete virulence determinants and also possess a specialized proteinaceous apparatus associated with the cell wall which is used to translocate toxins into eukaryotic cells. This is known as the type III secretion system (T3SS). The T3SS is a potent virulence mechanism shared by Pseudomonas and many other pathogenic Gram-negative bacteria that inject T3SS effector proteins into the cytosol of their host cells , . The T3SS is a complex syringe-like apparatus on the bacterial surface and consists of five groups of proteins: the needle complex, the translocation apparatus, regulator proteins, chaperones and effector toxins. The needle complex is responsible for the transport of effector toxins from the bacterial cytosol to the outside. The translocation apparatus is a membrane pore that accepts the effector proteins secreted by the needle complex and delivers them across the host cell plasma membrane. The T3SS of P. aeruginosa uses three proteins for translocation: PopB, PopD and PcrV . The latter is located at the distal end of the needle and serves as a molecular platform where PopB and PopD form the translocation pore by oligomerisation. The exact regulation of the polymerization is poorly understood. PopB, PopD and PcrV are secreted via the T3SS and are absolutely required for pore formation and translocation of effectors across the host cell plasma membrane , . In Yersinia YopB, it was demonstrated that secreted translocators cannot cross-complement a yopB null mutant, which suggests that pore formation requires that the secreted translocators remain in close proximity to the needle . The steps of triggering effector secretion upon cell contact have not been elucidated, but several events are known to occur. First, the bacterium makes contact with the cell, a process mediated by specific adhesins . Then, the T3SS is brought close to the plasma membrane and the translocator proteins PopB and PopD are inserted into the host membrane to form the translocation pore , . The needle tip protein PcrV is required for appropriate assembly and insertion of PopB and PopD into host membranes , . After formation of the translocation pore and docking of the needle to the pore, effector secretion is triggered. Transcription and secretion of the T3SS effector proteins are regulated by specific regulator proteins. In vitro, secretion can be triggered by calcium depletion or by contact with host cells . Proteins destined for secretion by T3SS are bound by chaperones that facilitate their storage in the cytosol and delivery to the secretion apparatus. P. aeruginosa has four known effector toxins: ExoS, ExoT, ExoY and ExoU. These proteins can modify signal transduction pathways and counteract innate immunity . ExoS and ExoT are bifunctional enzymes with GTPase activating protein (GAP) activity and ADP ribosyl transferase (ADPRT) activity, which target several proteins, including Ras and Ras-like GTPases. These two distinct enzymatic activities work redundantly to disrupt the actin cytoskeleton, resulting in profound effects on host cellular processes . While the ADPRT domains of ExoS and ExoT are highly homologous and both require the 14-3-3 family protein FAS as a cofactor, their targets are very different. In contrast to ExoS, which has poly-substrate specificity, ExoT ADP-ribosylates a more restricted subset of host proteins, including the Crk adaptor proteins. Expression of the ADPRT domain of ExoS is toxic to cultured cells, while expression of ExoT appears to interfere with host cell phagocytic activity . We previously reported that ExoS negatively regulates the P. aeruginosa induced interleukin-1β (IL-1β) maturation and secretion by a mechanism that is dependent on its ADPRT activity . ExoY is an adenylate cyclase that requires an unidentified host cell cofactor for it activity. Its role in virulence remains uncertain, though it can cause cell rounding upon cocultivation with cells  and is toxic when expressed in yeast . ExoU has been characterized as a member of the phospholipase family of enzymes and has at least phospholipase A2 activity . Similar to ExoS, ExoT and ExoY, ExoU requires either a eukaryote-specific cofactor for its activity and ubiquitinated proteins, as well as ubiquitin itself, have been suggested as being potential activators of the toxin . In mammalian cells, the direct injection of ExoU causes irreversible damage to cellular membranes and rapid necrotic death. ExoS and ExoU are rarely found together in one strain. Both genotypes (ExoS/ExoT and ExoU/ExoT) are associated with acute infections in humans, though ExoU-producing strains are under-represented in persistently infected cystic fibrosis patients .
Much effort has been put in the functional and structural characterization of the T3SS of P. aeruginosa and other Gram-negative bacteria, and its interaction with the host cell. These studies have focused mainly on the role of the T3SS effector proteins (ExoS, ExoT, ExoY, ExoU in the case of P. aeruginosa) in virulence. While this manuscript was under revision, T3SS rod and needle proteins were shown to trigger the activation of the NAIP/NLRC4 inflammasome when expressed in mammalian cells –, illustrating functions of the T3SS beyond the injection of T3SS effector proteins. In the present study, we compared the pathogenicity of P. aeruginosa WT bacteria (PAK strain, which is deficient for ExoU) with that of mutant bacteria ΔSTY (devoid of ExoS, ExoT and ExoY) and ΔSTY/ΔPopB (devoid of ExoS, ExoT, ExoY, and the T3SS translocator protein PopB). The ΔSTY/ΔPopB mutant has an intact needle complex but cannot form pores in host cells or translocate T3SS effector proteins. Using a murine model of acute lung infection as well as infection of cultured macrophages with the above described mutants, we provide evidence that the T3SS translocation pore plays an important role in P. aeruginosa pathogenicity that is independent of the injection of any of the known T3SS effector proteins. This is illustrated by the findings that compared to ΔSTY/ΔPopB, infection with ΔSTY leads to higher mortality, reduced bacterial clearance, and non-apoptotic killing of alveolar macrophages, which is associated with the production of pro-inflammatory IL-1β. Absence of IL-1β production in ΔSTY/ΔPopB infected mice might contribute to the more efficient clearance of the bacteria from the lungs and the lower mortality compared to ΔSTY infected mice. This is consistent with previous data showing that absence or reduction of endogenous IL-1β activity improves host defense against Pseudomonas pneumonia while suppressing the inflammatory response . The extended life of ΔSTY/ΔPopB infected mice compared to ΔSTY infected mice may therefore not only reflect a reduction in the bacterial burden but also the observed reduction in the amount of IL-1β, which is known to trigger several signaling cascades leading to massive inflammation and multiple organ failure. In this context, it was previously shown that several cytokines, among them IL-1β, were present at significantly higher levels in BALF from mice infected with bacteria possessing an intact T3SS than in mice infected with a PcrV mutant that is unable to form a translocation pore . We previously showed that ExoS interferes with the P. aeruginosa induced activation of caspase-1 and the production of IL-1β in a macrophage cell line . However, we did not observe a significant difference in survival between WT-infected and ΔSTY-infected mice in the current study. It is likely that the relative importance of ExoS in immune evasion depends on specific factors such as bacterial load and site of infection, or differs in acute versus chronic infection. The specific role of ExoS in immune evasion awaits further studies directly comparing infection under different conditions.