Pseudomonas aeruginosa Cytotoxin ExoU Is Injected into Phagocytic Cells during Acute Pneumonia

Abstract

ExoU, a cytotoxin translocated into host cells via the type III secretion system of Pseudomonas aeruginosa, is associated with increased mortality and disease severity. We previously showed that impairment of recruited phagocytic cells allowed survival of ExoU-secreting P. aeruginosa in the lung. Here we analyzed types of cells injected with ExoU in vivo using translational fusions of ExoU with a ␤-lactamase reporter (ExoU-Bla). Cells injected with ExoU-Bla were detectable in vitro but not in vivo, presumably due to the rapid cytotoxicity induced by the toxin. Therefore, we used a noncytotoxic ExoU variant, designated ExoU(S142A)-Bla, to analyze injection in vivo. We determined that phagocytic cells in the lung were frequently injected with ExoU(S142A). Early during infection, resident macrophages constituted the majority of cells into which ExoU was injected, but neutrophils and monocytes became the predominant types of cells into which ExoU was injected upon recruitment into the lung. We observed a modest preference for injection into neutrophils over injection into other cell types, but in general the repertoire of injected immune cells reflected the relative abundance of these cells in the lung. Our results indicate that phagocytic cells in the lung are injected with ExoU and support the hypothesis that ExoU-mediated impairment of phagocytes has a role in the pathogenesis of pneumonia caused by P. aeruginosa.

Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

“…Therefore, we set out to identify host cells that receive T4SS-translocated effectors during infection with L. pneumophila. BlaM reporter systems have been used during in vivo infection with Yersinia pseudotuberculosis (67,68), Yersinia pestis (69,70), Yersinia enterocolitica (71), Salmonella enterica serovar Typhimurium (72,73), and Pseudomonas aeruginosa (74,75) to detect the translocation of effectors into host cells by the type III and type IV secretion systems. We demonstrate in this study that by using ␤-lactamase (BlaM) translationally fused to the T4SS-translocated effector protein RalF, we can successfully track injection by the T4SS into host cells during both in vitro and in vivo infection, and we describe the first use of this BlaM reporter during in vivo pulmonary infection with L. pneumophila.…”

Mutations in the Pseudomonas aeruginosa Needle Protein Gene pscF Confer Resistance to Phenoxyacetamide Inhibitors of the Type III Secretion System

“…The resulting strain MDM1542 (Table 1) exhibits Gm r that is dependent on induction of T3SS and lost in the presence of T3SS inhibitors. A single copy of a gene encoding full-length ExoS (lacking adenosine diphosphate ribosyltransferase [ADPRT] activity due to two mutations, E379A/E381A) fused to TEM1 ␤-lactamase (BLA) on miniCTX1 (28) was constructed essentially as described previously for miniCTXexoU-BLA (29). First, the stop codon was removed from exoS using the primers exoSdeltastopF and exoSdeltastopR (Table 2).…”

PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

“…Second, we had previously demonstrated that PrtR is required for the expression of P. aeruginosa type III secretion system (T3SS) genes by repressing the expression of PtrB, a specific inhibitor of the T3SS (12). The T3SS plays an important role in the killing of neutrophils and is thus essential for survival and replication of the bacteria within the host environments (13)(14)(15).…”