Establishment and maintenance of chronic lung infections with mucoid Pseudomonas aeruginosa in patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoid P. aeruginosa to antibody-independent opsonic killing and is the molecular basis for the resistance of mucoid P. aeruginosa to normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical for P. aeruginosa resistance to host immune effectors in CF patients.The predominant bacterial pathogen in chronic pulmonary infection in cystic fibrosis (CF) patients is the mucoid variant of Pseudomonas aeruginosa, which is encapsulated by and overproduces mucoid exopolysaccharide (MEP), or alginate. That alginate is the major virulence factor of P. aeruginosa in CF lung infection is evident from the epidemiology of this disease. The pulmonary function of patients with CF declines only when mucoid P. aeruginosa is isolated and associated lung pathology develops (9, 32, 33). The growth of mucoid P. aeruginosa as a biofilm in the lungs of CF patients has been suggested to be a major factor in long-term bacterium survival. Biofilm formation by P. aeruginosa has been linked to genes involved in quorum sensing (7) and motility (31), with a recent demonstration that the acyl-homoserine lactone molecules involved in the quorum-sensing system (8) can be detected in the sputa of CF patients (42). However, the genes controlling alginate production appear to be independent of control by the known quorum-sensing genes of P. aeruginosa, including lasR and rhlR (8,44,45). Therefore, the question of whether there is a regulator or environmental cue common to both alginate production and quorum-sensing systems has not yet been answered.The conversion of P. aeruginosa to the mucoid state in CF patients is often associated with mutations at the mucA locus (23). MucA and MucB (also called AlgN) act as anti-sigma factors for the alternative sigma factor E (47), encoded by algT (25), also known as algU (22). Increased activity of this sigma factor results in hyperexpression of the alginate biosynthetic operon located at 34 min on the P. aeruginosa genome (25

Pseudomonas aeruginosa is the nosocomial bacterial pathogen most commonly isolated from the respiratory tract. Animal models of this infection are extremely valuable for studies of virulence and immunity. We thus evaluated the utility of a simple model of acute pneumonia for analyzing P. aeruginosa virulence by characterizing the course of bacterial infection in BALB/c mice following application of bacteria to the nares of anesthetized animals. Bacterial aspiration into the lungs was rapid, and 67 to 100% of the inoculum could be recovered within minutes from the lungs, with 0.1 to 1% of the inoculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin exclusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by P. aeruginosa is associated with a cytotoxic effect on epithelial cells in vitro and virulence in animal models. Insertional mutations in the exoU gene confer a noncytotoxic phenotype on mutant strains and decrease virulence for animals. We used the model of acute pneumonia to determine whether introduction of the exoU gene into noncytotoxic strains of P. aeruginosa lacking this gene affected virulence. Seven phenotypically noncytotoxic P. aeruginosa strains were transformed with pUCP19exoUspcU which carries the exoU gene and its associated chaperone. Three of these strains became cytotoxic to cultured epithelial cells in vitro. These strains all secreted ExoU, as confirmed by detection of the ExoU protein with specific antisera. The 50% lethal dose of exoU-expressing strains was significantly lower for all three P. aeruginosa isolates carrying plasmid pUCP19exoUspcU than for the isogenic exoU-negative strains. mRNA specific for ExoU was readily detected in the lungs of animals infected with the transformed P. aeruginosa strains. Introduction of the exoU gene confers a cytotoxic phenotype on some, but not all, otherwise-noncytotoxic P. aeruginosa strains and, for recombinant strains that could express ExoU, there was markedly increased virulence in a murine model of acute pneumonia and systemic spread.Pseudomonas aeruginosa infection occurs when normal defense mechanisms are impaired or in cases of extensive tissue damage. Extracellular virulence factors including proteases, cytotoxins, phospholipases, pili, flagella, and smooth lipopolysaccharides have been shown to contribute to virulence in various animal models (18,25,26). Proteins exported by the type III secretion system, notably, exoenzyme S (ExoS), ExoT, and ExoU, have toxic effects on cells in culture (3,7,14,24,27,28) and are thought to be important virulence factors of P. aeruginosa. Disruption of the pscC gene (a member of the secretin family of proteins needed for secretion of the exoenzyme proteins) by insertion of Tn1 (29) reduced the virulence of cytotoxic strain PA 388 in burn wound infections in mice (18). This disruption did not affect levels of the mutant strain in a rat model of chronic lung infection, although...

As children age, they become less susceptible to the diverse microbes causing pneumonia. These microbes are pathobionts that infect the respiratory tract multiple times during childhood, generating immunological memory. To elucidate mechanisms of such naturally-acquired immune protection against pneumonia, we modeled a relevant immunological history in mice by infecting their airways with mismatched serotypes of Streptococcus pneumoniae (pneumococcus). Previous pneumococcal infections provided protection against a heterotypic, highly virulent pneumococcus, as evidenced by reduced bacterial burdens and long-term sterilizing immunity. This protection was diminished by depletion of CD4+ cells prior to the final infection. The resolution of previous pneumococcal infections seeded the lungs with CD4+ resident memory T (TRM) cells, which responded to heterotypic pneumococcus stimulation by producing multiple effector cytokines, particularly IL-17A. Following lobar pneumonias, IL-17-producing CD4+ TRM cells were confined to the previously infected lobe, rather than dispersed throughout the lower respiratory tract. Importantly, pneumonia protection also was confined to that immunologically-experienced lobe. Thus, regionally localized memory cells provide superior local tissue protection to that mediated by systemic or central memory immune defenses. We conclude that respiratory bacterial infections elicit CD4+ TRM cells that fill a local niche to optimize heterotypic protection of the affected tissue, preventing pneumonia.