The galU Gene of Pseudomonas aeruginosa Is Required for Corneal Infection and Efficient Systemic Spread following Pneumonia but Not for Infection Confined to the Lung

Abstract

Acute pneumonias and corneal infections due to Pseudomonas aeruginosa are typically caused by lipopolysaccharide (LPS)-smooth strains. In cystic fibrosis patients, however, LPS-rough strains of P. aeruginosa, which lack O antigen, can survive in the lung and cause chronic infection. It is not clear whether an LPS-rough phenotype affects cytotoxicity related to the type III secretion system (TTSS). We previously reported that interruption of the galU gene in P. aeruginosa results in production of a rough LPS and truncated LPS core. Here we evaluated the role of the galU gene in the pathogenesis of murine lung and eye infections and in cytotoxicity due to the TTSS effector ExoU. We studied galU mutants of strain PAO1, of its cytotoxic variant expressing ExoU from a plasmid, and of the inherently cytotoxic strain PA103. The galU mutants were more serum sensitive than the parental strains but remained cytotoxic in vitro. In a corneal infection model, the galU mutants were significantly attenuated. In an acute pneumonia model, the 50% lethal doses of the galU mutants were higher than those of the corresponding wild-type strains, yet these mutants could cause mortality and severe pneumonia, as judged by histology, even with minimal systemic spread. These findings suggest that the galU gene is required for corneal infection and for efficient systemic spread following lung infection but is not required for infection confined to the lung. Host defenses in the lung appear to be insufficient to control infection with LPS-rough P. aeruginosa when local bacterial levels are high.

Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence

“…The galU gene product is responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP, and galU mutants are unable to synthesize UDP-glucose by this reaction (Priebe et al, 2004). We proved that A. hydrophila GalU is a UDPglucose pyrophosphorylase responsible for the chemical reaction mentioned above.…”

“…In the absence of GalU, not enough UDP-glucose would be present to form glucose 1-phosphate. In addition, the accumulation of galactose 1-phosphate could have a toxic effect and explain the inability to grow in minimal medium with galactose as sole carbon source (Priebe et al, 2004). Furthermore, the low amount of UDP-glucose found in Aeromonas galU mutants could not be explained by GalE (UDP-glucose epimerase) or by GalT, due to the inability of these mutants to grow in minimal medium with galactose as sole carbon source.…”

“…Besides its function as a substrate for glucosyltransferases resulting in glucosylated surface structures, UDP-glucose plays a well-established biochemical role as a glycosyl donor in the enzymic biosynthesis of carbohydrates (Csonka & Epstein, 1996;Stimson et al, 1995). The galU gene was found to be important for pathogenesis of infections due to a number of Gram-negative pathogens, including Actinobacillus pleuropneumoniae (Rioux et al, 1999), Escherichia coli (Komeda et al, 1977), Klebsiella pneumoniae (Chang et al, 1996), Pseudomonas aeruginosa (Evans et al, 2002;Priebe et al, 2004), Shigella flexneri (Sandlin et al, 1995) and Vibrio cholerae (Nesper et al, 2001). The phenotypes of the K. pneumoniae and V. cholerae galU mutants were dominated by abnormal capsule synthesis rather than LPS O-antigen abnormalities, and several of the galU mutants also had defects in other surface proteins, such as IscA for S. flexneri (Sandlin et al 1995) and flagella for E. coli (Komeda et al, 1977).…”

“…The phenotypes of the K. pneumoniae and V. cholerae galU mutants were dominated by abnormal capsule synthesis rather than LPS O-antigen abnormalities, and several of the galU mutants also had defects in other surface proteins, such as IscA for S. flexneri (Sandlin et al 1995) and flagella for E. coli (Komeda et al, 1977). Among all these studies, however, the common theme of impaired survival of galU mutants in the face of host factors such as complement does emerge, as for instance in the P. aeruginosa galU mutants (Priebe et al, 2004).…”

Relating the Physical Properties of Pseudomonas aeruginosa Lipopolysaccharides to Virulence by Atomic Force Microscopy

“…O-antigen-deficient mutants appear to be more attenuated than wild-type organisms in mouse burn, corneal, and pneumonia models of P. aeruginosa infections (11,36,41). They disseminate less readily from the initial site of infection, are more serum sensitive than wild-type strains (36), and also exhibit decreased adherence to epithelial cells in vitro (41).…”