Figure 4. Low glucose concentration impairs growth of P. aeruginosa in vitro. (A) Glucose concentration-dependent growth of P. aeruginosa. M9 medium with glucose concentrations as shown (0, 0.5, 2, 8 and 24 mM) was inoculated with P. aeruginosa (PAO1) and OD600 was followed. Data shown as mean 6 s.e.m. n = 3. (B) Growth of P. aeruginosa in ASL is nutrient-limited. ASL and M9 medium with and without added glucose (20 mM) as shown were inoculated with PAO1. OD600 was followed over time. Data shown as mean 6 s.e.m. n = 3. (***: p,0.0001 for ASL vs. ASL + glucose). (C) Growth of P. aeruginosa in human airway epithelia is limited by ASL glucose concentration. Cultures of human airway epithelia were inoculated apically with 2.3 or 3.3 log CFU of PAO1 in the absence (white bars) or presence (gray bars) of added glucose (20 mM). After 24 h, apical bacteria were quantified. Percentage of samples in which growth of bacteria occurred are shown. n = 5. (D) Facilitated diffusion transport in airway epithelia in vitro impairs growth of P. aeruginosa. Cultures of human airway epithelia were inoculated apically with 0.5, 1.5 or 2.5 log CFU of PAO1 after incubation with apical vehicle (white bars) or phloretin (gray bars). After 24 h, apical bacteria were quantified. Percentage of samples in which growth of bacteria occurred are shown. n = 12. (*: p,0.05 for vehicle vs. phloretin). doi:10.1371/journal.pone.0016166.g004 Hyperglycemia promotes bacterial growth in the lungs of mice To test the significance of low glucose concentrations in the ASL in vivo, we studied PAO1 growth in murine lungs. Collecting undiluted ASL from mouse airways is technically challenging. It has been shown [28] that attempts to collect ASL using filter paper techniques result in submucosal liquid collection with little ASL recovery. Therefore, to investigate the effect of hyperglycemia on ASL innate immunity, we used three complementary approaches that include two models of hyperglycemic mice and a mutant strain of P. aeruginosa that cannot metabolize glucose. First, we investigated the intrapulmonary growth of PAO1 (Figure 5, A) in ob/ob mice, which are genetically leptin deficient and become hyperglycemic and obese [29–33]. An intranasal inoculum of 7.69 log CFU of PAO1 was given to ob/ob mice (serum glucose = 20.764.7 mM) and their littermate controls (serum glucose = 5.761.2 mM). Bacterial counts in lung homog- enates 6 hours after inoculation were higher in hyperglycemic ob/ ob mice than in controls (median log CFU/mg of lung tissue = 1.28 for ob/ob vs. 0 for control mice). Second, since the observed differences in the previous experiment could be a direct consequence of leptin deficiency, we repeated the experiment (Figure 5, A) using db/db mice [30,34] and their littermate controls. These mice are leptin-receptor deficient and also become hyperglycemic and obese (serum Airway Glucose and Innate Immunity Figure 5. Hyperglycemia promotes growth of P. aeruginosa in the lungs of mice. (A and B) An intranasal inoculum (7.69 log CFU) of PAO1 (A) or edd2 (B) was given to ob/ob and db/db mice and their respective littermate controls (ob/ob ctrl and db/db ctrl). After 6 hours, lungs were homogenized for bacterial counts. n = 5 mice per group. (*: p,0.05, ns: p $ 0.05). doi:10.1371/journal.pone.0016166.g005 glucose = 22.665.3 mM). Bacterial counts in lung homogenates 6 hours after inoculation were higher in hyperglycemic db/db mice than in their littermate controls (median log CFU/mg of lung tissue = 2.3 for db/db vs. 0.35 for control mice). The data from ob/ ob and db/db mice show that the bacterial counts are significantly different between hyperglycemic and control mice (median log CFU/mg of lung tissue = 2.27, IQR 1.26 – 4.3 for hyperglycemic mice vs. median = 0.05, IQR 0 – 1.82 for control mice, p = 0.0176). The bacterial counts are not significantly different when the db/db genotype is compared to the ob/ob genotype (p = 0.848) excluding leptin or leptin-receptor deficiency as the cause of the difference in bacterial counts between control and hyperglycemic mice. Finally, we hypothesized that the higher susceptibility of hyperglycemic mice to infection is caused by elevated concentra- tions of glucose on the surface of the airway resulting in increased availability of glucose as a nutrient source for bacteria. We predicted that we would see no difference in bacterial growth if we used a mutant of PAO1 incapable of metabolizing glucose. Some artificially generated mutants [15] or clinical strains [12] of P. aeruginosa show variability in their capacity to preferentially use glucose as a carbon source, allowing to specifically assess the role of glucose in bacterial growth in vivo. We repeated the experiment using a transposon insertion mutant of PAO1 [15] with a defective edd gene, which codes for phosphogluconate dehydratase, a protein required for the catabolism of glucose and gluconate to glyceraldehyde-3-phosphate and pyruvate via the Entner-Doudor- off pathway [26,35,36]. The edd2 mutant was phenotypically similar to PAO1 in assays of swimming motility, twitching motility, protease activity and Type III secretion system protein (PcrV and ExoS) expression (Figure S3). In addition, lethality in a Drosophila melanogaster infection model was also similar following edd2 and PAO1 infection (Figure S4). When edd2 was used (Figure 5, B) to inoculate the airways of ob/ob mice and db/db mice and their respective littermate controls, neither showed increased bacterial growth when compared to their normoglycemic controls (median log CFU/mg of lung tissue = 0, IQR 0 – 0 for control mice vs. median = 0, IQR 0 – 0.67 for hyperglycemic mice, p = 0.5036). In contrast, when the edd2 mutant strain was injected into the peritoneal cavity (a nutrient-rich environment) of mice, the LD50 was similar to that of PAO1 (Figure S5). These data show that increased susceptibility to PAO1 infection in hyperglycemic mice is due to the elevated glucose concentration in ASL, which can be PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16166