The sputum (mucus) layer of the cystic fibrosis (CF) lung is a complex substrate that provides Pseudomonas aeruginosa with carbon and energy to support high-density growth during chronic colonization. Unfortunately, the CF lung sputum layer has been difficult to mimic in animal models of CF disease, and mechanistic studies of P. aeruginosa physiology during growth in CF sputum are hampered by its complexity. In this study, we performed chromatographic and enzymatic analyses of CF sputum to develop a defined, synthetic CF sputum medium (SCFM) that mimics the nutritional composition of CF sputum. Importantly, P. aeruginosa displays similar phenotypes during growth in CF sputum and in SCFM, including similar growth rates, gene expression profiles, carbon substrate preferences, and cell-cell signaling profiles. Using SCFM, we provide evidence that aromatic amino acids serve as nutritional cues that influence cell-cell signaling and antimicrobial activity of P. aeruginosa during growth in CF sputum.A key concept in bacterial pathogenesis is the ability of invading pathogens to obtain sufficient carbon and energy from the host for in vivo growth. Although Garber originally proposed the host as a growth medium over 40 years ago (12), the nutritional environment of most infection sites is poorly defined and often inadequately modeled by laboratory growth media. This lack of knowledge, combined with the limited utility of many animal models, provides significant challenges for mechanistic studies aimed at examining host nutrients as mediators of colonization and disease. To overcome these challenges, it is critical both to define the nutritional composition of key infection sites and to study bacterial physiology in the context of in vivo-relevant growth substrates.The heritable disease cystic fibrosis (CF) is an archetype for the development of nutritional models with which to study bacterial pathogenesis. A hallmark of CF disease is the accumulation of large volumes of sputum (mucus) within the lungs, which diminishes the host's ability to clear bacterial infections (17,31). The viscous CF lung sputum provides bacteria with a nutritionally rich growth environment composed of host-and bacterial-derived factors (17, 38). The opportunistic pathogen Pseudomonas aeruginosa chronically colonizes the CF lung, where it often grows to high cell densities in CF sputum (Ͼ10 9 cells/ml sputum). Although many other bacterial species persist and grow in the CF lung, chronic P. aeruginosa infection is likely the most clinically relevant, as it is correlated with declining lung function (17). Mechanistically, P. aeruginosa colonization and progression to chronic infection is poorly understood, although potential contributing factors are high-density growth and enhanced fitness of P. aeruginosa in CF sputum. P. aeruginosa fitness has been linked to nutritional components in CF sputum (39), thus necessitating the development of a versatile model that allows examination of CF sputum nutritional cues.The growth environment impacts several...

Volume 189, no. 22, p. 8079-8087, 2007. Page 8080, column 1, fifth paragraph, second and third lines, the parenthetical expression should read as follows: "(6.5 ml 0.2 M NaH 2 PO 4 , 6.25 ml 0.2 M Na 2 HPO 4 , 0.348 ml 1 M KNO 3 , 1.084 ml 0.25 M K 2 SO 4 , 0.122 g NH 4 Cl, 1.114 g KCl, 3.03 g NaCl, 10 mM MOPS, 779.6 ml deionized water)." 2906 on May 12, 2018 by guest

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand α-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFNγ to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4−/− mice and IFNγ−/− mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing α-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4−/− mice but did not confer protection. Finally, bone marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFNγ. NKT-derived IL-4 was required for OCH-enhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNγ also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by α-GC derivatives can be exploited to optimize anthrax vaccination.

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (␣-galactosylceramide [␣-GC]) at the time of immunization improves PA-specific Ab responses. We observed that ␣-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d ؊/؊ mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In J␣281 ؊/؊ mice expressing CD1d but lacking type I ␣-GC-reactive NKT cells, ␣-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.