Previous work has shown that lacZ fusions to the cysK, astD, tnaB, and gabT genes in Escherichia coli are activated by self-produced extracellular signals. Using a combination of ethyl acetate extraction, reversedphase C 18 chromatography, and thin-layer chromatography, we have purified an extracellular activating signal from E. coli supernatants. Mass spectrometry revealed a molecule with an m/z peak of 117, consistent with indole. Nuclear magnetic resonance analysis of the purified E. coli factor and synthetic indole revealed identical profiles. Using synthetic indole, a dose-dependent activation was observed with lacZ fusions to the gabT, astD, and tnaB genes. However, cysK::lacZ and several control fusions were not significantly activated by indole. Conditioned medium prepared from a tnaA (tryptophanase) mutant, deficient in indole production, supported 26 to 41% lower activation of the gabT and astD fusions. The residual level of activation may be due to a second activating signal. Activation of the tnaB::lacZ fusion was reduced by greater than 70% in conditioned medium from a tnaA mutant.The use of chemical signals for bacterial communication is a widespread phenomenon (10,11,20,23,33). In gram-negative bacteria, these signals can be N-acyl derivatives of homoserine lactone, cyclic dipeptides, and quinolones (3,8,17,(28)(29)(30)43). In gram-positive bacteria, small peptides appear to be the predominant signal (7,15,16,18,25,36). In some cases, small proteins can mediate signaling (22,40). These signals regulate a variety of functions, including bioluminescence, differentiation, virulence, DNA transfer, and biofilm maturation (1,2,4,5,9,19,24,27,31,32).Indole production is a common diagnostic marker for the identification of Escherichia coli (37). Among the Enterobacteriaceae, indole is produced by E. coli and certain members of the Proteeae, such as Proteus vulgaris, Providencia spp., and Morganella spp. (37). Indole is formed from tryptophan by the tryptophanase enzyme, encoded by the tnaA gene (35). At very high concentrations (5 mM), indole is toxic to E. coli, possibly by causing membrane changes that result in the generation of superoxide (12). However, the concentration at which indole is toxic is approximately 15-fold higher than the physiological concentration seen in stationary-phase supernatants of E. coli (see below). The efflux of indole from E. coli is mediated by the AcrEF pump, and acrEF mutants exhibit enhanced indole sensitivity (21). The primary pathway for indole transport into the cell is via the Mtr permease (42).For E. coli, the role of cell-to-cell signaling in a variety of functions, including regulation of ftsQAZ, expression of type III secretion systems, inhibition of DNA replication, and activation of degradative pathways, has been described (1,13,34,38,39,41). However, the extracellular signals involved in these processes are poorly understood. Previous studies from our lab have identified the E. coli genes cysK, astD, tnaB, and gabT, which are activated by extracellular signals...

Utilizing the bicistronic reporter transposon mini-Tn5 lacZ-tet͞1, we have identified lacZ fusions to four Escherichia coli genes͞operons that are strongly activated by the accumulation of self-produced extracellular signals. These fusions were designated cma9, cma48, cma113, and cma114 for conditioned medium activated. Each of the cma fusions was expressed in a growth phase-dependent manner, and the presence of conditioned medium from a stationary phase E. coli culture resulted in the premature activation of these fusions in cells at early to mid-logarithmic phase. The cma48 and cma114 fusions were dependent on RpoS for growth phase expression and response to extracellular factors. The extracellular factors that activated the cma9, cma48, and cma114 fusions were produced in both rich complex and defined minimal media. The cma fusions were shown to be within the cysK (cma9), astD (cma48), tnaB (cma113), and gabT (cma114) genes. These genes function in the uptake, synthesis, or degradation of amino acids that yield pyruvate and succinate.Bacteria are capable of regulating gene expression in response to a variety of extracellular signals. When the signal is produced by the bacterium itself, this type of regulation is termed autoinduction or quorum sensing (1-4). The composition of signaling molecules can include mixtures of amino acids, peptides, fatty acids, and acyl derivatives of homoserine lactone (1-4, 5-7). These signaling molecules can regulate gene expression by a number of mechanisms, including modulating the activity of members of the LuxR family, interacting with two-component systems, and inhibiting phosphatases (1,3,(8)(9)(10). The cellular processes regulated by quorum sensing are diverse, and some examples include spore formation, activation of luminescence, competence, conjugal transfer of plasmid DNA, regulation of virulence genes, regulation of peptidoglycan O-acetylation, and biofilm maturation (11)(12)(13)(14)(15)(16)(17)(18)(19)(20).In Escherichia coli, the regulation of gene expression by extracellular signals has also been established. Expression of the rpoS gene, encoding the alternate sigma factor S ( 38 ) involved in stationary-phase and osmoregulated gene expression is stimulated by the presence of a factor in conditioned medium (21,22). Studies by Huisman and Kolter (23) indicate that rpoS expression is decreased in a thrA, metL, lysC triple mutant that is defective in production of homoserine, and expression could be restored by exogenous homoserine lactone. However, the role of homoserine lactone or an acylated derivative in RpoS expression is unclear. Additional E. coli genes subject to regulation by extracellular factors include sdiA and the ftsQAZ cell division gene cluster (22,24). Recent studies by Surette and Bassler (25) have revealed that E. coli produces a signal that can substitute for AI-2, one of two Vibrio harveyi signals that control luminescence gene expression. The E. coli signal was heat labile, produced at mid-exponential phase of growth, and degraded at sta...

Antibodies to capsular polysaccharide (PS) are protective against systemic infection by Streptococcus pneumoniae, but the large number of pneumococcal serogroups and the age-related immunogenicity of pure PS limit the utility of PS-based vaccines. In contrast, cell wall-associated proteins from different capsular serotypes can be cross-reactive and immunogenic in all age groups. Therefore, we evaluated three pneumococcal proteins with respect to relative accessibility to antibody, in the context of intact pneumococci, and their ability to elicit protection against systemic infection by encapsulated S. pneumoniae. Sequences encoding pneumococcal surface adhesin A (PsaA), putative protease maturation protein A (PpmA), and the N-terminal region of pneumococcal surface protein A (PspA) from S. pneumoniae strain A66.1 were cloned and expressed in Escherichia coli. The presence of genes encoding PsaA, PpmA, and PspA in 11 clinical isolates was examined by PCR, and the expression of these proteins by each strain was examined by Western blotting with antisera raised to the respective recombinant proteins. We used flow cytometry to demonstrate that PspA was readily detectable on the surface of the pneumococcal strains analyzed, whereas PsaA and PpmA were not. Consistent with these observations, mice with passively or actively acquired antibodies to PspA or type 3 PS were equivalently protected from homologous systemic challenge with type 3 pneumococci, whereas mice with passively or actively acquired antibodies to PsaA or PpmA were not effectively protected. These experiments support the hypothesis that the extent of protection against systemic pneumococcal infection is influenced by target antigen accessibility to circulating host antibodies.

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 μg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.