Salmonella enterica is a facultative intracellular pathogen that is able to modify host cell functions by means of effector proteins translocated by the type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2). The SPI2-T3SS is also active in Salmonella after uptake by murine bone marrow-derived dendritic cells (BM-DC). We have previously shown that intracellular Salmonella interfere with the ability of BM-DC to stimulate antigen-dependent T-cell proliferation in an SPI2-T3SS-dependent manner. We observed that Salmonella-mediated inhibition of antigen presentation could be restored by external addition of peptides on major histocompatibility complex class II (MHC-II). The processing of antigens in Salmonella-infected cells was not altered; however, the intracellular loading of peptides on MHC-II was reduced as a function of the SPI2-T3SS. We set out to identify the effector proteins of the SPI2-T3SS involved in inhibition of antigen presentation and demonstrated that effector proteins SifA, SspH2, SlrP, PipB2, and SopD2 were equally important for the interference with antigen presentation, whereas SseF and SseG contributed to a lesser extent to this phenotype. These observations indicate the presence of a host cell-specific virulence function of a novel subset of SPI2-effector proteins.

The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electromobility shift assays with the purified Crp(Sco)-cAMP complex and the CRP-like cis-acting element of its own promoter failed. Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp(Sco). The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E. coli mutant suggests that the way Crp(Sco) interacts with DNA should mechanistically differ from its most similar members. This hypothesis was further supported by homology modelling of Crp(Sco) that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp(Eco).

Open reading frame SCO3571 of Streptomyces coelicolor encodes a protein of the cyclic AMP (cAMP) receptor protein (CRP) superfamily of regulatory proteins. A mutant revealed a dramatic defect in germination, followed by growth delay and earlier sporulation. This phenotype correlates with those of an adenylate cyclase (cya) mutant that cannot synthesize cAMP. This finding suggests that S. coelicolor may use a Cya-cAMP-CRP system to trigger complex physiological processes such as morphogenesis.Streptomycetes are gram-positive soil bacteria that undergo morphological differentiation. Their life cycle includes germination, vegetative mycelial growth, aerial mycelial growth, secondary metabolite synthesis, and eventually spore morphogenesis (5, 6). In the initial stages of germination, spores swell, resulting in an increase in size and decreased phase brightness followed by germ tube emergence (10). The growth of Streptomyces coelicolor in liquid media is characterized by a first phase of rapid growth (RG1), after which nutritional imbalances evoke a stress response and a shutdown of protein synthesis. This period is called the transition (T) phase and is followed by a second phase of rapid growth (RG2) and the onset of secondary metabolism (14).In S. coelicolor, cyclic AMP (cAMP) is synthesized throughout the developmental program (16). There is a peak of cAMP accumulation during germination, and then its level increases slowly during the growth of substrate mycelium. A maximal concentration is reached during the T phase and early stages of aerial mycelium formation. Later, during RG2, the cAMP level decreases rapidly. Characterization of the adenylate cyclase mutant (cya), unable to synthesize cAMP, demonstrated that cAMP facilitates developmental processes (16). The cya mutant is defective in germination and has a growth delay of 20 h. With regard to antibiotic production, cAMP determines the structure of the final product of the actinorhodin pathway (actinorhodin red or ␥-actinorhodin blue) through its effects on medium pH and also increases yield. All phenotypes specific to the cya mutant are suppressed by the exogenous addition of cAMP.In Escherichia coli, cAMP is the effecting molecule that modulates the DNA-binding ability of the pleiotropic transcription factor cAMP receptor protein (CRP). This protein activates and in some cases represses a vast number of genes that are implicated in diverse cellular functions, including carbon metabolism and starvation, resistance to stress and acid, and motility (2-4, 12, 15). In the same way, cAMP could trigger the streptomycete lifestyle by modulating proteins such as transcription factors of the CRP/fumarate and nitrate reduction regulator (FNR) superfamily (8). Hence, we raised the question whether a cAMP receptor protein exists in Streptomyces species. Such a potential crp-like gene (hereinafter called crp) within the S. coelicolor genome has been annotated as open reading frame SCO3571, located on cosmid SCH17 (1).Phylogenetic and comparative studies (data not shown...

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