Macrophage-infectivity potentiator of Trypanosoma cruzi (TcMIP) is a new pro-type 1 immuno-stimulating protein for neonatal human cells and vaccines in mice.

This work identifies the protein "macrophage infectivity potentiator" of Trypanosoma cruzi trypomastigotes, as supporting a new property, namely a pro-type 1 immunostimulatory activity on neonatal cells. In its recombinant form (rTcMIP), this protein triggers the secretion of the chemokines CCL2 and CCL3 by human umbilical cord blood cells from healthy newborns, after 24h in vitro culture. Further stimulation for 72h results in secretion of IFN-γ, provided cultures are supplemented with IL-2 and IL-18. rTcMIP activity is totally abolished by protease treatment and is not associated with its peptidyl-prolyl cis-trans isomerase enzymatic activity. The ability of rTcMIP to act as adjuvant was studied in vivo in neonatal mouse immunization models, using acellular diphtheria-tetanus-pertussis-vaccine (DTPa) or ovalbumin, and compared to the classical alum adjuvant. As compared to the latter, rTcMIP increases the IgG antibody response towards several antigens meanwhile skewing antibody production towards the Th-1 dependent IgG2a isotype. The amplitude of the rTcMIP adjuvant effect varied depending on the antigen and the co-presence of alum. rTcMIP did by contrast not increase the IgE response to OVA combined with alum. The discovery of the rTcMIP immunostimulatory effect on neonatal cells opens new possibilities for potential use as pro-type 1 adjuvant for neonatal vaccines. This, in turn, may facilitate the development of more efficient vaccines that can be given at birth, reducing infection associated morbidity and mortality which are the highest in the first weeks after birth.

NgcESco Acts as a Lower-Affinity Binding Protein of an ABC Transporter for the Uptake of N,N'-Diacetylchitobiose in Streptomyces coelicolor A3(2).

In the model species Streptomyces coelicolor A3(2), the uptake of chitin-degradation byproducts, mainly N,N'- diacetylchitobiose ([GlcNAc]2) and N-acetylglucosamine (GlcNAc), is performed by the ATP-binding cassette (ABC) transporter DasABC-MsiK and the sugar-phosphotransferase system (PTS), respectively. Studies on the S. coelicolor chromosome have suggested the occurrence of additional uptake systems of GlcNAc-related compounds, including the SCO6005-7 cluster, which is orthologous to the ABC transporter NgcEFG of S. olivaceoviridis. However, despite conserved synteny between the clusters in S. coelicolor and S. olivaceoviridis, homology between them is low, with only 35% of residues being identical between NgcE proteins, suggesting different binding specificities. Isothermal titration calorimetry experiments revealed that recombinant NgcESco interacts with GlcNAc and (GlcNAc)2, with Kd values (1.15 and 1.53 µM, respectively) that were higher than those of NgcE of S. olivaceoviridis (8.3 and 29 nM, respectively). The disruption of ngcESco delayed (GlcNAc)2 consumption, but did not affect GlcNAc consumption ability. The ngcESco-dasA double mutation severely decreased the ability to consume (GlcNAc)2 and abolished the induction of chitinase production in the presence of (GlcNAc)2, but did not affect the GlcNAc consumption rate. The results of these biochemical and reverse genetic analyses indicate that NgcESco acts as a (GlcNAc)2- binding protein of the ABC transporter NgcEFGSco-MsiK. Transcriptional and biochemical analyses of gene regulation demonstrated that the ngcESco gene was slightly induced by GlcNAc, (GlcNAc)2, and chitin, but repressed by DasR. Therefore, a model was proposed for the induction of the chitinolytic system and import of (GlcNAc)2, in which (GlcNAc)2 generated from chitin by chitinase produced leakily, is mainly transported via NgcEFG-MsiK and induces the expression of chitinase genes and dasABCD.

Unsuspected control of siderophore production by N-acetylglucosamine in streptomycetes.

Iron is one of the most abundant elements on earth but is found in poorly soluble forms hardly accessible to microorganisms. To subsist, they have developed iron-chelating molecules called siderophores that capture this element in the environment and the resulting complexes are internalized by specific uptake systems. While biosynthesis of siderophores in many bacteria is regulated by iron availability and oxidative stress, we describe here a new type of regulation of siderophore production. We show that in Streptomyces coelicolor, their production is also controlled by N-acetylglucosamine (GlcNAc) via the direct transcriptional repression of the iron utilization repressor dmdR1 by DasR, the GlcNAc utilization regulator. This regulatory nutrient-metal relationship is conserved among streptomycetes, which indicates that the link between GlcNAc utilization and iron uptake repression, however unsuspected, is the consequence of a successful evolutionary process. We describe here the molecular basis of a novel inhibitory mechanism of siderophore production that is independent of iron availability. We speculate that the regulatory connection between GlcNAc and siderophores might be associated with the competition for iron between streptomycetes and their fungal soil competitors, whose cell walls are built from the GlcNAc-containing polymer chitin. Alternatively, GlcNAc could emanate from streptomycetes' own peptidoglycan that goes through intense remodelling throughout their life cycle, thereby modulating the iron supply according to specific needs at different stages of their developmental programme.

The chitobiose-binding protein, DasA, acts as a link between chitin utilization and morphogenesis in Streptomyces coelicolor.

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such as cellulose, xylan and chitin for the colonization of their environmental niche and therefore these polysaccharides may play a critical role in monitoring the global nutritional status of the environment. In this work we analysed the role of DasA, the sugar-binding component of the chitobiose ATP-binding cassette transport system, in informing the cell of environmental conditions, and its role in the onset of development and in ensuring correct sporulation. The chromosomal interruption of dasA resulted in a carbon-source-dependent vegetative arrest phenotype, and we identified a second DasR-dependent sugar transporter, in addition to the N-acetylglucosamine phosphotransferase system (PTS(GlcNAc)), that relates primary metabolism to development. Under conditions that allowed sporulation, highly aberrant spores with many prematurely produced germ tubes were observed. While GlcNAc locks streptomycetes in the vegetative state, a high extracellular concentration of the GlcNAc polymer chitin has no effect on development. The striking distinction is due to a difference in the transporters responsible for the import of GlcNAc, which enters via the PTS, and of chitin, which enters as the hydrolytic product chitobiose (GlcNAc(2)) through the DasABC transporter. A model explaining the role of these two essentially different transport systems in the control of development is provided.

Conserved cis-acting elements upstream of genes composing the chitinolytic system of streptomycetes are DasR-responsive elements.

For soil-dwelling bacteria that usually live in a carbon-rich and nitrogen-poor environment, the ability to utilize chitin - the second most abundant polysaccharide on earth - is a decisive evolving advantage as it is a source for both elements. Streptomycetes are high-GC Gram-positive soil bacteria that are equipped with a broad arsenal of chitinase-degrading genes. These genes are induced when the streptomycetes sense the presence of chitooligosaccharides. Their expression is repressed as soon as more readily assimilated carbon sources become available. This includes for example glucose or N-acetylglucosamine, the monomer subunit of chitin. Historically, the first cis-acting elements involved in carbon regulation in streptomycetes were found more than a decade ago upstream of chitinase genes, but the transcriptional regulator had so far remained undiscovered. In this work, we show that these cis-acting elements consist of inverted repeats with multiple occurrences and are bound by the HutC/GntR type regulator DasR. We have therefore designated these sites as DasR-responsive elements (dre). DasR, which is also the repressor of the genes for the N-acetylglucosamine-specific phosphotransferase transport system, should therefore play a critical role in sensing the balance between the monomeric and polymeric forms of N-acetylglucosamine.

PREDetector: a new tool to identify regulatory elements in bacterial genomes.

In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory networks in bacteria. The main difficulty once a regulon prediction is available is to estimate its reliability prior to start expensive experimental validations and therefore trying to find a way how to identify true positive hits from an endless list of potential target genes of a regulatory protein. Here we introduce PREDetector (Prokaryotic Regulatory Elements Detector), a tool developed for predicting regulons of DNA-binding proteins in bacterial genomes that, beside the automatic prediction, scoring and positioning of potential binding sites and their respective target genes in annotated bacterial genomes, it also provides an easy way to estimate the thresholds where to find reliable possible new target genes. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php.