Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of Y. pestis genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of Y. pestis mutants, each lacking one or more of the genes previously identified as being up-regulated in vivo. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, “per-pool” screening method that we have developed. Our data showed that in addition to genes involved in physiological adaption and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (ympt1.66c, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of ympt1.66c reduced Y. pestis' ability to spread to the lymph nodes draining the dermal inoculation site – probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability.

In flea-borne plague, blockage of the flea's foregut by Yersinia pestis hastens transmission to the mammalian host. Based on microscopy observations, we first suggest that flea blockage results from primary infection of the foregut and not from midgut colonization. In this model, flea infection is characterized by the recurrent production of a mass that fills the lumen of the proventriculus and encompasses a large number of Y. pestis. This recurrence phase ends when the proventricular cast is hard enough to block blood ingestion. We further showed that ymt (known to be essential for flea infection) is crucial for cast production, whereas the hmsHFRS operon (known to be essential for the formation of the biofilm that blocks the gut) is needed for cast consolidation. By screening a library of mutants (each lacking a locus previously known to be upregulated in the flea gut) for biofilm formation, we found that rpiA is important for flea blockage but not for colonization of the midgut. This locus may initially be required to resist toxic compounds within the proventricular cast. However, once the bacterium has adapted to the flea, rpiA helps to form the biofilm that consolidates the proventricular cast. Lastly, we used genetic techniques to demonstrate that ribose-5-phosphate isomerase activity (due to the recent gain of a second copy of rpiA (y2892)) accentuated blockage but not midgut colonization. It is noteworthy that rpiA is an ancestral gene, hmsHFRS and rpiA2 were acquired by the recent ancestor of Y. pestis, and ymt was acquired by Y. pestis itself. Our present results (i) highlight the physiopathological and molecular mechanisms leading to flea blockage, (ii) show that the role of a gene like rpiA changes in space and in time during an infection, and (iii) emphasize that evolution is a gradual process punctuated by sudden jumps.

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Ligation of very late antigen (VLA)-4 (alpha 4 beta 1 integrin) with a cross-linked anti-alpha 4 subunit monoclonal antibody (mAb) triggered a biphasic Ca2+ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca2+ response. Tumor necrosis factor-alpha-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca2+ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca2+ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca2+ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (approximately 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca2+ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca2+ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca2+.

Mycolactone, a polyketide molecule produced by Mycobacterium ulcerans, is the etiological agent of Buruli ulcer. This lipid toxin is endowed with pleiotropic effects, presents cytotoxic effects at high doses, and notably plays a pivotal role in host response upon colonization by the bacillus. Most remarkably, mycolactone displays intriguing analgesic capabilities: the toxin suppresses or alleviates the pain of the skin lesions it inflicts. We demonstrated that the analgesic capability of mycolactone was not attributable to nerve damage, but instead resulted from the triggering of a cellular pathway targeting AT2 receptors (angiotensin II type 2 receptors; AT2R), and leading to potassium-dependent hyperpolarization. This demonstration paves the way to new nature-inspired analgesic protocols. In this direction, we assess here the hyperpolarizing properties of mycolactone on nociceptive neurons. We developed a dedicated medium-throughput assay based on membrane potential changes, and visualized by confocal microscopy of bis-oxonol-loaded Dorsal Root Ganglion (DRG) neurons. We demonstrate that mycolactone at non-cytotoxic doses triggers the hyperpolarization of DRG neurons through AT2R, with this action being not affected by known ligands of AT2R. This result points towards novel AT2R-dependent signaling pathways in DRG neurons underlying the analgesic effect of mycolactone, with the perspective for the development of new types of nature-inspired analgesics.

Background: Microarray analyses of stage-specific gene expression of Plasmodium falciparum require purification of RNAs from highly synchronized cultures. To date, no reliable method to control the quality of synchronization of P. falciparum cultures is available. Methods: A double-staining method using hydroethidine and thiazole orange for nucleic acid staining was carried out to compare by flow cytometric analysis the nucleic acid labeling of synchronized P. falciparum in cultures at different time points of the 48-h intraerythrocytic cycle. Results: With this method, we determined the quality of culture synchronization in schizont and ring stages. Nucleic acid analysis, based on thiazole orange fluorescence, clearly showed that low levels of schizonts in ring cultures results in a high contamination of ring nucleic acids by schizonts. Conversely, nucleic acids from trophozoite or

Ivy is required to counter lysozyme during infection, but its role as a virulence factor is species dependent. Our study also shows that a gene that is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.