A Widefield Light Microscopy-Based Approach Provides Further Insights into the Colonization of the Flea Proventriculus by Yersinia pestis.

Yersinia pestis (the agent of flea-borne plague) must obstruct the flea's proventriculus to maintain transmission to a mammalian host. To this end, Y. pestis must consolidate a mass that entrapped Y. pestis within the proventriculus very early after its ingestion. We developed a semiautomated fluorescent image analysis method and used it to monitor and compare colonization of the flea proventriculus by a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Our data suggested that flea blockage results primarily from the replication of Y. pestis trapped in the anterior half of the proventriculus. However, consolidation of the bacteria-entrapping mass and colonization of the entire proventricular lumen increased the likelihood of flea blockage. The data also showed that consolidation of the bacterial mass is not a prerequisite for colonization of the proventriculus but allowed Y. pestis to maintain itself in a large flea population for an extended period of time. Taken as the whole, the data suggest that a strategy targeting bacterial mass consolidation could significantly reduce the likelihood of Y. pestis being transmitted by fleas (due to gut blockage), but also the possibility of using fleas as a long-term reservoir. IMPORTANCE Yersinia pestis (the causative agent of plague) is one of the deadliest bacterial pathogens. It circulates primarily among rodent populations and their fleas. Better knowledge of the mechanisms leading to the flea-borne transmission of Y. pestis is likely to generate strategies for controlling or even eradicating this bacillus. It is known that Y. pestis obstructs the flea's foregut so that the insect starves, frantically bites its mammalian host, and regurgitates Y. pestis at the bite site. Here, we developed a semiautomated fluorescent image analysis method and used it to document and compare foregut colonization and disease progression in fleas infected with a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Overall, our data provided new insights into Y. pestis' obstruction of the proventriculus for transmission but also the ecology of plague.

Interplay between Yersinia pestis and its flea vector in lipoate metabolism.

To thrive, vector-borne pathogens must survive in the vector's gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway's lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium's lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles' heel that is exploited by pathogens.

A refined model of how Yersinia pestis produces a transmissible infection in its flea vector.

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.

Nutrient depletion may trigger the Yersinia pestis OmpR-EnvZ regulatory system to promote flea-borne plague transmission.

The flea's lumen gut is a poorly documented environment where the agent of flea-borne plague, Yersinia pestis, must replicate to produce a transmissible infection. Here, we report that both the acidic pH and osmolarity of the lumen's contents display simple harmonic oscillations with different periods. Since an acidic pH and osmolarity are two of three known stimuli of the OmpR-EnvZ two-component system in bacteria, we investigated the role and function of this Y. pestis system in fleas. By monitoring the in vivo expression pattern of three OmpR-EnvZ-regulated genes, we concluded that the flea gut environment triggers OmpR-EnvZ. This activation was not, however, correlated with changes in pH and osmolarity but matched the pattern of nutrient depletion (the third known stimulus for OmpR-EnvZ). Lastly, we found that the OmpR-EnvZ and the OmpF porin are needed to produce the biofilm that ultimately obstructs the flea's gut and thus hastens the flea-borne transmission of plague. Taken as a whole, our data suggest that the flea gut is a complex, fluctuating environment in which Y. pestis senses nutrient depletion via OmpR-EnvZ. Once activated, the latter triggers a molecular program (including at least OmpF) that produces the biofilm required for efficient plague transmission.

Polymorphism in the Yersinia LcrV Antigen Enables Immune Escape From the Protection Conferred by an LcrV-Secreting Lactococcus Lactis in a Pseudotuberculosis Mouse Model.

Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

Synergistic Effect of SN-38 in Combination with Cetuximab on Angiogenesis and Cancer Cell Invasion.

BACKGROUND: The combination of irinotecan, a topoisomerase I inhibitor with cetuximab, an antibody against epidermal growth factor receptor, produces synergistic and beneficial effects in patients with irinotecan-refractory colorectal cancer. Our hypothesis was that synergistic effects could be due to anti-angiogenesis and anti-invasion, but not to cytotoxicity. MATERIALS AND METHODS: Cytotoxicity was assessed by viability test and flow cytometry. Anti-angiogenesis, anti-invasion were studied by the endothelial cell capillary-like network formation and transmigration through an extracellular matrix. Protein kinase B (PKB, frequently cited as AKT), and extracellular signal-regulated kinases (ERK) activation was assayed by cell-based enzyme-linked immunosorbent assay (ELISA). RESULTS: Combinations of SN-38 (the active of irinotecan) and cetuximab did not induce any synergistic cytotoxicity confirmed by viability test and cell-cycle analyses. Interestingly, their combination produced synergistic anti-angiogenesis and anti-invasion activities revealed by endothelial cell capillary-like network formation and cell invasion tests. Subsequently, their combination attenuated either expression or phosphorylation of AKT and ERK1/2 using cell-based ELISA. CONCLUSION: SN-38/cetuximab combination has synergistic anti-angiogenesis and anti-invasion activities mediated by down-regulation of phosphatidylinositol-3-kinases/AKT and mitogen-activated protein kinase/ERK pathways.

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis.

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥ 37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.

New insights into how Yersinia pestis adapts to its mammalian host during bubonic plague.

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 adaptation 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.

Cilengitide targets pediatric glioma and neuroblastoma cells through cell detachment and anoikis induction.

The prognosis of children with high-grade glioma or high-risk neuroblastoma remains poor. Cilengitide is a selective antagonist of αvß3 and αvß5 integrins, which are involved in tumor growth and development of metastasis. We have evaluated the effects of cilengitide on pediatric glioma and neuroblastoma cell lines for the first time. Expression levels of αvß3 and αvß5 were determined by flow cytometry in three neuroblastoma and five pediatric glioma cell lines compared with adult U87-MG before and after irradiation. Cell detachment, cytotoxicity, and cell growth under nonadhesive conditions were measured using the MTS assay. Cell death and apoptosis were assessed by annexin-V/propidium iodide staining. The varying αvß3 and αvß5 expression levels were unrelated to tumor grade. Irrespective of the αvß5 expression level, the pediatric cells expressing αvß3 were dose dependently sensitive to cilengitide. UW479 cells expressed only αvß5 integrin and were not sensitive to cilengitide, suggesting that cilengitide's action largely depends on αvß3 inhibition. Cell detachment resulted in a higher cytotoxicity in pediatric glioma compared with U87-MG cells, which seem able to grow despite the significant cilengitide-induced cell detachment. Growth kinetics on polyHEMA showed that only pediatric glioma cells were sensitive to anoikis and so died after cilengitide-induced detachment. Furthermore, irradiation of glioma cells increased αvß3 expression slightly but not cilengitide sensitivity. Cilengitide's action on glioma and neuroblastoma cells appears to be dependent on αvß3 expression and sensitivity to anoikis. Cilengitide is able to target pediatric glioma and neuroblastoma cells in vitro directly and efficiently. Tumor context could validate these promising observations.

Circulating thrombospondin 1 level as a surrogate marker in patients receiving cyclophosphamide-based metronomic chemotherapy.

Several previous reports suggest that thrombospondin (TSP-1) may be a mediator of the antiangiogenic effects of low-dose metronomic cyclophosphamide-based chemotherapy (MC). We conducted a randomized phase II trial evaluating megestrol acetate (n = 44) versus MC (n = 44) in patients having exhausted all standard treatments. We measured the TSP-1 levels at baseline and D15. We did not observe significant differences in TSP-1 at baseline in the two arms (p = 0.07). TSP-1 levels decreased in patients receiving metronomic cyclophosphamide (from 16.6 ± 7.2 µg/ml to 12.8 ± 7.4 µg/ml; p = 0.057). The TSP-1 level was stable in patients receiving megestrol acetate. Nevertheless, the TSP-1 level driven by MC did not correlate to clinical benefit.

Mechanisms underlying resistance to cetuximab in the HNSCC cell line: role of AKT inhibition in bypassing this resistance.

EGFR is frequently overexpressed in head and neck squamous cell cancer (HNSCC). Cetuximab is a monoclonal antibody designed to interact with EGFR, block its activation, reduce the downstream signaling pathways and induce EGFR internalization. This study aims to investigate the role of the EGFR signaling pathway and EGFR internalization in a cetuximab-resistant cell line and to propose a new therapeutic strategy to optimize treatment of HNSCC. The HNSCC cell line, CAL33 was sensitive to gefitinib but resistant to cetuximab. Cetuximab induces an unexpected EGFR phosphorylation in CAL33 cells similarly to EGF but this EGFR activation does not trigger EGFR internalization/degradation, the process currently implicated in the response to cetuximab. Cetuximab inhibits ERK and AKT phosphorylation in cetuximab-sensitive A431 cells, whereas the level of AKT phosphorylation is unmodified in cetuximab-resistant cells. Interestingly, CAL33 cells harbor a PIK3CA mutation. The treatment of CAL33 cells with PI3K inhibitor and cetuximab restores the inhibition of AKT phosphorylation and induces growth inhibition. Our results indicate that EGFR internalization is impaired by cetuximab treatment in CAL33 cells and that the AKT pathway is a central element in cetuximab resistance. The combination of cetuximab with a PI3K inhibitor could be a good therapeutic option in PIK3CA-mutated HNSCC.