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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., Competing Interests: The authors declare that no competing interests exist.

Plague is transmitted by fleas or contaminated aerosols. To successfully produce disease, the causal agent (Yersinia pestis) must rapidly sense and respond to rapid variations in its environment. Here, we investigated the role of 2-component regulatory systems (2CSs) in plague because the latter are known to be key players in bacterial adaptation to environmental change. Along with the previously studied PhoP-PhoQ system, OmpR-EnvZ was the only one of Y. pestis' 23 other 2CSs required for production of bubonic, septicemic, and pneumonic plague. In vitro, OmpR-EnvZ was needed to counter serum complement and leukocytes but was not required for the secretion of antiphagocyte exotoxins. In vivo, Y. pestis lacking OmpR-EnvZ did not induce an early immune response in the skin and was fully virulent in neutropenic mice. We conclude that, throughout the course of Y. pestis infection, OmpR-EnvZ is required to counter toxic effectors secreted by polymorphonuclear leukocytes in the tissues., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

Potential benefits of combination antibiotic therapy for the treatment of plague have never been evaluated. We compared the efficacy of a ciprofloxacin (CIN) and gentamicin (GEN) combination therapy with that of each antibiotic administered alone (i) against Yersinia pestis in vitro and (ii) in a mouse model of bubonic plague in which animals were intravenously injected with antibiotics for five days, starting at two different times after infection (44 h and 56 h). In vitro, the CIN+GEN combination was synergistic at 0.5x the individual drugs' MICs and indifferent at 1x- or 2x MIC. In vivo, the survival rate for mice treated with CIN+GEN was similar to that observed with CIN alone and slightly higher than that observed for GEN alone 100, 100 and 85%, respectively when treatment was started 44 h post challenge. 100% of survivors were recorded in the CIN+GEN group vs 86 and 83% in the CIN and GEN groups, respectively when treatment was delayed to 56 h post-challenge. However, these differences were not statistically significant. Five days after the end of treatment, Y. pestis were observed in lymph nodes draining the inoculation site (but not in the spleen) in surviving mice in each of the three groups. The median lymph node log(10) CFU recovered from persistently infected lymph nodes was significantly higher with GEN than with CIN (5.8 vs. 3.2, p = 0.04) or CIN+GEN (5.8 vs. 2.8, p = 0.01). Taken as the whole, our data show that CIN+GEN combination is as effective as CIN alone but, regimens containing CIN are more effective to eradicate Y. pestis from the draining lymph node than the recommended GEN monotherapy. Moreover, draining lymph nodes may serve as a reservoir for the continued release of Y. pestis into the blood - even after five days of intravenous antibiotic treatment.