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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 bacteriaentrapping 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 fleablocking 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. [ABSTRACT FROM AUTHOR]

The adaptation of Yersinia pestis, the flea-borne plague agent, to fluctuating environmental conditions is essential for the successful colonization of the flea vector. A previous comparative transcriptomic analysis showed that the Cpx pathway of Y. pestis is up-regulated in infected fleas. The CpxAR two-component system is a component of the envelope stress response and is critical for maintaining the integrity of the cell. Here, a phenotypic screening revealed a survival defect of the cpxAR mutant to oxidative stress and copper. The measured copper concentration in the digestive tract contents of fed fleas increased fourfold during the digestive process. By direct analysis of phosphorylation of CpxR by a Phos-Tag gel approach, we demonstrated that biologically relevant concentrations of copper triggered the system. Then, a competitive challenge highlighted the role of the CpxAR system in bacterial fitness during flea infection. Lastly, an in vitro sequential exposure to copper and then H2O2 to mimic the flea suggests a model in which, within the insect digestive tract, the CpxAR system would be triggered by copper, establishing an oxidative stress response. [ABSTRACT FROM AUTHOR]