Biovar-related differences apparent in the flea foregut colonization phenotype of distinct Yersinia pestis strains do not impact transmission efficiency

Abstract

Background: Yersinia pestis is the flea-transmitted etiological agent of bubonic plague. Sylvatic plague consists of complex tripartite interactions between diverse flea and wild rodent species, and pathogen strains. Transmission by flea bite occurs primarily by the Y. pestis biofilm-mediated foregut blockage and regurgitation mechanism, which has been largely detailed by studies in the model interaction between Y. pestis KIM6+ and Xenopsylla cheopis. Here, we test if pathogen-specific traits influence this interaction by determining the dynamics of foregut blockage development in X. cheopis fleas among extant avirulent pCD1-Y. pestis strains, KIM6+ and CO92, belonging to distinct biovars, and a non-circulating mutant CO92 strain (CO92gly), restored for glycerol fermentation; a key biochemical difference between the two biovars. Methods: Separate flea cohorts infected with distinct strains were evaluated for (i) blockage development, bacterial burdens and flea foregut blockage pathology, and (ii) for the number of bacteria transmitted by regurgitation during membrane feeding. Strain burdens per flea was determined for fleas co-infected with CO92 and KIM6+ strains at a ratio of 1:1. Results: Strains KIM6+ and CO92 developed foregut blockage at similar rates and peak temporal incidences, but the CO92gly strain showed significantly greater blockage rates that peak earlier post-infection. The KIM6+ strain, however, exhibited a distinctive foregut pathology wherein bacterial colonization extended the length of the esophagus up to the feeding mouthparts in ~65% of blocked fleas; in contrast to 32% and 26%, respectively, in fleas blocked with CO92 and CO92gly. The proximity of KIM6+ to the flea mouthparts in blocked fleas did not result in higher regurgitative transmission efficiencies as all strains transmitted variable numbers of Y. pestis, albeit slightly lower for CO92gly. During competitive co-infection, strains KIM6+ and CO92 were equally fit maintaining equivalent infection proportions in fleas over time. Conclusions: We demonstrate that disparate foregut blockage pathologies exhibited by distinct extant Y. pestis strain genotypes do not influence transmission efficiency from X. cheopis fleas. In fact, distinct extant Y. pestis genotypes maintain equivalently effective blockage and transmission efficiencies which is likely advantageous to maintaining continued successful plague spread and establishment of new plague foci.

CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

“…Thus, an ∼50% reduction in blockage rate, as seen for the csrA mutant, is synonymous with a compromised ability to maintain natural plague transmission cycles. Coincidently, an hmsT mutant exhibits a similar ∼50% decrease in blockage rate ( 20 , 21 ), in agreement with the notion that csrA mutant blockage rates are due to the absence of CsrA-dependent enhancement of hmsT mRNA translation.…”

“…Cohorts of Xenopsylla cheopis fleas were artificially infected with Y. pestis strains, and infected flea maintenance, blockage, and CFU enumeration were performed as previously described ( 21 , 78 ). To account for growth defects in the Δ csrA strain, strains were grown overnight at room temperature and then moved to 37°C at 5 h prior to infection.…”

“…Conversely, HmsT is the predominant DGC for in vitro biofilms. An hmsT mutant produces little to no biofilm in vitro and exhibits intermediate flea blockage rates ( 17 , 20 , 21 ). HmsT protein abundance is regulated at the transcriptional and posttranscriptional levels, while HmsD protein abundance is regulated posttranslationally.…”

CsrA-Mediated Translational Activation of the hmsE mRNA Enhances HmsD-Dependent C-di-GMP-Enabled Biofilm Production in Yersinia pestis

“…The present work demonstrates that CsrA also binds to the 5′ leader region of hmsE mRNA to activate its translation, which in turn leads to greater HmsD-dependent biofilm production. Because HmsD is the predominant DGC in the flea, CsrA-dependent regulation of c-di-GMP levels through HmsD may serve as an important fine-tuning mechanism for the development of a transmissible Y. pestis infection from its flea vector and spread of plague by fleabite ( 9 , 28 ).…”

A Role for Early-Phase Transmission in the Enzootic Maintenance of Plague

“…When X . cheopis feed on highly bacteremic blood, as used for the early-phase experiments, however, several studies have shown that the blockage rate is 35 to 40% or higher [ 18 , 23 , 24 , 28 , 29 , 55 – 58 ].…”