Induction of a phagocytosis-resistant phenotype in the flea 
Soon after transmission, Y. pestis would be expected to encounter rapidly-responding phagocytic cells in the dermis.
To assess the overall effect of the flea-specific phenotype on this encounter, we compared the interaction of Y. pestis recovered from infected fleas and from in vitro cultures with murine bone marrow macrophages.
Bacteria from fleas showed significantly lower levels of phagocytosis (Fig. 4A).
We have previously reported analogous findings using human polymorphonuclear leukocytes (PMNs) [7].
The yit and yip genes in a Y. pestis locus (y0181-0191) that encode predicted insecticidal-like toxins of the toxin complex (Tc) family and three linked phage-related genes were upregulated 4- to 50-fold in the flea midgut (Tables 1 and S1).
We previously reported that the genes for these Tc-like proteins are highly expressed in fleas, but that their products are nontoxic to fleas [49].
yitR, a LysR-type regulator that activates the Tc-like yit genes [50], was upregulated >10-fold in the flea, but its expression was not detected in the rat bubo (Table 1).
The specific induction in the flea of yitR and genes in the adjacent Tc-like yit and yip loci suggests that they are involved in adaptation to and colonization of the flea.
However, deletion of yitR or yitA-yipB (y0183-y0191) does not affect the ability of Y. pestis KIM6+ to infect or block fleas (data not shown).
These observations, and the fact that the Yersinia Tc proteins have toxicity to certain eukaryotic cell lines in vitro [50],[51], prompted us to investigate a possible post-transmission antiphagocytic role for these proteins in the mammalian host.
To determine if the insecticidal-like toxins were involved in resistance to phagocytosis, we repeated the macrophage experiments with a Y. pestis DeltayitR mutant, which as expected showed greatly reduced expression of the yit and yip genes in vitro and in the flea (Fig. 4B).
Loss of yitR significantly reduced the increased resistance to phagocytosis of Y. pestis isolated from infected fleas (Fig. 4C).
Since the yit and yip genes are not required for Y. pestis to produce a transmissible infection in fleas, it was possible to compare the virulence of wild-type and DeltayitR Y. pestis following transmission by fleabite.
The incidence rate and time to disease onset were identical for both Y. pestis strains, demonstrating that expression of yit and yip is not essential for flea-borne transmission or disease (data not shown).
On average, the mice challenged with Y. pestis DeltayitR-infected fleas, both those that developed disease and those that did not, received a higher cumulative number of bites from blocked fleas than the mice challenged with Y. pestis-infected fleas, but this difference was not statistically significant (Fig. 5).
However, it was not possible to detect any relatively minor difference in LD50 because the number of bacteria transmitted by a blocked flea varies widely [1],[52].
Even a small decrease in LD50 provided by the Yit-Yip proteins would be significant at the ecological level in the maintenance of plague transmission cycles, because the transmission efficiency of blocked fleas is very low- often only a few or no bacterial cells are transmitted in an individual fleabite [52].
Because phoP is required by Y. pestis to produce a transmissible infection in fleas (unpublished data), it was not possible to similarly assess the effect on disease transmission of phoP induction in the flea.
