The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis

Abstract

Yersinia pestis, the agent of plague, has arisen from a less virulent pathogen, Yersinia pseudotuberculosis, by a rapid evolutionary process. Although Y. pestis displays a large number of virulence phenotypes, it is not yet clear which of these phenotypes descended from Y. pseudotuberculosis and which were acquired independently. Y. pestis is known to replicate in macrophages, but there is no consensus in the literature on whether Y. pseudotuberculosis shares this property. We investigated whether the ability to replicate in macrophages is common to Y. pestis and Y. pseudotuberculosis or is a unique phenotype of Y. pestis. We also examined whether a chromosomal type III secretion system (TTSS) found in Y. pestis is present in Y. pseudotuberculosis and whether this system is important for replication of Yersinia in macrophages. A number of Y. pestis and Y. pseudotuberculosis strains of different biovars and serogroups, respectively, were tested for the ability to replicate in primary murine macrophages. Two Y. pestis strains (EV766 and KIM10(+)) and three Y. pseudotuberculosis strains (IP2790c, IP2515c, and IP2666c) were able to replicate in macrophages with similar efficiencies. Only one of six strains tested, the Y. pseudotuberculosis YPIII(p(-)) strain, was defective for intracellular replication. Thus, the ability to replicate in macrophages is conserved in Y. pestis and Y. pseudotuberculosis. Our results also indicate that a homologous TTSS is present on the chromosomes of Y. pestis and Y. pseudotuberculosis and that this secretion system is not required for replication of these bacteria in macrophages.

FIG. 1.

Analysis of Y. pestis and Y. pseudotuberculosis replication in macrophages by microscopy. C57BL/6 macrophages were infected with KIM10⁺/GFP (a to f) or IP2790c/GFP (g to l) at an MOI of 5. The infected cells were incubated for 4 h (a to c and g to i) or 25 h (d to f and j to l) and then fixed, and bacteria were labeled with a rabbit anti-Yersinia antibody (red). One hour before fixation, IPTG was added to the cells to induce GFP expression in viable bacteria (green). The samples were visualized by phase (left panels) or epifluorescence (middle and right panels) microscopy. Representative images were captured using a digital camera. Macrophages containing large numbers of intracellular bacteria displayed an enlarged and vacuolated morphology under phase-contrast microscopy (arrowheads).

FIG. 2.

Kinetics of Y. pestis and Y. pseudotuberculosis replication in macrophages. C57BL/6 macrophages were infected with IP2790c/GFP (♦), KIM10⁺/GFP (▪), or YPIII(p⁻)/GFP (▴). Intracellular bacteria were enumerated at the indicated times postinfection by a CFU assay. The values represent averages ± standard deviations (error bars) of triplicate samples for IP2790c and KIM10⁺ and of duplicate samples for YPIII(p⁻).