Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin

Abstract

The causative agent of plague, Yersinia pestis, is regarded as being noninvasive for epithelial cells and lacks the major adhesins and invasins of its enteropathogenic relatives Yersinia enterocolitica and Yersinia pseudotuberculosis. However, there are studies indicating that Y. pestis invades and causes systemic infection from ingestive and aerogenic routes of infection. Accordingly, we developed a gentamicin protection assay and reexamined invasiveness of Y. pestis for HeLa cells. By optimizing this assay, we discovered that Y. pestis is highly invasive. Several factors, including the presence of fetal bovine serum, the configuration of the tissue culture plate, the temperature at which the bacteria are grown, and the presence of the plasminogen activator protease Pla-encoding plasmid pPCP1, were found to influence invasiveness strongly. Suboptimal combinations of these factors may have contributed to negative findings by previous studies attempting to demonstrate invasion by Y. pestis. Invasion of HeLa cells was strongly inhibited by cytochalasin D and modestly inhibited by colchicine, indicating strong and modest respective requirements for microfilaments and microtubules. We found no significant effect of the iron status of yersiniae or of the pigmentation locus on invasion and likewise no significant effect of the Yops regulon. However, an unidentified thermally induced property (possibly the Y. pestis-specific capsular protein Caf1) did inhibit invasiveness significantly, and the plasmid pPCP1, unique to Y. pestis, was essential for highly efficient invasion. pPCP1 encodes an invasion-promoting factor and not just an adhesin, because Y. pestis lacking this plasmid still adhered to HeLa cells. These studies have enlarged our picture of Y. pestis biology and revealed the importance of properties that are unique to Y. pestis.

FIG. 1

Time course for invasion of HeLa cells by Y. pestis. Pla⁺ Pgm⁻ Y. pestis KIM6 was grown at 26°C, washed, and used at an MOI of 10 to infect confluent HeLa cell monolayers in six-well cluster dishes. At various times, replicate infected cultures were analyzed for intracellular and total bacteria by the GM protection technique.

FIG. 2

Typical electron microscopic images of Pla⁺ and Pla⁻ Y. pestis interactions with HeLa cells at the time of harvest in a 60-min invasion assay. Prior to addition of the glutaraldehyde prefixative, the cultures for electron microscopy were handled exactly as in our optimized invasion assay. In parallel, replicate cultures were assayed for invasion by the GM protection assay. For the experiment depicted, the Pla⁺ Y. pestis showed 33% invasion, whereas the Pla⁻ strain showed 3.2% invasion. (Left panel) Pla⁻. Five adjacent frames are combined into one montage. (Right panel) Pla⁺. Five nonadjacent frames are presented. Bars, 5 μm.

FIG. 2

Typical electron microscopic images of Pla⁺ and Pla⁻ Y. pestis interactions with HeLa cells at the time of harvest in a 60-min invasion assay. Prior to addition of the glutaraldehyde prefixative, the cultures for electron microscopy were handled exactly as in our optimized invasion assay. In parallel, replicate cultures were assayed for invasion by the GM protection assay. For the experiment depicted, the Pla⁺ Y. pestis showed 33% invasion, whereas the Pla⁻ strain showed 3.2% invasion. (Left panel) Pla⁻. Five adjacent frames are combined into one montage. (Right panel) Pla⁺. Five nonadjacent frames are presented. Bars, 5 μm.