Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis

Abstract

Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37 degrees C. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is unique to Y. pestis. F1 is a surface polymer composed of a protein subunit, Caf1, with a molecular mass of 15.5 kDa. The secretion and assembly of F1 require the caf1M and caf1A genes, which are homologous to the chaperone and usher protein families required for biogenesis of pili. F1 has been implicated to be involved in the ability of Y. pestis to prevent uptake by macrophages. In this study we addressed the role of F1 antigen in inhibition of phagocytosis by the macrophage-like cell line J774. The Y. pestis strain EV76 was found to be highly resistant to uptake by J774 cells. An in-frame deletion of the caf1M gene of the Y. pestis strain EV76 was constructed and found to be unable to express F1 polymer on the bacterial surface. This strain had a somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, which is cured for the virulence plasmid common to the pathogenic Yersinia species, was, as expected, much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid and caf1M was even further hampered in the ability to prevent uptake and, in this case, essentially all bacteria (95%) were phagocytosed. Thus, F1 and the virulence plasmid-encoded type III system act in concert to make Y. pestis highly resistant to uptake by phagocytes. In contrast to the type III effector proteins YopE and YopH, F1 did not have any influence on the general phagocytic ability of J774 cells. Expression of F1 also reduced the number of bacteria that interacted with the macrophages. This suggests that F1 prevents uptake by interfering at the level of receptor interaction in the phagocytosis process.

FIG. 1.

Immunoelectron microscopy of Y. pestis strains with F1 antibodies. The indicated strains of Y. pestis were grown at 37°C and were treated as described in Materials and Methods.

FIG. 2.

Phagocytosis of different Y. pestis strains and Y. pseudotuberculosis by J774 cells. J774 cells were infected with different Y. pestis strains at a bacterium/cell ratio of 20:1 for 30 min at 37°C as described in Materials and Methods. All strains (except those labeled “23°C”) were grown 5 h at 37°C prior to the phagocytosis experiment. The diagram shows the average fraction (± the standard deviation from three experiments) of the bacteria that remained extracellular in the experiment.

FIG. 3.

Phagocytosis of IgG-opsonized Y. pestis strains by J774 cells. The experiment was performed as for Fig. 2. In this experiment, phagocytosis in the presence of opsonizing antibodies directed against whole bacteria (Y. pseudotuberculosis) was also included. Values for opsonized bacteria are indicated by black bars, and values for nonopsonized bacteria are indicated by shaded bars. The diagram shows the average fraction (± the standard deviation from three experiments) of the bacteria that remained extracellular in the experiment. For details, see Materials and Methods.

FIG. 4.

Effect of Y. pestis on uptake of IgG-opsonized yeast particles by J774 cells. The macrophages were first preinfected with the indicated Y. pestis strains for 30 min at 37°C and then analyzed for the ability to ingest yeast particles added at a ratio of 15:1 (yeast particles/cell). The cells were allowed to phagocytose for 30 min, and the number of the particles ingested was then determined as described in Materials and Methods. The results shown represent the means ± the standard error of three separate experiments.

FIG. 5.

Association of different Y. pestis with J774 cells. The cells were infected with bacteria (pregrown at 37°C for 5 h) without prior centrifugation to promote contact for 1 h at 37°C at a bacterium/cell ratio of 30:1. The number of bacteria associated with J774 cells was determined by fluorescent staining as described in Materials and Methods. The data given represent means ± the standard errors of the means of three separate experiments and are expressed as the percentage of total infected bacteria that were associated with J774 cells.