Protein aggregation in Ehrlichia chaffeensis during infection of mammalian cells

Abstract

Ehrlichia chaffeensis is an obligatory intracellular pathogen transmitted through infected ticks to humans and other vertebrates. We investigated the extent of protein aggregation in E. chaffeensis during infection of canine macrophage cell line, DH82. We discovered that the size of the aggregated fraction of E. chaffeensis proteins increased during the first 48 h post infection. We also incubated the infected cells with guanidinium chloride (GuHCl), a known inhibitor of the protein-disaggregating molecular chaperone ClpB. Up to 0.5 mM GuHCl had no impact on the host cells, whereas the viability of the pathogen was reduced by ∼60% in the presence of the inhibitor. Furthermore, we found that the size of the aggregated protein fraction in E. chaffeensis increased significantly in cultures supplemented with 0.5 mM GuHCl, which also resulted in the preferential accumulation of ClpB with the aggregated proteins. Altogether, our results suggest that an exposure of E. chaffeensis to the stressful environment of a host cell results in an increased aggregation of the pathogen's proteins, which is exacerbated upon inhibition of ClpB. Our studies establish a link between protein quality control and pathogen survival during infection of a host.

Keywords: ClpB; Ehrlichia chaffeensis; host–pathogen interaction; molecular chaperone; protein aggregation; protein folding.

Figure 1.

Mammalian cell environment induces aggregation of E. chaffeensis proteins. SDS-PAGE analysis followed by the Coomassie blue staining of the aggregated protein fraction in E. chaffeensis (A) and the total bacterial cell extracts (B) obtained at the indicated time post infection of macrophages. (C) The amount of the aggregated proteins in E. chaffeensis expressed as the percentage of the total protein concentration at the indicated time points post infection. The mean values and the standard deviations from three experiments are shown.

Figure 2.

Guanidinium chloride (GuHCl) inhibits the ClpB activity and E. chaffeensis growth, and increases protein aggregation in E. chaffeensis cells. (A) The ATPase activity of the purified E. chaffeensis ClpB was measured in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from two experiments are shown. (B) The growth of E. chaffeensis (right axis) in the infected DH82 cells and the host cell viability (left axis) were determined in cultures grown in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from three experiments are shown. (C) The amount of the aggregated proteins in E. chaffeensis expressed as the percentage of the total protein concentration after 24 and 48 h post infection of macrophages in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from three experiments are shown. (D) The amount of the aggregated proteins in E. chaffeensis expressed as the percentage of the total protein concentration as the function of time post infection of macrophages in the absence and presence of 0.5 mM GuHCl. The mean values and the standard deviations from three experiments are shown.

Figure 3.

ClpB is targeted to protein aggregates in E. chaffeensis upon the treatment with GuHCl. Immunodetection of ClpB (∼95 kDa) in the soluble (S) and aggregated (A) fractions of E. chaffeensis cells isolated from the infected macrophages at the indicated times post infection. The infected macrophages were cultured in the absence or presence of 0.5 mM GuHCl. The samples loaded onto the gel contained equal protein amounts, as determined with the BCA method.