Survival of Salmonella enterica serovar Typhimurium within late endosomal-lysosomal compartments of B lymphocytes is associated with the inability to use the vacuolar alternative major histocompatibility complex class I antigen-processing pathway

Abstract

Gamma interferon (IFN-gamma)-activated macrophages use an alternative processing mechanism to present Salmonella antigens to CD8(+) T lymphocytes. This pathway involves processing of antigen in a vacuolar compartment followed by secretion and loading of antigenic peptides to major histocompatibility complex class I (MHC-I) molecules on macrophage cell surface and bystander cells. In this study, we have shown that B lymphocytes are not able to process Salmonella antigens using this alternative pathway. This is due to differences in Salmonella enterica serovar Typhimurium-containing vacuoles (SCV) when comparing late endosomal-lysosomal processing compartments in B lymphocytes to those in macrophages. The IFN-gamma-activated IC21 macrophage cell line and A-20 B-cell line were infected with live or dead Salmonella enterica serovar Typhimurium. The SCV in B cells were in a late endosomal-lysosomal compartment, whereas SCV in macrophages were remodeled to a non-characteristic late endosomal-lysosomal compartment over time. Despite the difference in SCV within macrophages and B lymphocytes, S. enterica serovar Typhimurium survives more efficiently within the IFN-gamma-activated B cells than in activated macrophage cell lines. Similar results were found during in vivo acute infection. We determined that a lack of remodeling of late endosomal-lysosomal compartments by live Salmonella infection in B lymphocytes is associated with the inability to use the alternative MHC-I antigen-processing pathway, providing a survival advantage to the bacterium. Our data also suggest that the B lymphocyte late endosome-lysosome environment allows the expression of Salmonella virulence mechanisms favoring B lymphocytes in addition to macrophages and dendritic cells as a reservoir during in vivo infection.

FIG. 1.

Secreted peptides binding MHC-I through the alternative vacuolar pathway are present in IFN-γ-activated macrophages but not in activated B lymphocytes infected with Salmonella enterica serovar Typhimurium. IC21 macrophage monolayers or A-20 B lymphocytes in suspension were IFN-γ activated, infected with live (wt) or heat-killed (HK) Salmonella enterica serovar Typhimurium (MOI, 1:20), and cocultured with RMA-S cells. K^b molecules of RMA-S cells were detected by flow cytometry analysis using Y3 MAb. Macrophages and B lymphocytes were gated by Mac-1 and IgG surface antibodies, respectively. Cocultures of RMA-S cells in the presence of uninfected (Non Inf) macrophages or A-20 cells were run in parallel. Means and standard errors of the results from three different experiments are presented in panel A. The mean fluorescence channel is expressed on the y axis. The baseline of K^b expression fluorescence channel media in RMA-S is represented by the dotted horizontal line. Kinetics of bacterial survival within IFN-γ-activated IC21 and A-20 cells using a gentamicin protection assay were also determined in parallel experiments (B). Total numbers of CFU recovered per 5 × 10⁵ cells infected are presented on the y axis. Black columns represent IC21 macrophages, and white columns are B lymphocytes.

FIG. 2.

Late endosomal-lysosomal compartments of B lymphocytes lack Salmonella enterica serovar Typhimurium degradation and favor bacterial survival. After 1, 3, and 18 h postinfection with radiolabeled bacteria, intracellular SCV of activated macrophages and B lymphocytes were obtained by Percoll gradient separation. Salmonella-derived radioactivity within vacuoles was detected by beta scintillation analysis of the fractions. The distribution of late endosome-lysosome across the gradient was detected by β-hexosaminidase enzymatic activity. On the left y axis, we present the percentage of total radioactivity, the percentage of total β-hexosaminidase activity is displayed on the right y axis, and the x axis corresponds to the number of gradient density fractions. One representative experiment out of three is presented for β-hexosaminidase quantification and radioactive detection of SCV.

FIG. 3.

Confocal microscopy analysis of intracellular trafficking of Salmonella-containing vacuoles in macrophages and B lymphocytes. Monolayers of preactivated IC21 or bone marrow-derived macrophages from BALB/c mice or A-20 cells were infected with Salmonella enterica serovar Typhimurium (MOI, 1:10) for a period of 15 min. Selective images of colocalization with Lamp1 markers and bacteria within macrophages and B lymphocytes at 30 min and 24 h postinfection are shown.

FIG. 4.

Salmonella CFU recovered from intracellular vesicles from IFN-γ-activated macrophages and B lymphocytes. Aliquots of the Percoll gradient fractions were cultured in LB agar plates to detect bacterial survival. On the left y axis, we present the percentage of total β-hexosaminidase activity, CFU numbers are displayed on the right y axis, and the x axis corresponds to the number of density gradient fractions. Panels A and C show gradients obtained from B lymphocytes. Panels B and D show data obtained from macrophages. One representative experiment out of three is presented.

FIG. 5.

S. enterica serovar Typhimurium organisms survive in B lymphocytes during primary in vivo infection. BALB/c mice were infected orogastrically (OG) with 1,000 CFU or intraperitoneally (IP) with 50 CFU. On days (d) 3 and 5 postinfection, splenocytes were harvested and macrophages and B cells were sorted based on the expression of Mac-1 (macrophages) or B220 (B lymphocytes). In panel A, we exhibit a representative dot blot analysis of the sorted B lymphocytes using a CD19 marker. In panel B, we show Salmonela CFU obtained from splenic B cells and macrophages. The white bars correspond to CFU obtained from OG infected mice, and black bars correspond to IP infected mice. Data are presented as means and standard deviations of the results from three independent experiments.