Differential bacterial survival, replication, and apoptosis-inducing ability of Salmonella serovars within human and murine macrophages

Abstract

Salmonella serovars are associated with human diseases that range from mild gastroenteritis to host-disseminated enteric fever. Human infections by Salmonella enterica serovar Typhi can lead to typhoid fever, but this serovar does not typically cause disease in mice or other animals. In contrast, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, which are usually linked to localized gastroenteritis in humans and some animal species, elicit a systemic infection in mice. To better understand these observations, multiple strains of each of several chosen serovars of Salmonella were tested for the ability in the nonopsonized state to enter, survive, and replicate within human macrophage cells (U937 and elutriated primary cells) compared with murine macrophage cells (J774A.1 and primary peritoneal cells); in addition, death of the infected macrophages was monitored. The serovar Typhimurium strains all demonstrated enhanced survival within J774A.1 cells and murine peritoneal macrophages, compared with the significant, almost 100-fold declines in viable counts noted for serovar Typhi strains. Viable counts for serovar Enteritidis either matched the level of serovar Typhi (J774A. 1 macrophages) or were comparable to counts for serovar Typhimurium (murine peritoneal macrophages). Apoptosis was significantly higher in J774A.1 cells infected with serovar Typhimurium strain LT2 compared to serovar Typhi strain Ty2. On the other hand, serovar Typhi survived at a level up to 100-fold higher in elutriated human macrophages and 2- to 3-fold higher in U937 cells compared to the serovar Typhimurium and Enteritidis strains tested. Despite the differential multiplication of serovar Typhi during infection of U937 cells, serovar Typhi caused significantly less apoptosis than infections with serovar Typhimurium. These observations indicate variability in intramacrophage survival and host cytotoxicity among the various serovars and are the first to show differences in the apoptotic response of distinct Salmonella serovars residing in human macrophage cells. These studies suggest that nonopsonized serovar Typhimurium enters, multiplies within, and causes considerable, acute death of macrophages, leading to a highly virulent infection in mice (resulting in death within 14 days). In striking contrast, nonopsonized serovar Typhi survives silently and chronically within human macrophages, causing little cell death, which allows for intrahost dissemination and typhoid fever (low host mortality). The type of disease associated with any particular serovar of Salmonella is linked to the ability of that serovar both to persist within and to elicit damage in a specific host's macrophage cells.

FIG. 1

Survival of Salmonella strains in murine J774A.1 tissue-cultured macrophages. Bacterial CFU per 10⁵ macrophage cells (y axis) and time after addition of gentamicin (x axis) are indicated. All values are the means ± standard deviations of at least three experiments done in triplicate.

FIG. 2

Survival of Salmonella strains in human U937 tissue-cultured macrophages. Bacterial CFU per 10⁵ macrophage cells (y axis) and time after addition of gentamicin (x axis) are indicated. All values are the means ± standard deviations of at least three experiments done in triplicate.

FIG. 3

Survival of Salmonella strains in murine peritoneal macrophages. Bacterial CFU per 10⁵ macrophage cells (y axis) and time after addition of gentamicin (x axis) are indicated. All values are the means ± standard deviations of at least three experiments done in triplicate.

FIG. 4

Survival of Salmonella strains in elutriated human macrophages. Bacterial CFU per 10⁵ macrophage cells (y axis) and time after addition of gentamicin (x axis) are indicated. All values are the means ± standard deviations of at least three experiments done in triplicate.

FIG. 5

Fluorescent TUNEL reaction examination of macrophage cells undergoing programmed cell death following a 24-h infection with Salmonella. (A to C) Murine J774A.1 macrophage cells; (D to F) human U937 macrophage cells; (A and D) noninfected cells; (B and E) serovar Typhi strain Ty2-infected macrophages; (C and F) serovar Typhimurium LT2-infected macrophage cells. Weak, diffuse backround fluorescence staining of all cells was enhanced with Adobe Photoshop to allow differentiation between densely stained areas in apoptotic cells and total cells in the field.