Salmonella flagellin induces tumor necrosis factor alpha in a human promonocytic cell line

Abstract

During infection of the gastrointestinal tract, salmonellae induce cytokine production and inflammatory responses which are believed to mediate tissue damage in the host. In a previous study, we reported that salmonellae possess the ability to stimulate tumor necrosis factor alpha (TNF-alpha) accumulation in primary human monocytes, as well as in the human promonocytic cell line U38. In this model system, cytokine upregulation is not due to lipopolysaccharide but is mediated by a released protein. In the present study, TnphoA transposon mutagenesis was used to identify the TNF-alpha-inducing factor. A mutant Salmonella strain which lacks the ability to induce TNF-alpha was isolated from a TnphoA library. Genetic analysis of this mutant demonstrated that the hns gene has been interrupted by transposon insertion. The hns gene product is a DNA-binding protein that regulates the expression of a variety of unrelated genes in salmonellae. One of the known targets of histone-like protein H1 is flhDC, the master operon which is absolutely required for flagellar expression. Analysis of other nonflagellated mutant Salmonella strains revealed a correlation between the ability to induce TNF-alpha and the expression of the phase 1 filament subunit protein FliC. Complementation experiments demonstrated that FliC is sufficient to restore the ability of nonflagellated mutant Salmonella strains to upregulate TNF-alpha, whereas the phase 2 protein FljB appears to complement to a lesser extent. In addition, Salmonella FliC can confer the TNF-alpha-inducing phenotype on Escherichia coli, which otherwise lacks the activity. Furthermore, assembly of FliC into complete flagellar structures may not be required for induction of TNF-alpha.

FIG. 1

Southern blot of FC32 chromosomal DNA. A 5-μg sample of DNA was digested with SalI and separated on a 0.5% agarose gel. The DNA was then transferred to a nylon membrane and probed with a 1,169-bp BglII fragment containing transposon TnphoA labeled with the Genius system as described in Materials and Methods. DNA bands were visualized by using the chemiluminescent substrate Lumi-Phos. The sizes of known fragments are shown on the right.

FIG. 2

Effect of unpolymerized flagellin subunits on TNF-α activation in U38 cells. Serial dilutions of CM from SJW1103 or SJW2149 (a FliD⁻ strain that fails to polymerize FliC into filaments) were tested on U38 cells as described in Materials and Methods. The amount of TNF-α in the control samples (8 ± 2 pg) has been subtracted from the values shown.

FIG. 3

Effect of purified flagella on TNF-α expression in U38 cells and LPS-tolerant PBMC. Flagellar structures were isolated from Salmonella strain CD5 and tested on 3 × 10⁶ U38 cells or PBMC at various concentrations. The curves shown are from two representative experiments. Control TNF-α levels were <1 pg/3 × 10⁶ cells in unstimulated U38 cells and 7 ± 1 pg/3 × 10⁶ cells in unstimulated PBMC. TNF-α levels in LPS-stimulated PBMC were 13 ± 4 pg/3 × 10⁶ cells.