Identification and characterization of a novel bacterial virulence factor that shares homology with mammalian Toll/interleukin-1 receptor family proteins

Abstract

Many important bacterial virulence factors act as mimics of mammalian proteins to subvert normal host cell processes. To identify bacterial protein mimics of components of the innate immune signaling pathway, we searched the bacterial genome database for proteins with homology to the Toll/interleukin-1 receptor (TIR) domain of the mammalian Toll-like receptors (TLRs) and their adaptor proteins. A previously uncharacterized gene, which we have named tlpA (for TIR-like protein A), was identified in the Salmonella enterica serovar Enteritidis genome that is predicted to encode a protein resembling mammalian TIR domains, We show that overexpression of TlpA in mammalian cells suppresses the ability of mammalian TIR-containing proteins TLR4, IL-1 receptor, and MyD88 to induce the transactivation and DNA-binding activities of NF-kappaB, a downstream target of the TIR signaling pathway. In addition, TlpA mimics the previously characterized Salmonella virulence factor SipB in its ability to induce activation of caspase-1 in a mammalian cell transfection model. Disruption of the chromosomal tlpA gene rendered a virulent serovar Enteritidis strain defective in intracellular survival and IL-1beta secretion in a cell culture infection model using human THP1 macrophages. Bacteria with disrupted tlpA also displayed reduced lethality in mice, further confirming an important role for this factor in pathogenesis. Taken together, our findings demonstrate that the bacterial TIR-like protein TlpA is a novel prokaryotic modulator of NF-kappaB activity and IL-1beta secretion that contributes to serovar Enteritidis virulence.

FIG. 1.

Alignment and domain organization of the predicted protein sequence of serovar Enteritidis TlpA protein with TIR domains. (Top) Alignment of the TIR domains of serovar Enteritidis TlpA with the human TIR-containing proteins MyD88, TLR6, and TLR4, and plant TIR domain from a disease resistance protein shows strong sequence similarity in the highly conserved box 1 and box 2 sequences that are characteristic of all known TIR domains. (Bottom) The serovar Enteritidis TlpA protein has a putative TIR domain at the C terminus of the protein and a coil-coiled domain at the N terminus (19). The 236-amino-acid TlpA protein is similar in size to the TIR-containing cytoplasmic adapter proteins MyD88 and TIRAP but does not contain N-terminal death domain.

FIG. 2.

TlpA can suppress NF-κB activation induced by mammalian TIR-containing proteins. HEK293N cells were transfected with a 5X NF-κB binding site luciferase reporter plasmid for measurement of NF-κB activation. Cells were also transfected with the various amounts of pGFPTlpA plasmid (0, 50, 100, or 250 ng). To stimulate NF-κB activation, cells were transfected with plasmids encoding a constitutively active TLR4 construct (CD4:TLR4) or the MyD88 adaptor protein, or cells were stimulated by incubation of transfected cells with the IL-1β (50 ng/ml) or TNF-α (20 ng/ml) for 6 h. At 36 h posttransfection, cell lysates were prepared and used for measurement of luciferase activity (mean ± the standard deviation; n = 3) (a) or normalized for protein content and analyzed by immunoblotting (b).

FIG. 3.

TlpA inhibits TIR-induction of NF-κB DNA-binding activity. (a) HEK293N cells were transfected with equal amounts of either pEGFP vector alone or pGFPTlpA plasmid. Cells were left untreated (C), or NF-κB activation was stimulated either by incubation with TNF-α (20 ng/ml) or IL-1β (50 ng/ml). At 36 h after transfection, nuclear extracts were prepared and used for EMSAs, along with a ³²P-labeled oligonucleotide containing the DNA-binding site for NF-κB. Protein-DNA complexes are shown. The specificity of the band corresponding to NF-κB (arrowhead) was determined with a 100-fold molar excess of unlabeled consensus NF-κB oligonucleotide or mutant NF-κB oligonucleotide as competitors. Binding of STAT-1 to its an oligonucleotide containing its DNA-binding site served as a negative control. (b) Nuclear and cytoplasmic extracts from stimulated cells were also analyzed by immunoblotting with specific antibodies recognizing green fluorescent protein (GFP) fusion proteins (cytoplasmic) or the NF-κB subunits p65 and p50 (nuclear). Levels of actin and transcription factor TFII-I are shown to confirm equal protein loading of cytoplasmic and nuclear extracts, respectively.

FIG. 4.

TlpA contributes to virulence in both cell culture and murine infection models. (a) Serovar enteritidis LK5, tlpA::kan, and tlpA::kan strains carrying the pHATlpA expression plasmid were used to infect activated THP-1 macrophages at a multiplicity of infection of 10. At 2, 4, 8, and 12 h after bacterial addition, the numbers of intracellular bacterial CFU of LK5, tlpA::kan, and tlpA::kan and pHATlpA strains were determined by lysis of the mammalian cells and plating of the lysates on LB, LB-kanamycin, and LB-kanamycin+ampicillin, respectively. (b) Serovar Enteritidis LK5 and tlpA::kan strains were used to orally infect BALB/c mice at the same dose. Control animals were inoculated with saline alone. Animal survival was monitored for 10 days postinoculation. (c) BALB/c mice were inoculated with serovar Enteritidis strains. At 5 days postinfection, when ca. 40% of the mice that had been inoculated with the wild-type LK5 strain had succumbed to infection, representative mice were sacrificed, and their spleens were removed under sterile conditions. Spleens were homogenized, and the lysates were diluted and plated on LB or LB-kanamycin plates. The data represent mean ± the SE (n = 5).

FIG. 5.

TlpA regulates IL-1β secretion. (a) ΗΕΚ293Τ cells expressing recombinant murine caspase-1 and murine pro-IL-1β were transfected with either empty vector or vectors expressing TlpA, SipB, or MyD88 TIR domain (MyDMC). To inhibit caspase-1, cells were cotransfected with a CrmA expression construct, whereas to inhibit all caspases cells were incubated with the pan-caspase inhibitor zVAD-fmk (50 μg/ml). The presence of mature, cleaved IL-1β in the cell culture supernatant was used as a measure of caspase-1 activation and was quantified by ELISA (mean ± the standard deviation; n = 3). (b) Activated THP-1 macrophages were infected with serovar Enteritidis strains LK5, ΔtlpA::kan, and ΔtlpA::kan+pHATlpA. At 2, 4, 8, and 12 h after addition of bacteria, IL-1β secretion into the supernatant was measured by quantitative ELISA (mean ± the standard deviation; n = 3).