Phosphate groups of lipid A are essential for Salmonella enterica serovar Typhimurium virulence and affect innate and adaptive immunity

Abstract

Lipid A is a key component of the outer membrane of Gram-negative bacteria and stimulates proinflammatory responses via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. Its endotoxic activity depends on the number and length of acyl chains and its phosphorylation state. In Salmonella enterica serovar Typhimurium, removal of the secondary laurate or myristate chain in lipid A results in bacterial attenuation and growth defects in vitro. However, the roles of the two lipid A phosphate groups in bacterial virulence and immunogenicity remain unknown. Here, we used an S. Typhimurium msbB pagL pagP lpxR mutant, carrying penta-acylated lipid A, as the parent strain to construct a series of mutants synthesizing 1-dephosphorylated, 4'-dephosphorylated, or nonphosphorylated penta-acylated lipid A. Dephosphorylated mutants exhibited increased sensitivity to deoxycholate and showed increased resistance to polymyxin B. Removal of both phosphate groups severely attenuated the mutants when administered orally to BALB/c mice, but the mutants colonized the lymphatic tissues and were sufficiently immunogenic to protect the host from challenge with wild-type S. Typhimurium. Mice receiving S. Typhimurium with 1-dephosphorylated or nonphosphorylated penta-acylated lipid A exhibited reduced levels of cytokines. Attenuated and dephosphorylated Salmonella vaccines were able to induce adaptive immunity against heterologous (PspA of Streptococcus pneumoniae) and homologous antigens (lipopolysaccharide [LPS] and outer membrane proteins [OMPs]).

Fig 1

Lipid A structure of the S. Typhimurium msbB mutant and its derivatives. (A) Covalent modifications of lipid A in the msbB pagL pagP lpxR mutant. The known covalent modifications of lipid A are indicated. (B) Covalent modifications of lipid A in the msbB pagL pagP lpxR mutant in the presence of LpxE or LpxF. (C) Lipid A profiles from ESI-MS analysis of χ11065 (ΔmsbB48 ΔpagL7 ΔpagP8 ΔlpxR9), χ11067 (ΔmsbB48 ΔpagL7 ΔpagP81::P_lpp lpxE ΔlpxR9), χ11068 (ΔmsbB48 ΔpagL7 ΔpagP8 ΔlpxR93::P_lpp lpxF), and χ11069 (ΔmsbB48 ΔpagL7 ΔpagP81::P_lpp lpxE ΔlpxR93::P_lpp lpxF) grown in LB medium at 37°C. The msbB pagL pagP lpxR mutant makes a penta-acylated lipid A as indicated by the [M − 2H]²⁻ peak at m/z 792.55. Removal of one phosphate group shifts the lipid A [M − 2H]²⁻ peak by m/z −39.98. The addition of l-Ara4N to the 4′ phosphate, catalyzed by ArnT, shifts the lipid A [M − 2H]²⁻ peak by m/z +65.529. The addition of pEtN to the 1-phosphate, catalyzed by EptA, shifts the MS peak by m/z +61.505. See Table S3 in the supplemental material for detailed information on lipid A derivatives.

Fig 2

Colonization of mice by χ3761 and each of its lipid A mutants. Viable bacterial counts (log₁₀ CFU per gram) recovered from spleen (A and B) and liver (C and D) of BALB/c mice (n = 6) at 3 and 6 days after oral inoculation by the indicated S. Typhimurium mutant strains are shown. Strain χ3761 is the wild-type strain that was used as a control. (E) Weights of spleens (in grams) (E) from six BALB/c mice 6 days after oral inoculation by the indicated strains of S. Typhimurium. Horizontal bars show the means; error bars indicate SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (relative to the value for wild-type UK-1). ††, P < 0.01; †, P < 0.05 (relative to the value for χ11065 [ΔmsbB48 ΔpagL7 ΔpagP8 ΔlpxR9]).

Fig 3

LPS-induced cytokine production in cultured Mono Mac 6 (MM6) and RAW264.7 cells. IL-6 (A), IL-1β (B), and TNF-α (C) in the supernatant of the MM6 cell culture stimulated with 0.1 pmol/ml LPS for 24 h were quantified by Bioplex assay. TNF-α (D) and GM-CSF (E) released into the culture supernatant of RAW264.7 cells stimulated for 24 h with 0.1 pmol/ml or 10 pmol/ml of LPS, respectively, were quantified by Bioplex assay. Means and SEM of three independent experiments, each performed in triplicate, are given. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (relative to the levels obtained by stimulation with LPS from wild-type χ3761). †††, P < 0.001; ††, P < 0.01; †, P < 0.05 (relative to levels obtained by stimulation with LPS from the parent strain, χ11065 [ΔmsbB48 ΔpagL7 ΔpagP8 ΔlpxR9]).

Fig 4

Serum IgG and vaginal IgA immune responses in immunized and control mice. The total serum IgG against SOMP (A), Salmonella LPS (B), and rPspA (C), the total vaginal wash fluid IgA against rPspA (D), and the IgG2a/IgG1 ratio (E) were measured by ELISA. Mice (n = 10 or 13) received the first immunization on day 0 and a booster dose at week 5. Data represent reciprocal anti-IgG titers in pooled sera from mice orally immunized with attenuated Salmonella carrying either pYA4088 (pspA) or pYA3493 (control). No immune responses to PspA were detected in mice immunized with the S. Typhimurium vaccine strain carrying the control plasmid pYA3493 (reciprocal titers, <1:50 for IgG and <1:25 for IgA). Error bars represent variations between triplicate wells. ***, P < 0.001; **, P < 0.01; *, P < 0.05 [compared with the value for χ9241(pYA4088)]. †††, P < 0.001; ††, P < 0.01; †, P < 0.05 [compared with the value for χ11088(pYA4088)].