Mannose-binding lectin-deficient mice are susceptible to infection with Staphylococcus aureus

Abstract

Gram-positive organisms like Staphylococcus aureus are a major cause of morbidity and mortality worldwide. Humoral response molecules together with phagocytes play a role in host responses to S. aureus. The mannose-binding lectin (MBL, also known as mannose-binding protein) is an oligomeric serum molecule that recognizes carbohydrates decorating a broad range of infectious agents including S. aureus. Circumstantial evidence in vitro and in vivo suggests that MBL plays a key role in first line host defense. We tested this contention directly in vivo by generating mice that were devoid of all MBL activity. We found that 100% of MBL-null mice died 48 h after exposure to an intravenous inoculation of S. aureus compared with 45% mortality in wild-type mice. Furthermore, we demonstrated that neutrophils and MBL are required to limit intraperitoneal infection with S. aureus. Our study provides direct evidence that MBL plays a key role in restricting the complications associated with S. aureus infection in mice and raises the idea that the MBL gene may act as a disease susceptibility gene against staphylococci infections in humans.

Figure 1.

Generation and characterization of MBL-null mice. (a) MBL-C targeting construct. Genomic organization of MBL-C is shown and compared with the targeting vector and homologous recombinant. (b) RT-PCR analysis of transcript for MBL-A, MBL-C, and serum amyloid protein (SAP) in liver. (c) Serum levels of MBL-A and MBL-C in WT, MBL-C KO, and MBL-null mice. •, individual mice; bars, the mean value for each group. (d) C4 cleaving activity of serum. The capacity of serum to activate C4 via the MBL complement pathway (left) or classical pathway (right) was assayed as described above. •, individual mice; bars, the mean value for each group. (e) C4 cleaving activity. Comparison of rhMBL with purified MBL-A and MBL-C. •, rhMBL; ○, MBL-A; ▾, MBL-C.

Figure 2.

Increased mortality in MBL-null mice from S. aureus infection. S. aureus was inoculated i.v. and survival was followed as described in Materials and Methods. Numbers of mice used were 15 WT, 14 MBL-null, and 9 MBL-null plus rhMBL. *, P < 0.0001.

Figure 3.

Increased bacterial loads in blood and organs of MBL-null mice. Bacterial titers were assayed at 24 h after i.v. inoculation of S. aureus as described in Materials and Methods. Six mice were in each group. Open bars, WT; closed bars, MBL-null. Bars indicate mean ± SD. *, P ≤ 0.05; **, P < 0.01.

Figure 4.

Restricted bacterial growth in blood of WT mice and enhanced growth in blood but not in plasma of MBL-null mice. Open bars, at 10 min; closed bars, at 2 h. (a) Bacterial growth in plasma. Results are shown as a percentage of bacterial growth in WT plasma at 10 min. Pooled plasma was used and the assay was performed in triplicates as described in Materials and Methods. Bars indicate mean ± SD. (b) Results are shown as a percentage of bacterial growth in WT blood at 10 min. Blood was collected from four mice individually and the assay was performed in triplicates as described in Materials and Methods. Bars indicate mean ± SD. *, P < 0.05

Figure 5.

Cytokine production after S. aureus infection. Levels of cytokine induction was less at 2 h but more at 24 h in MBL-null mice compared with WT mice after S. aureus i.v. inoculation. Six mice were used in each group. Bars indicate mean ± SE. *, P < 0.05; **, P < 0.001.

Figure 6.

Increased S. aureus infection in MBL-null mice and rescued MBL complement pathway by rhMBL in MBL-null mice. (a) In vivo imaging of mice at 48 h after inoculation of the biolumi-S. aureus was performed as described in Materials and Methods. Representative pictures from WT, MBL-null, and MBL-null mice that were reconstituted with rhMBL (MBL null plus rhMBL) are shown. (b) Increased level of bacteria in organs from MBL-null mice. Organs were harvested at 96 h after the infection with biolumi-S. Aureus and bacterial load was measured as described in Materials and Methods. Bars indicate mean ± SD. Numbers of mice used: WT, 8; MBL-null, 7; MBL-null plus rhMBL, 7. (c) MBL complement pathway activity before and after S. aureus CP5 infection. Plasma was collected at 4 d before as a base line and 4 d after S. aureus inoculation and analyzed for C4 deposition activity on mannan as described in Materials and Methods. Numbers of mice used before infection: WT, 12; MBL-null, 19. Numbers of mice used after infection: WT, 12; MBL-null, 10; MBL-null plus rhMBL, 9. Two experiments were combined. Bars indicate mean ± SD. *, P = 0.002.

Figure 7.

Decreased macrophage phagocytosis in MBL-null mice. Phagocytosis was assayed in vivo (a) and in vitro (b) as described in Materials and Methods. Phagocytosis is shown by mean fluorescence intensity for the ingested FITC-S. aureus. Bars indicate mean ± SE. **, P = 0.003; *, P = 0.009.