Fig 1.
Depiction of apolipoprotein B.
Schematic of human apoB100 (UniProt P04114) indicating the region aligning with vitellogenin, for which a crystal structure is available [57], the C-terminus of apoB48 and the low density lipoprotein receptor (LDLR) recognition site which facilitates uptake and clearance of apoB100 by the LDLR.
Fig 2.
ApoB48-LP binds AIP and antagonizes agr-signaling.
(A) S. aureus agr-I strain AH1677 (LAC) (2 x 107 CFUs ml-1) was cultured for 2 hrs with 10 nM exogenous AIP1 and serum from apoE-/- or wild type (C57BL/6) mice (0.3%). agr::P3 promoter activation was measured by flow cytometry as mean channel fluorescence (MCF) and the MCF of the no serum control was normalized to 100%. (B) S. aureus was cultured as in (A) along with different concentrations of apoB48-LP or 50 nM human LDL (apoB100). agr::P3 promoter activation was measured by flow cytometry and the MCF of the no apoB control was normalized to 100%. Results shown are means ± SEM from three independent experiments performed in triplicate. (C-D) qRT-PCR analysis of (C) RNAIII and (D) hla expression relative to 16S rRNA under conditions described above. Results are means ± SEM from three independent experiments. (E) (Left) SDS-PAGE analysis of supernatants from 5 h cultures of LAC grown alone or with 50 nM AIP ± 50 nM apoB48-LP. Arrowhead indicates migration of Hla. (Right) Relative Hla concentration was determined by Western blot followed by quantification of band intensity compared to the + AIP control. Data are mean ± SEM of three experiments performed in triplicate. (F) Hla expression in supernatants grown as for (E), assessed via the rabbit red blood cell lysis assay. HA50 is the bacterial supernatant dilution factor required for lysis of 50% of the RBCs. Data are the mean ± SEM of triplicate experiments performed in duplicate. (G) Surface plasmon resonance (SPR) analysis of apoB48-LP binding to immobilized biotinylated AIP1. Binding was measured in resonance units (RU). (H) Anti-apoB antibody at 5-, 10- and 30-fold molar excess, but not IgG control, blocks apoB48-LP binding to AIP1. Results are the mean ± SEM of N = 3 to 5. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p≤0.0001.
Fig 3.
ApoB48- and apoB100-LP demonstrate equivalent inhibition of agr.
AH1677 (2x107 CFUs ml-1) was cultured with 50 nM AIP and increasing concentrations of mouse apoB48-LP or human LDL (apoB100). Data from three independent experiments are shown as normalized MCF versus the log of LP concentration.
Fig 4.
apoB48-LP antagonize agr-signaling and inhibit morbidity in an air-pouch model of S. aureus SSTI.
AH1677 (3x107 CFUs) and apoB48-LP (100 nM) or vehicle control, were injected into dorsal air-pouches of 4APP-treated, Nox2-/- mice. At 4 hrs post-infection, the mice were given a second dose of apoB48-LP or vehicle control. Twenty-four hrs post-infection, the following were determined: (A) percent weight loss; (B) clinical morbidity score (see Materials and Methods for details); (C) agr::P3 promoter activation in pouch lavage; and (D) bacterial burden in the pouch lavage and spleen. Results are the mean ± SEM of N = 7 mice/group from two independent experiments (A,B) and N = 3 mice/group (C,D). (E) Growth curves of USA300 isolate LAC grown in broth or broth with 10% serum from 4APP-treated, Nox2-/- mice ± 100 nM apoB48-LP. Data shown are mean ± SEM from at least 2 independent experiments performed in duplicate. ns, not significant; **, p<0.01; ****, p≤0.0001.
Fig 5.
Model of lipoprotein access to sites of infection.
(1) In a healthy subject, the liver releases HDL and VLDL, the latter of which is reduced to LDL by lipase activity. (2) Following oral feeding, enterocytes package dietary lipids and apoB48 into chylomicrons (CM). (3) Lipoproteins are available for host innate defense in the circulation or upon serum extravasation to sites of peripheral infection or inflammation. In a critically ill patient, LP release from the liver is limited as part of the APR and oral feeding may not be possible. The resulting reductions in serum LP levels may negatively impact both peripheral and systemic host innate defense against bacterial pathogens.