Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus

Abstract

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.

Figure 1. SapA is required for NTHI to directly counter host defensin lethality in vivo.

Chinchilla middle ears were pre-treated with (r)cBD-1 antiserum (B) or pre-immune serum (A) then co-infected with a mixture of wild type NTHI strain 86-028NP and the isogenic sapA mutant to determine the consequence of neutralization of native cBD-1 on persistence of the sapA mutant in vivo. A second dose of serum was delivered one day after bacterial challenge. (A) The sapA mutant strain (black triangles) was unable to persist in the middle ear, and showed a significant decrease in colonization relative to that of the wild type strain (gray circles) 14 days after infection (p<0.01). (B) Neutralization of cBD-1 restored the ability of the sapA mutant strain to persist in the middle ear, at levels equal to or greater than that of the wild type strain. (C) Competitive index ratios (wild type to sapA mutant strain) were determined for each cohort receiving either the pre-immune (gray squares) or (r)cBD-1 (black squares) neutralizing antiserum. The black line indicates a competitive index of 1 (equal wild type to sapA mutant ratio). Black and gray horizontal lines (panels A and B) represent the geometric mean value of each cohort (n = 5 animals per cohort). Statistical significance was determined by non-parametric Mann-Whitney U test of geometric means, significance at p≤0.05.

Figure 2. AMPs localize to cytoplasm-enriched fractions of NTHI.

NTHI was fractionated to obtain cytoplasm and periplasm-enriched fractions. (A) Two dimensional gel electrophoresis of enriched fractions confirmed the presence of unique proteins in the periplasm and cytoplasm-enriched fractions. (B) Enriched fractions were separated by SDS-PAGE (12%) and silver stained to determine unique proteomic profiles. (C) Confirmation of bacterial fractionation by immunodetection of the periplasmic enzyme, β – lactamase and the cytoplasmic ATPase protein, SapD. (D) NTHI strain 86-028NP was exposed to LL-37 or hBD3 at sublethal levels for 30 minutes, fractionated and cytoplasm-enriched fractions were separated on a 16.5% Tris-Tricine gel and AMPs were detected by Western immunoblot analysis. No AMPs were detected in cytoplasm-enriched fractions prepared from cells alone.

Figure 3. In vivo degradation of AMPs in NTHI cytoplasm.

The parental NTHI strain was exposed to AMPs for 30 minutes, washed, resuspended in buffer without AMPs, and incubated for a chase period of 0, 2, or 4 hours followed by cell fractionation. Enriched cytoplasmic fractions were separated by SDS-PAGE on a 16.5% Tris-Tricine gel and hBD-3 (A) or LL-37 (B) was detected by immunoblot analysis. Samples were matched for viability, normalized for protein amount, and confirmed by silver stain of lipopeptide [C] contained in enriched cytoplasmic samples. Average density of bands is represented graphically (bottom panel C). There was no signal for control samples from cells incubated in the absence of AMPs. Data are representative of three independent assays.

Figure 4. Degradation of AMPs by cytoplasmic peptidase activity.

The parental NTHI strain was fractionated to obtain cytoplasm-enriched fractions. This fraction was combined with hBD-3 or LL37 in the presence or absence of a protease inhibitor cocktail (−/+ Inhibitor) and incubated for 0, 2, 5 or 13 hours. Samples were separated by SDS-PAGE on a 16.5% Tris-Tricine gel, and hBD3 or LL-37 was detected by immunoblot analysis. Samples were matched for viability and normalized for protein amount, as shown [C] for both hBD3 and LL-37.

Figure 5. The Sap transporter inner membrane permease is required for AMP transport to the bacterial cytoplasm.

Parent and permease-deficient NTHI were incubated with sublethal concentrations of LL-37 or hBD3 for 30 minutes, washed, fixed, sectioned (50 nm) and immunogold labeled for AMP as visualized by transmission electron microscopy (TEM) analysis. Parallel samples not exposed to AMPs yet processed in parallel served as a labeling control. Scale bar = 200nm.

Figure 6. Model of Sap-dependent import of AMP molecules and subsequent degradation.

In the presence of low concentrations of AMP, NTHI are able to resist lethality by modification of the outer membrane (OM; ChoP and Lipid A acylation, red) and subsequent repulsion of cationic charged peptides away from the bacterial cell surface. An increase in local concentrations of AMP increases production of the Sap transporter which functions to bind and transport periplasmic AMPs across the NTHI cytoplasmic membrane (CM). Transported AMPs are susceptible to proteolytic degradation. A reduction in the critical threshold concentration of AMPs in the periplasm returns NTHI to a homestatic state of innate immune resistance.