Sap Transporter Mediated Import and Subsequent Degradation of Antimicrobial Peptides in Haemophilus

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

<i>Haemophilus</i> Responses to Nutritional Immunity: Epigenetic and Morphological Contribution to Biofilm Architecture, Invasion, Persistence and Disease Severity

<div><p>In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence <i>Haemophilus</i> biofilm formation and pathogenesis. Thus, we cultured <i>Haemophilus</i> through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted <i>Haemophilus</i>, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted <i>Haemophilus</i> was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites.</p></div

SapF-mediated heme iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus

Nontypeable Haemophilus influenzae (NTHI) is a common commensal bacterium that resides in the human upper respiratory tract of healthy individuals. NTHI is also a known causative agent of multiple diseases including sinusitis, otitis media as well as exacerbates disease severity of patients with cystic fibrosis and chronic obstructive pulmonary disease. We have previously shown that the Sap ABC transporter mediates resistance to host antimicrobial peptides (AMPs) and import of the iron-containing compound heme. Here, we analyzed the contribution of the Sap structural ATPase protein, SapF, in these essential functions. SapF was dispensable for NTHI survival when exposed to AMPs in vitro. SapF was responsible for heme utilization and recovery of depleted internal heme iron stores. Further, a loss of SapF resulted in morphological plasticity and enhanced community development and biofilm architecture, suggesting the potential role of heme iron availability in coordinating the complexity of NTHI biofilm architecture. SapF was required for colonization of the nasopharynx and acute infection of the middle ear, as SapF deficiency correlated with a statistically significant decrease in NTHI persistence in vivo. These data suggest that SapF is required for proper heme utilization which directly impacts NTHI survival. Thus, these studies further support a role for the Sap complex in the transport of multiple substrates and further defines substrate specificity for the two ATPase subunits. Given the multiple essential functions provided by the Sap ABC transporter, this complex could prove to be an effective therapeutic target for the treatment of NTHI diseases

Degradation of AMPs by cytoplasmic peptidase activity.

<p>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.</p

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

<p>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.</p

SapA is required for NTHI to directly counter host defensin lethality <i>in vivo.</i>

<p>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 <i>sapA</i> mutant to determine the consequence of neutralization of native cBD-1 on persistence of the <i>sapA</i> mutant in vivo. A second dose of serum was delivered one day after bacterial challenge. (A) The <i>sapA</i> 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 <i>sapA</i> 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 <i>sapA</i> 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 <i>sapA</i> 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.</p

Transient restriction of heme-iron promotes biofilm tower architecture.

<p>Schematic representation of environmental heme-iron restriction. 86-028NP was cultured in the presence (+) or absence (−) of heme-iron for 24 hours generate “replete” and “restricted” populations, respectively. These populations were subcultured into medium containing heme-iron to produce continuously exposed (CE) or transiently restricted (TR) populations (<b>A</b>). The height of biofilm base or tower formed by CE or TR NTHI was measured in 20 random fields of view from a representative 48-hour biofilm (<b>B</b>). Statistical analysis was performed using a two-tailed t-test. Biofilms were visualized with live/dead stain, imaged using optical sections and reconstructed to generate a 3D view from the top (large pane) including orthogonal views of the side (top and left side) (<b>C, E</b>). Series of optical sections from an independent experiment were used to generate a surface plot to depict biofilm architecture (<b>D, F</b>). Images were processed using Image J and the height of the biofilm is indicated in each panel.</p

AMPs localize to cytoplasm-enriched fractions of NTHI.

<p>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.</p

Robust towers arise from transiently restricted NTHI in a mixed culture.

<p>The reporter strains [86-028NP/pGM1.1 (production of GFP) and 86-028NP/pKM1.1 (production of mCherry)] were cultured for 24 hours in the presence or absence of heme-iron to generate “replete” and “restricted” populations. Restricted 86-028NP/pGM1.1 was co-cultured with equivalent numbers of replete 86-028NP/pKM1.1 for 48 hours in 2 µg heme mL⁻¹ (<b>A</b>) or 20 µg heme mL⁻¹ (<b>C</b>) in a glass chamber slide. Restricted 86-028NP/pKM1.1 was co-cultured with equivalent numbers of replete 86-028NP/pGM1.1 for 48 hours in 2 µg heme mL⁻¹ (<b>B</b>) in a glass chamber slide. Optical sections were rendered to form the top down and orthogonal views. Biofilms formed from co-culture of restricted 86-028NP/pKM1.1 with replete 86-028NP/pGM1.1 were subjected to disruption to enumerate the total numbers of each reporter strain present following 48 hours of biofilm growth (<b>D</b>). Restricted 86-028NP/pGM1.1 was co-cultured with 1000 fold more 86-028NP/pKM1.1 replete for heme-iron for 48 hours in 2 µg heme mL⁻¹ (<b>E</b>) in a glass chamber slide. Restricted 86-028NP/pKM1.1 was co-cultured with 1000 times more 86-028NP/pGM1.1 replete for heme-iron for 48 hours in 2 µg heme mL⁻¹ (<b>F</b>) in a glass chamber slide. Orthogonal views of 3D rendered image are depicted (<b>E, F</b>). Heights of the biofilm are indicated in each panel. Statistical analysis was performed using a two-tailed t-test.</p