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. 2013 Mar;81(3):684–689. doi: 10.1128/IAI.01362-12

Lipopolysaccharide-Deficient Acinetobacter baumannii Shows Altered Signaling through Host Toll-Like Receptors and Increased Susceptibility to the Host Antimicrobial Peptide LL-37

Jennifer H Moffatt a, Marina Harper a,b, Ashley Mansell c, Bethany Crane a, Timothy C Fitzsimons a, Roger L Nation d, Jian Li d, Ben Adler a,b, John D Boyce a,b,✉
Editor: L Pirofski
PMCID: PMC3584870  PMID: 23250952

Abstract

Infections caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious global health problem. We have shown previously that A. baumannii can become resistant to the last-line antibiotic colistin via the loss of lipopolysaccharide (LPS), including the lipid A anchor, from the outer membrane (J. H. Moffatt, M. Harper, P. Harrison, J. D. Hale, E. Vinogradov, T. Seemann, R. Henry, B. Crane, F. St. Michael, A. D. Cox, B. Adler, R. L. Nation, J. Li, and J. D. Boyce, Antimicrob. Agents Chemother. 54:4971–4977, 2010). Here, we show how these LPS-deficient bacteria interact with components of the host innate immune system. LPS-deficient A. baumannii stimulated 2- to 4-fold lower levels of NF-κB activation and tumor necrosis factor alpha (TNF-α) secretion from immortalized murine macrophages, but it still elicited low levels of TNF-α secretion via a Toll-like receptor 2-dependent mechanism. Furthermore, we show that while LPS-deficient A. baumannii was not altered in its resistance to human serum, it showed increased susceptibility to the human antimicrobial peptide LL-37. Thus, LPS-deficient, colistin-resistant A. baumannii shows significantly altered activation of the host innate immune inflammatory response.

INTRODUCTION

The Gram-negative bacterium Acinetobacter baumannii is a major cause of hospital-acquired infections, including meningitis, bacteremia, ventilator-associated pneumonia, and burn infections (1). Treatment of A. baumannii infections has been significantly hampered in recent years due to the increased prevalence of strains that are resistant to the most commonly used antibiotics (1). Resistance to fluoroquinolones, β-lactams, aminoglycosides, and tetracycline is now widespread, and many strains are now considered pan-resistant (2). A frequently used salvage therapy for patients infected with multidrug-resistant (MDR) A. baumannii is the antimicrobial peptide colistin, a polymyxin antibiotic that is structurally similar to polymyxin B (3). Colistin initially binds to the lipid A moiety of lipopolysaccharide (LPS), resulting in destabilization of the Gram-negative outer membrane (4). Alarmingly, as the use of colistin has increased, so have reports of colistin-resistant A. baumannii strains (5–10). Previously, we have shown that A. baumannii can become resistant to colistin via mutations in the lipid A biosynthesis genes lpxA, lpxC, and lpxD, leading to the complete absence of LPS from the outer membrane (11, 12); significantly, LPS loss has been observed in clinical isolates (11).

In order to establish infection, A. baumannii must first evade various components of the host innate immune response. However, despite the clinical significance of this pathogen, very little is known about the host response to A. baumannii infection. Recent in vitro studies have indicated that A. baumannii elicits a reduced cytokine response compared to those of other less pathogenic species of the genus, such as A. jejuni (13), and that recognition of the bacteria by CD14 and Toll-like receptor 4 (TLR4) is required for the host to mount an appropriate immune response (14). However, recent evidence indicates that in mouse bacteremia models, some strains of A. baumannii can cause death via septic shock in a TLR4-dependent manner (15). The key ligand for TLR4 is LPS, which is known to be a key stimulator of the host innate immune response (16). Due to the extreme rarity of LPS-deficient Gram-negative bacteria, little is known about the host immune response to such bacterial cells. While LPS-deficient mutants of Neisseria meningitidis (17) and Moraxella catarrhalis (18) have been constructed by directed mutagenesis, the mutant strains showed significantly reduced fitness due to the loss of LPS. However, in vitro studies indicate that LPS-deficient A. baumannii can grow at the same rate as the wild-type parent strain (11). LPS-deficient N. meningitidis was shown to stimulate significantly less tumor necrosis factor alpha (TNF-α) than wild-type cells, but it was still able to elicit a strong cytokine response despite the absence of LPS (19). Here, we have used a defined, LPS-deficient mutant of A. baumannii to determine the importance of LPS for the interaction of this significant pathogen with components of the host innate immune system. Importantly, we demonstrate that LPS-deficient cells show unaltered resistance to complement, reduced activation of the host inflammatory response, and increased susceptibility to killing by the human antimicrobial peptide LL-37.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The A. baumannii type strain ATCC 19606 was obtained from the American Type Culture Collection (ATCC). The colistin-resistant, LPS-deficient paired mutant of ATCC 19606, designated 19606R, was described previously (11), and it contains a single base deletion at nucleotide 90 in the lipid A biosynthesis gene lpxA (11). A. baumannii strains were maintained on Mueller-Hinton (MH) agar or cultured in cation-adjusted Mueller-Hinton broth (CAMHB; Oxoid) at 37°C with the addition of 10 μg/ml of colistin sulfate (Sigma) for the 19606R strain.

Cell culture and reagents.

Immortalized gene-deficient, bone marrow-derived macrophages were the kind gift of Eicke Latz (University of Bonn) and Douglas Golenbock (University of Massachusetts) (20). All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (DMEMFCS; Invitrogen), 2 mM glutamine and maintained in a 37°C humidified atmosphere with 5% CO2. The TLR2 ligand Pam3Cys was obtained from EMC Microcollections (Tubingen, Germany), while the TLR ligands poly(I·C) (TLR3) and LPS (TLR4) were purchased from InvivoGen (San Diego, USA).

NF-κB assay.

RAW264.7 mouse macrophage cells stably expressing the NF-κB-dependent ELAM-luciferase reporter construct (21) were seeded at 2 × 104 cells/well in 96-well flat-bottomed tissue culture plates in 200 μl of DMEMFCS and incubated overnight at 37°C with 5% CO2. Bacteria were grown to mid-logarithmic phase (optical density at 650 nm [OD650] of 0.6) from fresh overnight broth cultures, and viable bacterial cell numbers were determined by plating of appropriate dilutions onto MH agar. Bacteria were either left untreated (live samples) or heated at 70°C for 30 min (heat-killed samples) and then diluted appropriately in phosphate-buffered saline, pH 7.2 (PBS). RAW264.7 macrophage cells were stimulated at various multiplicities of stimulation (MOS) with either live or heat-killed wild-type (ATCC 19606) or LPS-deficient (19606R) bacteria for 6 h at 37°C. Following incubation, the medium was removed and the RAW264.7 macrophages lysed by incubation for 5 min at 20°C in 50 μl of Promega passive lysis buffer. A 20-μl aliquot from each well was transferred to a white opaque 96-well plate, and 30 μl of Luciferase assay reagent (Promega) was added. Luciferase activity was measured with a Fluro-Optima luminometer.

TNF-α assays.

Immortalized normal murine macrophages and immortalized TLR2-deficient, TLR4-deficient, or MyD88/Mal-deficient murine macrophages were seeded in 200 μl of DMEMFCS at 2 × 104 cells/well in 96-well flat-bottomed tissue culture plates and incubated overnight at 37°C with 5% CO2. Bacteria were grown to mid-logarithmic phase and cell numbers determined by plating of appropriate dilutions onto MH agar. Macrophages were stimulated with either heat-killed wild-type (ATCC 19606) or heat-killed LPS-deficient (19606R) bacteria for between 2 and 24 h. Following stimulation, cell supernatants were harvested and tested by enzyme-linked immunosorbent assay (ELISA) for TNF-α production using the BD OptEIA ELISA kit (BD Biosciences) with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma) for detection. Plates were read on a Tecan Infinite M200 plate reader using a test wavelength of 450 nm and a reference wavelength of 570 nm.

Serum resistance.

The susceptibility of wild-type and LPS-deficient A. baumannii strains to killing in pooled human serum, which was obtained from healthy volunteers, was determined by direct colony counts after incubation in serum concentrations ranging from 0 to 90% (diluted in PBS) for 30 min at 37°C. Bacteria were grown in CAMHB to exponential phase (OD650 of 0.5) and then inoculated directly into diluted serum.

LL-37 resistance.

The susceptibility of A. baumannii strains to the human antimicrobial peptide LL-37 was determined as described previously (22), with the following modifications. Bacterial cultures were grown to mid-logarithmic phase, and cells then were harvested by centrifugation and resuspended in 10 mM sodium phosphate buffer (pH 6.5) to give a final concentration of ∼2.5 × 106 CFU/ml. To determine actual numbers of viable cells, the cell suspension was serially diluted in 0.9% NaCl and plated onto MH agar. For the antimicrobial assay, a 25-μl aliquot of the cell suspension (containing ∼5 × 104 CFU) was added to 25 μl of H2O containing various concentrations of LL-37 (AnaSpec, Fremont, CA). Following incubation for 3 h at 37°C, serial 10-fold dilutions were made (in 0.9% NaCl), and 100 μl of each dilution was plated onto MH agar to determine cell viability.

Statistical analyses.

All experiments were performed in biological triplicate, and statistical significance was determined by paired t test using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA).

RESULTS

LPS-deficient A. baumannii stimulates reduced NF-κB activation and TNF-α secretion from macrophage cells.

To investigate the host innate immune response to LPS-deficient A. baumannii, we examined the ability of LPS-deficient cells to activate NF-κB, a prototypic inflammatory transcription factor that plays a critical role in modulating the host innate immune response. As the use of viable cells in macrophage stimulation assays can be problematic due to differential bacterial growth, macrophage toxicity, and macrophage uptake, we initially determined whether heat-killed bacteria were suitable for use in the stimulation assays and determined the optimal multiplicity of stimulation (MOS). We measured the response of RAW264.7 macrophages that stably express the NF-κB-dependent ELAM reporter construct (RAW-ELAM) to stimulation with between 1 and 200 live or killed A. baumannii cells (either wild-type ATCC 19606 or its LPS-deficient derivative, 19606R [11]) (Fig. 1). At each MOS, there was no significant difference in the response of the RAW-ELAM macrophages to stimulation with live 19606R or heat-killed 19606R. Furthermore, at an MOS of 10 or 50, there was no difference in the response of RAW-ELAM cells to stimulation with live or heat-killed wild-type ATCC 19606. At the higher MOS of 100 and 200, there was an increased response to live ATCC 19606; we predict that this was due to the rapid growth of the bacteria during the 6-h stimulation period. These results indicated that heat-killed bacteria at an MOS of 10 or 50 were appropriate for subsequent stimulation experiments.

Fig 1.

Fig 1

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LPS-deficient A. baumannii cells stimulate less NF-κB activation than wild-type cells. RAW264.7 cells stably transfected with an NF-κB-ELAM reporter construct were stimulated with the live or heat-killed A. baumannii wild-type strain (ATCC 19606) or the LPS-deficient derivative (19606R) for 6 h with between 1 and 200 bacterial cells per macrophage. NS, nonstimulated cells. Error bars represent standard errors of the means (SEM). *, P < 0.001.

Importantly, when the RAW-ELAM cells were stimulated with ≥10 bacteria per macrophage, the wild-type strain (either live or heat killed) stimulated significantly more NF-κB activation than the LPS-deficient strain (either live or heat killed) (P < 0.001) (Fig. 1). Indeed, the wild-type strain stimulated approximately 3- to 4-fold more NF-κB activation at an MOS of 10 and approximately 2-fold more at an MOS of 50. Therefore, for further experiments we chose to use heat-killed cells at an MOS of 25 cells per macrophage.

We next examined the role that A. baumannii LPS plays in stimulating production of the inflammatory cytokine TNF-α. Immortalized wild-type murine macrophages were incubated for between 2 and 24 h with 25 killed bacterial cells (ATCC 19606 or 19606R) per macrophage. For all stimulation times that were ≥4 h, macrophages stimulated with killed wild-type A. baumannii (ATCC 19606) secreted approximately 2-fold more TNF-α than those stimulated with the LPS-deficient mutant (19606R) (P < 0.05) (Fig. 2). However, at every MOS tested there was a significant response to the LPS-deficient mutant compared to the nonstimulated control. Thus, the LPS-deficient mutant was still able to stimulate the production of TNF-α but at significantly reduced levels compared to wild-type A. baumannii.

Fig 2.

Fig 2

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LPS-deficient A. baumannii stimulates less TNF-α secretion from macrophage cells. Immortalized wild-type murine macrophages were stimulated with wild-type ATCC 19606 or the LPS-deficient derivative 19606R with 25 cells per macrophage for between 2 and 24 h. Following stimulation, cell supernatants were harvested and the level of TNF-α determined by ELISA. NS, nonstimulated cells; LPS, lipopolysaccharide control; P3C, Pam3Cys control; poly(I·C), polyinosine-poly(C). Error bars represent SEM. *, P < 0.05; **, P < 0.01.

LPS-deficient A. baumannii initiates inflammatory signaling responses via TLR2 but not TLR4.

To characterize the TLR responses to LPS-deficient A. baumannii, we first stimulated an immortalized murine macrophage cell line deficient in the TLR adaptor molecule MyD88/Mal with killed ATCC 19606 or 19606R and measured TNF-α production. As expected, in the absence of MyD88/Mal there was no significant induction of TNF-α, indicating that TNF-α production is dependent on TLR recognition and subsequent signaling via MyD88/Mal (Fig. 3C). To determine the TLR-specific responses, we measured the TNF-α production from TLR2- or TLR4-deficient macrophages incubated with killed ATCC 19606 or 19606R. The LPS-deficient strain, 19606R, stimulated significantly more TNF-α secretion from the TLR4-deficient macrophages than did wild-type A. baumannii (Fig. 3A) (P < 0.05). Thus, LPS-deficient cells show increased stimulation of macrophages via non-TLR4 mechanisms. In contrast, the LPS-deficient mutant 19606R elicited approximately 5-fold less TNF-α secretion from the TLR2-deficient macrophages than those stimulated with the wild-type strain (Fig. 3B). Thus, LPS-deficient cells have a reduced ability to stimulate TNF-α secretion (Fig. 2) but do stimulate some secretion via TLR-2 (Fig. 3A).

Fig 3.

Fig 3

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LPS-deficient A. baumannii signals primarily through TLR2. TLR4-deficient macrophages (A), TLR2-deficient macrophages (B), and MyD88/Mal-deficient macrophages (C) were stimulated with 25 killed cells per macrophage of either wild-type ATCC 19606 or the LPS-deficient derivative 19606R strain for 6 h. Following stimulation, cell supernatants were harvested and the level of TNF-α determined by ELISA. NS, nonstimulated cells. Error bars represent SEM. *, P < 0.05; **, P < 0.01.

LPS does not contribute to serum resistance of A. baumannii.

To determine whether LPS plays a protective role against complement-mediated killing in A. baumannii, we incubated the wild-type strain (ATCC 19606) and the LPS-deficient mutant (19606R) in various concentrations of pooled human serum (0 to 90%) and measured bacterial survival. Both the wild-type strain and the LPS-deficient mutant were able to survive in up to 25% human serum for 30 min. However, both strains were rapidly killed in serum concentrations of 50% or above (Fig. 4). Importantly, there was no difference in the survival of the two strains at any of the serum concentrations tested (P > 0.05).

Fig 4.

Fig 4

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LPS does not contribute to A. baumannii ATCC 19606 serum resistance. Wild-type (ATCC 19606) and LPS-deficient (19606R) strains were incubated in various concentrations of pooled human serum for 30 min at 37°C and then plated onto Mueller-Hinton agar to determine percent survival. Error bars indicate SEM.

LPS plays a role in resistance to the human antimicrobial peptide LL-37.

The antimicrobial peptide LL-37 is a component of human serum and is produced by neutrophils and various epithelial cells. It has a direct bactericidal effect on a wide range of bacterial pathogens and also plays a role in innate immune modulation (23). It is believed that the initial target of LL-37 is the LPS molecule in the Gram-negative outer membrane (24), which is also the initial target of the antimicrobial peptide colistin. We therefore hypothesized that the colistin-resistant A. baumannii strain 19606R displays increased resistance to LL-37, as it is unable to synthesize LPS. To determine LL-37 susceptibility, we incubated the wild-type ATCC 19606 strain and 19606R in various concentrations of LL-37 and then measured bacterial viability. Neither the wild-type strain nor the LPS mutant showed reduced viability after incubation with a low concentration of LL-37 (0.625 μM). However, following incubation in 1.25, 2.5, and 5 μM LL-37, both strains showed reduced survival, demonstrating the bactericidal effect of LL-37. Interestingly, the LPS-deficient mutant showed reduced survival compared to the wild-type strain after incubation with LL-37 at ≥1.25 μM (P < 0.05).

DISCUSSION

In this study, we have shown that the loss of LPS from the outer membrane of A. baumannii significantly alters the interaction of this pathogen with the host innate immune system. During infection with Gram-negative pathogens, high levels of inflammatory cytokines, such as TNF-α, are released in direct response to the presence of LPS. This inflammatory response may be important for bacterial clearance, but if it is unregulated, it can result in a cytokine storm, ultimately leading to septic shock. Importantly, the host inflammatory response to infection may differ depending on tissue. We chose to analyze the cytokine response of macrophages to bacterial stimulation, as they express the entire complement of TLRs and act as the primary sentinels for immune recognition in bacterial infection, whereas other cell types, such as epithelial cells, may express only a subset.

For clearance of A. baumannii during lung infections, an appropriate proinflammatory response, including the recruitment of neutrophils, must occur. Critical for this response is the recognition of bacterial components by TLRs and subsequent NF-κB activation and TNF-α production. Indeed, A/J mice that secrete significantly lower levels of proinflammatory cytokines, including TNF-α, displayed delayed neutrophil recruitment to the lungs in response to A. baumannii infection and a higher mortality rate (25). Furthermore, for A. baumannii infections in C57/BL6 mice, the bacterial load in the lungs of TLR4 knockout or CD14 knockout mice was significantly higher than that in the lungs of normal mice (26). Therefore, reduced levels of NF-κB activation and TNF-α secretion in response to LPS-deficient cells may reduce the host response to A. baumannii pneumonic infections.

Recent evidence suggests that A. baumannii virulence in mouse models of bacteremia is mediated primarily through shedding of LPS and the induction of septic shock (15). In C3H and C57BL/6 mice, the virulence of different A. baumannii strains was correlated with their ability to shed LPS into culture supernatants. Additionally, TLR4 knockout mice were protected against lethal infection. Our results show that LPS-deficient A. baumannii induces significantly less activation of the transcription factor NF-κB and significantly less secretion of TNF-α from macrophages. This suggests that LPS-deficient A. baumannii is much less likely to induce septic shock, an outcome which can be associated with lethal A. baumannii infections (2, 27, 28).

The overall proinflammatory response to LPS-deficient A. baumannii was significantly reduced compared to the response to wild-type bacterial cells, and this correlated with highly reduced signaling through TLR4. However, the LPS-deficient cells stimulated increased activation via TLR2. These results correlate with similar findings for an LPS-deficient mutant of N. meningitidis, although that study utilized HeLa cells transfected with TLR2 and TLR4 reporter constructs, whereas we have used immortalized gene-deficient macrophages (29). TLR2 is associated with detection of a wide range of pathogen-associated molecules, including bacterial peptidoglycan, lipoteichoic acid, lipopeptides, and porins (16). In LPS-deficient A. baumannii, the expression of outer membrane components is significantly altered in response to the loss of LPS, characterized by increased expression of a range of surface components, including the polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG) and lipoproteins (30). Furthermore, in the absence of LPS, these surface components are likely to have increased surface exposure and therefore are likely to be more readily detected by TLR2. Indeed, LPS-deficient mutants of N. meningitidis signal the host innate immune response via TLR2 and show a significantly altered outer membrane composition, with reduced lipoproteins and a change in phospholipid composition (29, 31, 32).

In many bacterial species, LPS plays a protective role against complement-mediated killing (33–35). However, we have clearly shown that the LPS of A. baumannii plays little or no role in serum resistance, as the LPS-deficient mutant was as resistant to human serum as the wild-type strain (Fig. 4). Retaining full complement resistance would be important for the survival of LPS-deficient A. baumannii in vivo, as susceptibility to complement-mediated killing in vitro has been shown to correlate with A. baumannii clearance in a rat soft-tissue infection model (36).

The loss of LPS from the outer membrane of A. baumannii mediates high-level colistin resistance (11). Although the exact mechanism by which colistin exerts its bactericidal effect is unknown, it is widely accepted that the initial electrostatic interaction of colistin with lipid A is crucial. Like colistin, the human antimicrobial peptide LL-37 also displays a high affinity for LPS and binds to bacterial membranes via an electrostatic interaction (23, 24, 37). We have previously shown that the loss of LPS and lipid A from the surface of A. baumannii reduces the net negative charge on the surface of the bacteria (11, 38). However, the human antimicrobial peptide LL-37 was still able to efficiently kill LPS-deficient cells. Indeed, the LPS-deficient A. baumannii showed increased susceptibility to the bactericidal effect of LL-37 (Fig. 5). Thus, while LL-37 may bind LPS with high affinity, our results show that its bactericidal activity is not LPS dependent. This is perhaps unsurprising, as LL-37 is active against many Gram-positive bacteria, fungi, and viruses that do not elaborate LPS on their surface and which have differently charged membranes (23). Colistin, unlike LL-37, displays a more specific spectrum of bactericidal activity, being only effective against Gram-negative bacteria (3). As LPS-deficient A. baumannii shows increased membrane permeability (11), this may explain the increased sensitivity of LPS-deficient cells to LL-37; in Escherichia coli, bacterial growth is halted shortly after LL-37 reaches the periplasm (24).

Fig 5.

Fig 5

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LPS plays a role in resistance to LL-37. Wild-type (ATCC 19606) and LPS-deficient (19606R) strains were cultured in various concentrations of LL-37 for 3 h at 37°C and then were plated onto Mueller-Hinton agar to determine percent survival. Error bars indicate SEM. *, P < 0.05; ***, P < 0.001.

It is of interest that LpxC inhibitors, which have bactericidal effects against most Gram-negative bacteria, do not kill A. baumannii in vitro (15). This is likely because A. baumannii can survive without LPS; indeed, the LPS-deficient strain tested in this study is an lpxA mutant, and we have previously identified LPS-deficient A. baumannii with spontaneous lpxC mutations (11). Interestingly, treatment of A. baumannii-infected mice with the LpxC inhibitor Lpx-1 reduced the host inflammatory response, including the secretion of TNF-α, and protected the mice from lethal challenge. As A. baumannii can survive without LPS, it is likely that treatment of infected mice with Lpx-1 results in a significant reduction in the level of LPS produced by the bacteria. Indeed, the Lpx-1 treatment resulted in significantly reduced LPS shedding into the mouse serum, although total bacterial LPS was not measured in the recent study (15). In this report, we have shown that LPS-deficient lpxA mutant A. baumannii stimulates significantly reduced host inflammatory responses via TLR4, and this correlates well with the reduced inflammatory responses of A. baumannii-infected mice observed following Lpx-1 treatment (15).

Infections with MDR A. baumannii remain a significant problem around the globe. While colistin has been an effective antibiotic for treatment of MDR strains, LPS-deficient, colistin-resistant A. baumannii clinical isolates have been identified (11). Here, we have shown that the loss of LPS from the A. baumannii surface not only leads to high levels of colistin resistance but also can significantly alter the host proinflammatory response. Due to the loss of the initial interaction of TLR4 with LPS, LPS-deficient A. baumannii stimulates lower levels of NF-κB and TNF-α. LPS-deficient cells also retain full resistance to complement yet display increased sensitivity to the human antimicrobial peptide LL-37. Novel antimicrobial peptides are currently being investigated for their use as antimicrobial agents against A. baumannii, and to date several have proved to be promising candidates (39–41). Our results suggest that alternative antimicrobial peptides have the potential to be effective against colistin-resistant, LPS-deficient A. baumannii strains.

ACKNOWLEDGMENTS

This work was supported by the Australian National Health and Medical Research Council (NHMRC), Canberra, Australia, and the Victorian Operational Infrastructure Program. J.H.M. is supported by an Australian Postgraduate Award Scholarship. J.L. is an Australian NHMRC Senior Research Fellow.

Footnotes

Published ahead of print 17 December 2012

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