Broad-spectrum capture of clinical pathogens using engineered Fc-mannose-binding lectin enhanced by antibiotic treatment

FcMBL binding to bacteria

We first set out to determine the range of pathogens that FcMBL can capture by screening multiple species of bacteria, fungi, viral antigens, and parasites using the ELLecSA detection technology. In the FcMBL ELLecSA, pathogen materials in experimental samples are captured with FcMBL immobilized on superparamagnetic beads (1 µm diameter), magnetically separated, washed, detected with human MBL linked to horseradish peroxidase (HRP), magnetically separated again, washed, and then tetramethylbenzidine (TMB) substrate is added to measure the amount of pathogen material bound (Figure 1A). Results were quantified by interpolating against an internal standard curve generated using yeast mannan in buffer [50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, 5 mM CaCl₂, pH 7.4 (TBST 5 mM CaCl₂)] (Figure 1B). In addition, we show that while the curve sensitivity is reduced when mannan is spiked into whole human blood, the limit of detection remained similar (1 ng/ml) as it does in buffer (Figure 1B).

Figure 1. FcMBL ELLecSA.

(A) Diagrammatic representation of the FcMBL ELLecSA methodology. Live and fragmented bacteria are captured by FcMBL, which has been coated on superparamagnetic beads via an N-terminal aminooxy-biotin on the Fc to allow oriented attachment to streptavidin (FcMBL Bead). FcMBL beads with captured bacteria are then magnetically separated and detected using recombinant human MBL linked to horseradish peroxidase (Human MBL-HRP). Tetramethylbenzidine (TMB) substrate is added to quantify captured bacteria and results are read at OD 450nm. (B) Mannan standard curve showing FcMBL binding to mannan in buffer and spiked into whole human blood (<24 h from draw), measured at optical density 450 nm. The mannan standard curve in buffer serves as an internal assay control, and is used to determine FcMBL binding to pathogen material in units of PAMPs/ml (where 1 ng/ml of mannan bound by FcMBL is equivalent to 1 PAMP unit. PAMP units are then multiplied by the dilution factor of the sample volume to give PAMPs/ml).

Initially our focus was on screening bacteria and as such we compiled a comprehensive list of clinically relevant bacterial pathogens (Table 1)²³. When we screened 82 different species of bacteria to compare FcMBL binding to live versus fragmented cells, we found that FcMBL detected 59 out of 82 live microbes (72%) and that more could be detected (70 out of 82; 85%) after they were fragmented by treating with antibiotics, or mechanically disrupted using zirconia/silica beads in a mixer mill (Table 1)²³. The antibiotics we used in this study were clinical grade cefepime, ceftriaxone, meropenem, amikacin, gentamicin, and vancomycin, to provide enough coverage to target this diverse range of bacteria. We dosed each bacterial class with a single appropriate antibiotic dose (≤1 mg/ml) to obtain acute fragmentation within 4 hours.

To determine if inducing bacterial fragmentation via antibiotic treatment or bead milling would reduce variation in FcMBL binding between different strains of the same species, we screened 134 isolates from 21 of the 88 Gram-positive and Gram-negative bacterial species, including strains tested by Cartwright et al. (Figure 2)²³. As before, FcMBL bound a greater proportion of the pathogens when fragmented (113/134 = 84%) than when live and intact (77/134 = 57%). For some bacterial species such as Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae we found that mechanical disruption or antibiotic-induced fragmentation greatly increased FcMBL binding, whereas other bacteria like Pseudomonas aeruginosa, Yersinia pseudotuberculosis, and MRSA bound equally well when live and intact (Figure 2). With the exception of Proteus mirabilis and Enterococcus faecalis, which FcMBL did not bind at all, the capture of fragmented bacteria was equal to or greater than that of live bacteria (Figure 2).

Figure 2. FcMBL binding to bacterial isolates is equal to or enhanced upon fragmentation.

The graph is divided between Gram-negative bacterial isolates [Gram (-)] and Gram-positive bacterial isolates [Gram (+)]. Data are presented as the number of bacterial isolates bound live and fragmented within the total isolates tested for each species: A. baumannii (n = 3), E. aerogenes (n = 4), E. cloacae (n = 8), E. coli (n = 28), K. oxytoca (n = 7), K. pneumoniae (n = 9), P. mirabilis (n = 4), P. aeruginosa (n = 6), S. enteriditis (n = 2), S. paratyphi A (n = 2), S. typhimurium (n = 6), S. marcescens (n = 5), Y. pseudotuberculosis (n = 6), E. faecalis (n = 5), S. aureus (n = 17), S. aureus (MRSA) (n = 3), S. epidermidis (n = 4), S. hominis (n = 2), S. agalactiae (n = 2), S. pneumoniae (n = 9), S. viridans (n = 2).

These findings are consistent with past studies that showed the efficiency of MBL binding to live bacteria differs between isolates from the same bacterial genus and species, possibly due to differences in encapsulation^15,16. To illustrate that the heterogeneity of MBL binding to live isolates of the same species can be reduced by using antibiotics to disrupt previously cryptic binding sites, we used two different isolates of Escherichia coli, and two different isolates of Streptococcus pneumoniae. E. coli 41949 and S. pneumoniae 3 exhibited equivalent FcMBL binding whether they were live or fragmented with antibiotics (1 mg/ml cefepime or ceftriaxone, respectively, for 4 hours), whereas fragmented forms of E. coli RS218 and S. pneumoniae 19A isolates bound much more effectively to FcMBL than living forms (Figure 3A–D). This difference was further supported visually using scanning electron microscopy (SEM) in which magnetic FcMBL beads could be seen to bind both live and fragmented versions of E. coli 41949 and S. pneumoniae 3, but with E. coli RS218 and S. pneumoniae 19A, the FcMBL beads only bound to fragmented material (Figure 3E–H). FcMBL binding increases upon fragmentation, which is correlated with LPS release measured using a limulus amebocyte lysate (LAL) assay: equal amounts of LPS were detected for E. coli 41949 whether live or fragmented, whereas LPS levels were higher in antibiotic treated E. coli RS218 (Figure 3I, J). These results suggest that antibiotic treatment results in exposure of previously cryptic PAMPs in the cell wall, including toxins such as LPS and LTA, which leads to greatly increased binding of FcMBL.

Figure 3. FcMBL bacterial binding efficiency can be enhanced with antibiotic treatment.

(A–D) PAMPs/ml detection by FcMBL ELLecSA of both live (10⁷ CFU/ml) and fragmented bacteria using antibiotics (cefepime 1 mg/ml or ceftriaxone 1 mg/ml). (A) E. coli 41949, (B) S. pneumoniae 3, (C) E. coli RS218, and (D) S. pneumoniae 19A. (E–H) Scanning electron microscopy images showing FcMBL bead (128 nm) capture of both live and fragmented bacteria using antibiotics (cefepime 1 mg/ml or ceftriaxone 1 mg/ml). (E) E. coli 41949, (F) S. pneumoniae 3, (G) E. coli RS218, and (H) S. pneumoniae 19A. (I,J) LPS endotoxin measurement (LAL assay) using 10⁷ CFU/ml of both live and fragmented bacteria using antibiotics (cefepime 1 mg/ml). (I) E. coli 41949 and (J) E. coli RS218.

In these studies, we found that 12 bacterial species, including multiple species of Enterococcus and Proteus, failed to bind to FcMBL even when treated for 4 hours with combinations of antibiotics (500 µg/ml vancomycin and 500 µg/ml amikacin for Gram-positive isolates or 500 µg/ml cefepime and 500 µg/ml amikacin for Gram-negative isolates) (Table 1)²³. Importantly however, FcMBL was able to detect 84% (172 out of 204) of the bacterial isolates (Figure 4), which includes 9 of the 10 pathogens responsible for most healthcare-associated infections in acute care hospitals in the U.S., with Enterococcus species being the one exception²⁴.

Figure 4. Summary of FcMBL pathogen capture.

(Left) Percent of live and fragmented Gram-positive (n = 78) and Gram-negative (n = 117) bacterial isolates bound by FcMBL ELLecSA. (Right) Chart showing total number of isolates tested by FcMBL ELLecSA for bacteria, fungi, viral antigens, parasites, and bacterial antigens, total number FcMBL detected, and total percent detected overall. ***p-value < 0.0001; Pearson’s chi-squared test. NS, not significant.

FcMBL binding to bacterial cell wall components

We further explored FcMBL’s ability to bind bacterial cell wall components because when bead mill or antibiotics were used to disrupt the membranes of Gram-negative isolates (n = 117), there was a significant boost in FcMBL detection efficiency with fragmented cells (89%) versus live intact cells (58%) (Figure 4), which is likely due to exposure of LPS that is present in high concentrations in their cell wall²⁵. Similarly, FcMBL also detected a greater percentage (77%) of fragmented Gram-positive isolates (n = 78), versus live intact cells (65%) (Figure 4). Thus, to better understand some of the major targets that FcMBL binds when bacteria are fragmented, we extended our analysis using purified samples of LPS and LTA^25,26.

Using the ELLecSA, we screened LPS purified from Gram-negative bacteria (Serratia marcescens, Klebsiella pneumoniae, and Salmonella enterica serovar enteritidis), as well as LTA from Gram-positive bacteria (Enterococcus hirae, Staphylococcus aureus, and Streptococcus pyogenes) in TBST 5 mM CaCl₂, as well as in more clinically relevant human whole blood samples. We found that FcMBL was able to detect LPS from all 3 Gram-negative species, with S. marcescens being the best (1 ng/ml limit of detection in buffer and 3.9 ng/ml in blood) (Figure 5A–C). FcMBL also bound to E. hirae LTA very well (15.6 ng/ml limit of detect in buffer and 62.5 ng/ml in blood) (Figure 5D), which is consistent with past findings²⁷. Also consistent with past findings, we found that FcMBL binds S. aureus LTA through the carbohydrate recognition domain (Figure 5E and Supplementary Figure 1)^28,29. Notably, however, FcMBL also bound S. pyogenes LTA (Figure 5F), which is in contrast to the past finding that MBL binds well to E. hirae LTA but poorly to LTA from S. pyogenes due to lack of glycosyl substituents^27,30.

Figure 5. FcMBL ELLecSA screening of purified lipopolysaccharide (LPS) and lipoteichoic acid (LTA).

LPS from (A) S. marcescens, (B) K. pneumoniae, and (C) S. enterica serovar enteritidis, and LTA from (D) E. hirae, (E) S. aureus, and (F) S. pyogenes were spiked into either TBST 5 mM CaCl₂ buffer or whole human blood at indicated concentrations.

We next tested FcMBL’s ability to bind lipoarabinomannan (LAM) and its biosynthetic precursors, phosphatidylinositol mannoside 1 & 2 and 6 (PIM_1,2 and PIM₆) from Mycobacterium tuberculosis (TB) strain H37Rv^31,32. LAM released from metabolically replicating or degrading TB bacteria has been detected in both blood and urine^33,34. Thus, we assessed the ability of FcMBL to capture and detect LAM, as well as PIM_1,2 and PIM₆, spiked into both of these complex biological fluids as well as buffer. Our initial screen in buffer confirmed that FcMBL can detect LAM and PIM₆ at levels down to 4 ng/ml, but it did not detect PIM_1,2 (Figure 6A–C). FcMBL also bound to LAM in both blood and urine but its binding sensitivity was reduced as it could only detect 15.6 ng/ml and 250 ng/ml, respectively. FcMBL binding to PIM₆ exhibited a similar sensitivity in buffer, but it could only detect 62.5 ng/ml and 15.6 ng/ml in blood and urine, respectively.

Figure 6. FcMBL ELLecSA screening of Mycobacterium tuberculosis strain H37Rv glycolipids PIM_1,2, PIM₆, and LAM.

Glycolipids spiked into (A) buffer (TBST 5mM CaCl₂), (B) whole human blood, and (C) urine. FcMBL detected LAM and PIM₆ at 4 ng/ml in buffer, but not PIM_1,2. Sensitivity of LAM and PIM₆ is reduced in whole human blood and urine.

FcMBL binding to fungi, parasites, viral antigens, and bacterial cell wall antigens

In addition to screening multiple bacteria, we also tested FcMBL’s ability to bind to 12 different species of fungi, 10 viral antigens, 2 species of parasites, and 6 purified bacterial cell wall antigens (Table 1)²³. In contrast to studies with bacteria, FcMBL was found to bind 100% of live fungal cells from all 12 species and 13 isolates tested (Figure 4). Of the two parasites tested in this preliminary analysis, only Trichomonas vaginalis was bound by FcMBL, whereas 80% of the viral antigens screened and 92% of the purified bacterial cell wall antigens were detected (Figure 4 and Figure 7). The handful of pathogen material FcMBL did not detect included the E1 protein from Chikungunya virus, the NS1 protein from Tick-borne encephalitis virus, Plasmodium falciparum, and PIM_1,2 from TB. In total, the overall FcMBL binding profile respectively detected 85% (194 out of 229) of isolates, and 87% (97 out of 112) of the different pathogen species tested.

Figure 7. FcMBL binding to viral antigens.

FcMBL ELLecSA screening of Chikungunya E1 (3.0 µg/ml), Cytomegalovirus (CMV) (10⁷ PFU), Dengue serotype 1 VLP (0.36 µg/ml), Human immunodeficiency virus (HIV) gp120 (0.1 µg/ml), Ebola GP1 (0.2 µg/ml), Influenza H1N1 HA (0.1 µg/ml), Influenza H1N1 NA (1 µg/ml), Respiratory syncytial virus (RSV) glycoprotein g (10.0 µg/ml), Tick-borne Encephalitis NS1 (5 µg/ml), and Zika lysate (0.24 µg/ml).

Raw data for the present study are available on OSF²⁹.

Pathogen sources

Bacteria, fungi, viral antigens, parasites, and bacterial cell wall antigens were obtained from a multitude of sources which include: Abcam (Cambridge, USA), AERAS (Rockville, USA), American Type Culture Collection (Manassas, USA), Biodefense and Emerging Infections Resources (BEI Resources) (Manassas, USA), Boston Children’s Hospital (Boston, USA), Brigham and Women’s Hospital Crimson Biorepository (Boston, USA), Hospital Joseph-Ducuing (Toulouse, France), Sigma-Aldrich (St. Louis, USA), Sino Biological (Beijing, China), and The Native Antigen Company (Oxford, United Kingdom).

In addition, the following defined strains were used in this study: Streptococcus pneumoniae 3 (ATCC 6303), Streptococcus pneumoniae 19A (ATCC 700674), Escherichia coli 41949 (Multiple O antigens:H26) (Crimson Biorepository), and Escherichia coli RS218 (NMEC O18:H7) (Kindly provided by James R. Johnson from the University of Minnesota). LPS from Serratia marcescens (L6136), Klebsiella pneumoniae (L4268), Salmonella enterica serovar enteritidis (L6011), and LTA from Enterococcus hirae (L4015), Staphylococcus aureus (L2515), and Streptococcus pyogenes (L3140) were purchased through Sigma-Aldrich (St. Louis, USA). Mycobacterium tuberculosis H37Rv components, which include lipoarabinomannan (LAM, NR-14848) and phosphatidylinositol mannoside 1,2 and 6 (PIM_1,2, NR-14846 and PIM₆, NR-14847), were obtained from BEI Resources (Manassas, USA). Parasites, Trichomonas vaginalis (TV01-1000) and Plasmodium falciparum Circumsporozoite protein (ab73857), were purchased from The Native Antigen Company (Oxford, UK) and Abcam (Cambridge, USA), respectively. Viral antigens: Chikungunya E1 (CHIKV-E1), Dengue serotype 1 VLP (DENV1-VLP), Ebola GP1 (EBOVKW95-GP1-100), Tick-borne Encephalitis NS1 (TBEV-NS1-100), and Zika lysate (ZIKV-LYS-100) were purchased from The Native Antigen Company (Oxford, UK). Cytomegalovirus (CMV) was kindly provided by Brigham & Women’s Hospital (Boston, USA). Influenza H1N1 HA (11055-VNAB), Influenza H1N1 NA (11058-VNAHC), and Respiratory syncytial virus (RSV) glycoprotein g (11070-V08B2) were purchased from Sino Biological (Beijing, China). Human immunodeficiency virus (HIV) gp120 (ab174070) was purchased from Abcam (Cambridge, USA).

Preparation of bacteria

Bacteria were subcultured in RPMI (Thermo Fisher Scientific, USA) 10 mM glucose to a McFarland of 0.5 (equivalent to 10⁸ CFU/ml) (Becton Dickinson, USA). Bacteria were grown to this logarithmic phase to ensure cell viability, and RPMI was used because it does not contain interfering MBL-binding nutrients, such as yeast extract. The culture was then split—live bacteria were kept on ice while the other half were fragmented. Fragmented bacterial PAMPs were generated using antibiotics or mechanical disruption. Antibiotic treatment included the appropriate use of one of the following: cefepime (NDC 25021-121-20), ceftriaxone (NDC 60505-6104-4), meropenem (NDC 63323-507-20), amikacin (NDC 0703-9040-03), gentamicin (NDC 63323-010-02), or vancomycin (NDC 0409-4332-49), at ≤1 mg/ml for ≥4 hours at 37°C 225 rpm. Mechanical disruption consisted of bead mill treatment at 30 Hz for 10 min using 0.1 mm zirconia/silica beads (BioSpec Products, USA) in a Mixer Mill MM 400 machine (Verder Scientific, Inc., USA). Testing by FcMBL ELLecSA was performed on titers of live bacteria at ≤10⁷ CFU/ml, and on the same concentration of bacteria after fragmentation. LPS endotoxin from Gram-negative bacteria was quantified using a limulus amebocyte lysate (LAL) assay ([Endosafe®] Charles River Laboratories, USA).

Preparation of fungi, viral antigens, parasites, and bacterial cell wall antigens

Fungi species were primarily subcultured in RPMI 10 mM glucose; however other media, such as potato dextrose broth (Teknova, USA), were used to facilitate growth. In these cases, the fungal cells were pelleted at 3,000 × g for 5 minutes at 22°C (Eppendorf 5424, USA), washed 3x in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, 5 mM CaCl₂, pH 7.4 (TBST 5 mM CaCl₂) (Boston BioProducts, USA) to remove residual growth media, and then resuspended in TBST 5 mM CaCl₂. Testing by FcMBL ELLecSA was performed on titers of live fungi at ≤10⁷ CFU/ml. Purified or inactivated viral antigens, parasites, and bacterial antigens were resuspended or diluted in TBST 5 mM CaCl₂ for testing directly by FcMBL ELLecSA. Trichomonas vaginalis was screened at 0.46 µg/mL and Plasmodium falciparum Circumsporozoite protein was screened at 50 µg/mL. Concentrations of viral antigens tested are indicated in the legend of Figure 7.

Antibodies

Anti-protein A (catalog number ab19483) was purchased from Abcam (Cambridge, USA).

Biological reagents

Fresh whole human blood (sodium heparin) was purchased from Research Blood Components, LLC. (Boston, USA), and normal single donor human urine (IR100007) was purchased from Innovative Research Inc. (Novi, USA).

FcMBL ELLecSA

The key metric used to quantify direct FcMBL binding to pathogen-associated molecular patterns (PAMPs) from bacteria, fungi, viral antigens, parasites, and bacterial cell wall antigens is a 96 well ELLecSA, which has been previously published²³. The assay uses FcMBL coated superparamagnetic beads (1 µm MyOne Dynabead [Thermo Fisher Scientific, USA]) where FcMBL, biotinylated at the N termini of the Fc protein using an N-terminal amino-oxy reaction, is coupled to streptavidin beads in an oriented array (Figure 1A). Each sample was screened using 100 µl or 200 µl of test sample added to 900 µl or 800 µl of assay solution respectively, which contains 5 µg of the FcMBL beads at 5 mg/ml and 10 mM glucose in TBST 5 mM CaCl₂ to total 1 ml (50 µl heparin is added if testing blood). PAMPs in the test sample are captured by FcMBL for 20 minutes at 22°C 950 rpm in a plate shaker (Eppendorf, USA). Using an automated magnetic-handling system (KingFisher^TM Flex [not shown]) (Thermo Fisher Scientific, USA), captured PAMPs are washed two times using TBST 5 mM CaCl₂, and detected with human MBL (Sino Biological, China) linked to horseradish peroxidase (MBL-HRP). Non-specific MBL-HRP is removed by 4 washes in TBST 5 mM CaCl₂, and PAMPs are quantified with 1-step ultra tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, USA). Finally, the reaction is quenched with 1 M sulfuric acid and results are read at the optical density 450 nm wavelength. Quantification of bound PAMPs is determined using a standard curve generated using yeast mannan (catalog number M3640, Sigma-Aldrich, USA) in TBST 5 mM CaCl₂—a known target for MBL (1 ng/ml mannan = 1 PAMP unit)⁴³. PAMP units are multiplied back by the dilution factor of the test sample volume to give PAMPs/ml. Previously, a receiver operating characteristic comparison was performed for a small pilot sepsis patient study in which sepsis blood was analyzed versus non-infected controls to determine an optimal ELLecSA threshold of 0.45 PAMP units²³. Therefore, in this study we define and report FcMBL binding to a sample as having ≥5 PAMPs/ml. To confirm specificity of FcMBL binding, a negative control (FcMBL null) was used alongside FcMBL in the ELLecSA. FcMBL null was engineered by introducing two residue mutations, E347A and N349A, into aktFcMBL (GenBank accession: KJ710775.1) to remove functional binding of the CRD of MBL. FcMBL null was purified and used to coat beads in the same fashion as FcMBL described above for direct comparison. FcMBL null beads did not support any binding to yeast mannan, and supported less than half of binding to S. aureus compared with FcMBL, as the Fc portion of FcMBL binds S. aureus protein A²² (Supplementary Figure 1)²⁹.

Statistical analysis

Data analyses on FcMBL binding to Gram-positive and Gram-negative live and fragmented bacterial isolates was performed using the statistical R language. Categorical variables are described as frequency (percentage). Comparisons between nominal dichotomous variables were performed with Pearson’s chi-square, when all contingency table cells were >5. Results were deemed as statistically significant when the null hypothesis could be rejected with >95% confidence. An unpaired two-tailed t-test was performed on FcMBL and FcMBL null binding to S. aureus using GraphPad Prism 7.0b (OS X). A p-value of < 0.05 was determined to be statistically significant. Dataset analysis is indicated in the figure legends.

Scanning electron microscopy

For visualization of live and fragmented bacteria on FcMBL beads, bacteria were captured with 128 nm FcMBL beads (Ademtech, France), spun down onto 13=-mm coverslips and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, USA) for 1 hour. Cover slips were incubated in 1% osmium tetroxide in 0.1 M sodium cacodylate (Electron Microscopy Sciences, USA) for 1 hour. Ascending grades of ethanol dehydrated the sample before being chemically dried with hexamethydisilazane (Electron Microscopy Sciences, USA). Samples were then placed in a desiccator overnight. Dried samples were mounted on aluminum stubs, sputter-coated with a thin layer of gold particles, and imaged using a Zeiss Supra55VP microscope.

Underlying data

Data for this study on the broad-spectrum capture of clinical pathogens using engineered Fc-mannose-binding lectin (FcMBL) enhanced by antibiotic treatment are available from OSF. DOI: https://doi.org/10.17605/OSF.IO/GW4X7²⁹.

Extended data

Supplementary figure 1. FcMBL and FcMBL null binding to S. aureus. 10⁶ CFU/ml live S. aureus without and with treatment of 1 µg/ml anti-protein A, detected by ELLecSA. Anti-protein A antibody blocks the Fc-mediated binding of FcMBL null but has no significant effect on FcMBL binding. **p-value < 0.01; *p-value < 0.05; unpaired two-tailed t-test; NS: not significant. DOI: https://doi.org/10.17605/OSF.IO/GW4X7²⁹.

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).