The contact system—a novel branch of innate immunity generating antibacterial peptides
2006; Springer Nature; Volume: 25; Issue: 23 Linguagem: Inglês
10.1038/sj.emboj.7601422
ISSN1460-2075
AutoresInga‐Maria Frick, Per Åkesson, Heiko Herwald, Matthias Mörgelin, Martin Malmsten, Dorit K. Nägler, Lars Björck,
Tópico(s)Pediatric health and respiratory diseases
ResumoArticle9 November 2006free access The contact system—a novel branch of innate immunity generating antibacterial peptides Inga-Maria Frick Corresponding Author Inga-Maria Frick Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Per Åkesson Per Åkesson Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Heiko Herwald Heiko Herwald Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Matthias Mörgelin Matthias Mörgelin Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Martin Malmsten Martin Malmsten Department of Pharmacy, Uppsala University, Uppsala, Sweden Search for more papers by this author Dorit K Nägler Dorit K Nägler Department of Clinical Chemistry and Clinical Biochemistry, University Hospital of Surgery-City, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Lars Björck Lars Björck Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Inga-Maria Frick Corresponding Author Inga-Maria Frick Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Per Åkesson Per Åkesson Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Heiko Herwald Heiko Herwald Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Matthias Mörgelin Matthias Mörgelin Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Martin Malmsten Martin Malmsten Department of Pharmacy, Uppsala University, Uppsala, Sweden Search for more papers by this author Dorit K Nägler Dorit K Nägler Department of Clinical Chemistry and Clinical Biochemistry, University Hospital of Surgery-City, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Lars Björck Lars Björck Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden Search for more papers by this author Author Information Inga-Maria Frick 1, Per Åkesson1, Heiko Herwald1, Matthias Mörgelin1, Martin Malmsten2, Dorit K Nägler3 and Lars Björck1 1Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden 2Department of Pharmacy, Uppsala University, Uppsala, Sweden 3Department of Clinical Chemistry and Clinical Biochemistry, University Hospital of Surgery-City, Ludwig-Maximilians-University, Munich, Germany *Corresponding author. Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, BMC, B14, S-221 84 Lund, Sweden. Tel.: +46 46 2228569; Fax: +46 46 157756; E-mail: [email protected] The EMBO Journal (2006)25:5569-5578https://doi.org/10.1038/sj.emboj.7601422 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Activation of the contact system has two classical consequences: initiation of the intrinsic pathway of coagulation, and cleavage of high molecular weight kininogen (HK) leading to the release of bradykinin, a potent proinflammatory peptide. In human plasma, activation of the contact system at the surface of significant bacterial pathogens was found to result in further HK processing and bacterial killing. A fragment comprising the D3 domain of HK is generated, and within this fragment a sequence of 26 amino acids is mainly responsible for the antibacterial activity. A synthetic peptide covering this sequence kills several bacterial species, also at physiological salt concentration, as effectively as the classical human antibacterial peptide LL-37. Moreover, in an animal model of infection, inhibition of the contact system promotes bacterial dissemination and growth. These data identify a novel and important role for the contact system in the defence against invasive bacterial infection. Introduction Antimicrobial peptides (AMPs) were originally isolated from human leukocytes (Zeya and Spitznagel, 1963), but later identified in invertebrates (Steiner et al, 1981) and cold-blooded vertebrates (Zasloff, 1987). AMPs represent an important branch of innate immunity. They are ubiquitously present at biological boundaries prone to infection, where they provide a rapid and nonspecific defence against potentially invasive microorganisms (for references, see Lehrer and Ganz, 1999; Zasloff, 2002; Bevins, 2003; Boman, 2003; Peschel and Sahl, 2006), and the therapeutic potential of AMPs in clinical medicine has recently been further emphasized (Mygind et al, 2005). A large number of AMPs have been identified, and molecules previously not regarded as AMPs have been found to have antimicrobial activity: chemokines (Cole et al, 2001), neuropeptides (Vouldoukis et al, 1996; Goumon et al, 1998; Shimizu et al, 1998; Kowalska et al, 2002; Brogden et al, 2005), peptide hormones (Mor et al, 1994; Kowalska et al, 2002) and C3a of complement (Nordahl et al, 2004). The contact system (for references, see Bhoola et al, 1992; Colman and Schmaier, 1997; Joseph and Kaplan, 2005) comprises three serine proteinases: coagulation factors XI and XII, and plasma kallikrein, and the nonenzymatic co-factor high molecular weight kininogen (HK). In plasma, HK circulates in complexes with factor XI or plasma kallikrein. The system is activated when these complexes (via HK) and factor XII interact with negatively charged surfaces. As a consequence, plasma kallikrein is activated in the HK complex, and HK is cleaved to release the proinflammatory peptide bradykinin (BK). Factor XI is also activated in its complex with HK, whereby the intrinsic pathway of coagulation is initiated. HK consists of six domains: domains 1–3 are cystatin-like, D4 contains the BK peptide, D5 mediates binding to negatively charged surfaces, and D6 is responsible for the binding of plasma kallikrein and factor XI (Colman and Schmaier, 1997). There are two forms of kininogen, HK and low molecular weight kininogen (LK), resulting from alternative splicing of a single gene (Kitamura et al, 1985). The two forms have an identical heavy chain consisting of domains D1–D3, which through the BK-containing D4 domain is connected with the light chains that are unique for HK and LK, respectively (Müller-Esterl et al, 1988). In contrast to HK, LK is not cleaved as a result of contact activation. Previous work has demonstrated that HK and the other components of the contact system can bind to the surface of several important bacterial pathogens, leading to the assembly and activation of the system (Ben Nasr et al, 1996; Herwald et al, 1998; Persson et al, 2000; Mattsson et al, 2001). In addition, the BK peptide and domain D5 of HK were reported to have antimicrobial activity (Kowalska et al, 2002; Brogden et al, 2005; Nordahl et al, 2005), and a number of other human proteins also contain sequences that are antimicrobial when tested as synthetic peptides (Andersson et al, 2004). Finally, work in many laboratories has demonstrated that proteolytic activity at the site of infection, degrades bacterial surface proteins and human proteins associated with the bacterial surface (for references, see Rasmussen and Björck, 2002). This information raised the question whether HK itself, or fragments of HK generated at bacterial surfaces through contact activation or through degradation by proteinases released at the site of infection, could have antibacterial activity. The results identify a previously unknown role for the contact system in innate immunity. Results Domain D3 of kininogens is antibacterial Purified intact HK at a concentration of 40 μg/ml and LK at 80 μg/ml (corresponds to half of their concentrations in human plasma) showed no activity when tested against Streptococcus pyogenes in a bactericidal assay. Earlier studies have shown that neutrophil elastase acts on proteinase-sensitive regions in kininogens, whereby smaller fragments, such as the D3 and D5 domains, are generated (Vogel et al, 1988; Nordahl et al, 2005). HK was therefore incubated with supernatants from activated neutrophils or with purified human elastase, and cleavage products were separated by Tricine-SDS–PAGE for the detection of degradation products in the low molecular range. In both cases, HK was degraded into several fragments (Figure 1A, stain), and in Western blots a prominent band of approximately 15 kDa reacted with antibodies raised against a peptide sequence (NAT26) within the D3 domain of HK (Figure 1A, blot). Peptide sequencing by MALDI-TOF/TOF identified the 15 kDa band within the D3 domain, with a COOH-terminal extension containing the BK sequence. The peptides used for identification of the 15 kDa band are indicated in the schematic representation of the heavy chain of HK (D1–D3) shown in Figure 1B. In contrast to intact HK, the elastase-cleaved HK killed S. pyogenes, whereas elastase alone had no effect (data not shown). Figure 1.Proteolytic cleavage of HK in vitro generates antibacterial peptides. (A) HK (6 μg) was digested with supernatant from activated human neutrophils (1 μl) or purified human elastase (2.5 mU) for 1.5 h at 37°C, and subjected to Tricine-SDS/PAGE. One gel was stained with Coomassie blue (stain), and one was transferred to a PVDF membrane and probed with polyclonal antibodies raised against the NAT26 peptide derived from domain D3 of HK (blot). Lane 1: native HK (5 μg); lane 2: HK incubated with supernatant from activated neutrophils; lane 3: HK cleaved by elastase. The band at 15 kDa, marked with an arrow, was cut out and subjected to MALDI-TOF/TOF peptide sequencing. (B) Schematic representation of the heavy chain of HK (D1–D3). NAT26, BK and the peptides used for identification of the 15 kDa band by MALDI-TOF/TOF are indicated. Numbers refer to amino-acid residue positions (bars are not in scale). (C) Purified D3 domain (1 μg) was subjected to Tricine-SDS/PAGE analysis. One gel was stained with Coomassie Blue (stain), and one was transferred to a PVDF membrane and probed with antibodies against NAT26 (blot). (D) S. pyogenes AP1 bacteria (2 × 106 CFU/ml) were incubated for 1 h at 37°C in buffer with various concentrations of recombinant D3. Following incubation, appropriate dilutions of the mixtures were plated on TH agar plates. Plates were incubated at 37°C over night and CFUs were determined. The bars represent the mean±s.e.m. of at least three experiments. Download figure Download PowerPoint The D3 domain was recombinantly expressed in Escherichia coli and affinity purified using antibodies against a peptide (NAT26) sequence in the central part of D3. On SDS–PAGE purified D3 migrates as a 15 kDa band (Figure 1C), similar to the size of the HK fragment generated by incubation with supernatants from activated neutrophils or by treatment with purified elastase (Figure 1A, blot). The purified D3 was tested against S. pyogenes strain AP1 in a bactericidal assay, and a dose-dependent killing of the target bacteria was obtained (Figure 1D). The results show that the cleavage of HK by neutrophil proteinase(s) generates a D3-related peptide, and that D3 has antibacterial activity. Contact activation at bacterial surfaces generates antibacterial domain D3-related fragments of HK Several pathogenic bacteria such as S. pyogenes, Staphylococcus aureus, and Salmonella activate the contact system, resulting in the proteolytic cleavage of HK and the release of BK (for references, see Herwald et al, 2003). To investigate whether HK is further degraded at the bacterial surface, isolates of S. pyogenes (AP1), S. aureus (Cowan I) and Salmonella (SR11B) were grown to mid exponential phase, and separately incubated with human plasma for 1 h at 37°C. Proteins bound to the bacteria were eluted by low pH, followed by Western blot analysis using antibodies against the NAT26 peptide from domain D3. Multiple immuno-reactive fragments, including a peptide of approximately 14–15 kDa, were released from the surface of all strains (Figure 2A). When the contact system is activated, native HK (120 kDa) is cleaved by plasma kallikrein into a heavy chain of approximately 65 kDa, containing the D3 domain, and a light chain of approximately 55 kDa. HK eluted from the surface of AP1 was fully cleaved into its heavy and light chain. In contrast, a portion of HK bound to the surface of Cowan I and SR11B remained uncleaved (Figure 2A). Moreover, as judged from the presence of immuno-reactive bands in the high molecular weight range (45–66 kDa), the heavy chain of HK was not completely processed at the bacterial surface of the investigated strains (Figure 2A). The smaller bands (approximately 13–17 kDa) are D3-related fragments containing NAT26 epitopes, produced by further cleavage of HK (Figure 2A). The band pattern in the 13–17 kDa range differs between the three species, indicating variations in HK degradation and/or in the interactions between the generated fragments and the bacterial surfaces. Contact activation on bacterial surfaces is an immediate event as judged from incubations of AP1 bacteria in plasma for various time points (5–60 min). Already after 5 min of incubation, native HK was fully cleaved generating the fragments of 45–66 and 22 kDa (data not shown). Over time the smaller D3-related fragments (22 and 13–17 kDa) accumulated at the bacterial surface, as a result of further cleavage of HK. The band of 66 kDa in the plasma sample (Figure 2A, far left lane) represents LK. LK has the same heavy chain as HK and contains the D3 domain. However, LK is not part of the contact system and is not cleaved when the system is activated. Furthermore, Western blot analysis of wound fluid from a patient with a leg ulcer infected with S. aureus, showed bands in the low molecular range reacting with antibodies against NAT26, demonstrating that D3-related fragments are generated in vivo (Figure 2B). Figure 2.Domain D3-related antibacterial fragments of HK are generated at bacterial surfaces in plasma environment. (A) Suspensions of S. pyogenes strain AP1, S.aureus strain Cowan I and Salmonella strain SR11B in 1 ml of PBS (2 × 109 CFU/ml) were separately incubated with undiluted human plasma (1:1) for 1 h at 37°C. Bacteria were pelleted and washed with PBS. Plasma proteins bound to the bacteria were eluted with 0.1 M glycine buffer, pH 2.0, followed by TCA precipitation. This material was subjected to Tricine-SDS/PAGE followed by Western blot. Plasma diluted 1:25 and eluted material from the three bacterial strains was probed with anti-NAT26 antiserum. (B) Fluid from a wound infected with S. aureus was subjected to Tricine-SDS/PAGE followed by Western blot, and probed with anti-NAT26 antiserum. (C) Plasma preincubated with AP1 bacteria for indicated time points was subjected to anti-NAT26 Sepharose, and bound proteins were eluted with glycine buffer, pH 2.0. The samples were neutralized (pH 7.5) with 1 M Tris. S. pyogenes AP1 bacteria (2 × 106 CFU/ml) were incubated with equal volumes of eluates from the various time points for 1 h at 37°C, and CFUs were determined. Neutralized glycine buffer was used as a control. The bars represent the mean±s.e.m. of at least three experiments. Data representing the bactericidal activity of the eluates were statistically analysed using the Mann–Whitney U-test. (D) Plasma was preincubated with AP1 bacteria for 60 min at 37°C and subjected to anti-NAT26 affinity chromatography as described in (C). The protein content of the affinity purified material was determined by Bradford analysis and different amounts were tested for killing of AP1 bacteria. The bactericidal activity of LL-37 was used as a positive control. The bars represent the mean±s.e.m. of at least three experiments. Data were statistically analysed using the Mann–Whitney U-test. Download figure Download PowerPoint In plasma, contact activation on bacterial surfaces could also generate soluble HK fragments not bound to the bacteria. Plasma samples that had been incubated with AP1 bacteria for various time points were therefore subjected to affinity chromatography on anti-NAT26-Sepharose. When tested in the bactericidal assay, this purified material had antibacterial activity (Figure 2C). Maximum effect was obtained with material purified after 60 min of bacterial incubation with plasma, emphasizing the importance of the smaller D3-related fragments in bacterial killing. Plasma was again preincubated with AP1 bacteria for 1 h at 37°C, and subjected to affinity purification on anti-NAT26 Sepharose. This material contained HK fragments similar to those eluted from the surface of AP1 bacteria (see Figure 2A), including the 13–17 kDa D3-related peptides, and the material showed a dose-dependent killing of AP1 bacteria (Figure 2D). The data demonstrate that contact activation at the surface of pathogenic bacteria in plasma, results in the production of domain D3-related peptides that are antibacterial, suggesting that the contact system plays a role in the defence against invasive bacteria. Several human proteins contain heparin-binding sequences, and peptides spanning these sequences are often antibacterial (Andersson et al, 2004). The D5 domain of HK has such a sequence and is antibacterial (Nordahl et al, 2005). However, there are no data published suggesting that D5 is released during contact activation, and when antibodies against D5 were used in the Western blot experiment described above (Figure 2A), only intact HK but no D5-related fragments were identified (data not shown). It was reported that BK and other endogenous peptides (substance P and neurotensin) are antimicrobial in vitro (Kowalska et al, 2002), suggesting that BK could contribute to the antibacterial effect of contact activation. Complete cleavage of HK in plasma would result in a BK concentration of approximately 1 μg/ml. However, even at 20 μg/ml, BK had no effect in the bactericidal assay, which together with the short half-life of BK (<1 min) (Decarie et al, 1996) makes it highly unlikely that BK is antibacterial in vivo. In conclusion, the results suggest that domain D3-related peptides are solely responsible for the antibacterial effect of contact activation. Inhibition of bacterial growth by human plasma depends on contact activation The finding that antibacterial domain D3-related HK fragments are generated in plasma indicated that bacterial growth could be inhibited by plasma. The S. pyogenes, S. aureus and Salmonella strains described above (AP1, Cowan I and SR11B) are known to bind HK and activate the contact system (Ben Nasr et al, 1996; Herwald et al, 1998; Mattsson et al, 2001). However, strains of Enterococcus faecalis and E. coli strains not expressing fibrous surface proteins called curli (Olsén et al, 1989) do not have this property (Ben Nasr et al, 1996; Herwald et al, 1998). The growth of the three HK-binding strains mentioned above, a strain of E. faecalis (2374) and a non-curliated E. coli strain (B1351), was therefore investigated in plasma and in conventional TH medium. Figure 3A shows that the growth of bacteria that activate the contact system is dramatically reduced in plasma as compared to TH medium. In contrast, the E. faecalis and E. coli strains showed similar growth curves in plasma and TH medium (Figure 3B). Moreover, when grown in HK-free plasma, S. pyogenes AP1 bacteria multiplied significantly faster than in normal plasma (Figure 3C). Activation of the contact system is blocked by the synthetic peptide H-D-Pro-Phe-Arg-CMK, which is a specific inhibitor of FXII and plasma kallikrein (Ghebrehiwet et al, 1983). Addition of this inhibitor to human plasma, at concentrations known to block contact activation (Persson et al, 2000), enhanced the growth of AP1 bacteria (Figure 3C). The inhibitor also stimulated growth of Salmonella and S. aureus activating the contact system (data not shown). Taken together, the results demonstrate that contact activation at bacterial surfaces inhibits bacterial growth, and that the contact system is part of innate immunity. Figure 3.Contact activation in plasma inhibits bacterial growth. Various bacterial strains were grown in TH medium or in human plasma at 37°C. The optical density at 620 nm was measured at indicated time points. The multiplication factors at various time points are given. (A) Bacteria known to activate the contact system in plasma: S. pyogenes strain AP1 (□, ▪), S. aureus strain Cowan I (▵, ▴), and Salmonella strain SR11B (○, •). White symbols represent growth in TH medium and filled symbols represent growth in plasma. (B) Bacteria that do not activate the contact system in plasma: E. coli strain B1351 (□, ▪) and E. faecalis strain 2374 (○, •). White symbols represent growth in TH medium and filled symbols represent growth in plasma. (C) S. pyogenes AP1 bacteria were cultivated in undiluted human plasma (□), in human kininogen-free plasma (▪), or in human plasma supplemented with FXII/kallikrein inhibitor (100 μg/ml) (•), at 37°C. Bacterial growth was followed by plating appropriate dilutions of the bacterial cultures, at the indicated time points, on TH agar plates. Plates were incubated at 37°C over night, CFUs were determined, and the multiplication factors were calculated. Experiments were repeated three times and a representative experiment is shown. Download figure Download PowerPoint Contact activation inhibits bacterial growth in vivo To investigate the role of contact activation in vivo, we used a mouse model of S. pyogenes infection (Nordahl et al, 2004). In these experiments, mice were intraperitoneally injected with PBS or the FXII/kallikrein inhibitor mentioned above, at a dose where contact activation is completely blocked in mouse plasma, as judged by pronounced prolongation of the activated partial thromboplastin time. This was followed by intraperitoneal injection of S. pyogenes bacteria of the AP1 strain, and the dissemination of bacteria to the spleen after 18 h was determined. To demonstrate the effect of the inhibitor the bacterial load had to be carefully titrated. Thus, when small doses (50 × 103 colony-forming unit (CFU)/mouse) of bacteria were administered, no or very few bacteria were detected in the spleens, whereas high doses of bacteria (850 × 103 CFU/mouse) resulted in a massive spread to the spleens in both groups. However, at a bacterial load of 450 × 103 CFU/mouse, significantly higher number of bacteria were detected in the animals treated with the FXII/kallikrein inhibitor, as compared to the PBS control animals (P=0.024) (Figure 4). These results show that a functional contact system contributes to the defence against invasive bacteria in vivo. Figure 4.Inhibition of the contact system promotes bacterial growth in vivo. Female Balb/c mice were injected i.p. with PBS or FXII/kallikrein inhibitor (1 mg/animal), followed by i.p. injection of S. pyogenes AP1 bacteria (450 000 CFU). The mice were killed 18 h after injection and the total number of CFUs in the spleens was determined. The number of CFUs was significantly higher in animals treated with the inhibitor, compared to the control group (P=0.024, n=12). The P-value was determined by using the Mann–Whitney U-test. Bars represent the median value in each group. Download figure Download PowerPoint A central region of domain D3 is antibacterial In order to localize the antibacterial region of domain D3, six peptides spanning the domain were synthesized (Table I) and analysed in the bactericidal assay. Initial testing with S. pyogenes AP1 bacteria revealed that only the NAT26 peptide from the central part of D3 was bactericidal (Figure 5A) at the concentration used (12–13 μM). A partial antibacterial activity was recorded for the other peptides, except for KKY30 that had no effect (Figure 5A). NAT26 has physico-chemical properties typical of many well-characterized AMPs; small size, cationic charge (pI of 10.1 and net positive charge of +5), and a relatively high predicted helical content. When tested against the strains used previously in this study, plus a group G streptococcal strain (G41), NAT26 was bactericidal at 12.8 μM for all strains except Salmonella SR11B (Table II). However, also this strain was partially killed. LL-37 is a classical and significant human antibacterial peptide (Bals and Wilson, 2003). Notably, the bactericidal activity of NAT26 against S. pyogenes bacteria was comparable to LL-37 at low salt concentration, but superior to LL-37 at physiological salt concentration (Figure 5B). Figure 5.Mapping of the bactericidal region in domain D3. (A) S. pyogenes AP1 bacteria (2 × 106 CFU/ml) were incubated for 1 h at 37°C in buffer with synthetic peptides, covering various regions of the D3 domain (Table I), at a concentration of 12–13 μM. Following incubation, appropriate dilutions of the mixtures were plated on TH agar plates. Plates were incubated at 37°C over night and CFUs were determined. The bars represent the mean±s.e.m. of at least three experiments. (B) The bactericidal effect against AP1 bacteria of NAT26 (□) and LL-37 (○) at indicated concentrations in buffer without (left) or with 0.15 M NaCl (right). Experiments were repeated at least three times, and representative experiments are shown. Download figure Download PowerPoint Table 1. Synthetic peptides derived from the D3 domain used in this study Peptidea Sequenceb Positionc GKD25 GKDFVQPPTKICVGCPRDIPTNSPE 253–277 GCP28 GCPRDIPTNSPELEETLTHTITKLNAEN 266–293 NAT26 NATFYFKIDNVKKARVQVVAGKKYFI 294–319 KKY30 KKYFIDFVARETTCSKESNEELTESCETKK 315–344 RET27 RETTCSKESNEELTESCETKKLGQSLD 324–350 LDC27 LDCNAEVYVVPWEKKIYPTVNCQPLGM 349–375 a Peptides are identified by their first three NH2-terminal residues using the one-letter code, followed by the total number of residues constituting the peptide. b The one-letter code for amino-acid residues is used. c The amino-acid positions in the HK sequence. Table 2. Antibacterial activity of NAT26 against different bacterial speciesa Killing of bacteria (%) Peptide (μM) S. pyogenes AP1 GGSb G41 S. aureus Cowan I Salmonella SR11B E.coli B1351 E. faecalis 2374 12.8 100 100 100 90.1±17.9 100 100 6.4 100 100 100 76.3±33.0 93.3±11.5 100 0.64 100 47.5±30.5 87.3±15.4 3.8±5.3 37.6±9.5 89.3±7.6 0.064 6.0±6.6 12.1±17.5 15.6±16.5 3.3±5.7 2.3±2.6 1.1±1.8 a Bacteria (2 × 106 CFU/ml) were incubated with indicated concentrations of NAT26 for 1 h at 37°C. Appropriate dilutions were plated on TH agar plates, incubated over night at 37°C, and CFUs were determined. b Group G Streptococcus. The effect of NAT26 on S. pyogenes was analysed also by electron microscopy. AP1 bacteria grown in TH or in plasma over night were incubated with F(ab′)2 fragments of IgG antibodies against NAT26, labelled with colloidal gold. Bacterial cells grown in TH were also incubated with the NAT26 peptide for 1 h prior to the addition of the antibody fragments. Following negative staining, bacteria were analysed by electron microscopy. In contrast to bacteria grown in TH (Figure 6A and B), the cell wall architecture of bacteria grown in plasma (Figure 6C and D), or incubated with the NAT26 peptide (Figure 6E and F), was disintegrated as shown by ejected cytoplasmic material and membrane blebs. Furthermore, NAT26-containing peptides were detected at the bacterial surface by gold-labelled F(ab′)2 antibody fragments against NAT26 (Figure 6D and F, insets). Figure 6.Visualization of the antimicrobial affect of NAT26. AP1 bacteria were grown in TH broth (A, B), or in human plasma (C, D) over night at 37°C. Bacteria were washed and incubated with gold-labelled anti-NAT26 F(ab′)2-fragments. An aliquot of the bacteria grown in TH was incubated for 1 h at 37°C with 3.2 μM NAT26 (E, F) prior to the incubation with antibodies. Following negative staining, samples were analysed by electron microscopy. (A, C, E) Overviews at low magnification. The scale bar represents 2 μm. (B, D, F) Selected areas at higher magnification. The scale bar represents 200 nm. Arrows point to ejected cytoplasmic material due to exposure to plasma (C, D) or purified NAT26 (E, F). Note the intact bacterial membrane structure in (B) as compared to the disintegrated membranes in (D) and (F). Arrowheads in the insets point to gold-labelled F(ab′)2-fragments directed against NAT26. Scale bar, 20 nm. Download figure Download PowerPoint A liposome model was used to further investigate the effect of NAT26 on membranes. NAT26 displayed a high capacity to induce leakage in anionic liposomes at moderate ionic strength (Figure 7A), and leakage was
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