In this study, we focused on the clinical GGS isolate G45 expressing FOG and PG (G45WT) and isogenic FOG and PG deletion mutants of G45 (G45ΔFOG and G45ΔPG, respectively). PG is mainly responsible for the binding of IgG and albumin to the surface of GGS, whereas fibrinogen is bound to FOG (21
). These binding properties were used to investigate the surface expression of the two proteins during growth. Samples of G45WT collected at early, mid-log, and stationary growth phase were subjected to a binding assay in which the bacteria were incubated with either 125
I-labeled fibrinogen or albumin. In all samples, the bacteria bound ~50–55% of the two added radioactive probes, demonstrating that FOG and PG were expressed during the entire growth phase (data not shown). shows electron micrographs following negative staining of G45WT (A
), G45ΔFOG (B
), and G45ΔPG (C
) from the logarithmic growth phase, where the hair-like protrusions typical of M and M-like proteins, missing in G45ΔFOG but present in G45WT and G45ΔPG, represent FOG. PG with its globular and compact structure is not visualized in these micrographs.
Electron micrographs of G45WT (A) and mutants G45ΔFOG (B) and G45ΔPG (C) showing hair-like protrusions representing FOG in G45WT and G45ΔPG but not in G45ΔFOG.
Scale bar = 0.5 μm.
Next, the binding of FOG and PG to the proteins of the contact system was tested in slot binding experiments (A
). The contact factors were applied to Immobilon membranes, which were probed with radiolabeled FOG or PG. FOG bound to FXII and FXI, whereas PG showed affinity for HK, PK, FXI, and FXII, i.e.
all contact system components. When, instead, FOG and PG were applied to the membranes and radiolabeled HK was used as the probe, both proteins interacted with HK (data not shown). This kind of inconsistency, not uncommon in slot binding experiments and probably reflecting different exposure of immobilized proteins to the probe, makes it necessary to test possible protein-protein interactions with additional methods (see below). Because FXI and PK circulate in complex with HK (31
), the indicated binding of FXII and HK by FOG and PG suggested that both bacterial proteins can recruit the entire contact system to the bacterial surface. To further confirm and characterize the interactions between HK and the bacterial proteins (schematic representations of FOG, PG, and the fragments of the two proteins tested for HK binding are shown in B
), plasmon resonance experiments were performed (C
). Immobilized HK bound FOG1-D (amino acids 1–557) and FOG1-C (amino acids 1–493) with affinity constants of 6.1 and 14 nm
, respectively, whereas another FOG fragment, FOG1-B (amino acids 1–278), had no affinity for HK on the BIAcore chip. The affinity constant for the interaction between 35-kDa PG and HK was 0.92 nm
. In contrast, an IgG-binding fragment of PG (17 kDa) located in the C-terminal half showed no interaction with HK, suggesting that HK binding is located more C-terminally in FOG than in PG. Electron microscopy analyses following negative staining experiments were performed to visualize the proteins and the complexes between HK·FOG and HK·PG (). In line with the binding experiments described above, HK (red
) was associated with one end of the rod-shaped FOG (A
) and with the 35-kDa fragment of PG (B
). No complexes were formed with the 17-kDa PG fragment (C
). To investigate whether HK interacts also with FOG and PG at the bacterial surface, a number of clinical GGS isolates, including G45WT and its mutants G45ΔFOG and G45ΔPG, were tested for binding of 125
I-labeled HK (A
). The results show that HK had affinity for all of the strains and that the binding of HK was reduced in the strains lacking either FOG (G45ΔFOG and G148) or PG (G45ΔPG), suggesting that HK binding is a general property of GGS and that FOG and PG are responsible for the interaction. Whether the interaction data described above, indicating a more C-terminal binding of HK to FOG, mean that HK bound to FOG is located closer to the bacterial cell wall than HK in complex with PG remains an open question. Because of its fibrous structure (, A
, and A
), FOG protrudes farther from the bacterial cell wall than PG.
FIGURE 2. FOG and PG interact with contact system proteins.
A, various amounts of contact system components were applied to PVDF membranes, which were probed with 125I-labeled 35-kDa PG or FOG1-C. HSA and fibrinogen were included as positive controls for PG and (more ...)
FIGURE 3. Shown are electron micrographs after negative staining of HK in complex with FOG1-C (A) and 35-kDa PG (B). HK incubated with 17-kDa PG did not form any complex (C). The protein complexes visualized in the left panels are shown in pseudocolors in the (more ...)
FIGURE 4. A, isolates of GGS bind HK. Bacteria were grown overnight and incubated with 125I-labeled HK. After washing, the radioactivity of the pellets was measured. The values (percent of added radioactivity) represent the mean ± S.D. of at least three (more ...)
Previous work has shown that several pathogenic bacteria such as GAS, Staphylococcus aureus
, Escherichia coli
, and Bacteroides
) and Fusobacterium necrophorum
) bind and assemble HK and the other contact factors at their surface, leading to contact activation (for additional references, see Ref. 10
). The effect of G45WT, G45ΔFOG, and G45ΔPG on the coagulant FXI-dependent branch of the contact system was investigated in an aPTT assay. In this assay, bacteria are incubated with plasma, and after centrifugation, the clotting time, induced by kaolin, is measured in the supernatant. If procoagulant contact factors are bound to and activated at the bacterial surface, the resulting effect is a combination of binding and depletion of FXI and FXII, prolonging the aPTT, and FXI activation, shortening the aPTT. To more clearly define our experimental system, FXI-deficient plasma was employed. Bacteria were first incubated in normal plasma, followed by washing and reincubation in FXI-deficient plasma, in which the aPTT was measured. In this case, a decreased aPTT will require both binding and activation of FXI, and the results in B
show that G45WT and the two mutants shortened the aPTT compared with the control (FXI-deficient plasma + kaolin). These data and the experiments showing that FOG and PG bound FXI and FXII (A
) suggest that both surface proteins activate the intrinsic pathway of coagulation.
When the proinflammatory branch of the contact system is activated, native HK (120 kDa) is cleaved by PK into a heavy chain (65 kDa) and a light chain (55 kDa), and the nonapeptide BK is released (9
). This activation process was studied at the surface of GGS incubated with human plasma using a chromogenic assay in which the enzymatic activity of activated contact factors is determined by hydrolysis of a chromogenic substrate. Following incubation in plasma, bacteria were washed and incubated with the substrate, and the release of the chromophore was measured photometrically. Compared with G45WT, the FOG mutant exhibited reduced activity, whereas the G45ΔPG mutant, devoid of PG but expressing FOG, was not affected (A
). Activation of the contact system was blocked by the synthetic peptide H
-Pro-Phe-Arg-CMK, a specific FXII/PK inhibitor (34
), which was used as a control in these experiments (A
). The results show that the assay indeed measures contact activation and that FOG at the bacterial surface, in contrast to PG, activates the proinflammatory branch of the system. A consequence of the enzymatic activity observed in the chromogenic assay would be the release of the proinflammatory BK peptide. Thus, G45 bacteria and the isogenic mutants were incubated in plasma, and the BK release was measured by ELISA. Although G45WT and G45ΔPG caused a substantial release of BK, the amount of BK release caused by G45ΔFOG was reduced (B
). In the presence of the FXII/PK inhibitor H
-Pro-Phe-Arg-CMK, the BK release was completely abolished and similar to the negative control.
FIGURE 5. FOG, but not PG, activates the proinflammatory branch of the contact system.
A, analysis of contact activation by G45WT, G45ΔFOG, and G45ΔPG in human plasma using a chromogenic assay. Mid-log bacteria were incubated with plasma with or (more ...)
Previous work has shown that further processing of the heavy chain of HK upon contact activation generates smaller fragments, some of which contain the NAT-26 peptide from domain D3, and in contrast to intact HK, these fragments, as well as the synthetic NAT-26 peptide, are antibacterial. The antibacterial effect of NAT-26 against GAS (strain AP1) is more potent than that of LL-37 at physiological salt concentration and similar at lower salt concentrations (26
). Overlapping synthetic peptides covering domain D3 were analyzed in slot binding experiments using radiolabeled FOG or PG as the probe. Both streptococcal proteins showed affinity for the NAT-26 peptide but did not interact with the other peptides (D
). After incubation in 50% plasma, NAT-26-containing fragments were generated at the surface of G45WT and the two mutants as demonstrated by Western blot analyses of material eluted from the bacteria by low pH. However, the smaller NAT-26-containing fragments could not be eluted from G45ΔFOG bacteria following plasma incubation (C
). In 10% plasma, which may be more representative of inflammatory exudation, the processing of NAT-26-containing HK fragments into smaller peptides in the presence of FOG was confirmed (data not shown). Because NAT-26 has affinity for both FOG and PG, this suggests that smaller antibacterial NAT-26-containing HK fragments are not produced at the surface of G45ΔFOG, i.e.
PG is insufficient and FOG is required for a more complete activation of the contact system generating these peptides.
It has been reported that antibacterial NAT-26-containing fragments of HK are produced at the surface of GAS (26
). The molecular mass of these fragments is in the range of 13–17 kDa, which corresponds well with the peptides identified at the surface of FOG-expressing GGS (C
). To investigate whether FOG or PG could protect the bacteria against the NAT-26 peptide, G45WT, G45ΔFOG, and G45ΔPG were incubated with NAT-26 at different concentrations. The results demonstrate that the mutant lacking PG was more susceptible to killing by NAT-26 (A
). Apart from being attached to the bacterial cell wall, FOG and PG are also released into the growth medium (21
). Soluble FOG and PG both blocked (with PG more efficiently than FOG) the killing of G45ΔPG bacteria by NAT-26 (B
). In , electron microscopy was employed to study the morphology of G45WT and the two G45 mutants following incubation with NAT-26 at 2 μm
. The micrographs show that among the three isolates, the cell wall architecture of G45ΔPG bacteria was more disintegrated with ejected cytoplasmic material and membrane blebs. Combined, the results demonstrate that PG, in solution or associated with the bacterial cell surface, protects GGS against the killing activity of NAT-26.
FIGURE 6. PG protects GGS against antibacterial activity of NAT-26.
A, mid-log G45WT, G45ΔFOG, and G45ΔPG bacteria were incubated with various concentrations of NAT-26, and survival was determined by plating. Mean values and the range of three experiments (more ...)
Visualization of antibacterial effect of NAT-26. Electron micrographs show untreated G45WT and NAT-26-treated G45WT, G45ΔFOG, and G45ΔPG bacteria. Scale bar = 1 μm.
In conclusion, previous investigations have demonstrated that several bacterial pathogens activate the contact system, inducing coagulation and inflammation at the site of infection. The consequence(s) of this response for the bacteria is not clear. An inflammatory response will initiate host defense mechanisms such as complement activation and recruitment of phagocytes, and the fact that activation of the contact, complement, and coagulation systems generates antibacterial peptides (26
) should also be detrimental. On the other hand, a controlled induction of inflammation and coagulation could be advantageous for bacteria colonizing an epithelial surface. Increased vascular permeability will cause influx of plasma rich in nutrients and could facilitate spread of the infection, whereas the formation of a clot may promote adhesion and provide a protective shield. It is difficult to anticipate which of these contradictory effects prevail at different phases of infection.
The main finding of this study is that FOG and PG activate the contact system but also inhibit (PG) the antibacterial activity of peptides resulting from the activation. A question raised by the present data concerns the degree of inflammation in relation to the severity of streptococcal infection. Although invasive streptococcal infections and rheumatic fever kill hundreds of thousands of individuals annually, with GAS alone at >0.5 million (39
), it should be stressed that the vast majority of streptococcal infections are uncomplicated superficial cases of pharyngitis and skin infections and that asymptomatic colonization is even more common. A characteristic feature of severe invasive streptococcal infections is a systemic and massive inflammation, often connected with bleeding disorders, and in relation to the contact system, it is of interest that low levels of FXII and a prolonged aPTT are often seen in patients with septic shock (40
). It is unlikely that the rare but clinically highly significant condition with streptococci growing in the bloodstream represents a habitat to which the bacteria have adapted. In comparison, a fine-tuned and well controlled local induction of inflammation at the site of infection appears more adequate, a notion supported by this investigation.