Inga-Maria Frick

Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37

Effectors of the innate immune system, the anti‐bacterial peptides, have pivotal roles in preventing infection at epithelial surfaces. Here we show that proteinases of the significant human pathogens Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis and Streptococcus pyogenes, degrade the antibacterial peptide LL‐37. Analysis by mass spectrometry of fragments generated by P. aeruginosa elastase in vitro revealed that the initial cleavages occurred at Asn‐Leu and Asp‐Phe, followed by two breaks at Arg‐Ile, thus inactivating the peptide. Proteinases of the other pathogens also degraded LL‐37 as determined by SDS‐PAGE. Ex vivo, P. aeruginosa elastase induced LL‐37 degradation in human wound fluid, leading to enhanced bacterial survival. The degradation was blocked by the metalloproteinase inhibitors GM6001 and 1, 10‐phenantroline (both of which inhibited P. aeruginosa elastase, P. mirabilis proteinase, and E. faecalis gelatinase), or the inhibitor E64 (which inhibited S. pyogenes cysteine proteinase). Additional experiments demonstrated that dermatan sulphate and disaccharides of the structure [ΔUA(2S)‐GalNAc(4,6S)], or sucroseoctasulphate, in‐hibited the degradation of LL‐37. The results indicate that proteolytic degradation of LL‐37 is a common virulence mechanism and that molecules which block this degradation could have therapeutic potential.

The contact system--a novel branch of innate immunity generating antibacterial peptides.

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.

Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial alpha-defensin

Defensins represent an evolutionarily conserved group of small peptides with potent antibacterial activities. We report here that extracellular proteinases secreted by the human pathogens Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus pyogenes release dermatan sulphate by degrading dermatan sulphate‐containing proteoglycans, such as decorin. Dermatan sulphate was found to bind to neutrophil‐derived α‐defensin, and this binding completely neutralized its bactericidal activity. During infection, proteoglycan degradation and release of dermatan sulphate may therefore represent a previously unknown virulence mechanism, which could serve as a target for novel antibacterial strategies.

The dual role of the contact system in bacterial infectious disease

Hemostasis is a sensitive and tightly regulated process, involving the vascular endothelium and blood cells as well as factors of the coagulation and fibrinolytic cascades. Over the last four decades evidence has accumulated that during infection, inflammatory mediators from the microbe and/or host are capable to modulate the equilibrium between the procoagulant and anticoagulant status of the host. Dependent on the mode of activation, these changes can cause either local or systemic inflammatory reactions that may be beneficial or deleterious to the human host. The present review aims to present the state of the art with respect to the role of the contact system (also known as the intrinsic pathway of coagulation or the kallikrein/kinin system) in innate immunity and systemic inflammatory reactions.

Crystal structure and biological implications of a bacterial albumin binding module in complex with human serum albumin

Many bactericide species express surface proteins that interact with human serum albumin (HSA). Protein PAB from the anaerobic bacterium Finegoldia magna (formerly Peptostreptococcus magnus) represents one of these proteins. Protein PAB contains a domain of 53 amino acid residues known as the GA module. GA homologs are also found in protein G of group C and G streptococci. Here we report the crystal structure of HSA in complex with the GA module of protein PAB. The model of the complex was refined to a resolution of 2.7 Å and reveals a novel binding epitope located in domain II of the albumin molecule. The GA module is composed of a left-handed three-helix bundle, and residues from the second helix and the loops surrounding it were found to be involved in HSA binding. Furthermore, the presence of HSA-bound fatty acids seems to influence HSA-GA complex formation. F. magna has a much more restricted host specificity compared with C and G streptococci, which is also reflected in the binding of different animal albumins by proteins PAB and G. The structure of the HSA-GA complex offers a molecular explanation to this unusually clear example of bacterial adaptation.

Virulent aggregates of Streptococcus pyogenes are generated by homophilic protein-protein interactions.

Many strains of the important human pathogen Streptococcus pyogenes form aggregates when grown in vitro in liquid medium. The present studies demonstrate that this property is crucial for the adherence, the resistance to phagocytosis and the virulence of S. pyogenes. A conserved sequence of 19 amino acid residues (designated AHP) was identified in surface proteins of common S. pyogenes serotypes. This sequence was found to promote bacterial aggregation through homophilic protein–protein interactions between AHP‐containing surface proteins of neighbouring bacteria. A synthetic AHP peptide inhibited S. pyogenes aggregation, reduced the survival of S. pyogenes in human blood and attenuated its virulence in mice. In contrast, mutant bacteria devoid of surface proteins containing AHP‐related sequences did not aggregate or adhere to epithelial cells. These bacteria are also rapidly killed in human blood and show reduced virulence in mice, underlining the pathogenic significance of the AHP sequence and S. pyogenes aggregation.

The CXC Chemokine MIG/CXCL9 Is Important in Innate Immunity against Streptococcus pyogenes.

Pharyngitis caused by Streptococcus pyogenes is one of the most common bacterial infections in humans and is also a starting point for invasive S. pyogenes infection. Here, we describe that tonsil fluid from patients with streptococcal pharyngitis contains high amounts of the interferon (IFN)-dependent CXC chemokine known as monokine induced by IFN- gamma (MIG)/CXCL9. Also in vitro, inflamed pharyngeal epithelium produced large amounts of MIG/CXCL9 in the presence of bacteria. The CXC chemokines MIG/CXCL9, IFN-inducible protein-10/CXCL10, and IFN-inducible T cell alpha -chemoattractant/CXCL11 all showed antibacterial activity against S. pyogenes, and inhibition of MIG/CXCL9 expression reduced the antibacterial activity at the surface of inflamed pharyngeal cells. S. pyogenes of the clinically important M1 serotype secrets the protein streptococcal inhibitor of complement (SIC), which inhibited the antibacterial activity of the chemokines. As exemplified by S. pyogenes pharyngitis, the data identify a novel innate defense mechanism against invasive bacteria on epithelial surfaces.

Protein H--a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (lgGFc) region of immunoglobulin (lg) G. In absorption experiments with human plasma, protein H–sepharose could absorb not only lgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 × 109M−1, which is higher than the affinity between lgG and protein H (Ka= 1.6 × 109 M−1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein–protein interaction assays, the binding of albumin was mapped to three repeats (C1–C3) in the C‐terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin‐ and lgGFc‐binding bacterial surface protein. Aiso lgGFc‐binding could be mapped with the protein H fragments and the region was found N‐terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized lgG or lgGFc. This sequence was not found in previously described lgGFc‐binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify lgGFc‐ and albumin binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.

Gram-positive anaerobic cocci - commensals and opportunistic pathogens.

Among the Gram-positive anaerobic bacteria associated with clinical infections, the Gram-positive anaerobic cocci (GPAC) are the most prominent and account for approximately 25-30% of all isolated anaerobic bacteria from clinical specimens. Still, routine culture and identification of these slowly growing anaerobes to the species level has been limited in the diagnostic laboratory, mainly due to the requirement of prolonged incubation times and time-consuming phenotypic identification. In addition, GPAC are mostly isolated from polymicrobial infections with known pathogens and therefore their relevance has often been overlooked. However, through improvements in diagnostic and in particular molecular techniques, the isolation and identification of individual genera and species of GPAC associated with specific infections have been enhanced. Furthermore, the taxonomy of GPAC has undergone considerable changes over the years, mainly due to the development of molecular identification methods. Existing species have been renamed and novel species have been added, resulting in changes of the nomenclature. As the abundance and significance of GPAC in clinical infections grow, knowledge of virulence factors and antibiotic resistance patterns of different species becomes more important. The present review describes recent advances of GPAC and what is known of the biology and pathogenic effects of Anaerococcus, Finegoldia, Parvimonas, Peptoniphilus and Peptostreptococcus, the most important GPAC genera isolated from human infections.

Identification of a Streptococcal Octapeptide Motif Involved in Acute Rheumatic Fever

Acute rheumatic fever is a serious autoimmune sequela of pharyngitis caused by certain group A streptococci. One mechanism applied by streptococcal strains capable of causing acute rheumatic fever is formation of an autoantigenic complex with human collagen IV. In some geographic regions with a high incidence of acute rheumatic fever pharyngeal carriage of group C and group G streptococci prevails. Examination of such strains revealed the presence of M-like surface proteins that bind human collagen. Using a peptide array and recombinant proteins with targeted amino acid substitutions, we could demonstrate that formation of collagen complexes during streptococcal infections depends on an octapeptide motif, which is present in collagen binding M and M-like proteins of different β-hemolytic streptococcal species. Mice immunized with streptococcal proteins that contain the collagen binding octapeptide motif developed high serum titers of anti-collagen antibodies. In sera of rheumatic fever patients such a collagen autoimmune response was accompanied by specific reactivity against the collagen-binding proteins, linking the observed effect to clinical cases. Taken together, the data demonstrate that the identified octapeptide motif through its action on collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication of streptococci that express proteins with the collagen binding motif appears advisable for controlling rheumatic fever.