Katrin Pütsep

Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study

BACKGROUND Antibacterial peptides, such as defensins and LL-37, are natural bactericidal components similar in potency to classic antibiotics. These peptides are produced at mucosal linings in the body and the skin, and by leucocytes such as neutrophils and natural killer cells. Patients with morbus Kostmann-a severe congenital neutropenia-are treated by recombinant granulocyte-colony stimulating factor, which restores their levels of neutrophils. Despite this treatment, patients still have recurrent infections and periodontal disease. Our aim was to investigate if defensins and LL-37 are deficient in patients with morbus Kostmann. METHODS We studied samples of neutrophils, plasma, and saliva from six patients with congenital neutropenia and 22 healthy controls for presence of antibacterial peptides. Neutrophils were analysed by high-performance liquid chromatography and mass spectrometry for alpha-defensins. All samples were analysed by western blot for cathelin-LL-37 (precursor of LL-37) and LL-37. Neutrophils were also tested for lactoferrin and ability to produce oxidative burst. FINDINGS Neutrophils from patients with morbus Kostmann were deficient in cathelin-LL-37 and had reduced concentrations of a-defensins HNP1-3. No cathelin-LL-37 could be detected in plasma and saliva from patients. One patient with morbus Kostmann who had had bone-marrow transplantation had almost normal concentrations of LL-37. Lactoferrin concentrations and oxidative burst were normal in all patients. All patients with morbus Kostmann had severe periodontal disease, apart from the individual who had had a bone-marrow transplant, whose dental status was normal. INTERPRETATION Antibacterial peptides are a vital part of the first line of antibacterial immune defence. Deficiency in saliva LL-37 accords with occurrence of periodontal disease in patients with morbus Kostmann.

Secreted enteric antimicrobial activity localises to the mucus surface layer

Objectives: The intestinal mucosa is constantly exposed to a dense and highly dynamic microbial flora and challenged by a variety of enteropathogenic bacteria. Antibacterial protection is provided in part by Paneth cell-derived antibacterial peptides such as the α-defensins. The mechanism of peptide-mediated antibacterial control and its functional importance for gut homeostasis has recently been appreciated in patients with Crohn’s ileitis. In the present study, the spatial distribution of antimicrobial peptides was analysed within the small intestinal anatomical compartments such as the intestinal crypts, the overlaying mucus and the luminal content. Methods: Preparations from the different intestinal locations as well as whole mouse small intestine were extracted and separated by reversed-phase high-performance liquid chromatography. Antibacterial activity was determined in extracts, and the presence of antimicrobial peptides/proteins was confirmed by N-terminal sequencing, mass spectrometry analysis and immunodetection. Results: The secreted antibacterial activity was largely confined to the layer of mucus, whereas only minute amounts of activity were noted in the luminal content. The extractable activity originating from either crypt/mucus/lumen compartments respectively (given as a percentage) was for Listeria monocytogenes, 48 (4)/44 (4)/8 (8); Enterococcus faecalis, 44 (10)/49 (3)/7 (7); Bacterium megaterium, 56 (4)/42 (3)/2 (1); Streptococcus pyogenes, 48 (4)/46 (3)/6 (6); Escherichia coli, 46 (4)/47 (3)/7 (7); and Salmonella enterica sv. Typhimurium, 38 (3)/43 (7)/19 (10). A spectrum of antimicrobial peptides was identified in isolated mucus, which exhibited strong and contact-dependent antibacterial activity against both commensal and pathogenic bacteria. Conclusion: These findings show that secreted antimicrobial peptides are retained by the surface-overlaying mucus and thereby provide a combined physical and antibacterial barrier to prevent bacterial attachment and invasion. This distribution facilitates high local peptide concentration on vulnerable mucosal surfaces, while still allowing the presence of an enteric microbiota.

Antibacterial peptide from H. pylori.

Colonization of the human stomach by the bacterium Helicobacter pylori is a predisposing factor for gastrointestinal illnesses such as gastritis and peptic ulcers. But most infections are asymptomatic, and it has recently been suggested that H. pylori may actually have beneficial effects on infected carriers who are heavily exposed to other gastrointestinal pathogens. We find that H. pylori possesses antibacterial activity to which it is itself resistant. We have traced this activity to cecropin-like amino-terminal peptides derived from the ribosomal protein L1 (RpL1).

Increased diversity of intestinal antimicrobial peptides by covalent dimer formation.

Antimicrobial peptides are essential effector molecules of the innate immune system. Here we describe the structure, function and diversity of cryptdin-related sequence (CRS) peptides, a large family of antimicrobial molecules. We identified the peptides as covalent dimers in mouse intestinal tissue in amounts comparable to those of Paneth cell–derived enteric α-defensins. CRS peptides caused rapid and potent killing of commensal and pathogenic bacteria. The CRS peptides formed homo- and heterodimers in vivo, thereby expanding the repertoire of antimicrobial peptides and increasing the peptide diversity of Paneth cell secretions. CRS peptides might therefore be important in the maintenance of the microbial homeostasis within the intestinal tract.

Developmental switch of intestinal antimicrobial peptide expression

Paneth cell–derived enteric antimicrobial peptides provide protection from intestinal infection and maintenance of enteric homeostasis. Paneth cells, however, evolve only after the neonatal period, and the antimicrobial mechanisms that protect the newborn intestine are ill defined. Using quantitative reverse transcription–polymerase chain reaction, immunohistology, reverse-phase high-performance liquid chromatography, and mass spectrometry, we analyzed the antimicrobial repertoire in intestinal epithelial cells during postnatal development. Surprisingly, constitutive expression of the cathelin-related antimicrobial peptide (CRAMP) was observed, and the processed, antimicrobially active form was identified in neonatal epithelium. Peptide synthesis was limited to the first two weeks after birth and gradually disappeared with the onset of increased stem cell proliferation and epithelial cell migration along the crypt–villus axis. CRAMP conferred significant protection from intestinal bacterial growth of the newborn enteric pathogen Listeria monocytogenes. Thus, we describe the first example of a complete developmental switch in innate immune effector expression and anatomical distribution. Epithelial CRAMP expression might contribute to bacterial colonization and the establishment of gut homeostasis, and provide protection from enteric infection during the postnatal period.

Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract

BackgroundEnteric antimicrobial peptides secreted from Paneth cells, including α-defensins (in mice named cryptdins), are key effector molecules of innate immunity in the small intestine. The importance of Paneth cells α-defensins emerged from studies of enteric bacterial infection in genetically modified mice, as well as from recent studies linking reduced levels of these α-defensins to Crohns disease localized to the ileum. However, analysis of expression of Paneth cell α-defensins is incomplete. We therefore performed a comprehensive evaluation of the distribution of antimicrobial molecules along the mouse small intestinal tract to identify potential variations in regional expression.ResultsIn conventionally reared mice, the repertoire of Paneth cell antimicrobials differs between duodenum and ileum. In contrast to the uniform expression of most Paneth cell antimicrobials, both cryptdin 4 and cryptdin-related sequences (CRS) 4C peptides were expressed at progressively increasing amounts (101- and 104-fold, respectively) comparing duodenum and ileum. In tissues other than the small intestine, expression of CRS peptides was noted in thymus and caecum. Most Paneth cell products were also produced in the small intestine of germ-free mice at levels similar to those in controls, however CRS4C and RegIIIγ had reduced levels in the former (3- and 8-fold, respectively). No significant changes in expression levels of Paneth cell antimicrobial peptides was observed after oral challenge with either Salmonella enterica serovar typhimurium or Listeria monocytogenes, supporting current notions on the constitutive nature of this defensive system.ConclusionThe repertoire of antimicrobial peptides changes along the small intestinal tract, and a subset of these molecules are up-regulated upon colonization, but not in response to enteric bacterial pathogens. The changes detected upon colonization suggest that Paneth cell antimicrobial peptides may play an important role in commensal microbial homeostasis, in addition to their proposed role in protection against infection. In addition, the differential expression of CRS4C along the small intestine suggests mechanisms of regulation that are distinct from other Paneth cell derived antimicrobial peptides.

Benzyl isothiocyanate, a major component from the roots of Salvadora persica is highly active against Gram-negative bacteria.

Plants produce a number of antimicrobial substances and the roots of the shrub Salvadora persica have been demonstrated to possess antimicrobial activity. Sticks from the roots of S. persica, Miswak sticks, have been used for centuries as a traditional method of cleaning teeth. Diverging reports on the chemical nature and antimicrobial repertoire of the chewing sticks from S. persica led us to explore its antibacterial properties against a panel of pathogenic or commensal bacteria and to identify the antibacterial component/s by methodical chemical characterization. S. persica root essential oil was prepared by steam distillation and solid-phase microextraction was used to sample volatiles released from fresh root. The active compound was identified by gas chromatography-mass spectrometry and antibacterial assays. The antibacterial compound was isolated using medium-pressure liquid chromatography. Transmission electron microscopy was used to visualize the effect on bacterial cells. The main antibacterial component of both S. persica root extracts and volatiles was benzyl isothiocyanate. Root extracts as well as commercial synthetic benzyl isothiocyanate exhibited rapid and strong bactericidal effect against oral pathogens involved in periodontal disease as well as against other Gram-negative bacteria, while Gram-positive bacteria mainly displayed growth inhibition or remained unaffected. The short exposure needed to obtain bactericidal effect implies that the chewing sticks and the essential oil may have a specific role in treatment of periodontal disease in reducing Gram-negative periodontal pathogens. Our results indicate the need for further investigation into the mechanism of the specific killing of Gram-negative bacteria by S. persica root stick extracts and its active component benzyl isothiocyanate.

Studies on the Antibacterial Effects of Statins - In Vitro and In Vivo

Background Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections. Aim The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract. Methods MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert). Findings The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria. Conclusions Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.

The origin of cecropins; implications from synthetic peptides derived from ribosomal protein L1

We recently showed that Helicobacter pylori grown on plates produce cecropin‐like antibacterial peptides to which H. pylori is resistant. This antibacterial activity was traced to fragments from the N‐terminus of ribosomal protein L1 (Pütsep et al., Nature, April 22, 1999). The evolutionary suggestion from this finding has now been extended by the synthesis of eight peptides with sequences taken from the N‐terminus of ribosomal protein L1 (RpL1) of five different species. Two peptides of different length derived from H. pylori RpL1 showed a potent antibacterial activity, while a peptide with the sequence from Escherichia coli was 20 times less active. Like cecropins the H. pylori peptides were not cytolytic. We suggest that the cecropins have evolved from ribosomal protein L1 of an ancestral intracellular pathogen that developed to a symbiont ending as an organelle. When the R1 gene moved into the host nucleus, a duplication provided a copy from which today cecropins could have evolved.

Kostmann syndrome or infantile genetic agranulocytosis, part one : celebrating 50 years of clinical and basic research on severe congenital neutropenia

Congenital neutropenia in man was first reported 50 years ago by the Swedish paediatrician Rolf Kostmann. He coined the term “infantile genetic agranulocytosis” for this condition, which is now known as Kostmann syndrome. Recent studies have demonstrated a lack of antibacterial peptides and severe periodontitis in these patients despite recombinant growth factor treatment. Moreover, an increased degree of apoptosis of myeloid progenitor cells in the bone marrow has been shown.