Hans Tapper

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.

Role of Intracellular pH in Proliferation, Transformation, and Apoptosis

Both cellular proliferation and apoptosis (programmed cell death) have been claimed to be modulated, perhaps even triggered by, changes in intracellular pH. In this review, we summarize the evidence that gave rise to these hypotheses. To facilitate a critical appraisal of the existing data, we briefly review the main pathways involved in cytosolic pH homeostasis and their regulation by mitogens and by apoptosis-inducing agents. The information available at present suggests that cytosolic pH plays a permissive role in cellular growth and proliferation, but is neither a trigger nor an essential step in the mitogenic signal transduction cascade. Concerning apoptosis, it is clear that lowering the pH in vitro can activate DNase II. However, the evidence linking cytosolic acidification with DNA degradation in vivois presently not convincing. We conclude that the cytosolic pH, an essential physiological parameter that is tightly controlled by multiple, complementary, or redundant systems, is unlikely to play a role in signalling either cell growth or death.

Phagosome dynamics during phagocytosis by neutrophils

The neutrophil is a key player in immunity, and its activities are essential for the resolution of infections. Neutrophil‐pathogen interactions usually trigger a large arsenal of antimicrobial measures that leads to the highly efficient killing of pathogens. In neutrophils, the phagocytic process, including the formation and maturation of the phagosome, is in many respects very different from that in other phagocytes. Although the complex mechanisms that coordinate the membrane traffic, oxidative burst, and release of granule contents required for the microbicidal activities of neutrophils are not completely understood, it is evident that they are unique and differ from those in macrophages. Neutrophils exhibit more rapid rates of phagocytosis and higher intensity of oxidative respiratory response than do macrophages. The phagosome maturation pathway in macrophages, which is linked to the endocytic pathway, is replaced in neutrophils by the rapid delivery of preformed granules to nonacidic phagosomes. This review describes the plasticity and dynamics of the phagocytic process with a special focus on neutrophil phagosome maturation.

The secretion of preformed granules by macrophages and neutrophils.

The ability of macrophages and neutrophils to defend tissue homeostasis and participate in inflammatory responses depends on their ability to mobilize granule‐membrane proteins and granule content into their external milieu and into phagosomes by regulated secretory processes. Many laboratories have invested much time and effort into furthering our understanding of vesicular transport and secretion. A surge of interest in phagocytosis and phagosomal maturation is also apparent (e.g., the March 1995 issue of Trends in Cell Biology was entirely devoted to phagocytosis). The signaling and the regulation of the secretory response are most likely different for secretion into phagosomes than for secretion into the external milieu. However, these differentially targeted secretory processes rely both upon proteins in vesicular membranes, plasma membrane/phagosomal membrane, and cytosol and upon their interactions with cytoskeletal structures. It is the complex molecular interactions between these components that form the basis for regulation and control of secretion. In the following, the signaling role of granular and cytosolic pH in phagocyte lysosomal secretion is discussed and the current literature on regulated secretion by macrophages and neutrophils is reviewed.

Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils

Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of neutrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where all mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3‐ and CD177‐expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener’s granulomatosis and polycythemia vera.

Elastase-producing Pseudomonas aeruginosa degrade plasma proteins and extracellular products of human skin and fibroblasts, and inhibit fibroblast growth.

Leg ulcers of venous origin represent a disease affecting 0.1-0.2% of the population. It is known that almost all chronic ulcers are colonized by different bacteria, such as staphylococci, enterococci and Pseudomonas aeruginosa. We here report that P. aeruginosa, expressing the major metalloproteinase elastase, induces degradation of complement C3, various antiproteinases, kininogens, fibroblast proteins, and proteoglycans (PG) in vitro, thus mimicking proteolytic activity previously identified in chronic ulcer fluid in vivo. Elastase-producing P. aeruginosa isolates were shown to significantly degrade human wound fluid as well as human skin proteins ex vivo. Elastase-containing conditioned P. aeruginosa medium and purified elastase inhibited fibroblast cell growth. These effects, in conjunction with the finding that proteinase production was detected in wound fluid ex vivo, suggest that bacterial proteinases play a pathogenic role in chronic ulcers.

Streptococcus pyogenes expressing M and M-like surface proteins are phagocytosed but survive inside human neutrophils.

Strains of the Gram‐positive human pathogen Streptococcus pyogenes (group A streptococcus) that express surface‐associated M or M‐like proteins survive and grow in non‐immune fresh human blood. This is generally accepted to be caused by an antiphagocytic property of these proteins. However, in most previous studies, an inhibition of the internalization of the bacteria into host cells has not been studied or not directly demonstrated. Therefore, in the present paper, we used flow cytometry, fluorescence microscopy and electron microscopy to study phagocytosis by human neutrophils of wild‐type S. pyogenes and strains deficient in expression of M protein and/or the M‐like protein H. The results demonstrate that all strains of S. pyogenes tested, including the wild‐type AP1 strain, induce actin polymerization and are efficiently phagocytosed by human neutrophils. In addition, using classical bactericidal assays, we show that the wild‐type AP1 strain can survive inside neutrophils, whereas mutant strains are rapidly killed. We conclude that the ability of virulent S. pyogenes to survive and multiply in whole blood is most likely not possible to explain only by an antiphagocytic effect of bacterial surface components. Instead, our data suggest that bacterial evasion of host defences occurs intracellularly and that survival inside human neutrophils may contribute to the pathogenesis of S. pyogenes and the recurrence of S. pyogenes infections.

Localized exocytosis of primary (lysosomal) granules during phagocytosis: role of Ca2+-dependent tyrosine phosphorylation and microtubules.

The uptake and killing of bacteria by human neutrophils are dependent on the fusion of secretory granules with forming phagosomes. The earliest component of exocytosis was found to precede phagosome closure, so that granular membrane constituents were detectable on the plasmalemma. We show that during phagocytosis of IgG-opsonized particles, this early secretory response is highly polarized in the case of primary granules, but less so for specific granules. The vectorial discharge of primary granules was dependent on calcium, but no evidence was found that calcium is involved in determining the polarity of exocytosis. In particular, a redistribution of endomembrane calcium stores toward forming phagosomes could not be detected. Polarized granule exocytosis was accompanied by focal tyrosine phosphorylation and actin polymerization, although the latter was not required for the response. Instead, microtubules seemed to contribute to the vectorial nature of the response. During particle ingestion, the microtubule-organizing center relocated toward forming phagosomes, and colchicine treatment altered the pattern of exocytosis, reducing its directionality. We hypothesize that the focal activation of tyrosine kinases generates localized signals that induce exocytosis in a calcium-dependent manner, and that reorientation of microtubules facilitates preferential delivery of granules toward the forming phagosome.

Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes

We recently reported that the human pathogen Streptococcus pyogenes of the M1 serotype survives and replicates intracellularly after being phagocytosed by human neutrophils. These data raised the possibility that the generation of reactive oxygen metabolites by neutrophils, and the release of microbicidal molecules from their azurophilic and specific granules into phagosomes, can be modulated by S. pyogenes bacteria expressing surface‐associated M and/or M‐like proteins. We now demonstrate, using flow cytometry, immunofluorescence microscopy and transmission electron microscopy, that live wild‐type S. pyogenes, after internalization by human neutrophils, inhibits the fusion of azurophilic granules with phagosomes. In contrast, azurophilic granule‐content is efficiently delivered to phagosomes containing bacteria not expressing M and/or M‐like proteins. Also, when heat‐killed wild‐type bacteria are used as the phagocytic prey, fusion of azurophilic granules with phagosomes is observed. The inhibition caused by live wild‐type S. pyogenes is specific for azurophilic granule–phagosome fusion, because the mobilization of specific granules and the production of reactive oxygen species are induced to a similar extent by all strains tested. In conclusion, our results demonstrate that viable S. pyogenes bacteria expressing M and M‐like proteins selectively prevent the fusion of azurophilic granules with phagosomes.

The lipocalin alpha1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species.

α1-Microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of α1-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that α1-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of α1-microglobulin was up-regulated by these oxidants and silencing of the α1-microglobulin expression increased the cytosol oxidation. α1-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to α1-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, α1-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that α1-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.