Pseudomonas aeruginosa Elastase Provides an Escape from Phagocytosis by Degrading the Pulmonary Surfactant Protein-A

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitroand in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin

Deciphering Asthma Biomarkers with Protein Profiling Technology

Asthma is a chronic inflammatory disease of the airways, resulting in bronchial hyperresponsiveness with every allergen exposure. It is now clear that asthma is not a single disease, but rather a multifaceted syndrome that results from a variety of biologic mechanisms. Asthma is further problematic given that the disease consists of many variants, each with its own etiologic and pathophysiologic factors, including different cellular responses and inflammatory phenotypes. These facets make the rapid and accurate diagnosis (not to mention treatments) of asthma extremely difficult. Protein biomarkers can serve as powerful detection tools in both clinical and basic research applications. Recent endeavors from biomedical researchers have developed technical platforms, such as cytokine antibody arrays, that have been employed and used to further the global analysis of asthma biomarker studies. In this review, we discuss potential asthma biomarkers involved in the pathophysiologic process and eventual pathogenesis of asthma, how these biomarkers are being utilized, and how further testing methods might help improve the diagnosis and treatment strain that current asthma patients suffer

The Interferon-Induced Gene ISG15 Blocks Retrovirus Release from Cells Late in the Budding Process▿

The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process

Correction: Competence-Independent Activity of Pneumococcal Enda Mediates Degradation of Extracellular DNA and Nets and Is Important for Virulence.

[This corrects the article on p. e70363 in vol. 8.]

Competence-independent activity of pneumococcal EndA [corrected] mediates degradation of extracellular dna and nets and is important for virulence.

Membrane surface localized endonuclease EndA of the pulmonary pathogen Streptococcus pneumoniae (pneumococcus) is required for both genetic transformation and virulence. Pneumococcus expresses EndA during growth. However, it has been reported that EndA has no access to external DNA when pneumococcal cells are not competent for genetic transformation, and thus, unable to degrade extracellular DNA. Here, by using both biochemical and genetic methods, we demonstrate the existence of EndA-mediated nucleolytic activity independent of the competence state of pneumococcal cells. Pneumococcal mutants that are genetically deficient in competence development and genetic transformation have extracellular nuclease activity comparable to their parental wild type, including their ability to degrade neutrophil extracellular traps (NETs). The autolysis deficient ΔlytA mutant and its isogenic choline-treated parental wild-type strain D39 degrade extracellular DNA readily, suggesting that partial cell autolysis is not required for DNA degradation. We show that EndA molecules are secreted into the culture medium during the growth of pneumococcal cells, and contribute substantially to competence-independent nucleolytic activity. The competence-independent activity of EndA is responsible for the rapid degradation of DNA and NETs, and is required for the full virulence of Streptococcus pneumoniae during lung infection

Deciphering Asthma Biomarkers with Protein Profiling Technology

Asthma is a chronic inflammatory disease of the airways, resulting in bronchial hyperresponsiveness with every allergen exposure. It is now clear that asthma is not a single disease, but rather a multifaceted syndrome that results from a variety of biologic mechanisms. Asthma is further problematic given that the disease consists of many variants, each with its own etiologic and pathophysiologic factors, including different cellular responses and inflammatory phenotypes. These facets make the rapid and accurate diagnosis (not to mention treatments) of asthma extremely difficult. Protein biomarkers can serve as powerful detection tools in both clinical and basic research applications. Recent endeavors from biomedical researchers have developed technical platforms, such as cytokine antibody arrays, that have been employed and used to further the global analysis of asthma biomarker studies. In this review, we discuss potential asthma biomarkers involved in the pathophysiologic process and eventual pathogenesis of asthma, how these biomarkers are being utilized, and how further testing methods might help improve the diagnosis and treatment strain that current asthma patients suffer

Extracellular DNA degradation by pneumococcus EndA is independent of the competence regulon and cell autolysis.

<p>(A) The <i>rpsL</i> PCR products (30 µg/ml) were exposed to the wild-type D39 and isogenic mutants for the indicated time intervals, or in the presence or absence of ATA or 2% choline chloride. The integrity of DNA was visualized by agarose gel electrophoresis. (B) Competence-deficient pneumococcal mutants degrade salmon sperm DNA efficiently. Pneumococcal cells were streaked onto the THB agar supplemented with salmon sperm DNA in the presence or absence of ATA or choline chloride. After 24 hr, DNA degradation was visualized by flooding the plates with HCl. Three independent experiments were performed for both A and B with similar results. The data from one typical experiment are shown.</p

Competence-independent DNA degradation is caused by EndA secreted by pneumococcus into the culture medium.

<p>(A) The <i>rpsL</i> PCR products (30 µg/ml) were exposed to cell-free supernatants from D39 and its isogenic Δ<i>comD</i> and Δ<i>lytA</i> mutants collected during growth at the indicated time intervals. The integrity of DNA was visualized by agarose gel electrophoresis. (B) Bacterial cell lysates and cell-free supernatants from D39, Δ<i>comD</i> and Δ<i>lytA</i> cultured in THB were subjected to SDS-PAGE in a gel incorporated with 15 µg/ml of salmon sperm DNA. After renaturation, the gel was stained with ethidium bromide to visualize bands of DNA clearance by EndA. The experiments were performed independently three times. The results from one typical experiment are shown.</p