Molecular mechanism for the antigonococcal action of lysosomal cathepsin G

Shafer, William M.; Onunka, V C; Jannoun, M.; Huthwaite, L. W.

doi:10.1111/j.1365-2958.1990.tb00706.x

Cited by 15 publications

(9 citation statements)

References 35 publications

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“…These same investigators have recalled attention to the bactericidal activity of serine proteinases from PMNL, proteins which were among the earliest granule proteins analyzed for this biological activity (28,29). Cathepsin G has been the most active serine proteinase in this regard, but enzyme activity was not required for killing of any of the bacteria studied thus far (1,28,(38)(39)(40)(41). We present evidence * Corresponding author.…”

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confidence: 78%

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

1991

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Killing of Pseudomonas aeruginosa by a 55-kDa bactericidal protein (BP 55), a 30-kDa protein (BP 30), cathepsin G, elastase, and proteinase 3 has been compared. P. aeruginosa was resistant to killing by elastase and proteinase 3. BP 55 at a 50% lethal dose (LD50) of 0.23 ,ug of protein per 5 x 106 bacteria per ml killed P. aeruginosa and was far more active than BP 30 and cathepsin G. The LD5s of BP 30 and cathepsin G were 16.9 and 28.3 ,ug of protein per 5 x 106 bacteria per ml, respectively. Preincubation of BP 55 or BP 30 with lipopolysaccharide (LPS) from P. aeruginosa inhibited bactericidal activity. The N-terminal amino acid sequence of BP 55 and BP 30 revealed no relationship between the two proteins. However, a monoclonal antibody (AHN-15) reacted with both proteins by Western immunoblot. The bactericidal activity of cathepsin G toward P. aeruginosa appeared to be dependent on the availability of the active site of the enzyme; bactericidal activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and by the specific cathepsin G inhibitor, Z-Gly-Leu-Phe-CH2Cl. The enzyme and bactericidal activities of cathepsin G were also inhibited by LPS from P. aeruginosa. LPS from P. aeruginosa was shown to be a competitive inhibitor of the enzyme activity of cathepsin G. Elastase enzyme activity was also inhibited noncompetitively by LPS, but the enzyme was not bactericidal. We have concluded that all three bactericidal proteins (BP 55, BP 30, and cathepsin G) may bind to the LPS of the outer membrane of P. aeruginosa. It appears that the enzyme active site must be available for cathepsin G to kill P. aeruginosa and that the active site may be involved in the binding of cathepsin G to P. aeruginosa.Polymorphonuclear leukocytes (PMNL) occupy a central role in host defense and destroy intracellular microorganisms by both oxygen-dependent and oxygen-independent mechanisms. As reviewed by Spitznagel (45) and Thomas et al. (47), several proteins are implicated in oxygen-independent killing of microorganisms. Prominent among the cationic bactericidal proteins thus far purified from human PMNL is a 58to 60-kDa bactericidal/permeability-increasing protein (B/PI) described by Weiss et al. (51). B/PT is bactericidal toward Salmonella typhimurium and Escherichia coli. Shafer et al. have described two cationic antibacterial proteins (CAP) of 57 kDa (CAP57) and 37 kDa (CAP37) that are bactericidal toward S. typhimurium (37). We have described a 55-kDa glycoprotein (BP 55) that was isolated from normal human PMNL and was bactericidal toward Pseudomonas aeruginosa (19,20). The smaller cationic defensins (3.5 to 4.0 kDa) kill a spectrum of microorganisms, including P. aeruginosa (10, 16).More recently, reports by Gabay et al. (9) and Campanelli et al. (6) of a 30-kDa protein, called azurocidin, which is bactericidal for E. coli and Streptococcus faecalis and fungicidal for Candida albicans, have generated a great deal of interest. These same investigators have recalled attention to the bactericidal activity of serine proteinase...

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confidence: 78%

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

1991

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“…This is in agreement with our previous report that the fraction of Opa− Gc in primary granule‐positive, mature phagolysosomes is killed by exposure to PMN proteases (Johnson and Criss, ). Polymorphonuclear leukocyte serine proteases have direct antigonococcal activity, cleave the propeptide hCAP‐18 to produce the active LL‐37 antimicrobial peptide and enhance release of BPI from the primary granule matrix, all of which may contribute to the survival defect of Opa+ Gc (Rest and Pretzer, ; Shafer et al ., 1986; 1990; Shafer and Morse, ; Sorensen et al ., ; Edwards, ). While Sintsova et al .…”

Section: Discussionmentioning

confidence: 99%

Opa+Neisseria gonorrhoeaeexhibits reduced survival in human neutrophils via Src family kinase-mediated bacterial trafficking into mature phagolysosomes

et al. 2014

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Summary During gonorrheal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial content. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signaling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signaling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils’ full antimicrobial arsenal.

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“…TGF-β and IL-6 drive the development of Th17 cells, which secrete IL-17 and IL-22, leading to the recruitment or induction of innate defenses such as PMNs and anti-microbial peptides [ 18 ]. N. gonorrhoeae evades the immune system by PMNs and anti-microbial peptides while concomitantly suppressing the development of adaptive immune responses such as N. gonorrhoeae -specific antibodies that could enhance phagocytosis and intracellular clearance of gonococci by phagocytes and bacteriolysis through the classical complement pathway [ 4 , 9 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

Novel Whole-Cell Inactivated Neisseria Gonorrhoeae Microparticles as Vaccine Formulation in Microneedle-Based Transdermal Immunization

et al. 2018

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Neisseria gonorrhoeae is a strict human pathogen responsible for more than 100 million new sexually transmitted infections worldwide each year. Due to the global emergence of antibiotic resistance, the Center for Disease control (CDC) recently listed N. gonorrhoeae as an urgent threat to public health. No vaccine is available in spite of the huge disease burden and the possibility of untreatable gonorrhea. The aim of this study is to investigate the immunogenicity of a novel whole-cell-based inactivated gonococcal microparticle vaccine formulation loaded in dissolvable microneedles for transdermal administration. The nanotechnology-based vaccine formulation consists of inactivated whole-cell gonococci strain CDC-F62, spray dried and encapsulated into biodegradable cross-linked albumin matrix with sustained slow antigen release. The dry vaccine nanoparticles were then loaded in a dissolvable microneedle skin patch for transdermal delivery. The efficacy of the whole-cell microparticles vaccine formulation loaded in microneedles was assessed in vitro using dendritic cells and macrophages as well as in vivo mouse model. Antibody titers were measured using an enzyme immunosorbent assay (ELISA) and antigen-specific T lymphocytes were assessed in spleens and lymph nodes. Here we report that whole-cell-based gonococcal microparticle vaccine loaded in dissolvable microneedles for transdermal administration induced significant increase in antigen-specific IgG antibody titers and antigen-specific CD4 and CD8 T lymphocytes in mice compared to gonococcal antigens in solution or empty microneedles. Significant increase in antigen-specific IgG antibody levels was observed at the end of week 2 in groups that received the vaccine compared to the group receiving empty nanoparticles. The advantages of using formalin-fixed whole-cell gonococci that all immunogenic epitopes are covered and preserved from degradation. The spherical shaped micro and nanoparticles are biological mimics of gonococci, therefore present to the immune system as invaders but without the ability to suppress adaptive immunity. In conclusion, the transdermal delivery of microparticles vaccine via a microneedle patch was shown to be an effective system for vaccine delivery. The novel gonorrhea nanovaccine is cheap to produce in a stable dry powder and can be delivered in microneedle skin patch obviating the need for needle use or the cold chain.

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Molecular mechanism for the antigonococcal action of lysosomal cathepsin G

Cited by 15 publications

References 35 publications

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

Opa+Neisseria gonorrhoeaeexhibits reduced survival in human neutrophils via Src family kinase-mediated bacterial trafficking into mature phagolysosomes

Novel Whole-Cell Inactivated Neisseria Gonorrhoeae Microparticles as Vaccine Formulation in Microneedle-Based Transdermal Immunization

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