Motoyuki Sugai

A Novel Mechanism of Rapid Nuclear Neutrophil Extracellular Trap Formation in Response to Staphylococcus aureus

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3–4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5–60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton–Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton–Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.

Induction of Keratinocyte Migration via Transactivation of the Epidermal Growth Factor Receptor by the Antimicrobial Peptide LL-37

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 μg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.

Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B

ABSTRACT We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.

Enteropathogenic and enterohaemorrhagic Escherichia coli deliver a novel effector called Cif, which blocks cell cycle G2/M transition.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. Both use a type III secretion system (TTSS) encoded by the ‘locus of enterocyte effacement’ (LEE) to subvert and attach to epithelial cells through the injection of a repertoire of effector molecules. Here, we report the identification of a new TTSS translocated effector molecule called Cif, which blocks cell cycle G2/M transition and induces the formation of stress fibres through the recruitment of focal adhesions. Cif is not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface, but is defective in the ability to block mitosis. When expressed in TTSS competent LEE‐positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins that subvert the host eukaryotic cell cycle.

Efficient Elimination of Multidrug-Resistant Staphylococcus aureus by Cloned Lysin Derived from Bacteriophage ϕMR11

We report the successful purification of a cloned lysin encoded by the novel Staphylococcus aureus bacteriophage phi MR11. The lysin, designated MV-L, rapidly and completely lysed cells of a number of S. aureus strains tested, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus and a subset of vancomycin-intermediate S. aureus (VISA) in growing conditions. MV-L-mediated killing is specific to S. aureus and not to other species, except for S. simulans. MV-L exerted its staphylocidal effect synergistically with glycopeptide antibiotics against VISA. MV-L efficiently eliminated MRSA that had been artificially inoculated into the nares of mice. The intraperitoneal administration of MV-L also protected mice against MRSA septic death, without any harmful effects. Although MV-L evoked detectable levels of a humoral response in mice, the antibodies did not abolish the bacteriolytic activity. These results indicate that MV-L might be useful as a powerful therapeutic agent against multidrug-resistant S. aureus infections.

Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome

Bullous impetigo due to Staphylococcus aureus is one of the most common bacterial infections of man, and its generalized form, staphylococcal scalded skin syndrome (SSSS), is a frequent manifestation of staphylococcal epidemics in neonatal nurseries. Both diseases are mediated by exfoliative toxins (ETs), which show exquisite pathologic specificity in blistering only the superficial epidermis. We show that these toxins act as serine proteases with extremely focused molecular specificity to cleave mouse and human desmoglein 1 (Dsg1) once after glutamic acid residue 381 between extracellular domains 3 and 4. Mutation of the predicted catalytically active serine to alanine completely inhibits cleavage. The mutated ETs bind specifically to Dsg1 by immunofluorescence colocalization and by coimmunoprecipitation. Thus, ETs, through specific recognition and proteolytic cleavage of one structurally critical peptide bond in an adhesion molecule, cause its dysfunction and allow S. aureus to spread under the stratum corneum, the main barrier of the skin, explaining how, although they circulate through the entire body in SSSS, they cause pathology only in the superficial epidermis.

Permeation of Antimicrobial Agents through Pseudomonas aeruginosa Biofilms: A Simple Method

In this study, we evaluated the permeation of piperacillin (PIPC), imipenem (IPM), amikacin (AKM), gentamicin (GM), ofloxacin (OFLX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sparfloxacin (SPFX) through Pseudomonas aeruginosa biofilm with a simple new method. Bacteria used were a leucine-requiring mucoid mutant. Bacteria were grown on the membrane of a cell culture insert in chemically defined medium and incubated at 37 degrees C for 5 days. At days 0, 1, 3 and 5, the penetration rates through the biofilms were measured. PIPC and IPM demonstrated relatively high permeation both with penetration rates at day 5 of 50%, whereas AMK and GM, which are aminoglycosides, showed low permeation both with penetration rates after day 1 of less than 25%. Among the 4 fluoroquinolones, LVFX and SPFX demonstrated excellent permeation with penetration rates that reached 100% from day 0 to 5, while OFLX and CPFX showed almost the same permeation as IPM. This method of measuring penetration rates of antimicrobial agents through biofilm is very simple and useful for the evaluation of antibiotics against biofilm-forming bacteria.

Phage conversion of exfoliative toxin A production in Staphylococcus aureus

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (φETA) that encodes ETA and determined the complete nucleotide sequence of the φETA genome. φETA has a head with a hexagonal outline and a non‐contractile and flexible tail. The genome of φETA is a circularly permuted linear double‐stranded DNA, and the genome size is 43 081 bp. Sixty‐six open reading frames (ORFs) were identified on the φETA genome, including eta, which was found to be located very close to a putative attachment site (attP). φETA converted ETA non‐producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that φETA or related phages are responsible for the acquisition of eta genes in S. aureus.

Complete Nucleotide Sequence of a Staphylococcus aureus Exfoliative Toxin B Plasmid and Identification of a Novel ADP-Ribosyltransferase, EDIN-C

ABSTRACT The complete nucleotide sequence of pETB, a 38.2-kbStaphylococcus aureus plasmid encoding the exfoliative toxin B (ETB), was determined. A total of 50 open reading frames were identified on the plasmid genome and, among these, 32 showed sequence similarity to known proteins. pETB contains three copies of IS257, which divide the pETB genome into three regions: (i) a cadmium resistance operon-containing region, (ii) a lantibiotic production gene-containing region, and (iii) the remaining part where genes for plasmid replication and/or maintenance are dispersed. In the third region, genes of various kinds of functions are present among the replication- and maintenance-related genes. They include two virulence-related genes, the etb gene and a gene encoding a novel ADP-ribosyltransferase closely related to EDIN, which belongs to the C3 family of ADP-ribosyltransferases modifying Rho GTPases. They also include genes for a cell wall-anchoring surface protein and a phage resistance protein. Based on the determined sequence of pETB, the genome structures of etb-bearing plasmids (ETB plasmids) from various clinical isolates were analyzed by the PCR scanning method. The data indicate that, although the ETB plasmids are highly heterogeneous in genome size, the fundamental genome organization is well conserved. The size variation of the plasmid is mainly attributed to defined regions which may be hot spots for gene shuffling.

Identification and molecular characterization of an N-acetylmuramyl-l-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus

We purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N‐acetylmuramyl‐l‐alanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N‐acetylmuramyl‐l‐Ala bonds in dimeric cross‐bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus. The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus, which has been implicated in cell separation of S. aureus. Generation of an atl/sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus.