Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis.

Staphylococcus aureus (S. aureus) is a human pathogen that relies on the subversion of host phagocytes to support its pathogenic lifestyle. S. aureus strains can produce up to five beta-barrel, bi-component, pore-forming leukocidins that target and kill host phagocytes. Thus, preventing immune cell killing by these toxins is likely to boost host immunity. Here, we describe the identification of glycine-rich motifs within the membrane-penetrating stem domains of the leukocidin subunits that are critical for killing primary human neutrophils. Remarkably, leukocidins lacking these glycine-rich motifs exhibit dominant-negative inhibitory effects toward their wild-type toxin counterparts as well as other leukocidins. Biochemical and cellular assays revealed that these dominant-negative toxins work by forming mixed complexes that are impaired in pore formation. The dominant-negative leukocidins inhibited S. aureus cytotoxicity toward primary human neutrophils, protected mice from lethal challenge by wild-type leukocidin, and reduced bacterial burden in a murine model of bloodstream infection. Thus, we describe the first example of staphylococcal bi-component dominant-negative toxins and their potential as novel therapeutics to combat S. aureus infection.

Structure and Function of the Two-Component Cytotoxins of Staphylococcus aureus - Learnings for Designing Novel Therapeutics.

Staphylococcus aureus can produce up to five different bi-component cytotoxins: two gamma-hemolysins HlgAB and HlgCB, and leukocidins SF-PV (Panton Valentine leukocidin), ED (LukED) and GH (LukGH, also called LukAB). Their major function in S. aureus pathogenesis is to evade innate immunity by attacking phagocytic cells and to support bacterial growth by lysing red blood cells. The five cytotoxins display different levels of amino acid sequence conservation (30-82%), but all form a remarkably similar beta-barrel type pore structure (greatly resembling the mono-component toxin alpha-hemolysin) that inserts into the target cell membrane leading to necrotic cell death. This review provides an overview of the culmination of decades of research on the structure of these toxins, their unique sequence and structural features that helps to explain the observed functional differences, such as toxin potency towards different cell types and species, receptor specificity and formation of functional non-cognate toxin pairs. The vast knowledge accumulated in this field supports novel approaches and the design of therapeutics targeting these cytotoxins to tame virulence and fight S. aureus infections.

Structure-function analysis of heterodimer formation, oligomerization, and receptor binding of the Staphylococcus aureus bi-component toxin LukGH.

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30-40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Šresolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.

Prostatic abscesses and severe sepsis due to methicillin-susceptible Staphylococcus aureus producing Panton-Valentine leukocidin.

BACKGROUND: Prostatic abscesses are an uncommon disease usually caused by enterobacteria. They mostly occur in immunodeficient patients. It is thus extremely rare to have a Staphylococcal prostatic abscess in a young immunocompetent patient. CASE PRESENTATION: A 20-year-old patient was treated with ofloxacin for a suspicion of prostatitis. An ultrasonography was performed because of persisting symptoms and showed acute urinary retention and prostatic abscesses. So the empirical antibiotic therapy was modified with ceftriaxone/amikacin. The disease worsened to severe sepsis and the patient was admitted in ICU. CT-scan and MRI confirmed three abscesses with perirectal infiltration and the bacteriological samples (abscesses and blood cultures) were positive to methicillin-susceptible Staphylococcus aureus producing Panton-Valentine leukocidine. The treatment was changed with fosfomycin/ofloxacin which resulted in a general improvement and the regression of the abscesses. CONCLUSION: Staphyloccocus aureus producing Panton-Valentine leukocidin are most commonly responsible for skin and soft tissue infections. To this day, no other case of prostatic abscess due to this strain but susceptible to methicillin has been described.

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the ß-barrel pore formation mechanism by two components.

Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as water-soluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a ß-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting ß2 and ß3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal ß-strands (amino-latch) of the adjacent protomer, releasing the transmembrane stem domain folded into a ß-sheet in the monomer (prestem), and interaction with the adjacent protomer.

Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus (S. aureus) is an opportunistic infectious pathogen, which causes a high mortality rate during bloodstream infections. The early detection of virulent strains in patients' blood samples is of medical interest for rapid diagnosis. The main virulent factors identified in patient isolates include leukocidins that bind to specific membrane receptors and lyse immune cells and erythrocytes. Duffy antigen receptor for chemokines (DARC) on the surface of specific cells is a main target of leukocidins such as gamma-hemolysin AB (HlgAB) and leukocidin ED (LukED). Among them, HlgAB is a conserved and critical leukocidin that binds to DARC and forms pores on the cell membranes, leading to cell lysis. Current methods are based on ELISA or bacterial culture, which takes hours to days. For detecting HlgAB with faster response and higher sensitivity, we developed a biosensor that combines single-walled carbon nanotube field effect transistors (swCNT-FETs) with immobilized DARC receptors as biosensing elements. DARC was purified from a bacterial expression system and successfully reconstituted into nanodiscs that preserve binding capability for HlgAB. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed an increase of the DARC-containing nanodisc size in the presence of HlgAB, indicating the formation of HlgAB prepore or pore complexes. We demonstrate that this sensor can specifically detect the leukocidins HlgA and HlgAB in a quantitative manner within the dynamic range of 1 fM to 100 pM with an LOD of 0.122 fM and an LOQ of 0.441 fM. The sensor was challenged with human serum spiked with HlgAB as simulated clinical samples. After dilution for decreasing nonspecific binding, it selectively detected the toxin with a similar detection range and apparent dissociation constant as in the buffer. This biosensor was demonstrated with remarkable sensitivity to detect HlgAB rapidly and has the potential as a tool for fundamental research and clinical applications, although this sensor cannot differentiate between HlgAB and LukED as both have the same receptor.

Screening for methicillin-resistant Staphylococcus aureus (MRSA) in community-recruited injection drug users: are throat swabs necessary?

We examined and described colonization of MRSA in the anterior nares and throat from 184 community-recruited injection drug users. Thirty-seven (20%) were positive for MRSA: most (34, 92%) were carriers in the nares; while only three (8%) were carriers detected by throat swabs alone. The majority (29, 78%) of MRSA isolates were PVL positive.

Antigenic landscapes on Staphylococcus aureus pore-forming toxins reveal insights into specificity and cross-neutralization.

Staphylococcus aureus carries an exceptional repertoire of virulence factors that aid in immune evasion. Previous single-target approaches for S. aureus-specific vaccines and monoclonal antibodies (mAbs) have failed in clinical trials due to the multitude of virulence factors released during infection. Emergence of antibiotic-resistant strains demands a multi-target approach involving neutralization of different, non-overlapping pathogenic factors. Of the several pore-forming toxins that contribute to S. aureus pathogenesis, efforts have largely focused on mAbs that neutralize α-hemolysin (Hla) and target the receptor-binding site. Here, we isolated two anti-Hla and three anti-Panton-Valentine Leukocidin (LukSF-PV) mAbs, and used a combination of hydrogen deuterium exchange mass spectrometry (HDX-MS) and alanine scanning mutagenesis to delineate and validate the toxins' epitope landscape. Our studies identified two novel, neutralizing epitopes targeted by 2B6 and CAN6 on Hla that provided protection from hemolytic activity in vitro and showed synergy in rodent pneumonia model against lethal challenge. Of the anti-LukF mAbs, SA02 and SA131 showed specific neutralization activity to LukSF-PV while SA185 showed cross-neutralization activity to LukSF-PV, γ-hemolysin HlgAB, and leukotoxin ED. We further compared these antigen-specific mAbs to two broadly neutralizing mAbs, H5 (targets Hla, LukSF-PV, HlgAB, HlgCB, and LukED) and SA185 (targeting LukSF-PV, HlgAB, and LukED), and identified molecular level markers for broad-spectrum reactivity among the pore-forming toxins by HDX-MS. To further underscore the need to target the cross-reactive epitopes on leukocidins for the development of broad-spectrum therapies, we annotated Hla sequences isolated from patients in multiple countries for genomic variations within the perspective of our defined epitopes.

Chimeric mutants of staphylococcal hemolysin, which act as both one-component and two-component hemolysin, created by grafting the stem domain.

Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane ß-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component α-hemolysin (α-HL) and two-component γ-hemolysin (γ-HL). The α-HL forms a homo-heptamer, whereas γ-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a ß-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the ß-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT ß-barrels for use in further promising applications.

Membrane binding of pore-forming γ-hemolysin components studied at different lipid compositions.

Methicillin-resistant Staphylococcus aureus is among those pathogens currently posing the highest threat to public health. Its host immune evasion strategy is mediated by pore-forming toxins (PFTs), among which the bi-component γ-hemolysin is one of the most common. The complexity of the porogenesis mechanism by γ-hemolysin poses difficulties in the development of antivirulence therapies targeting PFTs from S. aureus, and sparse and apparently contrasting experimental data have been produced. Here, through a large set of molecular dynamics simulations at different levels of resolution, we investigate the first step of pore formation, and in particular the effect of membrane composition on the ability of γ-hemolysin components, LukF and Hlg2, to steadily adhere to the lipid bilayer in the absence of proteinaceous receptors. Our simulations are in agreement with experimental data of γ-hemolysin pore formation on model membranes, which are here explained on the basis of the bilayer properties. Our computational investigation suggests a possible rationale to explain experimental data on phospholipid binding to the LukF component, and to hypothesise a mechanism by which, on purely lipidic bilayers, the stable anchoring of LukF to the cell surface facilitates Hlg2 binding, through the exposure of its N-terminal region. We expect that further insights on the mechanism of transition between soluble and membrane bound-forms and on the role played by the lipid molecules will contribute to the design of antivirulence agents with enhanced efficacy against methicillin-resistant S. aureus infections.

The matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)-based protein peaks of 4448 and 5302 Da are not associated with the presence of Panton-Valentine leukocidin.

The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus plays an important role in the pathogenesis of necrotizing pneumonia and recurrent skin and soft tissue infections. The gene encoding for PVL, lukS/F-PV, is distributed by prophages and can thus spread between isolates. Molecular methods have normally been used to identify lukS/F-PV-positive strains. Recently, however, a matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)-based method has been described to identify PVL-positive S. aureus strains. The aim of this study was thus to investigate the association of distinct MALDI-TOF MS peaks to the toxin profile in molecularly characterized methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA) strains harbouring lukS/F-PV. In contrast to the previous results, the MALDI-TOF MS peaks were detected in all 104 recently described molecularly divergent MRSA irrespective of the presence of PVL. Our result indicates that these described peaks seem to be independent of the presence of PVL.

A histidine-to-arginine substitution in Panton-Valentine leukocidin from USA300 community-acquired methicillin-resistant Staphylococcus aureus does not impair its leukotoxicity.

Panton-Valentine leukocidin (PVL) is a synergohymenotropic toxin (SHT) produced by Staphylococcus aureus. At present, there are conflicting reports on the leukotoxic activity of PVL and its consequent role as a virulence factor in USA300. In this work, we compared the cytolytic effects induced by wild-type PVL and those of PVL harboring a histidine-to-arginine substitution at amino acid 176 in the S. aureus USA300 strain. We also investigated the capacity of wild-type and H176R LukS-PV to recruit and form pores with the F components of other SHTs. For this purpose, we assayed polymorphonuclear neutrophils for leukotoxicity after incubation with either culture supernatants from strains bearing different PVL haplotypes or recombinant toxins from different types of SHT. We show here that the H176R variation in the PVL sequence causes no change in leukotoxicity and that the R variant is as efficient as wild-type PVL at inducing pore formation in leukocytes.

Convergent Evolution of Neutralizing Antibodies to Staphylococcus aureus γ-Hemolysin C That Recognize an Immunodominant Primary Sequence-Dependent B-Cell Epitope.

Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved ß-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus.

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.

Identification of biologic agents to neutralize the bicomponent leukocidins of Staphylococcus aureus.

A key aspect underlying the severity of infections caused by Staphylococcus aureus is the abundance of virulence factors that the pathogen uses to thwart critical components of the human immune response. One such mechanism involves the destruction of host immune cells by cytolytic toxins secreted by S. aureus, including five bicomponent leukocidins: PVL, HlgAB, HlgCB, LukED, and LukAB. Purified leukocidins can lyse immune cells ex vivo, and systemic injections of purified LukED or HlgAB can acutely kill mice. Here, we describe the generation and characterization of centyrins that bind S. aureus leukocidins with high affinity and protect primary human immune cells from toxin-mediated cytolysis. Centyrins are small protein scaffolds derived from the fibronectin type III-binding domain of the human protein tenascin-C. Although centyrins are potent in tissue culture assays, their short serum half-lives limit their efficacies in vivo. By extending the serum half-lives of centyrins through their fusion to an albumin-binding consensus domain, we demonstrate the in vivo efficacy of these biologics in a murine intoxication model and in models of both prophylactic and therapeutic treatment of live S. aureus systemic infections. These biologics that target S. aureus virulence factors have potential for treating and preventing serious staphylococcal infections.

Rim domain loops of staphylococcal ß-pore forming bi-component toxin S-components recognize target human erythrocytes in a coordinated manner.

Staphylococcus aureus bi-component pore-forming toxins consist of S- and F-components, and form hetero-octameric beta-barrel pores on target blood cell membranes. Among them, γ-haemolysin (Hlg2 and F-component of Luk (LukF)) and LukED (LukE and LukD) possess haemolytic activity, whereas the Panton-Valentine leukocidin (LukS-PV and LukF-PV) does not lyse human erythrocytes. Here, we focussed on four loop structures in the rim domain of S-component, namely loops -1, -2, -3 and -4, and found that replacement of Loop-4 in both Hlg2 and LukE with that of LukS-PV abolished their haemolytic activity. Furthermore, LukS-PV gained haemolytic activity by Loop-4 exchange with Hlg2 or LukE, suggesting that Loop-4 of these S-components determined erythrocyte specificity. LOOP-1 and -2 enhanced the erythrocytes-binding ability of both components. Although Hlg2 and LukE recognize Duffy antigen receptor for chemokines on human erythrocytes, the ability of Loop-4 was not complementary between Hlg2 and LukE. Exchange of Hlg2 with LukE Loop-4 showed weaker activity than intact Hlg2, and LukE mutant with Hlg2 Loop-4 lost its haemolytic activity in combination of LukD. Interestingly, the haemolytic activities of these Loop-4 exchange mutants were affected by F-component, namely LukF enhanced haemolytic activities of these Hlg2 and LukE Loop-4 mutants, and also haemolytic activity of LukS-PV mutant with LukE Loop-4.

Intermolecular ionic interactions serve as a possible switch for stem release in the staphylococcal bi-component toxin for ß-barrel pore assembly.

The ß-strand stem release system of staphylococcal ß-barrel pore-forming toxin γ-hemolysin was investigated. Mutations at K15 and R16 in the cap domain of Hlg2 decreased hemolytic activity more markedly than their effect on erythrocyte binding. In addition, D122N mutation of LukF prestem lost the activity with Hlg2 R16A, indicating that electrostatic interactions between residues in the Hlg2 cap and prestem of adjacent LukF in the ring-shaped complex might serve as a switch for stem release.

Characterization of a leukocidin identified in Staphylococcus pseudintermedius.

Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry we identified two proteins, named "LukS-I" and "LukF-I", encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermedius LukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermedius LukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections.

Leukocidins: staphylococcal bi-component pore-forming toxins find their receptors.

Staphylococcus aureus is a major bacterial pathogen that causes disease worldwide. The emergence of strains that are resistant to commonly used antibiotics and the failure of vaccine development have resulted in a renewed interest in the pathophysiology of this bacterium. Staphylococcal leukocidins are a family of bi-component pore-forming toxins that are important virulence factors. During the past five years, cellular receptors have been identified for all of the bi-component leukocidins. The identification of the leukocidin receptors explains the cellular tropism and species specificity that is exhibited by these toxins, which has important biological consequences. In this Review, we summarize the recent discoveries that have reignited interest in these toxins and provide an outlook for future research.

Genomic comparison between Staphylococcus aureus GN strains clinically isolated from a familial infection case: IS1272 transposition through a novel inverted repeat-replacing mechanism.

A bacterial insertion sequence (IS) is a mobile DNA sequence carrying only the transposase gene (tnp) that acts as a mutator to disrupt genes, alter gene expressions, and cause genomic rearrangements. "Canonical" ISs have historically been characterized by their terminal inverted repeats (IRs), which may form a stem-loop structure, and duplications of a short (non-IR) target sequence at both ends, called target site duplications (TSDs). The IS distributions and virulence potentials of Staphylococcus aureus genomes in familial infection cases are unclear. Here, we determined the complete circular genome sequences of familial strains from a Panton-Valentine leukocidin (PVL)-positive ST50/agr4 S. aureus (GN) infection of a 4-year old boy with skin abscesses. The genomes of the patient strain (GN1) and parent strain (GN3) were rich for "canonical" IS1272 with terminal IRs, both having 13 commonly-existing copies (ce-IS1272). Moreover, GN1 had a newly-inserted IS1272 (ni-IS1272) on the PVL-converting prophage, while GN3 had two copies of ni-IS1272 within the DNA helicase gene and near rot. The GN3 genome also had a small deletion. The targets of ni-IS1272 transposition were IR structures, in contrast with previous "canonical" ISs. There were no TSDs. Based on a database search, the targets for ce-IS1272 were IRs or "non-IRs". IS1272 included a larger structure with tandem duplications of the left (IRL) side sequence; tnp included minor cases of a long fusion form and truncated form. One ce-IS1272 was associated with the segments responsible for immune evasion and drug resistance. Regarding virulence, GN1 expressed cytolytic peptides (phenol-soluble modulin α and δ-hemolysin) and PVL more strongly than some other familial strains. These results suggest that IS1272 transposes through an IR-replacing mechanism, with an irreversible process unlike that of "canonical" transpositions, resulting in genomic variations, and that, among the familial strains, the patient strain has strong virulence potential based on community-associated virulence factors.