Staphylococcal Leukocidin Research Articles

New targets for leukocidins.

This study shows that staphylococcal leukocidins target endothelial cells to cause host lethality.

Adaptation of the Staphylococcus aureus leukocidin LukGH for the rabbit host by protein engineering.

Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10-15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.

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.

LukMF' is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis.

Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal leukocidins in the pathogenesis of bovine S. aureus disease. We show that LukAB, in contrast to the γ-hemolysins, LukED, and LukMF′, was unable to kill bovine neutrophils, and identified CXCR2 as a bovine receptor for HlgAB and LukED. Furthermore, we assessed functional leukocidin secretion by bovine mastitis isolates and observed that, although leukocidin production was strain dependent, LukMF′ was most abundantly secreted and the major toxin killing bovine neutrophils. To determine the role of LukMF′ in bovine mastitis, cattle were challenged with high (S1444) or intermediate (S1449, S1463) LukMF′-producing isolates. Only animals infected with S1444 developed severe clinical symptoms. Importantly, LukM was produced in vivo during the course of infection and levels in milk were associated with the severity of mastitis. Altogether, these findings underline the importance of LukMF′ as a virulence factor and support the development of therapeutic approaches targeting LukMF′ to control S. aureus mastitis in cattle.

The staphylococcal Panton and Valentine Leukocidin and γ-haemolysin HlgC/HlgB share C5aR as a receptor, but operate diverse intracellular activities in human polymorphonuclear neutrophils

The staphylococcal Panton and Valentine Leukocidin and γ-haemolysin HlgC/HlgB share C5aR as a receptor, but operate diverse intracellular activities in human polymorphonuclear neutrophils

English

Staphylococcus aureus toxins represent a public health challenge all over the world. This study aims to analyze the prevalence of genes encoding the staphylococcal leukocidins and their correlation with antimicrobial susceptibility and the source of isolation. For this purpose, the susceptibility of 75 S. aureus isolates to 12 antimicrobial agents was investigated. The leukocidins genes (lukD, lukE, lukF and lukS) were detected by polymerase chain reaction (PCR). The ability to express these genes was assessed among 20 isolates by RT-PCR. The most prevalent luk genes were lukF gene (73.3%), followed by lukE (64%), lukD (44%) and lukS (34.7%). Expression of lukD, lukE and lukS genes were variable. lukF gene was not expressed by any of the tested isolates. A statistically significant association was found between lukF occurrence and burn isolates. Besides that, lukF gene was more prevalent among amoxicillin-clavulenic acid and amikacin resistant isolates, while lukE was predominant with gentamicin resistant isolates. High expression level of lukD was found in MRSA and MDR isolates. Key words: Staphylococcus aureus, leukocidins, antimicrobial sensitivity.

The staphylococcal toxins γ-haemolysin AB and CB differentially target phagocytes by employing specific chemokine receptors.

Evasion of the host phagocyte response by Staphylococcus aureus is crucial to successful infection with the pathogen. γ-Hemolysin AB and CB (HlgAB, HlgCB) are bicomponent pore-forming toxins present in almost all human S. aureus isolates. Cellular tropism and contribution of the toxins to S. aureus pathophysiology are poorly understood. Here, we identify the chemokine receptors CXCR1, CXCR2 and CCR2 as targets for HlgAB, and the complement receptors C5aR and C5L2 as targets for HlgCB. The receptor expression patterns allow the toxins to efficiently and differentially target phagocytic cells. Murine neutrophils are resistant to HlgAB and HlgCB. CCR2 is the sole murine receptor orthologue compatible with γ-Hemolysin. In a murine peritonitis model, HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. HlgAB-mediated targeting of CCR2+ cells highlights the involvement of inflammatory macrophages during S. aureus infection. Functional quantification identifies HlgAB and HlgCB as major secreted staphylococcal leukocidins.

Staphylococcal cassette chromosome mec (SCCmec) characterization and panton-valentine leukocidin gene occurrence for methicillin-resistant Staphylococcus aureus in Turkey, from 2003 to 2006

Methicillin-resistant Staphylococcus aureus (MRSA) cause serious community-acquired and nosocomial diseases all over the world. We determined the SCCmec types and occurrence of the PVL gene by using TaqMan real-time PCR method, and correlated these with phenotypic antibiotic susceptibility patterns for MRSA strains collected from Gulhane Military Medical Academy Hospital (GMMAH) during 4 years study period. To our knowledge, this is the first report from Turkey of molecular SCCmec typing analysis of MRSA stains. A total of 385 clinical MRSA isolates collected in the clinical and Microbiology Laboratory at GMMAH between 2003 and 2006 were included in the study. Overall, SCCmec types-I, II, III, IV, V, nontypeable and PVL occurrence were detected in 11 (2.8%), 3 (0.8%), 316 (82.1%), 20 (5.1%), 20 (5.1%), 15 (3.9%) and 5 (1.3%) isolates, respectively. A total of 330 (85.5%) were SCCmec-I/II/III and 40 (10.3%) were SCCmec IV/V. SCCmec-I/II/III isolates were recovered more from patients with serious infections in surgical departments especially those with intensive care units than the SCCmec-IV/V isolates (chi(2) = 13.560, P < 0.001). SCCmec-I/II/III MRSA strains were predominantly recovered from blood stream (53.0%, P = 0.014), while SCCmec-IV/V strains were predominately isolated from skin and soft tissue and abscess (55.0%, P < 0.001). The PVL gene was detected in 10.0% of SCCmec-IV/V isolates in contrast to 0.3% in SCCmec-I/II/III (chi(2) = 25.164, P < 0.001). SCCmec-I/II/III MRSA strains were more resistant to clindamycin (chi(2) = 5.078, P = 0.024), amoxicillin-clavulanate (chi(2) = 84.912, P < 0.001), erythromycin (chi(2) = 4.651, P = 0.031), gentamicin (chi(2) = 24.869, P < 0.001), and rifampin (chi(2) = 18.878, P < 0.001) than SCCmec-IV/V MRSA strains. This data indicates that SCCmec-III MRSA strains that do not carry the PVL gene are the predominant MRSA strains in our hospital setting in Ankara, capital of Turkey and that SCCmec-I/II/III MRSA strains may cause serious infections in surgical departments especially those with intensive care units.

Assembly of Staphylococcal Leukocidin into a Pore-Forming Oligomer on Detergent-Resistant Membrane Microdomains, Lipid Rafts, in Human Polymorphonuclear Leukocytes

Staphylococcal leukocidin (Luk) consists of LukS and LukF, which cooperatively lyse human polymorphonuclear leukocytes (HPMNLs), monocytes, and macrophages. Here we found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of HPMNLs. When HPMNLs were treated with LukS alone, 24% of the added LukS was localized in lipid rafts. Furthermore, in HPMNLs treated with both LukS and LukF simultaneously, about 90% of high molecular-mass complexes of 100 kDa, which consists of LukS and LukF, were detected in the lipid raft fractions. In contrast, in HPMNLs treated with LukF alone, LukF was not localized in lipid rafts despite binding to the target cell membranes. Ten mM methyl-beta-cyclodextrin, a dysfunctioning agent of lipid rafts, completely inhibited assembly of Luk on lipid rafts, and resulted in null leukocytolytic activity of Luk. Hence, we concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs and that LukF can bind to LukS, which had already bound to lipid rafts, to assemble into hetero-oligomers.

Assembly of the Bi-component Leukocidin Pore Examined by Truncation Mutagenesis

Staphylococcal leukocidin (Luk) and alpha-hemolysin (alphaHL) are members of the same family of beta barrel pore-forming toxins (betaPFTs). Although the alphaHL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on alphaHL suggest that, although the N termini of betaPFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.

In vitro effects of staphylococcal leukocidin LukE/LukD on the proliferative ability of lymphocytes isolated from common carp ( Cyprinus carpio L.)

Various strains of Staphylococcus aureus bacteria produce bicomponent toxins such as leukocidin LukE/LukD consisting of two subunits S (LukE) and F (LukD). The toxin subunits bind to leukocyte cell membrane inducing transmembrane pore formation and subsequent cell lysis. There is a lack of data on the effects of staphylococcal leukocidin LukE/LukD on fish immunocompetent cells in vitro. The purpose of the study was to determine the influence of leukocidin LukE/LukD on the proliferative ability of T and B lymphocytes isolated from head kidney and spleen of common carp ( Cyprinus carpio L.). The response of T and B lymphocytes was stimulated with concanavalin A (ConA) and lypopolysaccharide (LPS) respectively and measured by the MTT test. The results revealed the modulatory influence of staphylococcal leukocidin LukE/LukD on the proliferative ability of fish T and B lymphocytes. The highest concentrations (5000–25000 ng ml −1 of medium) of complete leukocidin LukE/LukD and its subunit LukE induced statistically significant suppression of both head kidney and splenic T and B lymphocyte proliferation. On the other hand, the lowest concentrations of the complete toxin (0.32-1.6 ng ml −1 of medium) caused slight but statistically significant stimulation of both head kidney and splenic T and B lymphocyte proliferative ability. No modulatory effects were observed at any concentration of subunit LukD used in the experiment. There is a need for further studies concerning the susceptibility of other immune cell populations isolated from fish to different types of staphylococcal leukocidin.

Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore.

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of beta-barrel pore-forming toxins (beta-PFTs). By contrast, other beta-PFTs form homo-oligomeric pores; for example, the staphylococcal alpha-hemolysin (alpha HL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the alpha HL pore. It is also pertinent to the mechanism of assembly of beta-PFT pores and suggests new possibilities for engineering these proteins.

Identification of serine138 residue in the 4-residue segment K135K1361137S138 of LukS-I component of Staphylococcus intermedius leukocidin crucial for the LukS-I-specific function of staphylococcal leukocidin.

Luk-I produced by Staphylococcus intermedius was found to be a new member of the staphylococcal bi-component pore-forming toxin family, in which staphylococcal leukocidin, Panton-Valentine leukocidin, and gamma-hemolysin are included. Luk-I consists of LukS-I and LukF-I. From the deduced amino acid sequence of LukS-I, a 4-residue sequence, K135K1361137S138, at the root of the stem region was found to be identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. A mutant of LukS-I (MLSI-SA), in which the Ser138 residue was replaced by an alanine residue, was created, purified, and assayed for its leukocytolytic and pore-forming activities with LukF-I. Both LukS-I and MLSI-SA formed a ring-shaped complex with LukF-I on rabbit erythrocytes and human polymorphonuclear leukocytes (HPMNLs) membrane. However, MLSI-SA showed no leukocytolytic activity with LukF-I. LukS-I was phosphorylated by protein kinase A in the presence of [gamma-32P] ATP in a cell-free system, but MLSI-SA was not phosphorylated significantly. A potent and selective inhibitor of protein kinase A (N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89)) showed 50% inhibition of the Luk-I-induced cell lysis at 0.5 nM. Thus, it is concluded that the phosphorylation of the Ser138 residue in the 4-residue segment K135K1361137S138 of LukS-I is important for the leukocytolysis of HPMNLs.

The staphylococcal leukocidin bicomponent toxin forms large ionic channels.

The genes encoding the F and S components of a leukocidin, LukF (HlgB) and LukS (HlgC), a pore-forming binary toxin, were amplified from the Smith 5R strain of Staphylococcus aureus both with and without sequences encoding 3'-hexahistidine tags. The His-tagged components were expressed in Escherichia coli and purified under nondenaturing conditions. In addition, the two unmodified proteins and the His-tagged versions were produced in an E. coli cell-free in vitro transcription and translation system. An SDS-stable oligomer of approximately 200 kDa appeared when both components were cotranslated in the presence of rabbit erythrocyte membranes. Hemolytic activity of the combined components against rabbit erythrocytes was measured for both in vitro- and in vivo-produced polypeptides, yielding similar HC(50) values of approximately 0.14 microg/mL. The pore-forming properties of the recombinant leukocidin were also investigated with planar lipid bilayers of diphytanoylphosphatidylcholine. Although leukocidins and staphylococcal alpha-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS [1 M KCl and 5 mM HEPES (pH 7.4)], which is more than 3 times greater than that of alpha-hemolysin measured under the same conditions. Therefore, if the leukocidin pore were a cylinder, its diameter would be almost twice that of alpha-hemolysin. In addition, the leukocidin pore is weakly cation selective and exhibits gating at low positive potentials, while alpha-hemolysin is weakly anion selective and gates only at high potentials. Taken together, these data suggest that the structure of the oligomeric pore formed by the leukocidin examined here has diverged significantly from that of alpha-hemolysin.

Construction of a LukS-PV mutant of a staphylococcal Panton-Valentine leukocidin component having a high LukS-like function.

A 2-residue (D12I13) segment of LukS of a staphylococcal leukocidin component is an essential region for the hemolytic function of LukS towards rabbit erythrocytes in the presence of LukF. Here, we report that insertion of D, I, or AA residue(s) between A11 and E12 residues of LukS-PV, in which the 2-residue D12I13 segment in LukS was absent, confers the full LukS function on LukS-PV, which has only 4% hemolytic activity of that of LukS towards rabbit erythrocytes.

Assembly of Staphylococcus aureus leukocidin into a pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit erythrocytes.

Staphylococcal leukocidin consists of two separate proteins, LukS and LukF, which cooperatively lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when polyethylene glycols with hydrodynamic diameters of > or = 2.1 nm were present in the extracellular space. (2) Electron microscopy showed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter of approximately 2 nm.

Phosphorylation of LukS by protein kinase A is crucial for the LukS-specific function of the staphylococcal leukocidin on human polymorphonuclear leukocytes.

Staphylococcal leukocidin (Luk) consists of two protein components, LukF and LukS, which cooperatively lyse human and rabbit polymorphonuclear leukocytes. Here, we demonstrate that the phosphorylation of LukS by protein kinase A is crucial for the LukS-specific leukocytolytic function of Luk on HPMNLs by using N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), which is a potent and selective inhibitor of protein kinase A. At 0.5 microM H-89 completely prevented the Luk-induced cell lysis accompanied by blocking of the incorporation of exogenous 32P-H3PO4 into LukS on HPMNLs. However, with LukS and LukF together, 0.5 microM H-89 did not inhibit the cell swelling which takes place before the cell lysis. HPMNLs also became swollen upon treating with both LukF and LukS mutants which could not be phosphorylated.

Identification of the minimum segment in which the threonine 246 residue is a potential phosphorylated site by protein kinase A for the LukS-specific function of staphylococcal leukocidin

Staphylococcal leukocidin and γ-hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for γ-hemolysin. In this report, we identify the minimum segment responsible for the LukS-specific function of leukocidin. After chemical analysis and homology study of the amino acid sequence of the C-terminal region between LukS and Hlg2, we found a unique 5-residue sequence I 242K 243R 244S 245T 246 in LukS in which the 4-residue KRST is identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. To elucidate whether the 5-residue segment is essential for the LukS function, we created plasmids containing a series of mutant genes corresponding to the 5-residue sequence and expressed them in Escherichia coli. The mutant proteins were purified and assayed for their leukocytolytic activity with LukF. The mutant MLS-TS, in which the T 246 in the 5-residue sequence was replaced by S, showed leukocidin activity 10 times higher than that of the intact LukS. However, neither mutant MLS-TY nor MLS-TA, in which T 246 was replaced by Y or A, respectively, showed leukocidin activity. The 5-residue segment was found to be deleted in Hlg2. The mutant of Hlg2, in which the 5-residue segment was inserted at the position that the segment is deleted, showed leukocidin activity. The boiled LukS, MLS-TS, and MHS-Z were strongly phosphorylated with [γ- 32P]ATP in the presence of protein kinase A in a cell-free system. Thus, we conclude that the 5-residue segment I 242K 243R 244S 245T 246 is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.

Identification of the essential regions for LukS- and H gamma II-specific functions of staphylococcal leukocidin and gamma-hemolysin.

Staphylococcal leukocidin and gamma-hemolysin consist of LukF and LukS for leudocidin and LukF and H gamma II for gamma-hemolysin. To identify the pivotal domain(s) of LukS and H gamma II responsible for their specific cytolytic activities, a series of chimeric genes (lukS/hlg2) were constructed and expressed in Escherichia coli. The results indicate that the C-terminal 24-residue and the N-terminal 57-residue segments of LukS and H gamma II, respectively, are the essential regions for the LukS and H gamma II functions.

Differentiation of HL-60 cells is promoted by H-toxin of Clostridium septicum

H-toxin of Clostridium septicum potentiated dimethyl sulfoxide(DMSO)-induced differentiation of human promyelocytic leukemia HL-60 cells which were monitored by nuclear morphology and production of oxidative radicals. But, H-toxin did not induce differentiation of HL-60 cells in the absence of DMSO. These phenomena were not observed by staphylococcal leukocidin, a cytotoxin affecting to HL-60 cells. In HL-60 cells, ADP-ribosylation of 118, 93, 75 and 58 kDa membrane proteins was observed, but the ADP-ribosylation was not detected either in differentiated HL-60 cells by DMSO or in normal polymorphonuclear leukocytes of human. When the membranes of HL-60 cells were incubated with H-toxin, ADP-ribosylation of the membrane proteins was inhibited. Such suppressive effects on ADP-ribosylation were not observed by DMSO or staphylococcal leukocidin. These data suggest that inhibition of the ADP-ribosylation by H-toxin may play an important role in potentiation of DMSO-induced differentiation of HL-60 cells.