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

ABSTRACT: The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The ?-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-Å resolution. 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.

SUBMITTER: Badarau A

PROVIDER: S-EPMC4281717 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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

Badarau Adriana A Rouha Harald H Malafa Stefan S Logan Derek T DT Håkansson Maria M Stulik Lukas L Dolezilkova Ivana I Teubenbacher Astrid A Gross Karin K Maierhofer Barbara B Weber Susanne S Jägerhofer Michaela M Hoffman David D Nagy Eszter E

The Journal of biological chemistry 20141103 1

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 ...[more]

PMID: 25371205

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Project description:Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases and is notorious for antibiotic resistance. S. aureus utilizes a family of pore-forming toxins, known as bi component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A, leukocidin B), a toxin that is secreted as a soluble heterodimer and assembles into an octameric beta barrel pore that is embedded in the host cell membrane, resulting in death of the host cell. The established cellular receptor for LukAB is CD11b of the Mac1 complex. LukAB variants from S. aureus clonal complexes (CC) 30 and 45 were recently described to use the proton channel hydrogen voltage gated channel 1 (HVCN1) as a receptor. Here we show that HVCN1 is an essential receptor used by all LukAB variants representing the major S. aureus clonal complexes, including CC8 LukAB, which belongs to the most prevalent lineage responsible for skin and soft tissue infections in the United States. We demonstrate that while each receptor is sufficient to recruit CC8 LukAB to the plasma membrane of phagocytes, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contribute to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow CC8 LukAB to bypass the requirement for CD11b. We discovered thirty mutations primarily localized in the stem domain of LukA and LukB that enabled LukAB to exhibit enhanced cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting experiments we show these mutations alter the solvent accessibility of the stem domain which prime LukAB for oligomerization. Together, our data allow us to introduce a role for CD11b beyond toxin recruitment to the target cell: we propose a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization, driving pore-formation. The mass spectrometric raw files for the hydroxyl radical footprinting experiment are included here.

Project description:Staphylococcus aureus is the major organism responsible for surgical implant infections. Antimicrobial treatment of these infections often fails, leading to expensive surgical intervention and increased risk of mortality to the patient. The challenge in treating these infections is associated with the high tolerance of S. aureus biofilm to antibiotics. MazEF, a toxin-antitoxin system, is thought to be an important regulator of this phenotype, but its physiological function in S. aureus is controversial. Here, we examined the role of MazEF in developing chronic infections by comparing growth and antibiotic tolerance phenotypes in three S. aureus strains to their corresponding strains with disruption of mazF expression. Strains lacking mazF production showed increased biofilm growth and decreased biofilm antibiotic tolerance. Deletion of icaADBC in the mazF::Tn background suppressed the growth phenotype observed with mazF-disrupted strains, suggesting the phenotype was ica dependent. We confirmed these phenotypes in our murine animal model. Loss of mazF resulted in increased bacterial burden and decreased survival rate of mice compared to its wild-type strain demonstrating that loss of the mazF gene caused an increase in S. aureus virulence. Although lack of mazF gene expression increased S. aureus virulence, it was more susceptible to antibiotics in vivo Combined, the ability of mazF to inhibit biofilm formation and promote biofilm antibiotic tolerance plays a critical role in transitioning from an acute to chronic infection that is difficult to eradicate with antibiotics alone.IMPORTANCE Surgical infections are one of the most common types of infections encountered in a hospital. Staphylococcus aureus is the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate, as the bacteria form biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria that are planktonic, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate that mazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allows S. aureus to transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host.

Dataset Information

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

Publications

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

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