Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N‐terminal fungalysin‐thermolysin‐propeptide domain

Nickerson, Nicholas N.; Joag, Vineet; McGavin, Martin J.

doi:10.1111/j.1365-2958.2008.06384.x

Cited by 44 publications

(47 citation statements)

References 67 publications

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“…The b-chain of C3 was not affected by aureolysin. N-terminal sequencing revealed that the aureolysincleaved a-chain starts with the amino acid sequence NH 2 -LDE-DII, indicating that aureolysin cleaves C3 between an asparagine (N751) and a leucine (L752), which is a typical cleavage site for aureolysin (38,39). This specific cleavage was also observed at lower molar ratios of aureolysin/C3 (Supplemental Fig.…”

Section: Aureolysin Acts As a C3 Convertasementioning

confidence: 88%

Staphylococcus aureus Metalloprotease Aureolysin Cleaves Complement C3 To Mediate Immune Evasion

Laarman

Ruyken

Malone

et al. 2011

The Journal of Immunology

173

126

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Complement is one of the first host defense barriers against bacteria. Activated complement attracts neutrophils to the site of infection and opsonizes bacteria to facilitate phagocytosis. The human pathogen Staphylococcus aureus has successfully developed ways to evade the complement system, for example by secretion of specific complement inhibitors. However, the influence of S. aureus proteases on the host complement system is still poorly understood. In this study, we identify the metalloprotease aureolysin as a potent complement inhibitor. Aureolysin effectively inhibits phagocytosis and killing of bacteria by neutrophils. Furthermore, we show that aureolysin inhibits the deposition of C3b on bacterial surfaces and the release of the chemoattractant C5a. Cleavage analyses show that aureolysin cleaves the central complement protein C3. Strikingly, there was a clear difference between the cleavages of C3 in serum versus purified conditions. Aureolysin cleaves purified C3 specifically in the α-chain, close to the C3 convertase cleavage site, yielding active C3a and C3b. However, in serum we observe that the aureolysin-generated C3b is further degraded by host factors. We pinpointed these factors to be factor H and factor I. Using an aureolysin mutant in S. aureus USA300, we show that aureolysin is essential and sufficient for C3 cleavage by bacterial supernatant. In short, aureolysin acts in synergy with host regulators to inactivate C3 thereby effectively dampening the host immune response.

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Section: Aureolysin Acts As a C3 Convertasementioning

confidence: 88%

Staphylococcus aureus Metalloprotease Aureolysin Cleaves Complement C3 To Mediate Immune Evasion

Laarman

Ruyken

Malone

et al. 2011

The Journal of Immunology

173

126

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“…2D). Interestingly, Arg80 belongs to a conserved RY motif that may contribute to the folding of the protease and its secretion (15,25). Two mutations R80V and R80D led to low solubility of the whole enzyme, suggesting that Arg80 may play a significant role in the folding of MCP-02.…”

Section: Resultsmentioning

confidence: 99%

“…2C). Although the two residues in the RY motif are not strictly conserved and can be replaced with TL, KL, RL, RF, or RM in different subfamilies (15), the first residue is always a hydrogen-bond-forming residue, and the second is a hydrophobic residue that can form a hydrophobic center. This relative conservation implies that the function of the RY motif is invariant.…”

Section: Resultsmentioning

confidence: 99%

“…How it functions, however, is still unknown. Biochemical studies have revealed that the maturation of TLPs occurs through autoprocessing and that this autoprocessing pathway is mediated by the propeptide (14,15). The propeptide in TLPs and in some serine proteases is also referred to as an intramolecular chaperone (IMC), as it is essential for the folding of the catalytic domain but is not required for its function (16).…”

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confidence: 99%

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Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Gao

Wang²,

Yu³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new β-strand, nestling into two other β-strands. Simultaneously, the apparent T m of the autoprocessed complex increases 20°C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.zymogen conversion | zinc metalloproteases inhibition | intramolecular chaperone T he thermolysin (M4) family is comprised of a large number of zinc metalloproteases in the subclan MA(E), most of which have similar sequences and domain structures (1). The representative member of this family is thermolysin (EC 3.4.24.27), a 34.6-kDa thermostable metalloprotease secreted by Bacillus thermoproteolyticus (2). The amino acid sequence and the threedimensional structure of thermolysin were first determined in 1972 (3, 4). Since then, thermolysin and thermolysin-like proteases (TLPs) have been used as models in studying zinc metalloproteases (5, 6).TLPs are widely distributed in many species of microorganisms, and many TLPs are regarded as key pathogenesis factors that are responsible for some severe bacterial infections (7). For example, λ-toxin from Clostridium perfringens can degrade various human immune defense proteins (8). Vibriolysin from Vibrio vulnificus (9) and pseudolysin from Pseudomonas aeruginosa (10) can be lethally blood-poisonous to humans. Therefore, understanding the catalytic and maturation mechanisms of TLPs is very important for developing therapeutics to treat such infectious diseases.TLPs are synthesized as a precursor with a propeptide. TLPs are divided into two distinct groups according to the primary structure of their propeptides. Less than 30% of the total TLPs have short propeptides (∼50 residues), and more than 70% of TLPs have long propeptides (∼200 residues) (11, 12). The long propeptide in TLPs contains two distinguished regions of conservation: an FTP (fungalysin/thermolysin propeptide) domain and a PepSY [peptidase propeptide and YPEB (a protein encoded by Bacillus subtilis ypeB gene)] domain. To date, the PepSY domain has been found not only in TLPs, but also in many nonpeptidase proteins, wherein the presence of this domain may prevent detrimental protease ...

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“…For comparative genome sequencing, genomic DNA was extracted from S. aureus using previously described protocols (28,29). All samples for comparative genome sequencing were processed at the London Regional Genomics Centre (Robarts Research Institute, London, Ontario, Canada [http://www.lrgc.ca]) using an Ion Torrent Personal Genome Machine (PGM) (Life Technologies, Carlsbad, CA) and 316 chips.…”

Section: Methodsmentioning

confidence: 99%

Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids

et al. 2015

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Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions, and in abscesses, a specific mechanism of inducible resistance to this important facet of innate immunity has not been identified. Here, we have sequenced the genome of S. aureus USA300 variants selected for their ability to grow at an elevated concentration of linoleic acid. The fatty acid-resistant clone FAR7 had a single nucleotide polymorphism resulting in an H 121 Y substitution in an uncharacterized transcriptional regulator belonging to the AcrR family, which was divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multidrug efflux pumps. We named these genes farR and farE, for regulator and effector of fatty acid resistance, respectively. Several lines of evidence indicated that FarE promotes efflux of antimicrobial fatty acids and is regulated by FarR. First, expression of farE was strongly induced by arachidonic and linoleic acids in an farRdependent manner. Second, an H 121 Y substitution in FarR resulted in increased expression of farE and was alone sufficient to promote increased resistance of S. aureus to linoleic acid. Third, inactivation of farE resulted in a significant reduction in the inducible resistance of S. aureus to the bactericidal activity of 100 M linoleic acid, increased accumulation of [ 14 C]linoleic acid by growing cells, and severely impaired growth in the presence of nonbactericidal concentrations of linoleic acid. Cumulatively, these findings represent the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen. IMPORTANCEStaphylococcus aureus colonizes approximately 25% of humans and is a leading cause of human infectious morbidity and mortality. To persist on human hosts, S. aureus must have intrinsic defense mechanisms to cope with antimicrobial fatty acids, which comprise an important component of human innate defense mechanisms. We have identified a novel pair of genes, farR and farE, that constitute a dedicated regulator and effector of S. aureus resistance to linoleic and arachidonic acids, which are major fatty acids in human membrane phospholipid. Expression of farE, which encodes an efflux pump, is induced in an farRdependent mechanism, in response to these antimicrobial fatty acids that would be encountered in a tissue abscess. Staphylococcus aureus has a dichotomous relation with human hosts, being able to establish an asymptomatic commensal relationship, but is also historically known as a leading cause of human infectious morbidity and mortality. Significantly, death attributed to S. aureus in the United States is now comparable to mortality rates for AIDS, tuberculosis, and viral hepatitis (1-3). Not surprisingly, therefore, S. aureus has been the subject of intensive research on mechanisms of pathogenesis and acquisition and transfer of antibiotic resistance and of efforts to identify potential vaccine antigens (4-6). Until the late ...

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Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N‐terminal fungalysin‐thermolysin‐propeptide domain

Cited by 44 publications

References 67 publications

Staphylococcus aureus Metalloprotease Aureolysin Cleaves Complement C3 To Mediate Immune Evasion

Staphylococcus aureus Metalloprotease Aureolysin Cleaves Complement C3 To Mediate Immune Evasion

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids

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