Martin J. McGavin

Staphylococcus δ-toxin induces allergic skin disease by activating mast cells

Atopic dermatitis is a chronic inflammatory skin disease that affects 15–30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca2+) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in KitW-sh/W-sh mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Molecular differentiation of historic phage-type 80/81 and contemporary epidemic Staphylococcus aureus

Staphylococcus aureus is a bacterial pathogen known to cause infections in epidemic waves. One such epidemic was caused by a clone known as phage-type 80/81, a penicillin-resistant strain that rose to world prominence in the late 1950s. The molecular underpinnings of the phage-type 80/81 outbreak have remained unknown for decades, nor is it understood why related S. aureus clones became epidemic in hospitals in the early 1990s. To better understand the molecular basis of these epidemics, we sequenced the genomes of eight S. aureus clinical isolates representative of the phage-type 80/81 clone, the Southwest Pacific clone [a community-associated methicillin-resistant S. aureus (MRSA) clone], and contemporary S. aureus clones, all of which are genetically related and belong to the same clonal complex (CC30). Genome sequence analysis revealed that there was coincident divergence of these clones from a recent common ancestor, a finding that resolves controversy about the evolutionary history of the lineage. Notably, we identified nonsynonymous SNPs in genes encoding accessory gene regulator C (agrC) and α-hemolysin (hla)—molecules important for S. aureus virulence—that were present in virtually all contemporary CC30 hospital isolates tested. Compared with the phage-type 80/81 and Southwest Pacific clones, contemporary CC30 hospital isolates had reduced virulence in mouse infection models, the result of SNPs in agrC and hla. We conclude that agr and hla (along with penicillin resistance) were essential for world dominance of phage-type 80/81 S. aureus, whereas key SNPs in contemporary CC30 clones restrict these pathogens to hospital settings in which the host is typically compromised.

Activation of the SspA serine protease zymogen of Staphylococcus aureus proceeds through unique variations of a trypsinogen-like mechanism and is dependent on both autocatalytic and metalloprotease-specific processing.

The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA29 ↓, releasing a Leu30 isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, 40QQTQSSKQQTPKIQ53. The preferred site is Gln43 with secondary processing at Gln47 and Gln53. This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu58 and then Val69 to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val69SspA promote rapid intermolecular processing of remaining zSspA at Glu65, producing an N-terminal 66HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His66 of this penultimate isoform blocks the active site by hydrogen bonding to Ser237 and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr232 proximal to Ser237, permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.

Evolutionary blueprint for host- and niche-adaptation in Staphylococcus aureus clonal complex CC30

Staphylococcus aureus clonal complex CC30 has caused infectious epidemics for more than 60 years, and, therefore, provides a model system to evaluate how evolution has influenced the disease potential of closely related strains. In previous multiple genome comparisons, phylogenetic analyses established three major branches that evolved from a common ancestor. Clade 1, comprised of historic pandemic phage type 80/81 methicillin susceptible S. aureus (MSSA), and Clade 2 comprised of contemporary community acquired methicillin resistant S. aureus (CA-MRSA) were hyper-virulent in murine infection models. Conversely, Clade 3 strains comprised of contemporary hospital associated MRSA (HA-MRSA) and clinical MSSA exhibited attenuated virulence, due to common single nucleotide polymorphisms (SNPs) that abrogate production of α-hemolysin Hla, and interfere with signaling of the accessory gene regulator agr. We have now completed additional in silico genome comparisons of 15 additional CC30 genomes in the public domain, to assess the hypothesis that Clade 3 has evolved to favor niche adaptation. In addition to SNPs that influence agr and hla, other common traits of Clade 3 include tryptophan auxotrophy due to a di-nucleotide deletion within trpD, a premature stop codon within isdH encoding an immunogenic cell surface protein involved in iron acquisition, loss of a genomic toxin–antitoxin (TA) addiction module, acquisition of S. aureus pathogenicity islands SaPI4, and SaPI2 encoding toxic shock syndrome toxin tst, and increased copy number of insertion sequence ISSau2, which appears to target transcription terminators. Compared to other Clade 3 MSSA, S. aureus MN8, which is associated with Staphylococcal toxic shock syndrome, exhibited a unique ISSau2 insertion, and enhanced production of toxic shock syndrome toxin encoded by SaPI2. Cumulatively, our data support the notion that Clade 3 strains are following an evolutionary blueprint toward niche-adaptation.

Induction of the Staphylococcal Proteolytic Cascade by Antimicrobial Fatty Acids in Community Acquired Methicillin Resistant Staphylococcus aureus

Community acquired methicillin resistant Staphylococcus aureus (CA-MRSA), and the USA300 strain of CA-MRSA in particular, are known for their rapid community transmission, and propensity to cause aggressive skin and soft tissue infections. To assess factors that contribute to these hallmark traits of CA-MRSA, we evaluated how growth of USA300 and production of secreted virulence factors was influenced on exposure to physiologic levels of unsaturated free fatty acids that would be encountered on the skin or anterior nares, which represent the first sites of contact with healthy human hosts. There was a sharp threshold between sub-inhibitory and inhibitory concentrations, such that 100 µM sapienic acid (C16∶1) and linoleic acid (C18∶1) were sufficient to prevent growth after 24 h incubation, while 25 µM allowed unrestricted growth, and 50 µM caused an approximate 10–12 h lag, followed by unimpeded exponential growth. Conversely, saturated palmitic or stearic acids did not affect growth at 100 µM. Although growth was not affected by 25 µM sapienic or linoleic acid, these and other unsaturated C16 and C18 fatty acids, but not their saturated counterparts, promoted robust production of secreted proteases comprising the Staphylococcal proteolytic cascade. This trait was also manifested to varying degrees in other CA-MRSA, and in genetically diverse methicillin susceptible S. aureus strains. Therefore, induction of the Staphylococcal proteolytic cascade by unsaturated fatty acids is another feature that should now be evaluated as a potential contributing factor in the aggressive nature of skin and soft tissue infections caused by USA300, and as a general virulence mechanism of S. aureus.

Colonization with community-acquired methicillin-resistant Staphylococcus aureus in children with atopic dermatitis: a cross-sectional study

Background  Bacterial infection with Staphylococcus aureus is a common complication of atopic dermatitis (AD). The incidence of community‐acquired methicillin‐resistant S. aureus infection (MRSA) in the AD population is unknown.

Comparison of Staphopain A (ScpA) and B (SspB) precursor activation mechanisms reveals unique secretion kinetics of proSspB (Staphopain B), and a different interaction with its cognate Staphostatin, SspC

The scpAB and sspABC operons of Staphylococcus aureus encode Staphopain cysteine proteases ScpA and SspB, and their respective Staphostatins ScpB and SspC, which are thought to protect against premature activation of Staphopain precursors during protein export. However, we found that the proSspB precursor was secreted and activated without detriment to S. aureus in the absence of SspC function. Our data indicate that this is feasible due to a restricted substrate specificity of mature SspB, a stable precursor structure and slow secretion kinetics. In contrast, mature ScpA had a broad substrate specificity, such that it was prone to autolytic degradation, but also was uniquely able to degrade elastin fibres. Modelling of proScpA relative to the proSspB structure identified several differences, which appear to optimize proScpA for autocatalytic activation, whereas proSspB is optimized for stability, and cannot initiate autocatalytic activation. Consequently, recombinant proSspB remained stable and unprocessed when retained in the cytoplasm of Escherichia coli, whereas proScpA initiated rapid autocatalytic activation, leading to capture of an activation intermediate by ScpB. We conclude that the status of sspBC in S. aureus, as paralogues of the ancestral scpAB genes, facilitated a different activation mechanism, a stable proSspB isoform and modified Staphostatin function.

Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1

Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis‐restricted Ilk gene inactivation and control integrin‐linked kinase (ILK)‐expressing litter‐mates, we investigated the role of ILK in epidermal S. aureus invasion. Heat‐killed, but not live, bacteria were internalized to Rab5‐ and Rab7‐positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5‐fold. ILK‐deficient mouse keratinocytes internalized bacteria 2‐ to 4‐fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK‐deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1‐deficient cells was 2.5‐fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin expiants with ILK‐deficient epidermis was 35‐fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK‐Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection.—Sayedyahossein, S., Xu, S. X., Rudkouskaya, A., McGavin, M. J., McCormick, J. K., Dagnino, L. Staphylococcus aureus keratinocyte invasion is mediated by integrin‐linked kinase and Rac1. FASEB J. 29, 711‐723 (2015). www.fasebj.org

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

UNLABELLED 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 H₁₂₁Y 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 farR-dependent manner. Second, an H₁₂₁Y 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 μM 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. IMPORTANCE Staphylococcus 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 farR-dependent mechanism, in response to these antimicrobial fatty acids that would be encountered in a tissue abscess.

Role of Lipase from Community-Associated Methicillin-Resistant Staphylococcus aureus Strain USA300 in Hydrolyzing Triglycerides into Growth-Inhibitory Free Fatty Acids

Part of the human host innate immune response involves the secretion of bactericidal lipids on the skin and delivery of triglycerides into abscesses to control invading pathogens. Two Staphylococcus aureus lipases, named SAL1 and SAL2, were identified in the community-associated methicillin-resistant S. aureus strain USA300, which, presumably, are produced and function to degrade triglycerides to release free fatty acids. We show that the SAL2 lipase is one of the most abundant proteins secreted by USA300 and is proteolytically processed from the 72-kDa proSAL2 to the 44-kDa mature SAL2 by the metalloprotease aureolysin. We show that spent culture supernatants had lipase activity on both short- and long-chain fatty acid substrates and that deletion of gehB, encoding SAL2, resulted in the complete loss of these activities. With the use of gas chromatography-mass spectrometry, we show that SAL2 hydrolyzed trilinolein to linoleic acid, a fatty acid with known antistaphylococcal properties. When added to cultures of USA300, trilinolein and, to a lesser extent, triolein inhibited growth in a SAL2-dependent manner. This effect was shown to be due to the enzymatic activity of SAL2 on these triglycerides, since the catalytically inactive SAL2 Ser412Ala mutant was incapable of hydrolyzing the triglycerides or yielding delayed growth in their presence. Overall, these results reveal that SAL2 hydrolyzes triglycerides of both short- and long-chain fatty acids and that the released free fatty acids have the potential to cause significant delays in growth, depending on the chemical nature of the free fatty acid.