Evidence for importance of the Staphylococcus hyicus lipase pro-peptide in lipase secretion, stability and activity

Abstract To investigate the function of the pro-peptide (PP) region of the Staphylococcus hyicus exolipase, restriction sites were created in the lipase gene to facilitate the construction of deletions in this region. Lipase gene expression was carried out in Staphylococcus carnosus . In the presence of the entire PP region, the 86-kDa pro-lipase was efficiently exported, had high lipolytic activity, and hardly any degradation products were seen in Western blot analysis. In addition to the 86-kDa pro-lipase, the membrane fraction contained a 106-kDa immunoreactive form. If the PP was completely or partially deleted, signal peptide processing, lipase secretion, lipase activity and/or lipase stability were impaired. The results obtained with lipase PP deletion mutants indicate that the PP region may have two functional domains. The N-terminal region of the lipase PP appears to be more important for lipase activity and the C-terminal portion for lipase secretion and proteolytic stability. In the presence of only the C-terminal part of the PP lipase, secretion was hardly affected. However, the activity of the extracellular lipase was markedly reduced. If only a small portion of the C-terminal part of the PP was present, lipase secretion was again markedly reduced and no lipase activity was detectable. In the presence of the N-terminal half of the PP region, lipase secretion was affected to a lesser extent. However, the resulting 60-kDa form, which showed comparably good specific lipase activity, suffered severe proteolytic degradation.     

Adaptive immune response to lipoproteins of Staphylococcus aureus in healthy subjects

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life‐threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll‐like receptors 2 and 6. This study addressed the specific B‐cell and T‐cell responses directed to lipoproteins in human S. aureus carriers and non‐carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.     

Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands

Staphylococcus aureus adhesion to the host''s skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.     

Polyclonal antibodies against Staphylococcus aureus ATCC 12 600 catalase do not recognize any protein in cellular extracts from S. aureus subsp. anaerobius

Rabbits were immunized with electrophoretically pure catalase from Staphylococcus aureus ATCC 12 600. The antiserum was used to study whether S. aureus subsp. anaerobius was able to synthesize the apoprotein of catalase. Proteins were separated on polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes and were detected by immunoblotting. No protein reacting with the purified immunoglobulins against S. aureus ATCC 12,600 catalase could be detected in crude and partially purified cellular extracts from S. aureus subsp. anaerobius or its aerotolerant mutants.     

Plasmid curing in Staphylococcus aureus by antibiotics affecting the bacterial cell wall

Abstract Strains of Staphylococcus aureus were converted into L-forms with β-lactam antibiotics, vancomycin and lysostaphin. Reverted bacteria obtained after several transfers in protoplast forms (unstable L-forms) as well as stable L-forms lost their plasmids. Curing was obtained whatever the plasmid size (from 3.4 to 28.2 kb) but complete curing required cell division. Elimination of plasmids in wall-less organisms could be the result of an inhibition of new rounds of plasmid replication following the loss of DNA-envelope interactions.     

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.     

2008-2010年舟山某院金黄色葡萄球菌的分布及耐药性变迁

目的 分析舟山医院三年来金黄色葡萄球菌分布及耐药性变迁,并对耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异做对比.方法 用ATB Expression半自动微生物分析仪进行菌株鉴定及药敏试验,用K-B法测红霉素、克林霉素、头孢西丁、苯唑西林直径,比较耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异.结果 金黄色葡萄球菌对苯唑西林、庆大霉素、红霉素、四环素和克林霉素的耐药率有上升的趋势;MRSA对苯唑西林、庆大霉素、复方新诺明、克林霉素、红霉素、青霉素、喹奴普汀-达福普汀、利福平和四环素的耐药率都明显高于MSSA的耐药率,二者间差异有统计学意义(P＜0.01),D-试验阳性71株,占72.45％.结论 金黄色葡萄球菌的耐药性逐渐升高,特别是对MRSA应引起临床的重视,检测克林霉素诱导型耐药具有重要的临床应用价值.     

Binding of type-I and type-II collagens to Staphylococcus aureus strains isolated from patients with toxic shock syndrome compared to other staphylococcal infections

Abstract Toxic shock syndrome toxin-1 (TSST-1) producing strains of Staphylococcus aureus isolated from 18 patients with toxic shock syndrome (TSS) and from 56 patients with other diagnoses were compared for capacity to interact with various serum and connective tissue proteins. TSS associated isolates showed significantly stronger binding of Type-I collagen (Cn-I) and Cn-II than non-TSS strains, in a particle agglutination assay (PAA) as well as in ¹²⁵I labelled Cn uptake experiments. ¹²⁵I Cn-IV binding, was similar between the two groups, whereas in PAA, a stronger interaction was observed for non-TSS than TSS associated strains. The median binding of ¹²⁵I Cn to TSS-associated strains were 52.2 (Cn-I), 30.6 (Cn-II) and 20.0 (Cn-IV) compared to 20.0 (Cn-I), 14.4 (Cn-II) and 24.4 (Cn-IV) values of non-TSS strains. A saturation with ¹²⁵I Cn-I and Cn-II binding was established for TSS (30 min) and non-TSS (15 min) strains. ¹²⁵I Cn-IV binding reached a saturation in 10 min and 90 min with TSS and non-TSS strains respectively. Finally, the binding profiles of TSS associated and non-TSS strains to fibronectin, fibrinogen, laminin and IgG did not differ in both PAA and radioisotope assays. In scanning electron microscopy, cells of TSS associated strains bound to the reprecipitated native Cn-I fibrils. In contrast, most cells of non-TSS strains were localized to the distal end or were trapped between the Cn fibrils. The stronger interaction with Cn-I and II in particular, shown by TSS associated strains, might enhance submucosal localization, thereby facilitating entry of toxins into the blood and establishment of TSS.     

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Iron depletion and virulence in Staphylococcus aureus

Abstract Staphylococcus aureus is able to grow in the presence of extremely low iron concentrations (0.04 μM). In iron-limiting conditions, this species develops alternative metabolic strategies such as highly efficient iron-uptake mechanisms which are only partially shared with S. epidermidis . Here we summarize the mechanisms induced by iron starvation in S. aureus in order to elucidate the virulence characteristics of this bacterium.     

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-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.     

金黄色葡萄球菌的耐药性分析及基因分型研究

目的通过分析上海地区院内分离金黄色葡萄球菌的药敏谱型及对耐甲氧西林的金黄色葡萄球菌（MRSA）进行基因谱型的研究,了解金黄色葡萄球菌的院内流行状况。方法对临床分离出的43株金黄色葡萄球菌进行药敏试验和SCCmec基因盒的多重PCR检测,并将结果整合后用MEGA3．1软件分析其进化相关关系。结果药敏结果显示43株金葡菌对青霉素和甲氧西林的耐药率最高。甲氧西林的耐药率达到62．8％。MecA阳性菌株SCCmec的分型显示均为Ⅱ型或Ⅲ型,且所占比例相近,未见Ⅰ型和Ⅳ型。进化树分析发现了在同一医院中亲缘关系相近的菌株,为院内感染流行株。结论MecA基因介导的MRSA在分离菌株中所占比例高,存在院内感染爆发性流行。     

Accumulation of porphyrins and pyrrole pigments by Staphylococcus aureus ssp. anaerobius and its aerobic mutant

Abstract The anaerobic, Gram-positive coccus Staphylococcus aureus ssp. anaerobius and its aerobic mutant MVF-SR, when kept under anaerobic conditions, excreted coproporphyrin (mainly type III) into the medium and enriched uroporphyrin (mainly type I) within the cells.
The rate of porphyrin synthesis stayed practically unaltered when the growth medium was supplemented with 50 μ g/ml 5-aminolevulinic acid (ALA), but was significantly enhanced upon supplementation with hemin (0.5 μ g/ml). When hemin and ALA were given simultaneously, a more than two-fold increase in porphyrin production compared to normal growth medium was observed. These observations indicate a stimulation of porphyrin synthesis in S. aureus by hemin.
An as yet unidentified violet pigment with an intense red-violet fluorescence under UV light ( λ = 366 nm) was found to be present in considerable amounts in cells of S. aureus ssp. anaerobius , whereas the supernatant medium of aerobically grown cells of the mutant MVF-SR contained an equally unidentified blue, non-fluorescing pigment.     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

Identification of a fmtA-like gene that has similarity to other PBPs and beta-lactamases in Staphylococcus aureus

We identified a gene from Staphylococcus aureus, flp (fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and beta-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.     

金黄色葡萄球菌对碘伏、乙醇、高铁酸钾和紫外线的抗性

目的了解临床分离的耐甲氧西林金黄色葡萄球菌（methicillin resistant Staphylococcus aureus,MR-SA）、甲氧西林敏感的金黄色葡萄球菌（methicillin sensitive Staphylococcus aureus,MSSA）及金黄色葡萄球菌CMCC（B）26003对碘伏、乙醇医院常用消毒剂及紫外线的抗性。方法以临床连续收集的67株金黄色葡萄球菌为实验菌株。测定碘伏、乙醇、高铁酸钾对耐甲氧西林金黄色葡萄球菌（MRSA）、甲氧西林敏感的金黄色葡萄球菌（MSSA）和金黄色葡萄球菌CMCC（B）26003作用1、2min后的最小抑菌浓度（MBC）及紫外线作用不同时间后的杀菌试验,观察其对碘伏、乙醇、高铁酸钾及紫外线抗性的变化趋势。结果碘伏对MRSA的MBC明显高于MSSA及CMCC（B）26003,而乙醇、高铁酸钾对3种菌的最小抑菌浓度均相近,3种菌对紫外线抗性相近。结论在正常使用浓度范围内碘伏、乙醇、高铁酸钾对金黄色葡萄菌中无论是MRSA、MSSA都有很好的灭菌作用,且乙醇作用时间越长,抑菌效果越显著。紫外线在作用75min以上时,3种菌全部不能存活。     

Caspase‐1 inflammasome activity in patients with Staphylococcus aureus bacteremia

The inflammasome is a multiprotein complex that mediates caspase‐1 activation with subsequent maturation of the proinflammatory cytokines IL‐1β and IL‐18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase‐1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent‐labeled inhibitor of caspase‐1), while IL‐1β and IL‐18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase‐1), and IL1B (pro‐IL‐1β) was analyzed by quantitative PCR. We found induced caspase‐1 activity in innate immune cells with subsequent release of IL‐18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase‐1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient.     

Immunization with heat‐inactivated Staphylococcus aureus induced an antibody response mediated by IgG1 and IgG2 in patients with recurrent tonsillitis

Currently Staphylococcus aureus is the predominant pathogen isolated from the respiratory tract of patients with recurrent tonsillitis. Because of an increase in multi‐drug resistant strains of S. aureus, there is a pressing need for effective treatments and preventive approaches to reduce the risk of invasive and life‐threatening infections. A preventive vaccine against S. aureus would have a tremendous clinical impact. However, multiple clinical trials have failed to identify an agent that can induce protective responses. Most trials have been based on subunit vaccines using one or a few purified antigens, which may not be enough to confer protection. Here, the impact of a whole‐cell vaccine comprised of heat‐inactivated S. aureus was investigated in patients with RT. The vaccine was well tolerated and had no significant local or systemic reactions. Immunization with heat‐inactivated S. aureus elicited a significant antibody response characterized by production of IgG1 and IgG2 antibodies and, to a lesser extent, of IgA antibodies. Notably, this response was associated with an important decrease in the incidence of tonsillitis and bacterial colonization of the oropharyngeal mucosa. Our results show that whole‐cell inactivated S. aureus is safe and capable of evoking specific antibody responses in patients with recurrent tonsillitis.