Subcellular Localization of the Major Autolysin,ATL and Its Processed Proteins in Staphylococcus aureus

The Staphylococcus aureus autolysin gene, atl, encodes a unique 138-kDa protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracellular peptidoglycan hydrolases, 51-kDa endo-β-N-acetylglucosaminidase (51-kDa GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (62-kDa AM). To investigate cell-associated bacteriolytic enzymes for atl gene products, proteins were extracted from the cells as follows. The cells were exposed to 3 M LiCl followed by 4% SDS. Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS-stable cell-associated bacteriolytic proteins were extracted without disrupting the cells. Exposure to 3 M LiCl released major 138-, 115-, 85-, 62- and 51-kDa bacteriolytic proteins, and subsequent 4% SDS extraction released major 138- and 115-kDa bacteriolytic proteins. These bacteriolytic proteins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 atl:: Tn551). Immunoblotting studies suggest that these are all atl gene products: the 138-kDa protein is an ATL with a cleaved signal sequence; the 115-and 85-kDa proteins are intermediates; and the 51- and 62-kDa proteins are cell-associated 51-kDa GL and 62-kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are located outside the cell membrane. Differences in extractability and immunoelectron microscopic studies suggest that atl gene products are associated with cells in two different ways, LiCl extractable and non extractable. We suggest that the 138-kDa ATL undergoes processing through intermediate proteins (115- and 85-kDa proteins) to mature as the active cell cluster-dispersing enzymes 51-kDa GL and 62-kDa AM on the cell surface.     

A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor

Staphylococcus aureus and Staphylococcus epidermidis can cause serious chronic and recurrent infections that are difficult to eradicate. An important pathogenicity factor in these infections caused by S. aureus is its ability to be internalized by non‐professional phagocytes thereby evading the host immune system and antibiotic treatment. Here, we report a novel mechanism involved in staphylococcal internalization by host cells, which is mediated by the major autolysin/adhesins Atl and AtlE from S. aureus and S. epidermidis respectively. In a flow cytometric internalization assay, atl and atlE mutants are significantly reduced in their capacities to be internalized by endothelial cells. Moreover, pre‐incubation of endothelial cells with recombinant Atl dose‐dependently inhibited internalization. As putative Atl‐host cell receptor, the heat shock cognate protein Hsc70 was identified by mass spectrometry. The importance of Hsc70 in internalization was demonstrated by the inhibition of S. aureus internalization with anti‐Hsc70 antibodies. In conclusion, this novel Atl‐ or AtlE‐mediated internalization mechanism may represent a ‘back‐up’ mechanism in S. aureus internalization, while it may represent the major or even sole mechanism involved in the internalization of coagulase‐negative staphylococci and thus may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens.     

A bacterial regulatory RNA attenuates virulence,spread and human host cell phagocytosis

Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation.     

Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface

Biofilm formation on a polymer surface which involves initial attachment and accumulation in multilayered cell clusters (intercellular adhesion) is proposed to be the major pathogenicity factor in Staphylococcus epidermidis foreign-body-associated infections. We have characterized two distinct classes of biofilm-negative Tn917 mutants in S. epidermidis affected in initial attachment (class A) or intercellular adhesion (class B). mut1 (class A mutant) lacks five surface-associated proteins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be complemented by transformation with a 16.4 kb wild-type DNA fragment. The complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1. Subcloning experiments revealed that the 60 kDa protein is sufficient for initial attachment. Immunofluorescence microscopy using an antiserum raised against the 60 kDa protein showed that this protein is located at the cell surface. DNA-sequence analysis of the complementing region revealed a single open reading frame which consists of 4005 nucleotides and encodes a deduced protein of 1335 amino acids with a predicted molecular mass of 148 kDa. The amino acid sequence exhibits a high similarity (61% identical amino acids) to the atl gene product of Staphylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE. By analogy with the S. aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by proteolytic processing. The 120 kDa protein missing from mut1 presumably represents the unprocessed amidase and glucosaminidase domain after proteolytic cleavage of the signal- and propeptide. The 45 and 38 kDa proteins are probably the degradation products of the 60 and 52 kDa proteins, respectively. Additionally, AtlE was found to exhibit vitronectin-binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein-coated polymer surfaces during later stages of adherence. Our findings provide evidence for a new function of an autolysin (AtlE) in mediating the attachment of bacterial cells to a polymer surface, representing the prerequisite for biofilm formation.     

Novel organization of the site-specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence-converting phages φ13 and φ42

Functions required for site-specific integration and excision of the Staphylococcus aureus serotype F virulence-converting phages φ13 and φ42 were localized and characterized. Like other temperate phages, integration of φ13 and φ42 sequences was found to require the product of an int gene located close to the phage attP site. Both int genes are almost identical, express proteins possessing characteristic features of the Int (integrase) family of recombinases, but share very little homology with previously described int genes, including those of the serotype B S. aureus phages L54a and φ11. Nevertheless, all four S. aureus phages share an almost identical short sequence located immediately 5′ to these distinct int genes, suggesting a common mechanism of int gene regulation. Upstream from these common sequences, the sequences of φ13 and φ42 are quite distinct from each other, and from the corresponding regions of φ11 and L54a which encode the Xis proteins that are required with Int to mediate site-specific excision of the latter phages. Surprisingly, φ13 and φ42 sequences encompassing the attP sites and int genes, but lacking either an adjacent or more distant phage excision protein gene, were sufficient to mediate site-specific excision of integrated phage DNA sequences.     

Isolation and Functional Characterization of a New Shrimp Ovarian Peritrophin with Antimicrobial Activity from <Emphasis Type="Italic">Fenneropenaeus merguiensis</Emphasis>

Shrimp ovarian peritrophin (SOP), a major protein in jelly layer (JL) and cortical rods (CRs), is proposed to play a role in the protection of spawned eggs. The full sequence of SOP cDNA from Fenneropenaeus merguiensis (Fm-SOP) shares approximately 50% identity with other SOP sequences and contains several putative chitin-binding or peritrophin-A domains. Interestingly, Fm-SOP contains a putative 61-amino acid propeptide located at the N-terminal end, downstream of a 19-amino acid signal peptide, which is unique among penaeid SOP sequences described so far. This 61-amino-acid sequence constitutes a putative chitin-binding domain with six conserved cysteines, and is cleaved at a dibasic recognition site for a furin (subtilisin-like endoprotease). Expression analyses indicated that Fm-SOP mRNA is abundant in early vitellogenic ovaries and scarce in late-vitellogenic ovaries. Conversely, Fm-SOP protein is the most abundant at the end of vitellogenesis. To investigate its biological function, a recombinant Fm-SOP was expressed to generate a glycosylated protein in Spodoptera frugiperda Sf9 cells (rSOP-Sf9) and a nonglycosylated protein (rSOP-Ec) in Escherichia coli. rSOP-Sf9 and rSOP-Ec were found to bind to chitin, similarly to the native protein extracted from F. merguiensis ovaries. Most interestingly, rSOP-Ec displayed a chitinase activity and efficiently inhibited the growth of Vibrio harveyi and Staphylococcus aureus, with minimum inhibitory concentrations of 2.4 and 15.7 μM, respectively. This first report shows that a major component of CR and JL is biologically active against known pathogens and predicts a significant role of JL in the protection of the spawned eggs against pathogens. The nucleotide sequence reported in this article has been submitted to the GeneBank™/EBI accession number AY775291 for Fm-SOP.     

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.     

Rapid screening for putative exported proteins from staphylococcus aureus using alkaline phosphatase as a reporter molecule

Staphylococcus aureus causes a wide range of infections in humans, ranging from superficial skin infections to the more serious toxin-mediated diseases such as toxic shock syndrome. Owing to the increasing resistance of this bacterium to a wide range of antibiotics, the need to determine the virulence factors involved in infection is becoming more important as these molecules are potential therapeutic targets. In this study, we have screened for putative exported proteins from S. aureus on the basis that these proteins are likely to be the first point of contact between the bacterium and host during infection. We have constructed gene fusions between S. aureus DNA and a truncated version of the Escherichia coli phoA gene, and we report on the characterization of the recombinants exhibiting alkaline phosphatase activity. As well as known S. aureus proteins, we have identified a number of putative open reading frames that encode proteins similar to those from nonstaphylococcal species and also unique proteins that do not have any homologues on the current databases.     

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction

The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minus-origin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria. Received: 25 November 1997 / Received last revision: 13 April 1998 / Accepted: 19 April 1998     

Identification of putative sex chromosomes in the blue tilapia, Oreochromis aureus, through synaptonemal complex and FISH analysis

Sex determination in the blue tilapia, Oreochromis aureus, is primarily a ZW female-ZZ male system. Here, by analysis of the pachytene meiotic chromosomes of O. aureus, we demonstrate the presence of two distinct regions of restricted pairing present only in heterogametic fish. The first, a subterminal region of the largest bivalent is located near to the region of unpairing found in the closely related species O. niloticus, while the second is in a small bivalent, most of which was unpaired. These results suggest that O. aureus has two separate pairs of sex chromosomes.     

金黄色葡萄球菌噬菌体vB_Sau_P68的基因组分析和裂解酶

【背景】金黄色葡萄球菌是常见的人畜共患条件致病菌,随着多耐药菌株分离率的增长,研发与抗生素作用模式不同的抗菌剂迫在眉睫。【目的】分离高效且特异性强的金黄色葡萄球菌噬菌体,对其进行功能注释,并对其编码的裂解酶进行功能验证。【方法】通过对噬菌体全基因组序列进行分析找到裂解酶基因,利用原核表达系统对其编码的2个裂解酶蛋白进行克隆,用SDS-PAGE与蛋白免疫印迹法(Western blotting)鉴定目的蛋白是否表达,并采用单斑法验证其裂解活性。【结果】本研究的噬菌体为一株新的金黄色葡萄球菌噬菌体,命名为vB＿Sau＿P68,该基因组全长为139 409 bp,GC含量为31.0%,编码220个开放阅读框(open reading frame,ORF),透射电镜观察具有正二十面体头部和收缩性尾部,形态学分类属于肌尾噬菌体。该噬菌体编码2个裂解酶基因,分别具有CHAP催化结构域与SH3＿5结合结构域,SDS-PAGE与Western blotting表明Lys161能够表达且有裂解活性,Lys163则无法外源表达。对Lys161序列进行分析,该裂解酶无信号肽,无跨膜区域,以无规则卷曲为主。【...     

Transcriptional analysis of the bglP gene from Streptococcus mutans

Background  

An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II.     

Phloretin reduces cell injury and inflammation mediated by Staphylococcus aureus via targeting sortase B and the molecular mechanism

Sortase B (SrtB) is a vital virulence factor that plays a critical role in Staphylococcus aureus (S. aureus) infections, indicating that it could be a latent target for S. aureus infections. In this study, phloretin, a natural compound that primarily exists in the pericarp and velamen of apples and pears, shows little anti-S. aureus activity, but significantly inhibited SrtB activity in vitro. The results of lactate dehydrogenase release and live/dead cell assays suggested that phloretin reduced human alveolar epithelial cell damage caused by S. aureus. Additionally, an adhesion assay confirmed that phloretin lowered the colony count of S. aureus in human alveolar cells. Phloretin treatment significantly attenuated the inflammatory response in macrophage cells (J774) co-cultured with S. aureus as determined by an enzyme-linked immune-sorbent assay. Furthermore, the results of molecular dynamics simulation, site-directed mutagenesis, and fluorescence spectroscopy quenching indicated that phloretin was directly located in the active pocket of SrtB and blocked substrate binding, leading to the loss of SrtB activity. These results indicate that phloretin is a possible candidate for treatment of S. aureus infections.

     

Prophage, φPV83-pro,Carrying Panton-Valentine Leukocidin Genes,on the Staphylococcus aureus P83 Chromosome: Comparative Analysis of the Genome Structures of φPV83-pro, φPVL, φ11, and Other Phages

Staphylococcus aureus P83 has Panton-Valentine leukocidin (PVL)-like genes, lukM and lukF-PV. Here, lukM and lukF-PV genes were found on the genome of a prophage, which was designated as φPV83-pro. The precise genome size was 45,636 bp with att core sequences of 10 base pairs. Sixty-four ORFs were identified on the φPV83-pro genome, including two extra operons, lukM-lukF-PV and orfs63-64. The lukM-lukF-PV cluster was located 2.1 kb upstream of the attL site. The most striking feature of the φPV83-pro genome was a constituent of at least 4 regions from φ11, φPVL, and other phages, i.e., (i) att sites identical with those of φ11, (ii) a cos sequence and the genes encoding packaging and head proteins of φPVL (occupied half region of φPV83-pro), and (iii) the other two regions which showed no significant similarity with known phages (occupied about 40% of φPV83-pro). Furthermore, two insertion sequences, ISSA1 and ISSA2 were integrated into attL site and orf44, respectively. φPV83-pro was not induced as phage particles from S. aureus P83 regardless of its treatment with mitomycin C. The insertion of ISSA1 into the attL site was one of the reasons of the failure of the induction of the phage particles by mitomycin C treatment of the strain P83.     

Subcellular Location of the Soluble Lytic Transglycosylase Homologue in Staphylococcus aureus

The immunodominant antigen A, IsaA, of Staphylococcus aureus was found to include a putative soluble lytic transglycosylase domain in its C-terminal region. Since the presence of this distinctive domain suggested that the protein might participate in peptidoglycan turnover, as indicated in Gram-negative bacteria, its cellular location was investigated. The protein was found not only in the culture supernatant but also in the cell wall fraction. To estimate its physiological role for the bacterium, its cell surface distribution was studied by immunoelectron microscopy. Protein A-gold particles binding to the immune complex were mainly located on the septal region of the bacterial cell surface. These data suggested that IsaA might be involved in bacterial cell separation through a preferential interaction with peptidoglycan chain.