Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion

Background

The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.

Methodology/ Principal Findings

Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca²⁺ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.

Conclusion/Significance

We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.     

Role of Surface Protein SasG in Biofilm Formation by Staphylococcus aureus

The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration of Zn²⁺. The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn²⁺-dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree, resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm formation.Staphylococcus aureus is a commensal bacterium that is carried persistently in the anterior nares of about 20% of the human population. The organism can cause superficial skin infections, such as abscesses and impetigo, and more dangerous and potentially life-threatening invasive infections, such as endocarditis, osteomyelitis, and septic arthritis (26). Staphylococcus epidermidis and S. aureus are the major causes of infections associated with indwelling medical devices, such as central venous catheters, cardiovascular devices, and artificial joints (34, 54). The ability to form a biofilm is crucial to the microbes'' success in device-related infections. Bacteria in the biofilm matrix are in a semidormant state, are difficult to inhibit with antibiotics, and are impervious to host neutrophils and macrophages (36, 43, 44, 51). Until recently biofilm formation by staphylococci was attributed to the ability to synthesize an extracellular polysaccharide called polysaccharide intercellular adhesin (PIA), which is composed of partially deacetylated poly-N-acetylglucosamine (15, 28, 50). Attachment of bacteria to biomedical devices is mediated by adhesion to the naked plastic or metal surface by a surface component such as the major autolysin Atl (2, 14). Alternatively, adhesion to surfaces that have been conditioned by fibronectin and fibrinogen from host plasma is mediated by surface proteins such as clumping factor A (ClfA) and fibronectin binding proteins (FnBPA/B) of S. aureus or SdrG/Fbe of S. epidermidis (17, 46, 47).Several surface proteins of staphylococci can also promote the accumulation phase of biofilm: (i) the biofilm-associated protein Bap, which is only expressed by bovine strains of S. aureus (8); (ii) the SasC surface protein of S. aureus (41); (iii) fibronectin binding proteins FnBPA and FnBPB, which are particularly associated with biofilm formation by some types of methicillin-resistant S. aureus (MRSA) (35, 48); (iv) the multifactorial virulence factor protein A, which promotes cell accumulation when expressed at high levels, for example,in mutants defective in the accessory gene regulator Agr (31); (v) the extracellular matrix binding protein (Embp) of S. epidermidis (4); (vi) the accumulation-associated protein (Aap) of S. epidermidis and the related protein SasG from S. aureus (7, 19, 40).Aap and SasG are typical LPXTG-anchored multidomain cell wall-associated proteins (see Fig. ​Fig.1A,1A, below). A signal sequence is removed from the N terminus during secretion across the cytoplasmic membrane. The C-terminal domains comprise a sorting signal (LPXTG) and hydrophobic membrane-spanning domain and positively charged residues that are required for covalent attachment of the proteins to cell wall peptidoglycan by sortase A. The N termini of the mature proteins (A domains) comprise related amino acid sequences that have been implicated in adhesion of bacteria to desquamated epithelial cells and could be involved in colonization of the nares and skin (7, 27, 39). The archetypal Aap protein of S. epidermidis RP62a has 12 repeats of almost identical sequences of 128 residues followed by a partial repeat of 68 residues (region B), while SasG from S. aureus strain 8325-4 and strain Newman has seven 128-residue repeats and one partial repeat. The B subunits of Aap and SasG are 64% identical.Open in a separate windowFIG. 1.(A) Schematic representation of SasG domain organization. The positions of the signal sequence (S), A domain, B region (B1 to -8), and the wall/membrane-spanning regions (W/M) are indicated. The LPKTG motif is recognized by the sortase A enzyme, which covalently anchors the protein to the cell wall peptidoglycan. (B) Whole-cell immunoblot validating expression of A domain and B regions of SasG variants. Serial dilutions of SH1000(pALC2073:sasG⁺) (row 1); SH1000(pALC2073sasG⁺ A⁺B⁻) (row 2); SH1000(pALC2073sasG⁺ A⁻B⁺) (row 3), and SH1000(pALC2073sasG⁺ A⁻B⁺) induced with tetracycline (90 ng/ml) (row 4) were applied to a nitrocellulose membrane and probed with anti-SasG A domain and anti-SasG B domain antibodies. (C) Biofilm formation by SH1000 constructs expressing SasG variants. Biofilm was allowed to form for 24 h at 37°C under static conditions in microtiter dishes. Biofilm was stained with crystal violet, and the absorbance was measured at 570 nm.The formation of biofilm by Aap in S. epidermidis is promoted by the removal of the A domain by cleavage by an as-yet-unidentified bacterial protease, an event that can also be precipitated by host proteases (40). The ability of the exposed Aap B domains of different bacterial cells to form homophilic interactions through a Zn²⁺-dependent zipper mechanism was proposed when it was shown that purified B domains formed dimers in vitro that were dependent on the presence of Zn²⁺ (6). Purified recombinant B domain protein, but not the A domain, inhibited biofilm formation, as did antibodies that specifically bound to the B domains (40). The Zn²⁺ chelator diethylenetriaminepentaacetic acid (DTPA) inhibited biofilm formation both by S. epidermidis RP62a (presumed to be due to Aap) and by community-associated MRSA (presumed to be due to SasG) (6).This study set out to investigate the molecular basis of biofilm accumulation promoted by the SasG protein of S. aureus. We demonstrate that processing of SasG occurs during growth and biofilm formation in a manner that is different from that reported for Aap, and we have investigated the mechanism.     

一种新型丝氨酸苏氨酸激酶CPK的初步克隆及功能

从人胎肝cDNA文库中分离到一种cDNA ,推测的蛋白氨基酸序列具有明显的丝氨酸苏氨酸激酶结构域,可能编码一种新的丝氨酸苏氨酸激酶(命名为CPK ,cellproliferationassociationkinase) .CPKmRNA全长约3 0kb .RT PCR检测表明CPKmRNA在增殖活跃组织或细胞如人胚胎组织、肿瘤细胞株中呈高丰度表达,在成人组织则呈低丰度或不表达.利用PCR技术扩增大鼠同源cDNA ,以此为探针,Northern杂交发现CPK表达于多种成年小鼠组织,以脑组织丰度最高.小鼠2 3肝部分切除可迅速诱导CPKmRNA的表达,在术后2 4～4 8h达高峰,与2 3肝部分切除后细胞增殖期吻合.以小鼠同源cDNA片段为模板,合成RNA探针,原位杂交显示在胚胎发育期CPK低丰度表达在大多数组织,以神经系统组织变化最大,在第8d胚胎神经管内皮细胞中即出现表达,11～13d在端脑、脑膜、间脑、脊神经节表皮细胞、海马、小脑神经胶质细胞等多种神经细胞中表达且丰度较高,16d后这些组织的表达迅速下降到较低水平,表明CPK可能与神经系统的生长发育有一定关联.利用信号通路检测技术,观察到CPK的表达对MAPK ,p38MAPK途径的激活有明显的影响,并可显著增强表皮生长因子对这两条途径的激活,提示该激酶可能参与细胞因子的信号转导.     

Molecular Cloning and Characterization of a Novel Retinoblastoma-Binding Protein

We describe the isolation and characterization of a novel cDNA encoding a polypeptide that interacts in a yeast two-hybrid system as well as in mammalian cells with the retinoblastoma (RB) protein. This new protein, which we call Rim, consists of 897 amino acids, has two leucine zipper motifs, and has a LECEE sequence previously identified as an RB-binding domain. Rim also has an E1A/CtBP-binding motif and four putative nuclear localization signals.RimmRNA is expressed ubiquitously at low levels in all human adult tissues tested and at much higher levels in several tumor cell lines. TheRimgene (HGMW-approved symbol RBBP8) is localized on human chromosome 18q11.2.     

Identification and Characterization of a Novel 38.5-Kilodalton Cell Surface Protein of Staphylococcus aureus with Extended-Spectrum Binding Activity for Extracellular Matrix and Plasma Proteins

The ability to attach to host ligands is a well-established pathogenic factor in invasive Staphylococcus aureus disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (srtA) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene emp (1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR, emp was demonstrated in all 240 S. aureus isolates of a defined clinical strain collection as well as in 6 S. aureus laboratory strains, whereas it is lacking in all 10 S. epidermidis strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50, a significantly decreased adhesion of mEmp50 to immobilized fibronectin and fibrinogen, and restoration of these characteristics upon complementation of mEmp50. Emp expression was also demonstrable upon heterologous complementation of S. carnosus. rEmp expressed in Escherichia coli interacted with fibronectin, fibrinogen, and vitronectin in surface plasmon resonance experiments at a K(d) of 21 nM, 91 nM, and 122 pM, respectively. In conclusion, the biologic characterization of Emp suggests that it is a member of the group of secreted S. aureus molecules that interact with an extended spectrum of host ligands and thereby contribute to S. aureus pathogenicity.     

Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.     

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325–4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325–4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325–4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325–4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325–4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.     

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

金黄色葡萄球菌A蛋白基因的克隆及序列分析

 我们构建了金黄色葡萄球菌Cowan1株(CMCC26111)的染色体文库,转化大肠杆菌后筛选出一株protein A的阳性克隆。SDS-PAGE及Western-blot结果显示该克隆株表达的重组protein A的分子量为30 000,较天然protein A的小。该克隆中的protein A基因片段的序列分析表明,它含有天然protein A基因的启动子、信号肽以及至少四个与IgGFc段结合的结构域,而不含天然protein A的胞壁结合区,并发现其中有24个碱基对与Uhlen报告的protein A基因不同,由此导致三个编码的氨基酸发生变化,但这些差异并不影响该重组protein A与IgG Fc段的特异结合。     

Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α₂-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.     

Las1 Is an Essential Nuclear Protein Involved in Cell Morphogenesis and Cell Surface Growth

Saccharomyces cerevisiae mutations that cause a requirement for SSD1-v for viability were isolated, yielding one new gene, LAS1, and three previously identified genes, SIT4, BCK1/SLK1, and SMP3. Three of these genes, LAS1, SIT4, and BCK1/SLK1, encode proteins that have roles in bud formation or morphogenesis. LAS1 is essential and loss of LAS1 function causes the cells to arrest as 80% unbudded cells and 20% large budded cells that accumulate many vesicles at the mother-daughter neck. Overexpression of LAS1 results in extra cell surface projections in the mother cell, alterations in actin and SPA2 localization, and the accumulation of electron-dense structures along the periphery of both the mother cell and the bud. The nuclear localization of LAS1 suggests a role of LAS1 for regulating bud formation and morphogenesis via the expression of components that function directly in these processes.     

Molecular Characterization of Desulfovibrio gigas Neelaredoxin, a Protein Involved in Oxygen Detoxification in Anaerobes

Desulfovibrio gigas neelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His(4)Cys coordination. Neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity. To further understand its role in anaerobes, its genomic organization and expression in D. gigas were studied and its ability to complement Escherichia coli superoxide dismutase deletion mutant was assessed. In D. gigas, neelaredoxin is transcribed as a monocistronic mRNA of 500 bases as revealed by Northern analysis. Putative promoter elements resembling sigma(70) recognition sequences were identified. Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O(2) or H(2)O(2). The neelaredoxin gene was cloned by PCR and expressed in E. coli, and the protein was purified to homogeneity. The recombinant neelaredoxin has spectroscopic properties identical to those observed for the native one. Mutations of Cys-115, one of the iron ligands, show that this ligand is essential for the activity of neelaredoxin. In an attempt to elucidate the function of neelaredoxin within the cell, it was expressed in an E. coli mutant deficient in cytoplasmic superoxide dismutases (sodA sodB). Neelaredoxin suppresses the deleterious effects produced by superoxide, indicating that it is involved in oxygen detoxification in the anaerobe D. gigas.     

Poly(C)-Binding Protein 1, a Novel Npro-Interacting Protein Involved in Classical Swine Fever Virus Growth

N^pro is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N^pro protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N^pro protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA suppressed CSFV growth, while overexpression of PCBP1 promoted CSFV growth. Furthermore, we showed that type I interferon was downregulated by PCBP1, as well as N^pro. Our results suggest that cellular PCBP1 positively modulates CSFV growth.     

小鼠新分泌蛋白基因mBolA1的克隆与鉴定

采用生物信息学工具预测与实验相结合的方法得到了一个新的小鼠分泌蛋白基因mBolA1。该基因定位于染色体3F2,cDNA全长为730bp,编码137个氨基酸的蛋白,该蛋白含有一个保守的BolA结构域,等电点为9.05。用RT-PCR方法从鼠的混合cDNA库中克隆到mBolA1。Western blot实验表明mBolA1能从瞬转的COS 7细胞中分泌到细胞培养液中。亚细胞定位显示mBolA1定位于细胞浆,且与高尔基体不共定位,提示它是个非经典分泌途径的分泌蛋白。RT PCR显示mBolA1在组织中广泛表达。它的具体功能有待进一步研究。