Pathogenic E. coli Exploits SslE Mucinase Activity to Translocate through the Mucosal Barrier and Get Access to Host Cells

SslE is a zinc-metalloprotease involved in the degradation of mucin substrates and recently proposed as a potential vaccine candidate against pathogenic E. coli. In this paper, by exploiting a human in vitro model of mucus-secreting cells, we demonstrated that bacteria expressing SslE have a metabolic benefit which results in an increased growth rate postulating the importance of this antigen in enhancing E. coli fitness. We also observed that SslE expression facilitates E. coli penetration of the mucus favouring bacteria adhesion to host cells. Moreover, we found that SslE-mediated opening of the mucosae contributed to the activation of pro-inflammatory events. Indeed, intestinal cells infected with SslE-secreting bacteria showed an increased production of IL-8 contributing to neutrophil recruitment. The results presented in this paper conclusively designate SslE as an important colonization factor favouring E. coli access to both metabolic substrates and target cells.     

Early Severe Inflammatory Responses to Uropathogenic E. coli Predispose to Chronic and Recurrent Urinary Tract Infection

Chronic infections are an increasing problem due to the aging population and the increase in antibiotic resistant organisms. Therefore, understanding the host-pathogen interactions that result in chronic infection is of great importance. Here, we investigate the molecular basis of chronic bacterial cystitis. We establish that introduction of uropathogenic E. coli (UPEC) into the bladders of C3H mice results in two distinct disease outcomes: resolution of acute infection or development of chronic cystitis lasting months. The incidence of chronic cystitis is both host strain and infectious dose-dependent. Further, development of chronic cystitis is preceded by biomarkers of local and systemic acute inflammation at 24 hours post-infection, including severe pyuria and bladder inflammation with mucosal injury, and a distinct serum cytokine signature consisting of elevated IL-5, IL-6, G-CSF, and the IL-8 analog KC. Mice deficient in TLR4 signaling or lymphocytes lack these innate responses and are resistant, to varying degrees, to developing chronic cystitis. Treatment of C3H mice with the glucocorticoid anti-inflammatory drug dexamethasone prior to UPEC infection also suppresses the development of chronic cystitis. Finally, individuals with a history of chronic cystitis, lasting at least 14 days, are significantly more susceptible to redeveloping severe, chronic cystitis upon bacterial challenge. Thus, we have discovered that the development of chronic cystitis in C3H mice by UPEC is facilitated by severe acute inflammatory responses early in infection, which subsequently are predisposing to recurrent cystitis, an insidious problem in women. Overall, these results have significant implications for our understanding of how early host-pathogen interactions at the mucosal surface determines the fate of disease.     

Production of the Escherichia coli Common Pilus by Uropathogenic E. coli Is Associated with Adherence to HeLa and HTB-4 Cells and Invasion of Mouse Bladder Urothelium

Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract.     

Rottlerin Inhibits Chlamydial Intracellular Growth and Blocks Chlamydial Acquisition of Sphingolipids from Host Cells

We report that rottlerin, a plant-derived compound known to inhibit various mammalian kinases, profoundly inhibited chlamydial growth in cell culture with a minimal inhibition concentration of 1 μM. The inhibition was effective even when rottlerin was added as late as the middle stage of chlamydial infection cycle, against multiple Chlamydia species, and in different host cell lines. Pretreatment of host cells with rottlerin prior to infection also blocked chlamydial growth, suggesting that rottlerin targets host factors. Moreover, rottlerin did not alter the chlamydial infection rate and did not directly target chlamydial protein synthesis and secretion. The rottlerin-mediated inhibition of chlamydial replication and inclusion expansion correlated well with the rottlerin-induced blockade of host cell sphingolipid trafficking from the Golgi apparatus into chlamydial inclusions. These studies not only allowed us to identify a novel antimicrobial activity for rottlerin but also allowed us to uncover a potential mechanism for rottlerin inhibition of chlamydial growth.     

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.     

Interplay between Clathrin and Rab5 Controls the Early Phagocytic Trafficking and Intracellular Survival of Brucella abortus within HeLa cells

Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.     

The Effect of Enterohemorrhagic E. coli Infection on the Cell Mechanics of Host Cells

Enterohaemorrhagic E. coli (EHEC) is a type of human pathogenic bacteria. The main virulence characteristics of EHEC include the formation of attaching and effacing lesions (A/E lesions) and the production of one or more Shiga-like toxins, which may induce human uremic complications. When EHEC infects host cells, it releases translocated intimin receptor (Tir) and effector proteins inside the host cells, inducing the rearrangement and accumulation of the F-actin cytoskeleton, a phenotype leading to the formation of pedestals in the apical cell surface, and the growth of stress fibers at the base of the cells. To examine the effect of EHEC infection on cell mechanics, we carried out a series of experiments to examine HeLa cells with and without EHEC infection to quantify the changes in (1) focal adhesion area, visualized by anti-vinculin staining; (2) the distribution and orientation of stress fibers; and (3) the intracellular viscoelasticity, via directional video particle tracking microrheology. Our results indicated that in EHEC-infected HeLa cells, the focal adhesion area increased and the actin stress fibers became thicker and more aligned. The cytoskeletal reorganization induced by EHEC infection mediated a dramatic increase in the cytoplasmic elastic shear modulus of the infected cells, and a transition in the viscoelastic behavior of the cells from viscous-like to elastic-like. These changes in mechanobiological characteristics might modulate the attachments between EHEC and the host cell to withstand exfoliation, and between the host cell and the extracellular matrix, and might also alter epithelial integrity.     

Immune Evasion,Stress Resistance,and Efficient Nutrient Acquisition Are Crucial for Intracellular Survival of Candida glabrata within Macrophages

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes'' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.     

Pertussis Toxin Exploits Specific Host Cell Signaling Pathways for Promoting Invasion and Translocation of Escherichia coli K1 RS218 in Human Brain-derived Microvascular Endothelial Cells

Pertussis toxin (PTx), an AB5 toxin and major virulence factor of the whooping cough-causing pathogen Bordetella pertussis, has been shown to affect the blood-brain barrier. Dysfunction of the blood-brain barrier may facilitate penetration of bacterial pathogens into the brain, such as Escherichia coli K1 (RS218). In this study, we investigated the influence of PTx on blood-brain barrier permissiveness to E. coli infection using human brain-derived endothelial HBMEC and TY10 cells as in vitro models. Our results indicate that PTx acts at several key points of host cell intracellular signaling pathways, which are also affected by E. coli K1 RS218 infection. Application of PTx increased the expression of the pathogen binding receptor gp96. Further, we found an activation of STAT3 and of the small GTPase Rac1, which have been described as being essential for bacterial invasion involving host cell actin cytoskeleton rearrangements at the bacterial entry site. In addition, we showed that PTx induces a remarkable relocation of VE-cadherin and β-catenin from intercellular junctions. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx not only facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it also affects intercellular barriers to increase paracellular translocation.     

Uropathogenic E. coli induce different immune response in testicular and peritoneal macrophages: implications for testicular immune privilege

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells.     

Importance of Glutathione for Growth and Survival of Escherichia coli Cells: Detoxification of Methylglyoxal and Maintenance of Intracellular K+

The role of the tripeptide glutathione in the growth and survival of Escherichia coli cells has been investigated. Glutathione-deficient mutants leak potassium and have a reduced cytoplasmic pH. These mutants are more sensitive to methylglyoxal than the parent strain, indicating that in the absence of glutathione-dependent detoxification, acidification of the cytoplasm cannot fully protect cells. However, increasing the intracellular pH of the glutathione-deficient strain resulted in enhanced sensitivity to methylglyoxal. This suggests that acidification of the cytoplasm can provide some protection to E. coli cells in the absence of glutathione. In the presence of the Kdp system, glutathione-deficient mutants are highly sensitive to methylglyoxal. This is due to the higher intracellular pH in these cells. In the absence of methylglyoxal, the presence of the Kdp system in a glutathione-deficient strain also leads to an extended lag upon dilution into fresh medium. These data highlight the importance of glutathione for the regulation of the K⁺ pool and survival of exposure to methylglyoxal.     

Surfactant Protein D Inhibits Adherence of Uropathogenic Escherichia coli to the Bladder Epithelial Cells and the Bacterium-induced Cytotoxicity: A POSSIBLE FUNCTION IN URINARY TRACT*

The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca²⁺-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.     

Characterization and Intracellular Distribution of Pea Phytochrome I Polypeptides Expressed in E. coli

The pea phytochrome I (PI) cDNA clone, pPP1001, was expressedin E. coli. The plasmid pPP1001 contains pea PI cDNA which coversthe entire coding region with the Shine-Dalgarno consensus sequencejoined upstream of the cDNA in an expression vector pNUT6. ThepPP1001 transformants formed typical inclusion bodies when culturedat 32?C. However, when cultured at 37?C or in the presence ofisopropyl-ß-D-thiogalactopyranoside (IPTG) at 32?C,the bacteria lysed before inclusion body formation. Immuno-stainingwith anti-PI monoclonal antibody, mAP5, of transformants fixedby cold methanol showed that stainable materials were distributedin whole cytoplasmic region. When the inclusion bodies wereobserved clearly, the regions corresponding to the inclusionbodies became difficult to stain. Western blot analysis, however,showed that a ca. 100 kDa PI polypeptide was detected in thefraction from inclusion bodies and a ca. 90 kDa PI polypeptidefrom the soluble fraction. The amino acid sequence analysisof purified 100 kDa PI sample indicated that its amino terminusis blocked. However, minor signals in one experiment yieldeda sequence corresponding to the expected amino terminus of peaPI except for the initiation methionine. One of the anti-peaPI monoclonal antibodies, mAP9, that recognizes the near N-terminusof pea phytochrome was reactive to the 100 kDa polypeptide. (Received June 22, 1990; Accepted November 18, 1990)     

Uropathogenic Escherichia coli strains generally lack functional Trg and Tap chemoreceptors found in the majority of E. coli strains strictly residing in the gut

The prevalence and function of four chemoreceptors, Tsr, Tar, Trg, and Tap, were determined for a collection of uropathogenic, fecal-commensal, and diarrheagenic Escherichia coli strains. tar and tsr were present or functional in nearly all isolates. However, trg and tap were significantly less prevalent or functional among the uropathogenic E. coli strains (both in 6% of strains) than among fecal-commensal strains (both in > or =50% of strains) or diarrheal strains (both in > or =75% of strains) (P < 0.02).     

Survival of E. coli O157:H7 in organic wastes destined for land application

AIM: To determine the persistence of Escherichia coli O157 in contrasting organic wastes spread to land and to assess the potential environmental risk associated with the disposal of these wastes to land. METHODS AND RESULTS: Twenty-seven organic wastes originating from slaughterhouses, wastewater treatment plants (raw and treated sewage), creameries and farms (bovine slurry), were inoculated with E. coli O157:H7 and incubated at 10 degrees C. Although pathogen numbers gradually declined in all the wastes, albeit at different rates even in the same waste type, E. coli O157:H7 was still viable in 77% of organic wastes tested after 2 months. CONCLUSIONS: Long-term storage of organic wastes led to a significant and gradual decline in E. coli O157:H7 numbers. Consequently, storage may be a useful means of reducing the pathogen load of wastes destined for land application. However, in most cases, long-term storage cannot be expected to completely eliminate E. coli O157:H7 from waste. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that current legislation may be insufficient to protect the environment from E. coli O157:H7 contamination from untreated wastes spread to land.     

Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation-mimicking SEPT9 mutant rescued these effects. Collectively, this study provides the first global analysis of phosphorylation-mediated processes during infection with an extracellular, diarrheagenic bacterial pathogen.Diarrheagenic E. coli are a major global health burden and cause much morbidity and mortality worldwide. Enteropathogenic E. coli (EPEC)¹ is the causative agent of potentially fatal infantile diarrhea and remains an endemic health threat for children in developing countries. EPEC and the closely related enterohemorrhagic E. coli (EHEC) belong to the group of attaching and effacing (A/E) pathogens that form distinct A/E lesions on the surface of intestinal epithelial cells causing the loss of the characteristic intestinal brush border architecture (1).Upon attachment to intestinal cells, EPEC uses a syringe-like molecular apparatus, the type III secretion system (T3SS), to inject at least 25 unique bacterial effector proteins into the host cell (2–4). Once translocated into mammalian cells, these effectors manipulate a wide range of host signaling pathways, thereby subverting host cell function and promoting virulence (5). The bacterial translocated intimin receptor (Tir) is one of the first and most abundant effectors injected into the host cell: it mediates intimate attachment of EPEC to the enterocyte apical surface via its interaction with the bacterial surface adhesin intimin (6, 7). In concert with other effectors, Tir also provokes an expansive cytoskeletal rearrangement leading to the formation of actin-rich protrusions, termed pedestals, beneath the site of bacterial attachment (8). Besides altering the host cell cytoskeleton, EPEC effectors also manipulate cellular trafficking, host immune response and ion and water homeostasis to cause disease (5). Although significant effort in recent years has led to the identification of multiple key players in both the host and the pathogen, the complex interactions between EPEC and the epithelial host cell, and the underlying molecular mechanisms, are still collectively not well understood.There is increasing evidence that hijacking host post-translational mechanisms such as protein phosphorylation is a key strategy for bacterial pathogens to efficiently subvert host cell function (9) and there are several indications that this may be the case for EPEC. For example, Tir is phosphorylated upon insertion into the host cell membrane and this event plays a role in the rearrangement of the actin cytoskeleton (10). Another EPEC-encoded effector, NleH, contains a functional kinase domain suggesting the potential of directly phosphorylating host cell targets (11). Moreover, the phosphorylation profiles of a few specific host proteins such as cortactin, CT10 regulator of kinase (CRK) adaptors, focal adhesion kinase (FAK) and mitogen-activated protein kinase 1 (MAPK1), as well as alterations in tyrosine phosphorylation of host proteins, are impacted in an EPEC effector-dependent manner (12–18). These are selective observations though. Thus, a more comprehensive, system-level analysis is needed to better understand how and to what extent EPEC hijacks host cell phosphorylation to cause disease.Recent advances in quantitative phosphoproteomics have made it possible to successfully profile the changes in host protein phosphorylation following infection by the invasive, diarrheagenic bacterial pathogens Shigella and Salmonella (19–21). To our knowledge, no such analysis has been reported for a noninvasive, diarrheagenic bacterial pathogen such as EPEC. In this study, we applied a stable isotope labeling by amino acids in cell culture (SILAC)-based (22) quantitative phosphoproteomics approach to assess the impact of EPEC infection on the host cell phosphoproteome. The integration of time course experiments and the use of an EPEC mutant deficient in type III secretion (T3S) provided further insights into the dynamics as well as the effector dependence of these processes. This experimental approach enabled identification of both stable and transient interactions between EPEC bacterial effectors and host proteins. Additional infection studies focusing on a newly identified host target, septin-9, further emphasizes the biological significance of the manipulation of host protein phosphorylation in EPEC pathogenesis.     

白细胞介素12的p35和p40在大肠埃希菌中的克隆表达

目的用基因工程的方法在大肠埃希菌中克隆表达白细胞介素12(IL-12)的两个亚单位基因p35和p40。方法从乳腺癌患者全血中分离淋巴细胞,利用RT—PCR扩增获得了IL-12的2个亚单位基因p35和p40,分别转化到大肠埃希菌中,获得了高效表达。结果薄层扫描分析结果表明,在BL21(DE3)中表达的重组蛋白IL-12P35和IL-12P40分别占菌体裂解液中蛋白总量的39％和30％。结论该实验结果为IL-12在体外的批量生产奠定了基础。     

Creating Carotenoid Diversity in E. coli Cells using Combinatorial and Directed Evolution Strategies

Carotenoids represent a structurally diverse class of pigments with important biological functions and commercial applications. Biosynthesis of carotenoids has been studied on a molecular level for the core pathways and recombinant hosts have been engineered for heterologous carotenoid production. This paper summarizes our efforts on accessing novel carotenoid compounds in engineered E. coli by altering the catalytic activities of enzymes using in vitro evolution, by exploring the catalytic promiscuity of known carotenoid enzymes and by mining for novel enzymes in microbial genome sequences.