Changes in expression of the 44-kilodalton outer surface membrane antigen (p44 kD) for monitoring progression of infection and antimicrobial susceptibility of the human granulocytic ehrlichiosis (HGE) agent in HL-60 cells

Changes in human granulocytic ehrlichiosis (HGE)-specific major outer membrane protein (p44 kD) were assayed by Western blot analysis in HL-60 cells in vitro infected by the HGE agent. Time course study demonstrated that the expression of p44 preceded the rise in cell infection as determined by the presence of intracellular morulae. To test whether the expression of p44 may be suitable for evaluating the effects of antibiotics in vitro, three recent isolates of the HGE agent were exposed to doxycycline and ampicillin during culture with HL-60 cells. Loss of infection concurrent with disappearance of the 44 kD protein was found with doxycycline treatment. In contrast, ampicillin treatment had no discernible effects on infection or 44 kD expression. There was excellent agreement between infection, as measured by morulae, and 44 kD expression (coefficient of correlation r = 0.97, p < 0.01). Following treatment with doxycycline, the 44 kD protein disappeared with an estimated t1/2 of approximately 24-30 h, which was considerably shorter than a t1/2 of >60 h calculated for loss of morulae. Measurement of p44 expression may be a more rapid and simple assay to determine antibiotic susceptibility of the HGE agent in cell culture. Furthermore, it may be used to indicate the presence of infection before morulae are apparent.     

Specific processing of poly(ADP-ribose) polymerase, accompanied by activation of caspase-3 and elevation/reduction of ceramide/hydrogen peroxide levels, during induction of apoptosis in host HL-60 cells infected by the human granulocytic ehrlichiosis (HGE) agent

We studied the mechanism by which the human granulocytic ehrlichiosis (HGE) agent induces programmed cell death (apoptosis) in human promyelocytic HL-60 leukemia cells. Using several New York HGE isolates, we show that the HGE agent-elicited apoptosis is accompanied by increased processing of nuclear enzyme poly(ADP-ribose) polymerase (PARP), concurrent with a noticeable increase in caspase 3 activities. A marked increase in the amounts of the signaling molecule ceramide but not of diacylglycerol was also observed in HGE agent-infected HL-60 cells, compared with the amounts in uninfected controls. Simultaneous or prior treatment of infected HL-60 cells with the ceramide synthase inhibitor fumonisin B1 did not affect the magnitude of infection by the intracellular pathogen, as determined by both the presence of morulae and the expression of its outer surface membrane protein, p44. These results suggest that the observed changes in ceramide are generated through the sphingomyelinase pathway and not by way of de novo synthesis of ceramide. We also assayed for changes in intracellular hydrogen peroxide and show that the HGE agent causes a decrease in its concentrations in infected cells.     

Tobacco-induced alterations to Porphyromonas gingivalis–host interactions

Smokers are more susceptible than non-smokers to persistent infection by Porphyromonas gingivalis , a causative agent of periodontitis. Patients who smoke exhibit increased susceptibility to periodontitis and are more likely to display severe disease and be refractory to treatment. Paradoxically, smokers demonstrate reduced clinical inflammation. We show that P. gingivalis cells exposed to cigarette smoke extract (CSE) induce a lower proinflammatory response (tumour necrosis factor-α, interleukin-6, interleukin-12 p40) from monocytes and peripheral blood mononuclear cells than do unexposed bacteria. This effect is reversed when CSE-exposed bacteria are subcultured in fresh medium without CSE. Using microarrays representative of the P. gingivalis genome, CSE-exposure resulted in differential regulation of 6.8% of P. gingivalis genes, including detoxification and oxidative stress-related genes; DNA repair genes; and multiple genes related to P. gingivalis virulence, including genes in the major fimbrial and capsular operons. Exposure to CSE also altered the expression of outer membrane proteins, most notably by inducing the virulence factors RagA and RagB, and a putative lipoprotein cotranscribed with the minor fimbrial antigen. Therefore, CSE represents an environmental stress to which P. gingivalis adapts by altering gene expression and outer membrane proteins. These changes may explain, in part, the altered virulence and host–pathogen interactions that have been documented in vivo in smokers with periodontal disease.     

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.     

The gusBC genes of Escherichia coli encode a glucuronide transport system

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.     

Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system

Expression in mammalian COS cells and an efficient microtiter-based strategy for immunoselection was used in a novel approach to identify genes encoding plant membrane proteins. COS cells were transfected with an Arabidopsis thaliana root cDNA library constructed in a bacterial mammalian shuttle vector and screened with an antiserum raised against purified deglycosylated integral plasma membrane proteins from A. thaliana roots. Antibodies directed against a prominent 27 kDa antigen led to the identification of five different genes. They comprised two subfamilies related to the major intrinsic protein (MIP) superfamily and were named plasma membrane intrinsic proteins, PIP1 and PIP2, since the cellular localization of PIP1 and most probably PIP2 proteins in the plasma membrane was independently confirmed by their co-segregation with marker enzymes during aequeous two-phase partitioning. Surprisingly, expression in Xenopus laevis oocytes revealed that all five PIP mRNAs coded for Hg²⁺-sensitive water transport facilitating activities. There had been no previous evidence of the existence of water channels in the plasma membrane of plant cells and the high diffusional water permeability of the lipid bilayer was considered to be sufficient for water exchange. Nevertheless, Northern and Western analyses showed that the PIP genes are constitutively and possibly even redundantly expressed from the small A. thaliana genome.     

Using Codon Usage to Predict Genes Origin: Is the Escherichia coli Outer Membrane a Patchwork of Products from Different Genomes?

Analysis of the codon usage of genes coding for the structuralcomponents of the outer membrane in Escherichia coli, is consistentwith the requirement for high expression of these genes. Becauseporins (which constitute the major protein component of theouter membrane), and LPS (which constitute the major outermostconstituent of the outer membrane), are synthesized from genesdisplaying widely different codon usage, it is possible to investigatethe origin of the outer membrane. The analysis predicts thatthe outer membrane might originate from a genome other thanthe genome coding for the major part of the cell. Such a specialorigin would explain in structural terms, the likely lethalityof porins if they were inadvertently inserted within the innermembrane, giving rise to the Gram-negative bacterial type, havingan envelope comprising two membranes, instead of a single cytoplasmicmembrane and a murein sacculus.     

In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.     

Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage.     

Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study

A main pathway for energy ATP production inhuman body is by tricarboxylic acid cycle (Krebs cy-cle). Sodium-dependent dicarboxylate co-transporterprotein (SDCT, NaDC, NaC) is an organic aniontransporter protein family responsible for trans-mem- for 30 s, and extension 72℃ for 2 min; followed bybrane transport of Krebs cycle intermediate metabolite final extension 72℃ for 7 min. PCR products weresuch as succinate and citrate. They predominantly lo- …     

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。【...     

Cloning, characterisation and expression of Aeromonas hydrophila major adhesin

Aeromonas hydrophila, an important pathogen in fish, is believed to cause diseases by adhesive and enterotoxic mechanisms. The adhesion is a prerequisite for successful invasion. In this study, the gene of a 43 kDa major adhesin (designated as AHA1) was cloned and expressed. Nucleotide sequence analysis of AHA1 revealed an open reading frame encoding a polypeptide of 373 amino acids with a 20-amino-acid putative signal peptide (molecular weight 40,737 Da). The amino acid sequences of Aha1p showed a very high homology with the other two outer membrane proteins of A. hydrophila. Using the T-5 expression system, this major adhesin Aha1p was expressed in Escherichia coli. The purified recombinant adhesin could competitively inhibit A. hydrophila from invading fish epithelial cells in vitro. Western-blot analysis showed that this major adhesin is a very conserved antigen among various strains of Aeromonas. When used to immunise blue gourami, the recombinant adhesin could confer significant protection to fish against experimental A. hydrophila challenge.