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1.
Listeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10-100-fold lower affinity for ferric siderophores (Km approximately 1-10 nM) than Gram-negative bacteria, and generally lower uptake rates. Vmax for [59Fe]-enterobactin (0.15 pMol per 10(9) cells per minute) was 400-fold lower than that of Escherichia coli. For [59Fe]-corynebactin, Vmax was also low (1.2 pMol per 10(9) cells per minute), but EGD-e transported [59Fe]-apoferrichrome similarly to E. coli (Vmax=24 pMol per 10(9) cells per minute). L. monocytogenes encodes potential Fur-regulated iron transporters at 2.031 Mb (the fur-fhu region), 2.184 Mb (the feo region), 2.27 Mb (the srtB region) and 2.499 Mb (designated hupDGC region). Chromosomal deletions in the fur-fhu and hupDGC regions diminished iron uptake from ferric hydroxamates and haemin/haemoglobin respectively. In the former locus, deletion of fhuD (lmo1959) or fhuC (lmo1960) strongly reduced [59Fe]-apoferrichrome uptake. Deletion of hupC (lmo2429) eliminated the uptake of haemin and haemoglobin, and decreased the virulence of L. monocytogenes 50-fold in mice. Elimination of srtB region genes (Deltalmo2185, Deltalmo2186, Deltalmo2183), both sortase structural genes (DeltasrtB, DeltasrtA, DeltasrtAB), fur and feoB did not impair iron transport. However, deletion of bacterioferritin (Deltafri, lmo943; 0.97 Mb) decreased growth and altered iron uptake: Vmax of [59Fe]-corynebactin transport tripled in this strain, whereas that of [59Fe]-apoferrichrome decreased 20-fold.  相似文献   

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The Staphylococcus aureus pckA gene was identified and characterized. A pckA mutant lacked detectable phosphoenolpyruvate carboxykinase activity and grew poorly in the absence of glucose. Both enzymatic activity and pckA promoter activity in wild-type cells grown in the absence of glucose were at least 22-fold greater than activities in cells grown in the presence of glucose.  相似文献   

4.
Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.  相似文献   

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Enterobactin-mediated iron transport in Pseudomonas aeruginosa.   总被引:21,自引:9,他引:12       下载免费PDF全文
K Poole  L Young    S Neshat 《Journal of bacteriology》1990,172(12):6991-6996
A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin. Apparently, at least two uptake systems for ferrienterobactin exist in P. aeruginosa: one of higher affinity which is specifically inducible by enterobactin under iron-limiting conditions and the second, of lower affinity, which is also inducible under iron-limiting conditions but is independent of enterobactin for induction.  相似文献   

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为了研究单核细胞增生李斯特菌毒力基因启动子的结构特点与转录调控因子PrfA蛋白之间的关系,应用PCR定点突变和重组PCR技术缺失了该菌毒力基因inlC启动子上可能与PrfA蛋白结合以及诱发转录起始相关的碱基序列,构建了一系列突变启动子与lacZ报告基因融合表达质粒,使lacZ基因的表达置于inlC突变启动子下,并分别电转化单核细胞增生李斯特菌野生株P14、PrfA蛋白高表达突变株P14a和prfA基因等位缺失突变株A42中,检测相应的β-半乳糖苷酶活性。结果表明:位于inlC启动子转录起始点下游22bp处的一段17bp的类似PrfA蛋白结合序列TTAACAGCGTTTGTTAA并没有增强和抑制PrfA转录调控活性的功能;甚至将其改造成“完美的”PrfA蛋白结合序列TTAACATTTGTTAA后,也不影响inlC依赖于PrfA的转录活性地表达;但是,如果缺失inlC启动子上原始的PrfA蛋白结合序列,则使inlC依赖于PrfA的转录活性完全丧失;另外,单核细胞增生李斯特菌毒力基因inlC和plcA依赖于PrfA的转录活性的表达也与启动子上PrfA蛋白结合区(PrfA-box)距离-10区的碱基个数有关:最适为22或23bp,长于23bp或短于22bp的突变启动子的依赖PrfA的转录活性大大降低,甚至没有活性。说明除PrfA蛋白结合序列外,受PrfA调控的毒力基因启动子上还可能存在其它尚未阐明的结构和序列影响PrfA蛋白的结合以及启动转录表达。  相似文献   

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Most bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins. Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes. The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity). We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single- and double-chromosomal knock-out mutants. Inactivation of sipX did not affect intracellular multiplication of L. monocytogenes but significantly reduced bacterial virulence (approximately 100-fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC-PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD(50) of 10(8.3)), inactivation of sipY had no detectable effects. Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD(50) of 10(7.2)), whereas, a mutant expressing only SipZ behaved like wild-type EGD in all the assays performed. The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L. monocytogenes. This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post-translational control mechanism of virulence factors expression.  相似文献   

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The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32-50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wild-type protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.  相似文献   

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The integrity of the genetic material of bacteria is guaranteed by a set of distinct repair mechanisms. The participation of these repair systems in bacterial pathogenicity has been addressed only recently. Here, we study for the first time the participation in virulence of the MutSL mismatch repair system of Listeria monocytogenes. The mutS and mutL genes, which are contiguous in the L. monocytogenes chromosome, were identified after in silico analysis. The deduced MutS shares 62% identity with MutS of Bacillus subtilis and 50% identity with HexA, its homologue in Streptococcus pneumoniae; MutL shares 59% identity with MutL of B. subtilis and 47% identity with HexB of S. pneumoniae. Functional analysis of the mutSL locus was studied by constructing a double knock-out mutant. We showed that the deletion DeltamutSL induces: (i) a 100- to 1000-fold increase in the spontaneous mutation rate; and (ii) a 10- to 15-fold increase in the frequency of transduction, thus demonstrating the role of mutSL of L. monocytogenes in both mismatch repair and homologous recombination. We found that the deletion DeltamutSL moderately affected bacterial virulence, with a 1-log increase in the lethal dose 50% (LD50) in the mouse. Strikingly, repeated passages of the mutant strain in mice reduced virulence further. Competition assays between wild-type and mutant strains showed that the deletion DeltamutSL reduced the capacity of L. monocytogenes to survive and multiply in mice. These results thus demonstrate that, for the intracellular pathogen L. monocytogenes, a hypermutator phenotype is more deleterious than profitable to its virulence.  相似文献   

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Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data.  相似文献   

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Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an approximately 5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron-deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm-) strains, comprises five genes arranged in two distinct operons (yfeA-D and yfeE ). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm-, Yfe- (DeltayfeAB ) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2'-dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt-, DeltayfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 x 107 cfu) compared with its parental ybt-, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, DeltayfeAB mutant of Y. pestis had an approximately 100-fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.  相似文献   

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Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by trans-duction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enter-ocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.  相似文献   

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Sortase B, a new class of sortase in Listeria monocytogenes   总被引:5,自引:0,他引:5       下载免费PDF全文
Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The DeltasrtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.  相似文献   

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Graminaceous plants acquire iron by secreting mugineic acid family phytosiderophores into the rhizosphere and taking up complexes of iron and phytosiderophores through YSL (yellow stripe 1-like) transporters. Rice OsYSL15 is a transporter of the iron(III)-2'-deoxymugineic acid complex. OsYSL16 has 85?% similarity to both OsYSL15 and the iron(II)-nicotianamine transporter OsYSL2. In the present study, we show that OsYSL16 functionally complemented a yeast mutant defective in iron uptake when grown on medium containing iron(III)-deoxymugineic acid, but not when grown on medium containing iron(II)-nicotianamine. OsYSL16-knockdown seedlings were smaller than wild-type seedlings when only iron(III)chloride was supplied as an iron source. The iron concentration in shoots of OsYSL16-knockdown plants was similar to that of the wild type; however, they showed more severe chlorosis than wild-type plants under iron-deficient conditions. Furthermore, OsYSL16-knockdown plants accumulated more iron in the vascular bundles of the leaves. Expression of the OsYSL16 promoter fused to the β-glucuronidase gene showed that OsYSL16 is expressed in the root epidermis and vascular bundles of whole plants. The expression was typically observed around the xylem. In the vascular bundles of unelongated nodes, it was detected in the xylem of old leaves and the phloem of new leaves. Graminaceous plants translocate iron from the roots to old leaves mainly via the xylem and to new leaves mainly via the phloem. Our results suggest that OsYSL16 plays a role in the allocation of iron(III)-deoxymugineic acid via the vascular bundles.  相似文献   

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Vibrio cholerae has multiple iron transport systems, one of which involves haem uptake through the outer membrane receptor HutA. A hutA mutant had only a slight defect in growth using haemin as the iron source, and we show here that V. cholerae encodes two additional TonB-dependent haem receptors, HutR and HasR. HutR has significant homology to HutA as well as to other outer membrane haem receptors. Membrane fractionation confirmed that HutR is present in the outer membrane. The hutR gene was co-transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. A hutA, hutR mutant was significantly impaired, but not completely defective, in the ability to use haemin as the sole iron source. HasR is most similar to the haemophore-utilizing haem receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three haem receptors was unable to use haemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2, but haemin transport through either receptor was more efficient in strains carrying the tonB1 system genes. In contrast, haemin uptake through HasR was TonB2 dependent. Efficient utilization of haemoglobin as an iron source required HutA and TonB1. The triple haem receptor mutant exhibited no defect in its ability to compete with its Vib- parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae used haem derived from marine invertebrate haemoglobins, suggesting that haem may be available to V. cholerae growing in the marine environment.  相似文献   

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