A New Role for Escherichia coli DsbC Protein in Protection against Oxidative Stress

We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the l-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing l-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress.     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes. Received: 9 March 1999 / Accepted: 31 May 1999     

Role of a differentially expressed cAMP phosphodiesterase in regulating the induction of resistance against oxidative damage in Leishmania donovani

Differentiation-coupled induction of resistance of Leishmania parasites to macrophage oxidative damage was shown to be associated with an increased cAMP response. This study explores the significance of the cAMP response in the parasite by identifying a differentially expressed cAMP phosphodiesterase (LdPDEA) and deciphering its role in regulating antioxidant machineries in the parasite. LdPDEA, a high K_M class I cytosolic cAMP phosphodiesterase, was expressed maximally in log-phase promastigotes, but was significantly reduced in stationary-phase promastigotes and amastigotes. Chemical inhibition or silencing of PDEA conferred enhanced resistance to pro-oxidants in these cells and this led to studies on trypanothione biosynthesis and utilization, as trypanothione is one of the major modulators of antioxidant defense in kinetoplastidae. Despite enhanced arginase and ornithine decarboxylase activity, trypanothione biosynthesis seemed to be unaffected by PDEA blockage, whereas significant elevations in the expression of tryparedoxin peroxidase, ascorbate peroxidase, and tryparedoxin were detected, suggesting a definite shift of trypanothione-pool utilization bias toward antioxidant defense. Moreover, parasites that overexpressed PDEA showed reduced resistance to oxidative damage and reduced infectivity toward activated macrophages. This study reveals the significance of a cAMP phosphodiesterase in the infectivity of Leishmania parasites.     

Role of collectin-11 in innate defence against uropathogenic Escherichia coli infection

Classical collectins (surfactant protein A and D) play a significant role in innate immunity and host defence in uropathogenic Escherichia coli (UPEC)-induced urinary tract infection (UTI). However, the functions of collectin-11 (CL-11) with respect to UPEC and UTI remain largely unexplored. This study aimed to investigate the effect of CL-11 on UPEC and its role in UTI. We further examined its modulatory effect on inflammatory reactions in proximal tubular epithelial cells (PTECs). The present study provides evidence for the effect of CL-11 on the growth, agglutination, binding, epithelial adhesion and invasion of UPEC. We found increased basal levels of phosphorylated p38 MAPK and human cytokine homologue (keratinocyte-derived chemokine) expression in CL-11 knockdown PTECs. Furthermore, signal regulatory protein α blockade reversed the increased basal levels of inflammation associated with CL-11 knockdown in PTECs. Additionally, CL-11 knockdown effectively inhibited UPEC-induced p38 MAPK phosphorylation and cytokine production in PTECs. These were further inhibited by CD91 blockade. We conclude that CL-11 functions as a mediator of innate immunity via direct antibacterial roles as well as dual modulatory roles in UPEC-induced inflammatory responses during UTI. Thus, the study findings suggest a possible function for CL-11 in defence against UTI.     

The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2,4-dicarboxylic acid

Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor sigmas and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog L-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.     

Protection against oxidative stress in Escherichia coli stationary phase by a phosphate concentration-dependent genes expression

Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37 mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD⁺ ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.     

番茄SlTpx原核表达蛋白的体外功能分析*

H₂O₂是一种重要的信号分子,参与植物体内多种生理代谢活动,但过量的H₂O₂破坏生物大分子,从而使细胞受到毒害。硫氧还蛋白过氧化物酶(thioredoxin peroxidase,Tpx)通过清除H₂O₂在保护植物免受氧化损伤方面起着重要作用。为进一步研究番茄Tpx基因(SlTpx)的功能,构建了番茄SlTpx原核表达载体,并诱导和纯化了SlTpx蛋白,发现该蛋白质大小约为21 kDa。为检测SlTpx的抗氧化功能,通过体外的混合功能氧化酶(MFO)实验、过氧化氢清除实验和SlTpx蛋白体外抗重金属和H₂O₂实验,证明SlTpx可以保护DNA不受有害活性氧切割,并且提高大肠杆菌抵抗重金属和H₂O₂胁迫的能力。为揭示SlTpx在植物中的功能和作用机制奠定基础。     

大肠埃希菌耐药性水平传播实验研究

目的研究重症监护病房（ICU）患者标本中分离的大肠埃希菌的耐药情况以及耐药性水平传播的实验研究。方法采取双纸片法（K-B）检测细菌的耐药性;产超广谱β-内酰胺酶（ESBLs）大肠埃希菌为供体菌,耐利福平大肠埃希菌（对其他抗生素敏感）作为受体菌进行接合实验;采用聚合酶链反应（PCR）技术扩增整合子和耐药基因。结果30株大肠埃希菌中产ESBLs菌株检出率为46．7％;接合培养后,接合菌携带23kb和25kb大质粒,而无供体菌中一系列小质粒;供体菌和接合菌均携带I型整合子。结论大肠埃希菌耐药性严重,且呈多重耐药性;产ESBLs菌株可通过质粒和整合子将耐药基因转移给敏感菌,导致耐药性传播。     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Role of oxidative damage in tulip bulb scale micropropagation

The activation of oxygen stress-related enzymes was compared in regenerating and non-regenerating tulip bulb scale explants and regenerating stalk explants. The phospholipid composition of scale explants showed an increase of linolenic acid (1–15%) and a decrease in linoleic acid (70–55%). After incubation it was comparable to that of stalk explants in which no changes were observed. In all tested systems an increase in activity of catalase, peroxidase, SOD, lipoxygenase, polyphenoloxidase and phenylalanine ammonia lyase, was observed during incubation of the explants. The reaction can be divided into two phases. The first one (observed for scale explant lipoxygenase and to a lesser extent for SOD) occurs rapidly (1–2 h) after cutting the explants and appears to be wounding related. In the second phase (observed for all enzymes), starting during the first week of incubation, wound healing and regeneration can be observed. The activation of catalase, peroxidase and phenylalanine ammonia lyase was comparable in all tested systems and appears not to be related with the differences in tissue culture performance. In the second phase, the activity of lipoxygenase, peroxidase, catalase and phenylalanine ammonia lyase decreases in regenerating explants, while in non-regenerating explants they remain high. Our conclusion from these results is that oxidative damage is not the prime cause of the low regenerability of tulip bulb scale explants.     

质粒介导的大肠埃希菌对喹诺酮类抗菌药物耐药机制的研究

目的了解临床分离的耐环丙沙星的大肠埃希菌质粒介导的喹诺酮类耐药基因的携带情况,并进行相关耐药机制的分析。方法采用VITEK-2全自动微生物检测系统鉴定细菌,用K-B法检测细菌对16种常用抗生素的敏感性,采用聚合酶链反应检测喹诺酮类耐药基因qnrS、qnrA、qnrB、qepA和aac（6′）-Ib,并对阳性的aac（6′）-Ib结果进行测序分析。结果 30株耐环丙沙星的大肠埃希菌中,2株（6.67%）检出qepA基因,8株（26.67%）检出aac（6′）-Ib基因,经测序证实其中6株为aac（6）′-Ib-cr（20.0%）。未检出qnrS、qnrA和qnrB基因。结论对环丙沙星耐药的大肠埃希菌携带aac（6′）-Ib-cr和qepA基因,引起质粒介导的对喹诺酮类抗菌药物的低水平耐药。     

sucAB and sucCD are mutually essential genes in Escherichia coli

sucAB and sucCD of Escherichia coli encode enzymes that generate succinyl-CoA from 2-oxoglutarate and succinate, respectively. Their mutual essentiality was studied. sucAB and sucCD could be deleted individually, but not simultaneously. The mutual essentiality of sucAB and sucCD was further confirmed by the conditional expression of sucABCD, sucAB, and sucCD under the control of a P(BAD) in E. coli MG1655, E. coli MG1655 (DeltasucCD), and E. coli MG1655 (DeltasucAB), respectively. These strains grew well in Luria-Bertani medium containing 0.1% arabinose, but not in the absence of arabinose unless the medium was supplemented with succinyl-CoA. Our results indicate that either sucAB or sucCD is enough to produce succinyl-CoA that is essential for cell viability.     

Role of verocytotoxigenic Escherichia coli in cattle and buffalo calf diarrhoea

Abstract 273 Strains of Escherichia coli isolated from diarrhoeic and healthy control cattle and buffalo calves in Sri Lanka, were tested for Verocytotoxin (VT) and for heat-stable (ST) and heat-labile (LT) enterotoxins. VT and ST toxigenic E. coli were significantly associated with diarrhoea, accounting for 28% and 18% of diarrhoeic episodes, respectively. LT toxigenic E. coli were not significantly associated with diarrhoea.     

recO and recR mutations delay induction of the SOS response in Escherichia coli

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage; therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of -galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.     

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of a large number of genes in Escherichia coli under stress conditions. The present study investigated the growth characteristics and metabolic pathways of rpoS gene knockout mutant of E. coli growing in LB media under aerobic condition. The analyses were made based on gene expressions obtained by DNA microarray and RT-PCR, enzyme activities and intracellular metabolite concentrations at the exponential and early stationary phases of growth. Although the glucose utilization pattern of the mutant was similar to the parent strain, the mutant failed to utilize acetate throughout the cultivation period. Microarray data indicated that the expression levels of several important genes of acetate metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in the absence of rpoS gene. Interestingly, there was an increased activity of TCA cycle during the exponential growth phase, which was gradually diminished at the onset of stationary phase. Moreover, rpoS mutation had profound effect on the expression of several other genes of E. coli metabolic pathways that were not described earlier. The changes in the gene expressions, enzyme activities and intracellular metabolite concentrations of the rpoS mutant are discussed in details with reference to the major metabolic pathways of E. coli.     

Escherichia coli from retail meats carry genes associated with uropathogenic Escherichia coli, but are weakly invasive in human bladder cell culture

Aims: The aim of this study was to determine the uropathogenic potential of Escherichia coli isolated from retail meats. Methods and Results: Two hundred E. coli isolates recovered from retail meats, which were previously identified molecularly as extraintestinal pathogenic E. coli, were investigated for the presence of 21 uropathogenic E. coli (UPEC) virulence‐associated genes. Twenty‐three E. coli isolates were selected based on their serogroups and the number of virulence genes they contained, and further characterized using multilocus sequence typing, and by tissue culture assays for adherence to and invasion of T‐24 human bladder cells and for their induction of interleukin (IL)‐6 secretion. All virulence genes tested, except afa/dra and hlyD, were detected among the E. coli isolates. Multilocus sequence typing analysis of 23 selected isolates revealed that 17 isolates belonged to STs associated with human UPEC. Nearly all 23 isolates exhibited lower level of adherence and invasion compared to a clinical strain, UPEC CFT073. Conclusions: These observations suggested that a small proportion of E. coli isolates from retail meats carry uropathogenic associated virulence genes and thus may serve as a reservoir of these genes to UPEC in the human intestine. Their virulence potential seemed limited as they were only weakly invasive in human bladder cell culture. Significance and Impact of the Study: These findings support the hypothesis that retail meat E. coli may play a role in relation to urinary tract infection (UTI) and may be considered in development of a UTI prevention strategy.     

Effect of finishing diets on Escherichia coli populations and prevalence of enterohaemorrhagic E. coli virulence genes in cattle faeces

AIM: To determine the effect of different carbohydrate-based finishing diets on fermentation characteristics and the shedding of Escherichia coli and enterohaemorrhagic E. coli (EHEC) virulence genes in cattle faeces. METHODS AND RESULTS: The size of faecal E. coli populations and fermentation characteristics were ascertained in three experiments where cattle were maintained on a range of finishing diets including high grain, roughage, and roughage + molasses (50%) diets. Increased E. coli numbers, decreased pH and enhanced butyrate and lactate fermentation pathways were associated with grain diets, whereas roughage and roughage + molasses diets resulted in decreased concentrations of ehxA, eaeA and stx(1) genes, this trend remaining at lairage. In one experiment, faecal E. coli numbers were significantly lower in animals fed roughage and roughage + molasses, than animals fed grain (4.5, 5.2 and 6.3 mean log10 g(-1) digesta respectively). In a second experiment, faecal E. coli numbers were 2 log lower in the roughage and roughage + molasses diets compared with grain-fed animals prior to lairage (5.6, 5.5 and 7.9 mean log10 g(-1) digesta respectively) this difference increasing to 2.5 log at lairage. CONCLUSIONS: The type of dietary carbohydrate has a significant effect on E. coli numbers and concentration of EHEC virulence genes in faeces of cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a better understanding of the impact finishing diet and commercial lairage management practices may have on the shedding of E. coli and EHEC virulence factors, thus reducing the risk of carcass contamination by EHEC.