Identification of three ABC transporter genes in Trypanosomabrucei spp.

ABC transporters are key players in the multi-drug resistance of cancer cells and yeast, and they appear to be involved in the drug resistance of various pathogenic protozoa. No member of this ubiquitous protein family has yet been described in Trypanosoma brucei spp., the causative agents of African sleeping sickness and animal trypanosomiases. However, different cases of artificially induced drug resistance were shown to be linked to a reduction in net drug uptake. We used polymerase chain reaction with degenerate oligonucleotide primers corresponding to particularly conserved regions within the ATP-binding cassette to probe the genome of T.brucei spp. for the presence of ABC transporter genes. Three different sequence segments encoding ATP-binding cassettes were identified, which, upon Southern blotting, appeared to belong to distinct genes designated Tbabc1, Tbabc2, and Tbabc3. They appear to be single-copy genes in both drug-susceptible and drug-resistant stocks of T.brucei spp., expressed in bloodstream forms as well as in the procyclic life stage. Whereas Tbabc3 shows moderate homology to various known ABC transporters, Tbabc1 and Tbabc2 are highly homologous to P-glycoprotein A of Leishmaniatarentolae and to the multidrug resistance protein 1 of L.donovani, respectively. Received: 18 July 1997 / Accepted: 13 August 1997     

双重实时荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌

目的 建立一种快速、灵敏、特异的鉴定克柔念珠菌和光滑念珠菌的双重实时荧光定量PCR方法.方法 以核糖体基因内转录间隔区Ⅱ(ITSⅡ)为靶目标,设计并合成分别针对克柔念珠菌、光滑念珠菌的种特异引物和探针.建立双重实时荧光定量PCR反应体系,并用该体系对呼吸道相关致病菌进行检测.鉴定结果与临床常规鉴定方法对照,评价其敏感度、特异度及重复性.结果 通过对100例样品的检测,结果显示该双重实时荧光定量PCR法检测标本的鉴定结果与常规鉴定方法的结果对照,特异度为100%,敏感度为100%;最小能检测到10个拷贝数的重组质粒;批内重复实验和批间重复实验结果均与常规鉴定方法结果相符.结论 双重荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌,特异度和敏感度高,重复性好,且快速、简便,该方法将有助于念珠菌病的早期诊断和针对性治疗.     

ABC转运体介导肿瘤多药耐药机制的研究进展

多药耐药(multidrug resistance,MDR)是导致肿瘤化疗失败的主要原因之一,而过表达三磷酸腺苷结合盒(ATP binding cassette,ABC)转运蛋白是导致MDR产生的主要机制.因此,筛选ABC转运蛋白的抑制剂,在逆转肿瘤MDR提高肿瘤化疗疗效中具有重要的意义.     

Increased incidence of fungemia caused by Candida krusei.

Candida krusei colonized 12.4% of 868 patients undergoing episodes of therapy-induced granulocytopenia over a 9-year period. The gastrointestinal tract was most frequently colonized, followed by the respiratory tract and urinary tract. Ten patients developed systemic infections with C. krusei; all 10 had two or more positive blood cultures. Nine of the 10 patients were colonized with C. krusei, and 6 were receiving systemic antifungal agents at the time of development of the infection. Seven patients died within 1 month of C. krusei sepsis; systemic candidiasis was seen in the autopsies of the four patients on whom autopsies were performed. Therefore, C. krusei should be recognized as an emerging pathogen in select patient populations.     

Identification of an immunodominant ABC transporter in methicillin-resistant Staphylococcus aureus infections

Immunoblotting sera from 26 patients with septicemia due to an epidemic strain of methicillin-resistant Staphylococcus aureus (EMRSA-15), 6 of whom died, revealed an immunodominant EMRSA-15 antigen at 61 kDa. There was a statistically significant correlate (P < 0.001) between survival and immunoglobulin G to the 61-kDa band. The antigen was identified by sequencing positive clones obtained by screening a genomic expression library of EMRSA-15 with pooled sera from patients taken after the septicemic episode. Eluted antibody reacted with the 61-kDa antigen on immunoblots. The amino terminus was obtained by searching the S. aureus NCTC 8325 and MRSA strain COL databases, and the whole protein was expressed in Escherichia coli TOP 10F'. The derived amino acid sequence showed homology with ABC transporters, with paired Walker A and Walker B motifs and 73% homology to YkpA from Bacillus subtilis. Epitope mapping of the derived amino acid sequence with sera from patients who had recovered from EMRSA-15 septicemia delineated seven epitopes. Three of these epitopes, represented by peptides 1 (KIKVYVGNYDFWYQS), 2 (TVIVVSHDRHFLYNNV), and 3 (TETFLRGFLGRMLFS), were synthesized and used to isolate human recombinant antibodies from a phage antibody display library. Recombinant antibodies against peptides 1 and 2 gave logarithmic reductions in organ colony counts, compared with control groups, in a mouse model of the infection. This study suggests the potential role of an ABC transporter as a target for immunotherapy.     

Immunochemical determinant and serological specificity of Candida krusei

We have investigated the chemical structure of the antigenic determinant of Candida krusei cell-wall mannan. Acetolysis of the mannan, obtained by extraction with alkali and purified as copper complex, gave five oligosaccharides (from mono- to pentasaccharide) and a minor amount of an octasaccharide. Partial acetolysis of the mannan gave a large amount of mannooctaose. We have examined the inhibition by these oligosaccharides of the precipitin reaction between anti-Candida krusei serum and homologous mannan, and found that the mannooctaose was the most effective inhibitor. Results obtained by proton magnetic resonance spectroscopy, methylation analysis, and other structural studies of Candida krusei mannan, suggest that the octasaccharide possesses six alpha (1-2) linkages and one alpha (1-6) linkage located in the middle of the chain, and that this mannooctaose may be responsible for the specificity of Candida krusei cell-wall mannan.     

Experimental hematogenous candidiasis caused by Candida krusei and Candida albicans: species differences in pathogenicity.

Hematogenous infections caused by Candida krusei have been noted with increasing frequency, particularly in cancer patients receiving prophylaxis with antifungal triazoles. Progress in understanding the pathogenesis of this emerging infection has been limited by the lack of an animal model. We developed a CF1 mouse intravenous inoculation model of candidiasis to evaluate the pathogenicity of C. krusei in normal and immunosuppressed mice and to compare it with that of Candida albicans. Several inocula (10(6) to 10(8) CFU per animal) of two clinical strains of C. krusei and three American Type Culture Collection strains of C. albicans were tested. Groups of 20 mice each were injected with a single intravenous dose of one inoculum. Animals randomized to receive C. krusei were immunosuppressed by intraperitoneal injection of cyclophosphamide or the combination of cyclophosphamide plus cortisone acetate or they did not receive immunosuppressive agents (normal mice). One hundred percent mortality was observed in normal mice injected with 10(6) CFU of C. albicans per mouse compared with no mortality in normal mice that received 10(8) CFU of C. krusei per mouse (P < 0.01). Resistance to C. krusei infection was markedly lowered by immunosuppression, particularly by the combination of cyclophosphamide plus cortisone acetate, with a significantly shorter survival and a higher organ fungal burden in immunosuppressed than in normal animals (P < 0.01). Tissue infection was documented by culture and histopathologic findings in all examined organs.     

Strain variation among and antifungal susceptibilities of isolates of Candida krusei.

Candida krusei is an emerging pathogen that is well known for its propensity to develop resistance to fluconazole and other azoles. Despite the potential clinical significance of C. krusei, little is known of its epidemiology and genetic diversity as defined by the newer DNA-based typing methods. We investigated the genotypic diversity and antifungal susceptibility of 67 clinical isolates from 44 patients and 5 health care workers from six different medical centers. Strain delineation was performed by restriction endonuclease analysis of genomic DNA (REAG) with the restriction enzyme HinfI followed by conventional electrophoresis. The susceptibility of the isolates to the antifungal agents amphotericin B, flucytosine, fluconazole, and itraconazole was determined by methods recommended by the National Committee for Clinical Laboratory Standards. The MICs at which 90% of the isolates were inhibited ranged from 1.0 microgram/ml for itraconazole to 64 micrograms/ml for fluconazole. In general, isolates from a given patient, or epidemiologically related isolates from a single institution, were identical by molecular typing methods. Epidemiologically unrelated isolates were distinctly different by the REAG typing method employed. These data document the genetic diversity and antifungal susceptibility of clinical isolates of C. krusei.     

肺耐药相关蛋白及其介导的多药耐药研究进展

肺耐药相关蛋白 (lungresistance─relatedprotein ,LRP)是一人类主要穹窿蛋白 ,分子量为12 0KD ,它定位于核孔复合物上 ,作为该复合物的转运单位LRP基因控制着多种底物的双向转运。本文对肺耐药相关蛋白的分子生物学特征和生理功能及其在肿瘤细胞中的过度表达所产生的临床意义作一综述     

Characteristics of a thermotolerant strain of Candida krusei

A novel thermotolerant strain of Candida krusei was distinguished on the basis of its typical enzymatic, growth and respiratory properties. Unlike other thermotolerant Candida species and other strains of the same species, this strain exhibited diauxy on glucose. The strain also exhibited prolonged lag during growth on citrate and very low levels of NAD-linked isocitrate dehydrogenase in comparison to the NADP linked enzyme. The capability of the strain to grow over a wide temperature range, relatively high exponential growth rate and short lag period indicate the potential of the organism for industrial applications.     

Identification of a fourth half ABC transporter in the human peroxisomal membrane

Three half ATP-binding cassette transporters (ALDP, ALDR, PMP70) are known to be present in the human peroxisome membrane. Mutations in the gene encoding ALDP cause X-linked adrenoleukodystrophy; the role of ALDR and PMP70 in human disease is unclear. We report the cloning and characterization of a fourth human gene encoding a peroxisomal half ABC transporter. The gene, designated P70R, maps to chromosome 14q24, encodes a 73 kDa transporter most similar to PMP70, and is expressed in all human tissues examined. Because half ABC transporters heterodimerize to form functional transporters, the identification of a fourth member of this family in the peroxisome membrane has implications for our understanding of mammalian peroxisomes and the genetic disorders of peroxisomal function.      

Fluconazole resistance mechanisms in Candida krusei: the contribution of efflux-pumps.

The main resistance mechanism for fluconazole in Candida krusei is the diminished sensitivity of the target enzyme cytochrome P450 sterol 14 alpha-demethylase (CYP51) to inhibition by azole agents. An alternative mechanism of resistance, efflux-pump activity, has been proposed. The aim of our study was to find out the possible contribution of efflux-pumps in conferring resistance to fluconazole in 33 C. krusei isolates from different clinical sources. The activity of efflux-pumps was checked using the inhibitor CCCP (carbonyl cyanide 3-chloro-phenylhydrazone), which decreases the minimum inhibitory concentration (MIC) when resistance is attributed. We established a concentration of 0.5 microg/ml of CCCP. The susceptibility patterns of our isolates for five antifungal drugs (amphotericin B, fluconazole, itraconazole, flucytosine and voriconazole) were determined according to an NCCLS M27-A2 protocol modification (Sensititre Yeast One). We tested all the strains before and after adding CCCP to the RPMI medium. The MIC90s and ranges of the drugs were identical before and after addition of CCCP. The MIC for fluconazole was higher than for the other antifungals. The new triazoles were active and the MICs were lower, although this should be interpreted carefully as the drugs showed different cut-offs. Only one isolate showed a two-fold decrease in MIC to fluconazole when CCCP was added. We did not find any multi-resistant strains. According to our study with C. krusei, CCCP-inhibited efflux-pumps do not play a significant role in resistance to fluconazole.     

Successful treatment of Candida krusei fungemia with amphotericin B and caspofungin.

We report a leukemic patient with C. krusei fungemia who failed to respond to liposomal amphotericin B therapy alone. The addition of caspofungin eradicated infection and was well tolerated. Our report is the first to describe successful treatment of a patient with invasive C. krusei infection using this combination of antifungals. Combination therapy could be a useful treatment option for invasive candidosis, particularly when caused by more resistant species such as C. krusei.     

Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity

Multidrug resistance ABC transporter Pdr5p of Saccharomyces cerevisiae is particularly important due to its ability to export a wide range of unrelated substrates. To clarify its function, we generated Pdr5p mutants by random mutagenesis and screened for mutants with altered drug specificity in vivo by using 5 drug compounds. Nine point mutations that caused significant changes in drug specificity distributed throughout the length of Pdr5p, namely, in the extracellular, transmembrane or cytoplasmic regions of the transporter. We then investigated their effects upon drug resistance, using 36 chemically related or distinct substrates. From this study, overall geometry of the Pdr5p was suggested to contribute in acquiring the enormous range of drug specificity. Based on their ability to inhibit the growth of the mutant strains, the 36 tested drugs were classified into: drugs to which the mutants responded differently (Group 1), drugs to which all the mutants showed sensitivity (Group 2), and drugs to which all the mutants exhibited resistance (Group 3). The ability of the compounds to be partitioned to the plasma membrane seemed an important factor for recognition by Pdr5p.     

Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata.

CHROMagar Candida is a new differential culture medium that allows selective isolation of yeasts and simultaneously identifies colonies of Candida albicans, C. tropicalis, and C. krusei. We evaluated the use of this medium with 316 yeast isolates including 247 isolated directly on CHROMagar from clinical material. Over 95% of stock and clinical isolates of C. albicans, C. tropicalis, and C. krusei were correctly identified on the basis of colony morphology and pigmentation on CHROMagar. Additionally, CHROMagar also allowed the identification of C. (Torulopsis) glabrata at a similar level of accuracy. The overall agreement between two observers in reading the CHROMagar plates was 95%. Growth of Candida sp. isolates on CHROMagar had no adverse effect on antifungal MICs or Vitek identification results. In parallel, cultures of 548 stool and rectal swab specimens set up on CHROMagar and Sabouraud glucose agar (SGA) were positive in 234 instances. CHROMagar was positive and SGA was negative for 11 specimens, and CHROMagar was negative and SGA was positive for 18 specimens. A single yeast species was isolated on both media from 162 specimens, and in 146 (90%) of these specimens the same species was detected on both CHROMagar and SGA. A total of 43 of the 234 positive cultures contained mixtures of yeast species. Twenty (47%) of these mixed cultures were detected only on CHROMagar. CHROMagar is extremely useful in making a rapid presumptive identification of common yeast species. This capability plus the ability to detect mixed cultures of Candida spp. promises to improve and streamline the work flow in the mycology and clinical microbiology laboratory.