BMC microbiology update, may 20
Written by admin on May 22nd, 2008 | Filed under: mikrobiology UPDATE
Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species
Background: The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far. Results: The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both CpCnh1 and CdCnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; CpCnh1p provided cells with a much higher salt tolerance than the CdCnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters. Conclusions: We have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations.
Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis
Background: The incidence and diversity of human methanogens are insufficiently characterised in the gastrointestinal tract of both health and disease. A PCR and clone library methodology targeting the mcrA gene was adopted to facilitate the two-fold aim of surveying the relative incidence of methanogens in health and disease groups and also to provide an overview of methanogen diversity in the human gastrointestinal tract. Results: DNA faecal extracts (207 in total) from a group of healthy controls and five gastrointestinal disease groups were investigated. Colorectal cancer, polypectomised, irritable bowel syndrome and the control group had largely equivalent numbers of individuals positive for methanogens (range 45-50%). Methanogen incidence in the inflammatory bowel disease groups was reduced, 24% for ulcerative colitis and 30% for Crohn’s disease. Four unique mcrA gene restriction fragment length polymorphism profiles were identified and bioinformatic analyses revealed that the majority of all sequences (94%) retrieved from libraries were 100% identical to Methanobrevibacter smithii mcrA gene. In addition, mcrA gene sequences most closely related to Methanobrevibacter oralis and members of the order Methanosarcinales were also recovered. Conclusions: The mcrA gene serves as a useful biomarker for methanogen detection in the human gut and the varying trends of methanogen incidence in the human gut could serve as important indicators of intestinal function. Although Methanobrevibacter smithii is the dominant methanogen in both the distal colon of individuals in health and disease, the diversity of methanogens is greater than previously reported. In conclusion, the low incidence of methanogens in Inflammatory Bowel Disease, the functionality of the methanogens and impact of methane production in addition to competitive interactions between methanogens and other microbial groups in the human gastrointestinal tract warrants further investigation.
Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake
Fosmidomycin is a phosphonic antibiotic which acts by inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme [1], mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration. Results: We demonstrated that dxr (Rv2780c) is an essential gene in M. tuberculosis, since we could only delete the chromosomal copy when a second functional copy was provided on an integrating vector. This confirmed that the intracellular target of fosmidomycin was essential as well as sensitive. We looked at the uptake of fosmidomycin in two mycobacterial species, the slow-growing pathogenic M. tuberculosis and the fast-growing, saprophytic Mycobacterium smegmatis; both species were resistant to fosmidomycin to a high level. Fosmidomycin was not accumulated intra-cellularly in M. tuberculosis or M. smegmatis but remained in the extra-cellular medium. In contrast, fosmidomycin uptake was confirmed in the sensitive organism, Escherichia coli. We established that the lack of intra-cellular accumulation was not due to efflux, since efflux pump inhibitors had no effect on fosmidomycin resistance. Finally, we demonstrated that fosmidomycin was not modified by mycobacterial cells or by extracts but remained in a fully functional state. Conclusions: Taken together, these data demonstrate that fosmidomycin resistance in M. tuberculosis and M. smegmatis results from a lack of penetration of the antibiotic to the site of the sensitive target.
High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation
Background: Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5′nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated. Results: To determine the minimum number of genome equivalents (geq) that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 geq/ml [95%CI, 2,188-4,745] with a manual extraction procedure and 4,235 geq/ml (95%CI, 3,143-7,428 geq/ml) with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction. A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR. Conclusion: A sensitive and thoroughly evaluated real-time PCR was established. Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.
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