AcuD Gene Knockout Attenuates the Virulence of Talaromyces marneffei in a Zebrafish Model

Talaromyces marneffei is the only dimorphic species in its genus and causes a fatal systemic mycosis named talaromycosis. Our previous study indicated that knockdown of AcuD gene (encodes isocitrate lyase of glyoxylate bypass) of T. marneffei by RNA interference approach attenuated the virulence of T. marneffei, while the virulence of the AcuD knockout strains was not studied. In this study, T. marneffei-zebrafish infection model was successfully established through hindbrain microinjection with different amounts of T. marneffei yeast cells. After co-incubated at 28°C, the increasing T. marneffei inoculum doses result in greater larval mortality; and hyphae generation might be one virulence factor involved in T. marneffei-zebrafish infection. Moreover, the results demonstrated that the virulence of the ΔAcuD was significantly attenuated in this Zebrafish infection model.

Effect of overexpressing isocitrate lyase on succinate production in ldh(-1) Corynebacterium glutamicum / 生物工程学报

Corynebacterium glutamicum SA001 is a mutant with lactate dehydrogenase (ldhA) deletion. In order to increase metabolic flux from isocitrate to succinate, and to improve the production of succinate under anaerobic conditions,we transducted the gene aceA coding isocitrate lyase (ICL) from Escherichia coli K12 into Corynebacterium glutamicum SA001 (SA001/pXMJ19-aceA). After 12 h aerobic induction by adding 0.8 mmol/L of IPTG, the recombinant strain was transferred to anaerobic fermentation for 16 h. Succinate reached 14.84 g/L, with a productivity of 0.83 g/(L x h). Compared to C. glutamicum SA001, the activity of ICL of the recombinant strain was increased 5.8-fold, and the succinate productivity was increased 48%. Overexpression of isocitrate lyase will increase the metabolic flux of glyoxylate bypass flowing to succinate.

Metabolic regulation of isocitrate lyase regulator in Escherichia coli based on metabolic flux information / 生物工程学报

Gene expression is regulated by different transcriptional regulators. The transcriptional regulator isocitrate lyase regulator (IclR) of Escherichia coli represses the expression of the aceBAK operon that codes for the glyoxylate pathway enzymes. In this study, physiological and metabolic responses of the deletion of the ic1R gene in E. coli BW25113 were investigated based on the quantification and analysis of intracellular metabolic fluxes. The knockout of the iclR gene resulted in a decrease in the growth rate, glucose uptake rate and the acetate secretion rate, but a slight increase in biomass yield. The latter could be attributed to the lowered metabolic fluxes through several CO2 generating pathways, including the redirection of 33% of isocitrate directly to succinate and malate without CO2 production as well as the reduced flux through the pentose phosphate pathway. Furthermore, although the glyoxylate shunt was activated in the iclR mutant, the flux through phosphoenolpyruvate (PEP) carboxykinase kept almost unchanged, implying an inactive PEP-glyoxylate cycle and no extra loss of carbon atoms in the mutant strain. Both the reduced glucose uptake rate and the active glyoxylate shunt were responsible for the minor decrease in acetate secretion in the ic1R knockout strain compared to that in the wild-type E. coli strain.

Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Isocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.</p><p><b>METHODS</b>MTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.</p><p><b>RESULTS</b>RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.</p><p><b>CONCLUSIONS</b>MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.</p>

Differential expression of isocitrate lyase in P. marneffei phagocytized by nonstimulated and stimulated murine macrophages / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the differential expression of isocitrate lyase in Penicillium marneffei phagocytized by nonstimulated and stimulated murine macrophages, and explore the role of glyoxylate pathway in pathogenesis of Penicilliosis marneffei.</p><p><b>METHODS</b>Penicillium marneffei conidia and Raw264.7 cells were incubated in 16 cultures, which were divided to 4 groups for treatment with N-monomethyl-L-arginine (LNMMA, CI group), murine interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) (T group), IFN-gamma plus LPS and LNMMA (TI group), or the same volume of culture medium (C group). The transcriptional levels of isocitrate lyase were detected using real-time RT-PCR, and its expression levels detected biochemically.</p><p><b>RESULTS</b>The transcriptional levels of isocitrate lyase in C, CI, T, TI groups were 1.00, 1.42, 33.09, and 74.88 (P<0.05), while the expression levels were 0.06, 0.07, 0.18, and 0.93, respectively (P<0.05). The content of nitric oxide in T group was significantly higher than that in the other groups (P<0.01), but the CFU of T group was the lowest (P<0.01).</p><p><b>CONCLUSION</b>Reactive nitrogen intermediates induced by stimulated murine macrophages restrain the expression of isocitrate lyase of Penicillium marneffei and development of Penicillium marneffei, in which process the glyoxylate pathway may play an important role.</p>

Glyoxylate cycle is required for the overproduction of glutamate but is not essential for Corynebacterium glutamicum growth on glucose / 生物工程学报

The glyoxylate cycle was hypothesed to be indispensable for glutamate overproduction in coryneform bacteria, for it was thought to fulfill anaplerotic functions and to supply energy during the growth phase. During glutamate overproduction phase, however, it has been noted that the high level of the cycle is detrimental to the glutamate production. In order to clarify the relationship between the glutamate production and the glyoxylate cycle, a chromosomal aceA-disrupted mutant of wild-type C. glutamicum ATCC 13032 was constructed. The isocitrate lyase (ICL) activity of the parental strain was 0.011 u/mg of protein and reached 1.980 u/mg of protein after acetate induction; the mutant strain WTdeltaA, however, had no detectable ICL activity and was no longer able to grow on minimal medium with acetate as the sole carbon source. Compared with the wild-type C. glutamicum WT, the mutant strain WTdeltaA, exhibited the same growth rate with glucose as the sole carbon source, indicating glyoxylate cycle is not required for its growth on glucose. On the contrary, the glutamate production in WTdeltaA was severely impaired and more residual glucose was found in the fermentation broth at the end of fermentation with the mutant strain than with the wild-type strain. Further investigations into the relationship between the glutamate production and the glyoxylate cycle are under the way, which may help to elucidate the mechanism of glutamate overproduction.

Clone and expression of isocitrate lyase gene in Mycobacterium tuberculosis H37Rv / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct recombinant plasmid with isocitrate lyase (ICL) gene of Mycobacterium tuberculosis H37Rv for stable and high level expression of ICL in prokaryotic expression system.</p><p><b>METHODS</b>The recombinant plasmid with ICL gene (pET30 (a)-Rv0467) was constructed by polymerase chain reaction and cloning. The fusion protein was expressed in E. coli host strain BL21 (DE3). Activity of the fusion protein was studied after it was purified with metal chelating chromatography.</p><p><b>RESULTS</b>We constructed the plasmid which could highly express Mycobacterium tuberculosis H37Rv ICL. The plasmid was highly expressed in E. coli BL21 (DE3), in which the fusion protein accounted for 30% of total protein content. After having been purified by metal chelating chromatography, the purity of the soluble fusion protein was 90%. The fusion protein had activity of ICL.</p><p><b>CONCLUSION</b>Using the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point.</p>

Efecto de metales traza sobre la morfología celular, actividad enzimßtica y producción de ácido cítrico en una cepa de Aspergillus wentii / Effect of trace metals on cell morphology, enzyme activation, and production of citric acid in a strain of Aspergillus wentii

Data concerning the effect of very low concentrations of metals on citric acid production by microorganisms, as well as on the activity of enzymes presumptively involved in the process, are confuse. The bulk of information was obtained mainly studying selected strains of Aspergillus niger. Information concerning other citric acid producer filamentous fungi, such as A. wentii, is scanty. In the present article we report the effect of different cations on the growth pattern of A. wentii P1 as well as on the related citric acid production and the activity of several enzymes. It was found that without any addition to the culture medium the fungus developed a pelleted form of growth, pellets being about 1.5 mm in diameter. The citric acid yield was about 90. The addition of Cu2+ impaired the sugar uptake, as well as the production of citric acid and biomass. The uptake of sugar increased in the presence of Zn2+, and there was a marked increase of the biomass production, which could account for the low citric acid production. The addition of Fe2+ impaired the citric acid production and, as sulfate, the sugar uptake. The presence of Fe3+ markedly impaired the citric acid production and increased the sugar uptake. There is no agreement about the enzymes involved in the accumulation of citric acid by microorganisms. In spite of this, aconitase (Ac), isocitrate lyase (IL), isocitrate dehydrogenase NAD(+)-dependent (ICDH- NAD+) and isocitrate dehydrogenase NADP(+)-dependent (ICDH-NADP+) are often postulated as key enzymes. In our case, these enzymes were active during the standard fermentation, although with variations, particularly concerning Ac and IL. The behavior of enzymes might be different when tested in vivo or in vitro, mainly from the quantitative point of view. Nevertheless, the activity determined in vitro might give some indication concerning the effect on fermentation of substances present in the medium. It was found that all the enzymes tested increased their activity in the presence of Fe2+ and Fe3+. The addition of Cu2+ improved the activity of Ac, IL and ICDH-NADP+, while that of ICDH-NAD+ was impaired. The presence of Zn2+ decreased the activity of the enzymes excepting that of ICDH-NADP+ which increased. It must be pointed out that according to these results there are differences concerning the behavior of ICDH-NAD+ and ICDH-NADP+ in the presence of Cu2+ or Zn2+.

Site-site heterogeneity in isocitrate lyase of castor seed endosperm: evidence from kinetics of inactivation by tetranitromethane.

Inactivation of isocitrate lyase (native and EDTA-dialysed) by excess tetranitromethane (TNM) exhibits, biphasic kinetics, in which half of the initial activity is lost in a fast and the remaining half in a slow phase each following the pseudo-first order kinetics. Rate constants of the two phases are proportional to the TNM concentration. High succinate concentration protects the enzyme against TNM inactivation only in the slow phase without any effect on the fast phase. With the EDTA-dialysed enzyme, no such protection (against inactivation by TNM) is observed in the presence of succinate or Mg2+ ions. Addition of both these ligands together brings about protection against the slow phase (as with the native enzyme). It has been proposed that the site-site heterogeneity of isocitrate lyase is a consequence of its quaternary structure constraints.

Metabolic studies on mycobacteria. IV. Assay of isocitrate lyase and malate synthase activity in M. leprae.

Cell free extracts from M. tuberculosis H37 Rv, M. smegmatis armadillo derived M. leprae and normal armadillo liver homogenates were assayed for the presence of isocitrate lyase and malate synthase activity. It was observed that significant amount of isocitrate lyase and malate synthase activity was present in M. tuberculosis H37 Rv, M. smegmatis and armadillo derived M. leprae. No such activity was demonstrable in cell free extracts of normal armadillo liver. It is concluded that M. leprae like other mycobacteria has the capability to metabolise via glyoxylate bypass of TCA cycle. These findings may be relevant for understanding the energy metabolism of M. leprae under stress conditions and possibly the 'persister' stage.

Atividade da isocitrato liase em Mucor rouxii / Activity of isocitrate liase in mucor rouxii

O ficomiceto dimórfico Mucor rouxii foi cultivado em meio que continha glucose a 2% (YPG), tendo-se verificado um crescimento bifásico característico. A isocitrato liase, enzima do ciclo do glioxilato, apareceu sob estas condiçöes somente após a glucose do meio ter sido totalmente esgotada. Altos níveis de atividade da isocitrato liase foram encontrados durante o metabolismo gluconeogenético. Isso mostra a participaçäo do ciclo do glioxilato como fonte de succinato e de oxalacetato para as vias biossintéticas do fungo. Em meio complexo sem glucose (YP), estritamente gluconeogenético, foram encontradas altas atividades de isocitrato liase

Metabolic studies on mycobacteria--II. Glyoxylate by-pass (TCA cycle) enzymes of slow and fast growing mycobacteria.

Glyoxylate by-pass of tricarboxylic acid cycle (TCA) comes into prominence during survival of microorganisms under oxygen limitations and study of these enzymes may contribute to understanding of physiology of 'persisters' in various mycobacterial diseases. The enzymes of glyoxylate by-pass have been assayed in the extracts of various mycobacterial species, namely, M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. flavescens, M. vaccae, M. smegmatis and Mycobacteria strain w (M.w.). M.w. has been included because of its close antigenic resemblance to M. leprae. It has been found that all of the above investigated species possess isocitrate lyase and malate synthetase, the key enzymes of glyoxylate by-pass. The presence of the enzymes is being reported for the first time in M. flavescens, M. vaccae and M.w. whereas these were earlier shown to be present in M. tuberculosis and M. smegmatis. It was also demonstrated in M.w. where acetate alone could not serve as sole source of carbon, but in the presence of glycerol stimulates the activity of glyoxylate pathway enzymes. The importance of these findings has been discussed.