The expression profile of the <Emphasis Type="Italic">Tuber borchii</Emphasis> nitrite reductase suggests its positive contribution to host plant nitrogen nutrition

Ectomycorrhizal symbiosis is a ubiquitous association between plant roots and numerous fungal species. One of the main aspects of the ectomycorrhizal association are the regulation mechanisms of fungal genes involved in nitrogen acquisition. We report on the genomic organisation of the nitrate gene cluster and functional regulation of tbnir1, the nitrite reductase gene of the ectomycorrhizal ascomycete Tuber borchii. The sequence data demonstrate that clustering also occurs in this ectomycorrhizal fungus. Within the TBNIR1 protein sequence, we identified three functional domains at conserved positions: the FAD box, the NADPH box and the two (Fe/S)-siroheme binding site signatures. We demonstrated that tbnir1 presents an expression pattern comparable to that of nitrate transporter. In fact, we found a strong down-regulation in the presence of primary nitrogen sources and a marked tbnir1 mRNA accumulation following transfer to either nitrate or nitrogen limited conditions. The real-time PCR assays of tbnir1 and nitrate transporter revealed that both nitrate transporter and nitrite reductase expression levels are about 15-fold and 10-fold higher in ectomycorrhizal tissues than in control mycelia, respectively. The results reported herein suggest that the symbiotic fungus Tuber borchii contributes to improving the host plant’s ability to make use of nitrate/nitrite in its nitrogen nutrition.     

Validation of a Low-cost Optic Nerve Sheath Ultrasound Phantom: An Educational Tool

Objective

To validate an ocular phantom as a realistic educational tool utilizing in vivo and phantom optic nerve sheath (ONS) images obtained by ultrasound.

Structural and functional characterization of a maltose/maltodextrin ABC transporter comprising a single solute binding domain (MalE) fused to the transmembrane subunit MalF

Canonical ATP-binding cassette import systems rely on extracellular substrate binding proteins (SBP) for function. In gram-negative bacteria, SBPs are usually freely diffusible in the periplasm and, where studied, exist in excess over their cognate transporters. However, in vitro studies with the maltose transporter of Escherichia coli (MalFGK₂) have demonstrated that mechanistically one copy of its SBP (MalE) per transport complex is sufficient for activity. To address whether such a condition is physiologically relevant, we have characterized a homolog of the E. coli system from the gram-negative bacterium Bdellovibrio bacteriovorus which has a single copy of a maltose binding domain fused to the MalF subunit. Both transporters share substrate specificity for maltose and linear maltodextrins. Specific ATPase and transport activities of the B. bacteriovorus transporter were comparable to those of the E. coli system assayed at a 1:1 M ratio of MalE to the transport complex. While MalE_Ec was able to additionally increase ATPase activity of MalFGK_2Bb, the isolated MalE domain of B. bacteriovorus failed to stimulate the E. coli system. Strikingly, interactions of the MalE domain with the transmembrane subunits during the transport cycle as studied by site-specific cross-linking were found to differ from those observed for E. coli MalE-FGK₂.     

Cover Picture: J. Basic Microbiol. 3/2007

Actinobacteria of the genus Frankia are able to form a symbiosis with plant roots able to fix nitrogen. The actinorhizal plant Discaria trinervis was co‐inoculated with Frankia ‐strain BCU 110501 and in additon with a helper streptomycete, Micromonospora ‐strain BCRU‐MM18 and grown for 12 weeks. The root nodules are easily visible by the naked eye. In the current issue this symbiotic interaction of gram‐positive bacteria and plant roots is described in more detail (Foto: Gernot Vobis, San Carlos de Bariloche, Argentina). (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Enhanced efficacy of imipenem-colistin combination therapy against multiple-drug-resistant Enterobacter cloacae: in vitro activity and a Galleria mellonella model

Background/Purpose

To investigate the in vitro and in vivo activity of imipenem-colistin combination against multidrug-resistant Enterobacter cloacae infections in order to determine whether it should be explored further.

Co-occurrence of heterozygous CREB3L3 and APOA5 nonsense variants and polygenic risk in a patient with severe hypertriglyceridemia exacerbated by estrogen administration

We describe a case of a 36-year-old woman with severe hypertriglyceridemia likely caused by double heterozygosity of a known pathogenic APOA5 nonsense variant (p.Q275X) and a novel CREB3L3 nonsense variant (p.C296X) on a background of very strong polygenic susceptibility. Her clinical course worsened with development of eruptive xanthomata after oral administration of 2 mg estradiol twice daily for 2 weeks as part of a medical protocol for intrauterine embryo transfer following in vitro fertilization. Her triglyceride levels decreased to baseline and xanthomata resolved without treatment after discontinuation of hormonal therapy, which also resulted in termination of pregnancy. Before undergoing a second embryo transfer using her natural cycle and no exogenous hormones, the patient started combination therapy with eicosapentaenoic acid ethyl ester and gemfibrozil, leading to an ～80% decrease in triglyceride levels. She continued treatment throughout pregnancy, which progressed to term with the delivery of healthy twins.     

Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa

Summary A mutant of Neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. In contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. However, glutamine still represses these nitrate assimilation enzymes in mea-1. The nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. In addition, mea-1; nit-2 double mutants cannot utilize ammonium as a nitrogen source. We suggest therefore that nit-2 and mea-1 loci play a role in ammonia/methylamine uptake.     

The regulatory protein NIT4 that mediates nitrate induction inNeurospora crassa contains a complex tripartite activation domain with a novel leucine-rich,acidic motif

Expression ofnit-3 andnit-6, the structural genes which encode nitrate reductase and nitrite reductase inNeurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys₆/Zn₂ zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo inN. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.     

Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model

Objectives

Streptococcus pyogenes causes life-threatening invasive infections including necrotizing fasciitis (NF). Current treatment guidelines recommend the use of a cell-wall–active antibiotic combined with a protein synthesis inhibitor and surgical debridement in NF patients. Adjunctive therapy with intravenous immunoglobulin (IVIG) has been proposed for superantigen-associated streptococcal toxic shock syndrome. So far, benefits of IVIG treatment remain unclear and prospective clinical studies are scarce. Thus, we aimed to assess the effects of IVIG on virulence factor activity in vitro, ex vivo in patients and in vivo in a NF mouse model.

The impact of inoculum size on the activity of cefoperazone-sulbactam against multidrug resistant organisms

Objectives

This study aims to assess the in vitro activity of cefoperazone alone and different cefoperazone-sulbactam ratios against different inoculum sizes of multidrug resistant organisms.

Upregulated expression of Tim-3 involved in the process of toxoplasmic encephalitis in mouse model

Toxoplasma gondii can establish chronic infection and is characterized by the formation of tissue cysts in the brain. The cysts may remain throughout the life of the host but can reactivate and cause life-threatening toxoplasmic encephalitis (TE) in immunocompromised patients. T cell-mediated immune responses are essential for preventing the reactivation of chronic infection of T. gondii in the brain. The immunoinhibitory receptor T cell immunoglobulin and mucin domain (Tim)-1 and Tim-3 are expressed on terminally differentiated T helper (Th) 2 and Th1 cells, respectively, participating in the regulation of Th immune response. However, there is no report concerning the role of Tim genes in TE. In this study, Kunming outbred mice were infected with Prugniaud (Pru), a type II strain of T. gondii by oral gavage. Compared with the uninfected controls, there were mild brain inflammations at 3 weeks postinfection (p.i.), moderate brain inflammations at 5 weeks p.i., and aggravated brain inflammations and necrosis at 7 and 9 weeks p.i. The expressions of tachyzoite stage-specific genes in brains were consistent with the severity of brain histopathology of TE at 5 and 7 weeks p.i., while the expressions of bradyzoite stage-specific genes in brains were significantly increased at 7 and 9 weeks p.i. Using quantitative real-time PCR detection and immunohistochemistry staining, our results showed that the expressions of Tim-3 were significantly upregulated in both brains and spleens at 5 weeks p.i. and in spleens at 9 weeks p.i., which showed the similar dynamic tendency as that of interferon-γ expressions in both brains and spleens at the same times. In contrast, the Th2-specific marker Tim-1 expressions were significantly downregulated in both brains and spleens at 3 weeks p.i. and upregulated in both brains and spleens at 7 and 9 weeks p.i., which showed the similar dynamic tendency as that of interleukin-4 expressions in both brains and spleens at the same time. Our data indicate that Tim-3 may involve in the process of TE in mice infected with T. gondii Pru strain.     

Gene expression profile of LPS-stimulated dendritic cells induced by a recombinant Sj16 (rSj16) derived from Schistosoma japonicum

Sj16, a 16-kDa protein secreted from Schistosoma japonicum, has been demonstrated an anti-inflammatory effect in vitro and in vivo, but its mechanism is still not clear. In this study, microarray analysis was performed to investigate the effects of recombinant Sj16 (rSj16) on the gene expression of the lipopolysaccharide (LPS)-stimulated dendritic cells (DCs). Immature DCs were treated with LPS, LPS?+?recombinant Sj16 (rSj16), or rSj16 alone for 24 h, and the gene expression profiles were examined using complementary DNA (cDNA) microarrays. With the cutoff value of 2-fold change in the expression, 509 genes were affected, 226 genes upregulated, and 283 genes downregulated after adding rSj16. Analysis by functional annotation clustering tool showed that rSj16-affected genes mainly associated with inflammatory response, defense response, regulation of immune system process, apoptosis, and cell migration. The results revealed that rSj16 reduced the LPS-induced pro-inflammatory genes such as cytokines (e.g., IL6, IL18, IFN-γ, IL12a, IL1b), chemokines, and receptors (e.g., CXCL1, CXCL9, CCL5, CCR5, CCR1, CCR2, CXCR3) and increased the anti-inflammatory gene IL-10. Further data mining of these genes by pathway analysis showed that genes regulated by rSj16 were significantly involved in cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, antigen processing and presentation, and Jak-STAT signaling pathway. In addition, quantitative real-time PCR (qRT-PCR) and Western blot analysis showed that rSj16 downregulated the expression of inhibitor of nuclear factor kappa-β kinase subunit beta (IKKβ) and nuclear factor-kappa β p65 (NF-κβ) messenger RNA (mRNA) and inhibited the phosphorylation of IKKβ and the NF-κB p65 protein, which implied that rSj16 exerting immunomodulatory effects by suppressing NF-κB signaling pathway. These results provide useful information in further understanding of the immunoregulation mechanisms of Sj16, and it is indicated that Sj16 could be as a potential molecule for the immunosuppressant drug development.     

Update on fosfomycin-modified genes in Enterobacteriaceae

The long-used antibiotic fosfomycin has recently been re-evaluated as a potential regimen for treating extended-spectrum β-lactamases (ESBLs) and carbapenem-resistant Enterobacteriaceae (CRE). Fosfomycin is known for its robust bactericidal effect against ESBL-producing Enterobacteriaceae and CRE. However, fosfomycin-modified genes have been reported in transposon elements and conjugative plasmids, resulting in fosfomycin resistance in parts of East Asia. Here we review reports of fosfomycin-modified (fos) genes in Enterobacteriaceae and assess the efficacy of fosfomycin against multidrug-resistant Enterobacteriaceae infections. At least 10 kinds of fos genes have been identified in the past decade; of these, fosA (and fosA subtypes) and fosC2 are primarily found in Enterobacteriaceae. All fosA subtypes except fosA2 are found in plasmids and transposons, nearby insertion sequence elements, or integrons, indicating that mobilizing elements also play an important role in plasmid-mediated fos genes in Enterobacteriaceae. fosA3, which is prevalent in East Asia, has been transmitted (mostly by animals) within and across continents via IS26 mobile elements. The acquisition of multiple antibiotic resistance genes via plasmids and mobile elements has resulted in a need for combined treatments for Enterobacteriaceae cases. The combination of fosfomycin and carbapenem has been the focus of many in vitro studies, but there is a clear need for additional in vivo investigations involving pharmacokinetics.