Members of the ferric/zinc uptake regulator (Fur/Zur) family are the central metal-dependent regulator proteins in many Gram-negative and -positive bacteria. They are responsible for the control of a ...wide variety of basic physiological processes and the expression of important virulence factors in human pathogens. Therefore, Fur has gathered significant interest as a potential target for novel antibiotics. Here we report the crystal structure of FurB from Mycobacterium tuberculosis at a resolution of 2.7Å, and we present biochemical and spectroscopic data that allow us to propose the functional role of this protein. Although the overall fold of FurB with an N-terminal DNA binding domain and a C-terminal dimerization domain is conserved among the Zur/Fur family, large differences in the spatial arrangement of the two domains with respect to each other can be observed. The biochemical and spectroscopic analysis presented here reveals that M. tuberculosis FurB is Zn(II)-dependent and is likely to control genes involved in the bacterial zinc uptake. The combination of the structural, spectroscopic, and biochemical results enables us to determine the structural basis for functional differences in this important family of bacterial regulators.
Summary Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis . Moreover, the recent isolation of M. tuberculosis strains resistant to both ...first- and second-line antitubercular drugs (XDR–TB) threatens to make the treatment of this disease extremely difficult and becoming a threat to public health worldwide. Recently, it has been shown that azoles are potent inhibitors of mycobacterial cell growth and have antitubercular activity in mice, thus favoring the hypothesis that these drugs may constitute a novel strategy against tuberculosis disease. To investigate the mechanisms of resistance to azoles in mycobacteria, we isolated and characterized several spontaneous azoles resistant mutants from M. tuberculosis and Mycobacterium bovis BCG. All the analyzed resistant mutants exhibited both increased econazole efflux and increased transcription of mmpS5 – mmpL5 genes, encoding a hypothetical efflux system belonging to the resistance–nodulation–division (RND) family of transporters. We found that the up-regulation of mmpS5 – mmpL5 genes was linked to mutations either in the Rv0678 gene, hypothesized to be involved in the transcriptional regulation of this efflux system, or in its putative promoter/operator region.
Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic ...antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.
To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed.
Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.
Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of ...benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-β- d-ribose 2′-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.
The ESAT-6 (early secreted antigenic target, 6 kDa) family collects small mycobacterial proteins secreted by Mycobacterium tuberculosis, particularly in the early phase of growth. There are 23 ESAT-6 ...family members in M. tuberculosis H37Rv. In a previous work, we identified the Zur- dependent regulation of five proteins of the ESAT-6/CFP-10 family (esxG, esxH, esxQ, esxR, and esxS). esxG and esxH are part of ESAT-6 cluster 3, whose expression was already known to be induced by iron starvation.
In this research, we performed EMSA experiments and transcriptional analysis of ESAT-6 cluster 3 in Mycobacterium smegmatis (msmeg0615-msmeg0625) and M. tuberculosis. In contrast to what we had observed in M. tuberculosis, we found that in M. smegmatis ESAT-6 cluster 3 responds only to iron and not to zinc. In both organisms we identified an internal promoter, a finding which suggests the presence of two transcriptional units and, by consequence, a differential expression of cluster 3 genes. We compared the expression of msmeg0615 and msmeg0620 in different growth and stress conditions by means of relative quantitative PCR. The expression of msmeg0615 and msmeg0620 genes was essentially similar; they appeared to be repressed in most of the tested conditions, with the exception of acid stress (pH 4.2) where msmeg0615 was about 4-fold induced, while msmeg0620 was repressed. Analysis revealed that in acid stress conditions M. tuberculosis rv0282 gene was 3-fold induced too, while rv0287 induction was almost insignificant.
In contrast with what has been reported for M. tuberculosis, our results suggest that in M. smegmatis only IdeR-dependent regulation is retained, while zinc has no effect on gene expression. The role of cluster 3 in M. tuberculosis virulence is still to be defined; however, iron- and zinc-dependent expression strongly suggests that cluster 3 is highly expressed in the infective process, and that the cluster contributes to the antigenic profile during the course of infection. Moreover, cluster 3 induction in acid stress conditions strengthens the hypothesis that cluster 3 is expressed in the course of infection.In M. smegmatis, the expression of msmeg0615 and msmeg0620 genes is broadly similar in differing growth phases and in stress conditions, with the exception of acid stress (pH 4.2). Differences in expression between cluster 3 genes can be explained by the presence of internal promoters, both in M. smegmatis and M. tuberculosis.
The effects of fish farming on bacterial density, biomass, community structure and their possible link to antibiotic resistance have been investigated in coastal sediments of the Ligurian Sea ...(Western Mediterranean). The top 2 cm of the sediment under a fish farm and control areas were analysed during summer 2000. Bacterial density and biomass were three folds higher (up to 3×10
10 cells/g and 2602.7 μC/g) (ANOVA,
p<0,01) in sediments beneath the fish cages compared to the control sediments. The number of Colony Forming Units (CFUs) for culturable bacteria and microbiological investigations indicated a shift in the relative importance of the Gram-negative bacteria in impacted sediments. Gram-positive bacteria increased their relevance in the control site where they represented up to 90% of total isolates.
Antibiotic sensitivity tests showed a high percentage of resistant strains in both control and impacted sediments, which indicates a widespread antibiotic resistance within bacterial populations in areas surrounding fish farms. A high frequency of antibiotic resistance was observed for ampicillin (AMP) in impacted sediments (ANOVA,
p<0.05). Gram-negative bacteria displayed the highest resistance to ampicillin, and streptomycin (STR) (ANOVA,
p<0.05) and the shift in the structure of microbial assemblage was apparently related to the presence of Gram-negative resistant strains in fish-farm sediments. The incidence of multiple resistance patterns in bacterial isolates was also greater in impacted sediments and the presence of
Bacillus strains producing antimicrobial compounds may be related to the high level of drug resistance.
This study highlighted a major change occurring in the structure of the benthic bacterial community most probably due to fish farming and its close association with antibiotic resistance patterns.
Background: Chronic low-grade inflammation of the visceral adipose tissue (VAT) in obesity leads to insulin resistance (IR) and type 2 diabetes (T2D). Growing data from preclinical models of obesity ...indicate that, in the cascade of events leading to T2D, T cells are engaged early and precede macrophage recruitment in the VAT. It is yet to be explored, however, why only a fraction of obese patients fails to control glucose metabolism resulting in the development of T2D. Here we aimed to assess profile and spatial dynamics of T cells localized in the VAT of obese patients with T2D. Methods: Characterization of T cell subsets in the stromal vascular fraction (SVF) obtained from VAT of obese patients with prediabe- tes or T2D (OB-PreD&D) and without T2D (OB-ND) undergoing bar- iatric surgery was performed using flow cytometry. RNA and T cell receptor (TCR) sequencing of VAT-derived PD-1+ CD4 conventional T cells (Tconv) was performed in comparison with the PD-1- counter- part. TCR sequencing was also performed on lean donors (LC) as well as peripheral blood samples. Results: Unsupervised and supervised analysis of flow cytometry data showed that a subset of resident CD4 Tconv cells expressing PD-1 is reduced in the VAT of OB-PreD&D compared to OB-ND. Increased frequency of TNF-a-producing PD-1+, but not PD-1- , CD4 Tconv was evident in OB-PreD&D. In line with this, transcriptomic analy- sis showed selective upregulation of pro-i nflammatory pathways in PD-1+ CD4 Tconv from OB-PreD&D. Furthermore, clonotypes of PD1+ CD4 T cells from OB-PreD&D tend to recirculate between PB and VAT and their clonality correlates with the level of IR. Conclusions: These data indicate that impairment of glucose metab- olism fosters the generation of pro-inflammatory PD1+ CD4 Tconv in the VAT with high potential to recirculate between blood and tissue(s). The PD1+ CD4 Tconv cell subset has the potential to be the mediator of an "infectious inflammatory state" that drives IR in insulin-sensitive tissues.
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