Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB, and its role in benzothiazinone resistance.

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-ß-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.

Transcriptional analysis of ESAT-6 cluster 3 in Mycobacterium smegmatis.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Genomic analysis of zinc homeostasis in Mycobacterium tuberculosis.

Zn2+ is involved in several cellular processes and the maintenance of cellular Zn2+ status is essential for life. Therefore, an improved understanding of zinc acquisition and metabolism is of great significance, especially in important pathogens such as Mycobacterium tuberculosis whose capacity to survive within the phagosomal compartment is fundamental for its pathogenicity. A crucial point is the bacterial ability to compete with the host for nutrients, and the acquisition of metal ions, such as iron and zinc.

A naturalistic study on the relationship among resilient factors, psychiatric symptoms, and psychosocial functioning in a sample of residential patients with psychosis.

OBJECTIVE: Resilience is a multidimensional process of adaptation aimed to overcome stressful or traumatic life experiences; only in the last few years it has been considered as a personal resource in psychosis and schizophrenia. This study aimed to assess the relationship between intrapersonal and interpersonal resilience factors and schizophrenia, particularly whether and how resilience can improve the course of psychotic illness. PATIENTS AND METHODS: In this observational study, all patients recruited had to fulfill the following inclusion criteria: diagnosis of schizophrenia spectrum disorder (Diagnostic and Statistical Manual of Mental Disorders-5); aged between 18 and 65 years; provided written informed consent; to be clinically stable (Clinical Global Impression Scale <3); history of illness ≥5 years; to be compliant with antipsychotic therapy over the last year; and regular submission to periodic monthly psychiatric visits. Patients were evaluated through the following scales: Resilience Scale for Adults (RSA) for resilience; Brief Psychiatric Rating Scale-Anchored version (BPRS-A), Scale for the Assessment of Negative Symptoms (SANS), and Scale for the Assessment of Positive Symptoms (SAPS) for psychotic symptomatology; and Life Skills Profile (LSP) for psychosocial functioning. Statistical analysis was performed by SPSS. Partial correlations were evaluated to assess the relationship between RSA total scores and subscores and BPRS-A, SANS, SAPS, and LSP total scores, removing the common variance among variables. Then, a series of hierarchical multiple linear regression models were used to examine the association between resilience, psychopathology, and psychosocial functioning. RESULTS: A statistically significant negative correlation among intrapersonal resilience factors and BPRS-A total score emerged, predicting psychiatric symptoms severity and explaining approximately 31% of the BPRS-A variance; otherwise, only the interpersonal resilience factors associated with social support were statistically and positively correlated with LSP total score, predicting psychosocial functioning and explaining the 11% of LSP variance. CONCLUSION: The specific contribution that resilience factors may have in predicting the severity of symptoms and the extent of psychosocial functioning emphasizes the importance of personalizing treatment for patients affected by schizophrenia, promoting personal resources, and translating them into better outcomes.

The Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc.

The Mycobacterium tuberculosis genome encodes two ferric uptake regulator homologues, furA and furB, the function of which is under investigation. Using Mycobacterium smegmatis as a model system, we investigated the transcriptional pattern of Rv(Ms)2358-furB genes. Transcripts covering the two genes could be identified by northern blotting and by reverse transcriptase PCR. The transcriptional start point was mapped at one base upstream of the Ms2358 start codon by the RACE technique. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of one promoter, located immediately upstream of the Rv(Ms)2358 gene. Promoter induction was tested on several cultures grown under different conditions of pH and temperature, and in the presence of different concentrations of metallic ions. The promoter was found to be specifically induced by zinc. The recombinant M. tuberculosis FurB protein typically contained two zinc ions per protein monomer. Complete removal of zinc could not be obtained, even with strong denaturation treatment. Our data are in favour of the hypothesis that Rv2358 and FurB are transcriptional regulators involved in zinc homeostasis.

Azole resistance in Mycobacterium tuberculosis is mediated by the MmpS5-MmpL5 efflux system.

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.

Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

Crystal structure and function of the zinc uptake regulator FurB from Mycobacterium tuberculosis.

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 2.7A, 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.

Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.

Rv2358 and FurB: two transcriptional regulators from Mycobacterium tuberculosis which respond to zinc.

In a previous work, we demonstrated that the Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc. In this study, the orthologous genes from Mycobacterium smegmatis mc(2)155 were inactivated and mutants analyzed. Rv2358 protein was purified and found to bind upstream of the Rv2358 gene. Binding was inhibited by Zn(2+) ions.

Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase.

The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real M. tuberculosis NAD(+) synthetase. These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.

Mycobacterium tuberculosis FurA autoregulates its own expression.

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.