The Orphan Response Regulator Rv3143 Modulates the Activity of the NADH Dehydrogenase Complex (Nuo) in Mycobacterium tuberculosis via Protein-Protein Interactions.

Two-component signal transduction systems enable mycobacterial cells to quickly adapt and adequately respond to adverse environmental conditions encountered at various stages of host infection. We attempted to determine the role of the Rv3143 "orphan" response regulator in the physiology of Mycobacterium tuberculosis and its orthologue Msmeg_2064 in Mycobacterium smegmatis. We identified the Rv3143 protein as an interaction partner for NuoD, a member of the type I NADH dehydrogenase complex involved in oxidative phosphorylation. The mutants Δrv3143 and Δmsmeg_2064 were engineered in M. tuberculosis and M. smegmatis cells, respectively. The Δmsmeg_2064 strain exhibited a significant reduction in growth and viability in the presence of reactive nitrogen species. The Rv3143-deficient strain was sensitive to valinomycin, which is known to reduce the electrochemical potential of the cell and overexpressed genes required for nitrate respiration. An increased level of reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) electron acceptor in Δrv3143 and Δmsmeg_2064 cells was also evident. The silencing of ndh expression using CRISPRi/dCas9 affected cell survival under limited oxygen conditions. Oxygen consumption during entry to hypoxia was most severely affected in the double-mutant Δmsmeg_2064 ndhCRISPRi/dCas9 . We propose that the regulatory protein Rv3143 is a component of the Nuo complex and modulates its activity.

Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis.

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen that can adapt to the various nutrients available during its life cycle. However, in the nutritionally stringent environment of the macrophage phagolysosome, Mtb relies mainly on cholesterol. In previous studies, we demonstrated that Mtb can accumulate and utilize cholesterol as the sole carbon source. However, a growing body of evidence suggests that a lipid-rich environment may have a much broader impact on the pathogenesis of Mtb infection than previously thought. Therefore, we applied high-resolution transcriptome profiling and the construction of various mutants to explore in detail the global effect of cholesterol on the tubercle bacillus metabolism. The results allow re-establishing the complete list of genes potentially involved in cholesterol breakdown. Moreover, we identified the modulatory effect of vitamin B12 on Mtb transcriptome and the novel function of cobalamin in cholesterol metabolite dissipation which explains the probable role of B12 in Mtb virulence. Finally, we demonstrate that a key role of cholesterol in mycobacterial metabolism is not only providing carbon and energy but involves also a transcriptome remodeling program that helps in developing tolerance to the unfavorable host cell environment far before specific stress-inducing phagosomal signals occur.

The NnaR orphan response regulator is essential for the utilization of nitrate and nitrite as sole nitrogen sources in mycobacteria.

Nitrogen is an essential component of biological molecules and an indispensable microelement required for the growth of cells. Nitrogen metabolism of Mycobacterium smegmatis is regulated by a number of transcription factors, with the glnR gene product playing a major role. Under nitrogen-depletion conditions, GlnR controls the expression of many genes involved in nitrogen assimilation, including the msmeg_0432 gene encoding NnaR, the homologue of a nitrite/nitrate transport regulator from Streptomyces coelicolor. In the present study, the role of NnaR in the nitrogen metabolism of M. smegmatis was evaluated. The ∆glnR and ∆nnaR mutant strains were generated and cultured under nitrogen-depletion conditions. Total RNA profiling was used to investigate the potential role of NnaR in the GlnR regulon under nitrogen-depletion and in nitrogen-rich media. We found that disruption of MSMEG_0432 affected the expression of genes involved in nitrite/nitrate uptake, and its removal rendered mycobacteria unable to assimilate nitrogen from those sources, leading to cell death. RNA-Seq results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assays (EMSAs). The ability of mutants to grow on various nitrogen sources was evaluated using the BIOLOG Phenotype screening platform and confirmed on minimal Sauton's medium containing various sources of nitrogen. The ∆glnR mutant was not able to convert nitrates to nitrites. Interestingly, NnaR required active GlnR to prevent nitrogen starvation, and both proteins cooperated in the regulation of gene expression associated with nitrate/nitrite assimilation. The ∆nnaR mutant was able to convert nitrates to nitrites, but it could not assimilate the products of this conversion. Importantly, NnaR was the key regulator of the expression of the truncated haemoglobin trHbN, which is required to improve the survival of bacteria under nitrosative stress.

PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics.

Two-component regulatory systems (TCSSs) are key regulatory elements responsible for the adaptation of bacteria to environmental stresses. A classical TCSS is typically comprised of a sensory histidine kinase and a corresponding response regulator. Here, we used homologous recombination to construct a Mycobacterium smegmatis mutant defective in the synthesis of cytosolic histidine kinase PdtaS (Msmeg_1918). The resulting ΔpdtaS mutant strain was tested in the Phenotype Microarray screening system, which allowed us to identify aminoglycoside antibiotic sensitivity, tetracyclines antibiotic resistance as well as membrane transport and respiration, as the main processes affected by removal of pdtaS. The antibiotic sensitivity profiles were confirmed by survival assessment and complementation studies. To gain insight into the molecular mechanisms responsible for the observed phenotype, we compared ribosomal RNA and protein profiles of the mutant and wild-type strains. We carried out Northern blotting and qRT-PCR to compare rRNA levels and analyzed ribosome sedimentation patterns of the wild-type and mutant strains on sucrose gradients. Isolated ribosomes were further used to estimate relative abundance of individual proteins in the ribosomal subunits using label free mass spectrometry analysis. Additionally, the ΔpdtaS mutant revealed lower activity of the respiratory chain as measured by the rate of TTC (triphenyltetrazolium chloride) reduction, while at the same time showing only insignificant changes in the uptake of aminoglycosides. We postulate that deficiency of PdtaS affects the oxidative respiration rates and ribosomal composition causing relevant changes to intrinsic resistance or susceptibility to antibiotics targeting ribosomes, which are commonly used to treat mycobacterial infections.

Anti-mycobacterial activity of thymine derivatives bearing boron clusters.

A series of novel thymine derivatives bearing lipophilic, electron-neutral 1,2-dicarba-closo-dodecaborane, 1,12-dicarba-closo-dodecaborane or hydrophilic 7,8-dicarba-nido-undecaborate anions were synthesized. Synthesis was performed via copper(I)-catalysed Huisgen-Meldal-Sharpless 1,3-dipolar cycloaddition of N(1)-propargylthymine or N(1),N(3)-bispropargylthymine to 1-(3-azidopropyl)-1,2-dicarba-closo-dodecaborane. The obtained compounds were tested in vitro against Mycobacterium tuberculosis thymidylate kinase (TMPKmt) and as inhibitors of mycobacteria growth in culture using both saprophytic Mycobacterium smegmatis (M. smegmatis) and pathogenic Mycobacterium tuberculosis (M. tuberculosis) strains. The most potent TMPKmt inhibitor in the series contained two negatively charged 7,8-dicarba-nido-undecaborate modifications at positions 1 and 3 of thymine (9) and exhibited a Ki value of 1.5 µM. The most potent inhibitors of mycobacteria growth was compound 5 with one electron-neutral 1,2-dicarba-closo-dodecaborane modification at position 1 of thymine, and compound 8 with two modifications, at position 1 and 3. Both compounds completely inhibited M. smegmatis proliferation at a concentration of 100 µg/mL (0.25 mM and 0.15 mM, respectively).

ChoD and HsdD can be dispensable for cholesterol degradation in mycobacteria.

Cholesterol degradation is achieved through a complex metabolic pathway that starts with the oxidation of the 17-alkyl side chain and the steroid ring system. In bacteria, the oxidation of the 3ß-hydroxyl group and isomerization of the resulting cholest-5-en-3-one to cholest-4-en-3-one is catalyzed by hydroxysteroid dehydrogenase (HsdD) or cholesterol oxidase (ChoD). Genes encoding both enzymes were annotated in both fast and slow growing mycobacteria, however the enzymatic activity was confirmed for HsdD, exclusively. Here, we used homologous recombination to engineer multiple mutants, and directly show that both ChoD and HsdD are dispensable for cholesterol degradation in fast-growing Mycobacterium smegmatis mc(2)155 and slow-growing Mycobacterium tuberculosis H37Rv strains. The mutants deffective in the synthesis of ChoD, HsdD or both enzymes were able to grow in minimal media supplemented with cholesterol as a sole source of carbon and energy. Multiple mutants, defective in synthesis of ChoD, HsdD and ketosteroid dehydrogenase (KstD), showed attenuated growth in minimal medium supplemented with cholesterol and accumulated cholesterol degradation intermediates: androstendion (AD) and 9-hydroxy androstendion (9OHAD).

Influence of cigarette smoking on endoscopic sinus surgery long-term outcomes.

OBJECTIVE: The purpose of this study was to explore the influence of smoking on long-term outcomes of endoscopic sinus surgery for chronic rhinosinusitis. METHODS: The study prospectively enrolled 274 patients at the Department of Otolaryngology of the Warsaw Medical University from 1993 to 2000. All patients were diagnosed with chronic rhinosinusitis and scheduled for the endoscopic sinus surgery. We evaluated subgroups of patients with respect to bronchial asthma, allergy, aspirin triad, gastro-esophageal reflux disease and nasal septal deviation. Patients were divided into smokers and non-smokers. Patient CT scan results were recorded according to the four-grade classification system by Kennedy. Patients were observed over a period between 2 to 9 years following the surgical intervention and had their surgery revised if the severity of symptoms were at the same level or worsened. RESULTS: Prior to endoscopic sinus surgery, 23% of smokers and 20% of non-smokers scored III or IV on the Kennedy Scale. The revision ESS was carried out in 27 patients. In this group there were 20% smokers and 7% non-smokers, with the difference being significant. There was no significant difference in the postoperative quality of life scale scores. CONCLUSIONS: The study shows that while smoking did not influence preoperative symptoms, smokers had worse postoperative outcomes.

AccD6, a key carboxyltransferase essential for mycolic acid synthesis in Mycobacterium tuberculosis, is dispensable in a nonpathogenic strain.

Acetyl coenzyme A carboxylase (ACC) is a key enzyme providing a substrate for mycolic acid biosynthesis. Although in vitro studies have demonstrated that the protein encoded by accD6 (Rv2247) may be a functional carboxyltransferase subunit of ACC in Mycobacterium tuberculosis, the in vivo function and regulation of accD6 in slow- and fast-growing mycobacteria remain elusive. Here, directed mutagenesis demonstrated that although accD6 is essential for M. tuberculosis, it can be deleted in Mycobacterium smegmatis without affecting its cell envelope integrity. Moreover, we showed that although it is part of the type II fatty acid synthase operon, the accD6 gene of M. tuberculosis, but not that of M. smegmatis, possesses its own additional promoter (P(acc)). The expression level of accD6(Mtb) placed only under the control of P(acc) is 10-fold lower than that in wild-type M. tuberculosis but is sufficient to sustain cell viability. Importantly, this limited expression level affects growth, mycolic acid content, and cell morphology. These results provide the first in vivo evidence for AccD6 as a key player in the mycolate biosynthesis of M. tuberculosis, implicating AccD6 as the essential ACC subunit in pathogenic mycobacteria and an excellent target for new antitubercular compounds. Our findings also highlight important differences in the mechanism of acetyl carboxylation between pathogenic and nonpathogenic mycobacterial species.

Mycobacterium tuberculosis is able to accumulate and utilize cholesterol.

It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.

The composition of cell wall skeleton and outermost lipids of Mycobacterium vaccae is modified by ethambutol treatment.

Ethambutol (EMB) is a first line drug in tuberculosis treatment inhibiting the biosynthesis of arabinogalactan, which is a component of the mycobacterial cell wall. The growth of Mycobacterium vaccae cells in the presence of EMB increases cell wall permeability, which was monitored by beta-sitosterol biotransformation. GC/MS and GLC/MS (gas chromatography/mass spectrometry) analysis revealed dramatic changes in the content of covalently bound mycolic acids and in molar ratio galactose (Gal) to arabinose (Ara) in the cell envelopes of EMB-treated cells. The detected variations in the compositions of fatty acids indicate that both the cell wall skeleton and outer layer (free lipids) are decomposed due to EMB treatment.

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis.

Bacterial chromosomes (though not Escherichia coli and some other gamma-proteobacterial chromosomes) contain parS sequences and parAB genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial parABS elements were characterized for the first time. parAB genes are not essential in Mycobacterium smegmatis; however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of parB also leads to a rather severe chromosome segregation defect: up to 10% of the cells were anucleate. Mycobacterial ParB protein uses three oriC-proximal parS sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB-ParB interactions and is assisted by ParA protein.

Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis.

Recent reports have indicated that cholesterol plays a crucial role during the uptake of mycobacteria by macrophages. However, the significance of cholesterol modification enzymes encoded by Mycobacterium tuberculosis for bacterial pathogenicity remains unknown. Here, the authors explored whether the well-known cholesterol modification enzyme, cholesterol oxidase (ChoD), is important for virulence of the tubercle bacillus. Homologous recombination was used to replace the choD gene from the M. tuberculosis genome with a nonfunctional copy. The resultant mutant (delta choD) was attenuated in peritoneal macrophages. No attenuation in macrophages was observed when the same strain was complemented with an intact choD gene controlled by a heat shock promoter (delta choDP(hsp)choD). The mice infection experiments confirm the significance of ChoD in the pathogenesis of M. tuberculosis.

Evaluation of NAD(+) -dependent DNA ligase of mycobacteria as a potential target for antibiotics.

Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD(+)-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD(+)-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD(+)-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD(+)-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.

The effect of ethambutol on mycobacterial cell wall permeability to hydrophobic compounds.

Ethambutol (EMB), the first line drug in the treatment of tuberculosis, is an inhibitor of the biosynthesis of the cell wall compound - arabinogalactan. It was found that EMB at sub-inhibitory concentration increases the permeability of the M. vaccae cell wall, which was monitored by cell sensitization to erythromycin and rifampicin. The high permeability of the cell wall to hydrophobic compounds allows enhanced intracellular bioconversion of beta-sitosterol to 4-androsten-3,17-dione (AD) and 1,4-androstadien-3,17-dione (ADD).

Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis.

The catabolic potential for sterol degradation of fast-growing mycobacteria is well known. However, no genes or enzymes responsible for the steroid degradation process have been identified as yet in these species. One of the key enzymes required for degradation of the steroid ring structure is 3-ketosteroid Delta(1)-dehydrogenase (KsdD). The recent annotation of the Mycobacterium smegmatis genome (TIGR database) revealed six KsdD homologues. Targeted disruption of the MSMEG5898 (ksdD-1) gene, but not the MSMEG4855 (ksdD-2) gene, resulted in partial inactivation of the cholesterol degradation pathway and accumulation of the intermediate 4-androstene-3,17-dione. This effect was reversible by the introduction of the wild-type ksdD-1 gene into M. smegmatis DeltaksdD-1 or overexpression of ksdD-2. The data indicate that KsdD1 is the main KsdD in M. smegmatis, but that KsdD2 is able to perform the cholesterol degradation process when overproduced.

[Acute invasive fungal rhinosinusitis--case report]. / Grzybica inwazyjna zatok przynosowych o piorunujacym przebiegu--opis przypadku.

A case report of acute invasive fungal rhinosinusitis in 28 year old woman with acute myeloid leukemia is described in this paper. The diagnosis of the fungal disease was based on clinical presentation, endoscopic evaluation of nasal cavity, computed tomography and magnetic resonance imaging of the paranasal sinuses and histopathological findings. An aggressive treatment including antifungal therapy (amphotericin B), antibiotics and the surgery of paranasal sinuses was implemented. Unfortunately the underlying disease and the fungal invasion progressed rapidly and the patient died on the forth week post-op due to cardiorespiratory failure.

Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds.

Polycations [protamine, polymyxin B nonapeptide (PMBN) and polyethyleneimine (PEI)] have been shown to increase the cell wall permeability of Mycobacterium vaccae to highly hydrophobic compounds, as manifested in enhanced intracellular bioconversion of beta-sitosterol to 4-androsten-3,17-dione (AD) and 1,4-androstadien-3,17-dione (ADD), and cell sensitization to erythromycin and rifampicin. The quantity of AD(D) formed per biomass unit was twice as high in the presence of PMBN and PEI, and three times higher with protamine. The sensitization factor, i.e. the MIC(50) ratio of the control bacteria to those exposed to polycations, ranged from 4 to 16, depending on the polycation/antibiotic combination. Non-covalently bound free lipids were extracted from the control and polycation-treated cells and fractionated with the use of chloroform, acetone and methanol. Chloroform- and acetone-eluted fractions (mainly neutral lipids and glycolipids, respectively) showed significant polycation-induced alterations in their quantitative and qualitative composition. The fatty acid profile of neutral lipids was reduced in comparison to control, whereas acetone-derived lipids were characterized by a much higher level of octadecenoic acid (C(18:1)) and a considerably lower content of docosanoic acid (C(22:0)), the marker compound of mycolate-containing glycolipids. Methanol-eluted fractions remained unaltered. Cell-wall-linked mycolates obtained from delipidated cells were apparently unaffected by the action of polycations, as judged from the TLC pattern of mycolic acid subclasses, the mean weight of mycolate preparations and the C(22:0) acid content in the mycolates, determined by GC/MS and pyrolysis GC. The results suggest the involvement of the components of non-covalently bound lipids in the outer layer in the M. vaccae permeability barrier.