From Antagonism to Enhancement: Triton X-100 Surfactant Affects Phenanthrene Interfacial Biodegradation by Mycobacteria through a Shift in Uptake Mechanisms.

Polycyclic aromatic hydrocarbons (PAHs), as persistent environmental pollutants, often reside in nonaqueous-phase liquids (NAPLs). Mycobacterium sp. WY10, boasting highly hydrophobic surfaces, can adsorb to the oil-water interface, stabilizing the Pickering emulsion and directly accessing PAHs for biodegradation. We investigated the impact of Triton X-100 (TX100) on this interfacial uptake of phenanthrene (PHE) by Mycobacteria, using n-tetradecane (TET) and bis-(2-ethylhexyl) phthalate (DEHP) as NAPLs. Interfacial tension, phase behavior, and emulsion stability studies, alongside confocal laser scanning microscopy and electron microscope observations, unveiled the intricate interplay. In surfactant-free systems, Mycobacteria formed stable W/O Pickering emulsions, directly degrading PHE within the NAPLs because of their intimate contact. Introducing low-dose TX100 disrupted this relationship. Preferentially binding to the cells, the surfactant drastically increased the cell hydrophobicity, triggering desorption from the interface and phase separation. Consequently, PAH degradation plummeted due to hindered NAPL access. Higher TX100 concentrations flipped the script, creating surfactant-stabilized O/W emulsions devoid of interfacial cells. Surprisingly, PAH degradation remained efficient. This paradox can be attributed to NAPL emulsification, driven by the surfactant, which enhanced mass transfer and brought the substrate closer to the cells, despite their absence at the interface. This study sheds light on the complex effect of surfactants on Mycobacteria and PAH uptake, revealing an antagonistic effect at low concentrations that ultimately leads to enhanced degradation through emulsification at higher doses. These findings offer valuable insights into optimizing bioremediation strategies in PAH-contaminated environments.

Transporters Involved in the Biogenesis and Functionalization of the Mycobacterial Cell Envelope.

The biology of mycobacteria is dominated by a complex cell envelope of unique composition and structure and of exceptionally low permeability. This cell envelope is the basis of many of the pathogenic features of mycobacteria and the site of susceptibility and resistance to many antibiotics and host defense mechanisms. This review is focused on the transporters that assemble and functionalize this complex structure. It highlights both the progress and the limits of our understanding of how (lipo)polysaccharides, (glyco)lipids, and other bacterial secretion products are translocated across the different layers of the cell envelope to their final extra-cytoplasmic location. It further describes some of the unique strategies evolved by mycobacteria to import nutrients and other products through this highly impermeable barrier.

Seeing and Touching the Mycomembrane at the Nanoscale.

Mycobacteria have unique cell envelopes, surface properties, and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate, and resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometer-scale ultrastructural, adhesive, and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unraveled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope, and cellular processes leading to division. The unique capabilities of AFM, which include subdiffraction-limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties, and growth dynamics of this important class of pathogens.

Metabolic labeling probes for interrogation of the host-pathogen interaction.

Bacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need for novel classes of antibiotic drugs. One particularly troublesome class of bacteria are those that have evolved highly efficacious mechanisms for surviving inside the host. These contribute to their virulence by immune evasion, and make them harder to treat with antibiotics due to their residence inside intracellular membrane-limited compartments. This has sparked the development of new chemical reporter molecules and bioorthogonal probes that can be metabolically incorporated into bacteria to provide insights into their activity status. In this review, we provide an overview of several classes of metabolic labeling probes capable of targeting either the peptidoglycan cell wall, the mycomembrane of mycobacteria and corynebacteria, or specific bacterial proteins. In addition, we highlight several important insights that have been made using these metabolic labeling probes.

Investigating Bacterial Volatilome for the Classification and Identification of Mycobacterial Species by HS-SPME-GC-MS and Machine Learning.

Species of Mycobacteriaceae cause disease in animals and humans, including tuberculosis and leprosy. Individuals infected with organisms in the Mycobacterium tuberculosis complex (MTBC) or non-tuberculous mycobacteria (NTM) may present identical symptoms, however the treatment for each can be different. Although the NTM infection is considered less vital due to the chronicity of the disease and the infrequency of occurrence in healthy populations, diagnosis and differentiation among Mycobacterium species currently require culture isolation, which can take several weeks. The use of volatile organic compounds (VOCs) is a promising approach for species identification and in recent years has shown promise for use in the rapid analysis of both in vitro cultures as well as ex vivo diagnosis using breath or sputum. The aim of this contribution is to analyze VOCs in the culture headspace of seven different species of mycobacteria and to define the volatilome profiles that are discriminant for each species. For the pre-concentration of VOCs, solid-phase micro-extraction (SPME) was employed and samples were subsequently analyzed using gas chromatography-quadrupole mass spectrometry (GC-qMS). A machine learning approach was applied for the selection of the 13 discriminatory features, which might represent clinically translatable bacterial biomarkers.

The ambruticins and jerangolids - chemistry, biology and chemoenzymatic synthesis of potent antifungal drug candidates.

Covering: 1977 to 2020The ambruticins and jerangolids are myxobacterial reduced polyketides, which are produced via highly unusual biosynthetic pathways containing a plethora of non-canonical enzymatic transformations. Since the discovery of the first congeners in the late 1970s, they have been in the focus of drug development due to their good antifungal activity and low toxicity in mammals, which result from interaction with an unusual innercellular target in fungi. Despite significant efforts, which have led to the development of various total syntheses, their structural complexity has yet avoided full exploitation of their pharmacological potential. This article summarises biological, total and semisynthetic as well as biosynthetic studies on both compounds. An outlook on the biosynthesis-based approaches to them and their derivatives is presented. Due to the structural and biosynthetic characteristics of the ambruticins and jerangolids, chemoenzymatic processes that make use of their biosynthetic pathway enzymes are particularly promising to gain efficient access to derivative libraries for structure activity relationship studies.

Revisiting intrinsic transcription termination in mycobacteria: U-tract downstream of secondary structure is dispensable for termination.

In Escherichia coli, G/C-rich hairpin structure followed by a U-tract in the 3' region of the nascent RNA are crucial determinants for intrinsic or factor independent transcription termination. In mycobacteria, there is a scarcity of such intrinsic terminators. However, secondary structures having G/C-rich stem devoid of any U's or with suboptimal U-tracts were identified earlier as terminators and found to be functional both in vitro and in vivo. Two different observations - that a mycobacterial RNA polymerase (RNAP) does not function at intrinsic terminators devoid of U-tracts and the identification of an altogether new motif for termination in mycobacteria necessitated re-examining a number of putative terminators for their function as terminators. When these in silico identified non-canonical terminators were subjected to experimental validation, they were found to dissociate RNA from the elongating RNAP. Termination is observed when the U-tracts were reduced, or totally absent both in vitro and in vivo. Our results, thus indicate that the presence of U-tract following the G/C-rich stem in an intrinsic terminator may not be an essential determinant for transcription termination in mycobacteria.

Immune Recognition of Pathogen-Derived Glycolipids Through Mincle.

Mincle (macrophage inducible C-type lectin, Clec4e, Clecsf9) was originally identified as a member of the C-type lectin receptor family in 1999. Then, the function of Mincle to control antifungal immunity by binding to Candida albicans was reported in 2008. Around the same time, it was reported that Mincle recognized damaged cells and induced sterile inflammation by coupling with the ITAM-adaptor molecule FcRγ. In the following year, a breakthrough discovery reported that Mincle was an essential receptor for mycobacterial cord factor (trehalose-6,6'-dimycolate, TDM). Mincle gained increasing attention immediately after this critical finding. Although our understanding of the recognition of Mycobacteria has been advanced significantly, it was also revealed that Mincle interacts with pathogens other than Mycobacteria. In addition, endogenous ligands of Mincle were identified recently. Therefore, Mincle is now considered a danger receptor both for self and non-self ligands, so-called damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). This chapter will give an overview of the accumulated knowledge of the multi-task danger receptor Mincle from its discovery to the latest findings.

Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii.

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.

Induction of tumor-specific CD8+ cytotoxic T lymphocytes from naïve human T cells by using Mycobacterium-derived mycolic acid and lipoarabinomannan-stimulated dendritic cells.

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.

Simultaneous isolation and label-free identification of bacteria using contactless dielectrophoresis and Raman spectroscopy.

The traditional bacterial identification method of growing colonies on agar plates can take several days to weeks to complete depending on the growth rate of the bacteria. Successfully decreasing this analysis time requires cell isolation followed by identification. One way to decrease analysis time is by combining dielectrophoresis (DEP), a common technique used for cell sorting and isolation, and Raman spectroscopy for cell identification. DEP-Raman devices have been used for bacterial analysis, however, these devices have a number of drawbacks including sample heating, cell-to-electrode proximity that limits throughput and separation efficiency, electrode fouling, or inability to address sample debris. Presented here is a contactless DEP-Raman device to simultaneously isolate and identify particles from a mixed sample while avoiding common drawbacks associated with other DEP designs. Using the device, a mixed sample of bacteria and 3 µm polystyrene spheres were isolated from each other and a Raman spectrum of the trapped bacteria was acquired, indicating the potential for cDEP-Raman devices to decrease the analysis time of bacteria.

Synthesis of a Di-Mycoloyl Tri-Arabinofuranosyl Glycerol Fragment of the Mycobacterial Cell Wall, Based on Synthetic Mycolic Acids.

Fragments of mycobacterial cell walls such as arabinoglycerol mycolate and dimycoloyl diarabinoglycerol, comprising complex mixtures of mycolic acids, have immunostimulatory and antigenic properties. A related di-mycoloyl tri-arabinofuranosyl glycerol fragment has been isolated from cell wall hydrolysates. An effective stereoselective synthesis of tri-arabinofuranosyl glycerol, followed by coupling with stereochemically defined mycolic acids of different structural classes, to provide unique di-mycoloyl tri-arabinofuranosyl glycerols is now described.

Structural Characterization of the Hydratase-Aldolases, NahE and PhdJ: Implications for the Specificity, Catalysis, and N-Acetylneuraminate Lyase Subgroup of the Aldolase Superfamily.

NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might differentiate them from other NAL subgroup members. The individual specificities of NahE and PhdJ are governed by Asn-281/Glu-285 and Ser-278/Asp-282, respectively. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.

Performance of Vitek MS v3.0 for Identification of Mycobacterium Species from Patient Samples by Use of Automated Liquid Medium Systems.

The accuracy and robustness of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system was evaluated by identifying mycobacteria from automated liquid-medium systems using patient samples. This is the first report to demonstrate that proteins within the liquid medium, its supplements, and decontamination reagents for nonsterile patient samples do not generate misidentification or false-positive results by use of the Vitek MS v3.0 system. Prior to testing with patient samples, a seeded-culture study was conducted to challenge the accuracy of the Vitek MS system at identifying mycobacteria from liquid medium by mimicking a clinical workflow. Seventy-seven Mycobacterium strains representing 21 species, seeded in simulated sputum, were decontaminated, inoculated into BacT/Alert MP liquid culture medium, incubated until positivity, and identified using the Vitek MS system. A total of 383 liquid cultures were tested, of which 379 (99%) were identified correctly to the species/complex/group level, 4 (1%) gave a "no-identification" result, and no misidentifications were observed. Following the simulated-sputum study, a total of 73 smear-positive liquid-medium cultures detected using BD BBL MGIT and VersaTREK Myco liquid media were identified by the Vitek MS system. Sixty-four cultures (87.7%) were correctly identified to the species/complex/group level; 7 (9.6%) resulted in no identification; and 2 (2.7%) were misidentified at the species level. These results indicate that the Vitek MS v3.0 system is an accurate tool for routine diagnostics of Mycobacterium species isolated from liquid cultures.

Investigation of mycobacteria fatty acid profile using different ionization energies in GC-MS.

Gas chromatography (GC) coupled with electron ionization (EI) mass spectrometry (MS) is a well-established technique for the analysis of volatile and semi-volatile compounds. The main advantage is the highly repeatable fragmentation of the compounds into the ion source, generating intense and diagnostic fragmentation when the ionization is performed at 70 eV; this is considered the standard ionization condition and has been used for creating many established databases, which are of great support in the analyte identification process. However, such an intense fragmentation often causes the loss of the molecular ion or more diagnostic ions, which can be detrimental for the identification of homologous series or isomers, as for instance fatty acids. To obtain this information chemical or soft ionization can be used, but dedicated ion sources and conditions are required. In this work, we explored different ionization voltages in GC-EI-MS to preserve the intensity of the molecular ion using a conventional quadrupole MS. Twenty, 30, 50, and 70 eV were tested using a mixture of fatty acid methyl esters standards. Intensity and repeatability of the most informative ions were compared. Twenty and 70 eV were then used to analyze the fatty acid composition of six different strains of mycobacteria. Two approaches were used for elaborating the data: (1) a single average spectrum of the entire chromatogram was derived, which can be considered (in terms of concept) as a direct EI-MS analysis; (2) the actual chromatographic separation of the compounds was considered after automatic alignment. The results obtained are discussed herein. Graphical abstract ᅟ.

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD.

The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin- and flavodoxin reductase-based reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.

Insight into Structure-Function Relationships and Inhibition of the Fatty Acyl-AMP Ligase (FadD32) Orthologs from Mycobacteria.

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is one of the targets of first-line antituberculous drugs. This pathway contains a number of potential targets, including some that have been identified only recently and have yet to be explored. One such target, FadD32, is required for activation of the long meromycolic chain and is essential for mycobacterial growth. We report here an in-depth biochemical, biophysical, and structural characterization of four FadD32 orthologs, including the very homologous enzymes fromMycobacterium tuberculosisandMycobacterium marinum Determination of the structures of two complexes with alkyl adenylate inhibitors has provided direct information, with unprecedented detail, about the active site of the enzyme and the associated hydrophobic tunnel, shedding new light on structure-function relationships and inhibition mechanisms by alkyl adenylates and diarylated coumarins. This work should pave the way for the rational design of inhibitors of FadD32, a highly promising drug target.

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media.

During the last decade, many investigators have studied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification of mycobacteria. Diverse and contradictory results indicated that optimal level for routine testing has not been reached yet. This work aimed to assess Vitek MS through two distinct versions, Saramis v4.12 RUO and the IVD v3.0, under conditions close to routine laboratory practice. Overall, 111 mycobacterial isolates were subjected to protein extraction and same spectra were matched against both databases. The IVD v3.0 database proved to be superior to Saramis v4.12 and its identification rates remarkably increased, from 67% to 94% for isolates grown on Middlebrook 7H10 solid medium and from 62% to 91% for isolates grown on mycobacterial growth indicator tube (MGIT) liquid medium. With this new version, IVD v3.0, MALDI-TOF MS might be integrated into routine clinical diagnostics, although molecular techniques remain mandatory in some cases.

Bi-PSSM: Position specific scoring matrix based intelligent computational model for identification of mycobacterial membrane proteins.

Mycobacterium is a pathogenic bacterium, which is a causative agent of tuberculosis (TB) and leprosy. These diseases are very crucial and become the cause of death of millions of people every year in the world. So, the characterize structure of membrane proteins of the protozoan play a vital role in the field of drug discovery because, without any knowledge about this Mycobacterium's membrane protein and their types, the scientists are unable to treat this pathogenic protozoan. So, an accurate and competitive computational model is needed to characterize this uncharacterized structure of mycobacterium. Series of attempts were carried out in this connection. Split amino acid compositions, Unbiased-Dipeptide peptide compositions (Unb-DPC), Over-represented tri-peptide compositions, compositions & translation were the few recent encoding techniques followed by different researchers in their publications. Although considerable results have been achieved by these models, still there is a gap which is filled in this study. In this study, an evolutionary feature extraction technique position specific scoring matrix (PSSM) is applied in order to extract evolutionary information from protein sequences. Consequently, 99.6% accuracy was achieved by the learning algorithms. The experimental results demonstrated that the proposed computational model will lead to develop a powerful tool for anti-mycobacterium drugs as well as play a promising rule in proteomic and bioinformatics.

A universal stress protein (USP) in mycobacteria binds cAMP.

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.