Protease domain and transmembrane domain of the type VII secretion mycosin protease determine system-specific functioning in mycobacteria.

Mycobacteria use type VII secretion systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to ESX-5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, mycosin protease (MycP), is not part of the core complex but is essential for secretion, as it stabilizes this membrane complex. Here we investigated which MycP domains are required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane domain are required for the ESX system-specific function of mycosins. In addition, we observed that the transmembrane domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in interaction with and stabilization of their respective ESX complexes.

Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose.

The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.

Inhibitory Potential of Antifungal Drugs on ATP-Binding Cassette Transporters P-Glycoprotein, MRP1 to MRP5, BCRP, and BSEP.

Inhibition of ABC transporters is a common mechanism underlying drug-drug interactions (DDIs). We determined the inhibitory potential of antifungal drugs currently used for invasive fungal infections on ABC transporters P-glycoprotein (P-gp), MRP1 to MRP5, BCRP, and BSEP in vitro Membrane vesicles isolated from transporter-overexpressing HEK 293 cells were used to investigate the inhibitory potential of antifungal drugs (250 µM) on transport of model substrates. Concentration-inhibition curves were determined if transport inhibition was >60%. Fifty percent inhibitory concentrations (IC50s) for P-gp and BCRP were both 2 µM for itraconazole, 5 and 12 µM for hydroxyitraconazole, 3 and 6 µM for posaconazole, and 3 and 11 µM for isavuconazole, respectively. BSEP was strongly inhibited by itraconazole and hydroxyitraconazole (3 and 17 µM, respectively). Fluconazole and voriconazole did not inhibit any transport for >60%. Micafungin uniquely inhibited all transporters, with strong inhibition of MRP4 (4 µM). Anidulafungin and caspofungin showed strong inhibition of BCRP (7 and 6 µM, respectively). Amphotericin B only weakly inhibited BCRP-mediated transport (127 µM). Despite their wide range of DDIs, azole antifungals exhibit selective inhibition on efflux transporters. Although echinocandins display low potential for clinically relevant DDIs, they demonstrate potent in vitro inhibitory activity. This suggests that inhibition of ABC transporters plays a crucial role in the inexplicable (non-cytochrome P450-mediated) DDIs with antifungal drugs.

A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins.

Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90ß, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.

The HtrA-like protease CD3284 modulates virulence of Clostridium difficile.

In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that the C. difficile 630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its role in vivo and in vitro through the creation of an isogenic ClosTron-based htrA mutant of C. difficile strain 630Δerm (wild type). In contrast to the attenuated phenotype seen with htrA deletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acute C. difficile infection. Microarray data analysis showed a pleiotropic effect of htrA on the transcriptome of C. difficile, including upregulation of the toxin A gene. In addition, the htrA mutant showed reduced spore formation and adherence to colonic cells. Together, our data show that htrA can modulate virulence in C. difficile.

A Chimeric EccB-MycP Fusion Protein is Functional and a Stable Component of the ESX-5 Type VII Secretion System Membrane Complex.

The mycosin protease (MycP) is widely conserved in type VII secretion (T7S) systems throughout Actinobacteria. Within the T7S systems of mycobacteria, also known as the ESX systems, MycP is essential for secretion, which is probably linked to its stabilizing effect on the ESX membrane complex. However, it is unknown how this is mediated, as MycP is not a stable component of this complex. In this study, we set out to create a chimeric fusion protein of EccB5 and MycP5, based on a chimeric gene of eccB and mycP in the T7S locus of Bifidobacterium dentium. We show that this fusion protein is functional and capable of complementing ESX-5 secretion in both an eccB5 and a mycP5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB5 and mycP5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis, with the chimeric gene. The EccB5-MycP5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC5 showed that under these conditions, the EccB5-MycP5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP5 carries out its essential function in secretion in close proximity to EccB5, indicating that EccB5 is the direct interaction partner of MycP5.

Protein Export into and across the Atypical Diderm Cell Envelope of Mycobacteria.

Mycobacteria, including the infamous pathogen Mycobacterium tuberculosis, are high-GC Gram-positive bacteria with a distinctive cell envelope. Although there is a typical inner membrane, the mycobacterial cell envelope is unusual in having its peptidoglycan layer connected to a polymer of arabinogalactan, which in turn is covalently attached to long-chain mycolic acids that help form a highly impermeable mycobacterial outer membrane. This complex double-membrane, or diderm, cell envelope imparts mycobacteria with unique requirements for protein export into and across the cell envelope for secretion into the extracellular environment. In this article, we review the four protein export pathways known to exist in mycobacteria: two conserved systems that exist in all types of bacteria (the Sec and Tat pathways) and two specialized systems that exist in mycobacteria, corynebacteria, and a subset of low-GC Gram-positive bacteria (the SecA2 and type VII secretion pathways). We describe the progress made over the past 15 years in understanding each of these mycobacterial export pathways, and we highlight the need for research to understand the specific steps of protein export across the mycobacterial outer membrane.

Mycosins Are Required for the Stabilization of the ESX-1 and ESX-5 Type VII Secretion Membrane Complexes.

Pathogenic mycobacteria contain up to five type VII secretion (T7S) systems, ESX-1 to ESX-5. One of the conserved T7S components is the serine protease mycosin (MycP). Strikingly, whereas MycP is essential for secretion, the protease activity of MycP1 in Mycobacterium tuberculosis has been shown to be dispensable for secretion. The essential role of MycP therefore remains unclear. Here we show that MycP1 and MycP5 of M. marinum have similar phenotypes, confirming that MycP has a second unknown function that is essential for its T7S system. To investigate whether this role is related to proper functioning of the T7S membrane complex, we first analyzed the composition of the ESX-1 membrane complex and showed that this complex consists of EccBCDE1, similarly to what was previously shown for ESX-5. Surprisingly, while mycosins are not an integral part of these purified core complexes, we noticed that the stability of both the ESX-1 complex and the ESX-5 complex is compromised in the absence of their MycP subunit. Additional interaction studies showed that, although mycosins are not part of the central ESX membrane complex, they loosely associate with this complex. We hypothesize that this MycP association with the core membrane complex is crucial for the integrity and functioning of the T7S machinery. IMPORTANCE: Among the major virulence factors of pathogenic mycobacteria are the type VII secretion (T7S) systems. Three of these systems, ESX-1, ESX-3, and ESX-5, have been shown to be crucial for virulence or viability. Here we describe the function of mycosin proteases, which are conserved components within these systems. We show that MycP1 and MycP5 have a second, proteolytic-independent function which is essential for the T7S system. We additionally found that this second essential role is related to the stabilization and proper functioning of their respective ESX membrane core complexes. Finally, we found that this is mediated by a loose association of MycP with the complex. Understanding the essential role of mycosins in type VII secretion systems, which play central roles in the virulence and viability of pathogenic mycobacteria, may provide new intervention strategies to treat tuberculosis.

Drug-interactions of azole antifungals with selected immunosuppressants in transplant patients: strategies for optimal management in clinical practice.

The management of drug-drug interactions (DDIs) between azole antifungals (fluconazole, itraconazole, posaconazole and voriconazole) and immunosuppressants (cyclosporine, tacrolimus, everolimus and sirolimus) in transplant patients remains challenging, as the impact of altered immunosuppressant concentrations puts the patient at high risk for either toxicity or transplant rejection. As a result, it is a complex task for the clinician to maintain immunosuppressant concentrations within the desired therapeutic range and this requires a highly individualized patient approach. We provide important tools for adequate assessment of the drug interactions that cause this pharmacokinetic variability of immunosuppressants. A stepwise approach for the evaluation and subsequent management options, including a decision flow chart, are provided for optimal handling of these clinically relevant DDIs.

Impact of special patient populations on the pharmacokinetics of echinocandins.

Echinocandins belong to the class of antifungal agents. Currently, three echinocandin drugs are licensed for intravenous treatment of invasive fungal infections: anidulafungin, caspofungin and micafungin. While their antifungal activity overlaps, there are substantial differences in pharmacokinetics (PK). Numerous factors may account for variability in PK of echinocandins including age (pediatrics vs adults), body surface area and body composition (normal weight vs obesity), disease status (e.g., critically ill and burn patients) and organ dysfunction (kidney and liver impairment). Subsequent effects of altered exposure might impact efficacy and safety. Knowledge of PK behavior is crucial in optimal clinical utilization of echinocandin in a specific patient or patient population. This review provides up-to-date information on PK data of anidulafungin, caspofungin and micafungin in special patient populations. Patient populations addressed are neonates, children and adolescents, obese patients, patients with hepatic or renal impairment, critically ill patients (including burn patients) and patients with hematological diseases.

Influence of stiripentol on cytochrome P450-mediated metabolic pathways in humans: in vitro and in vivo comparison and calculation of in vivo inhibition constants.

OBJECTIVE: The spectrum of cytochrome P450 inhibition of stiripentol, a new anticonvulsant, was characterized in vitro and in vivo. METHODS: Stiripentol was incubated in vitro with (R)-warfarin, coumarin, (S)-warfarin, (S)-mephenytoin, bufuralol, p-nitrophenol, and carbamazepine as probes for CYPs 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4, respectively. Caffeine demethylation and the 6 beta-hydroxycortisol/cortisol ratio were monitored in vivo before and after 14 days of treatment with stiripentol as measures of CYP1A2 and CYP3A4 activity, and dextromethorphan O- and N-demethylation were used to measure CYP2D6 and CYP3A4 activity, respectively. In vivo inhibition constants for CYP3A4 were calculated with use of data that previously documented the interaction between stripentol and carbamazepine. RESULTS: In vitro, stiripentol inhibited CYPs 1A2, 2C9, 2C19, 2D6, and 3A4, with inhibition constant values at or slightly higher than therapeutic (total) concentrations of stiripentol, but it did not inhibit CYPs 2A6 and 2E1 even at tenfold therapeutic concentrations. In vivo inhibition of caffeine demethylation and dextromethorphan N-demethylation were consistent with inhibition of CYP1A2 and CYP3A4, respectively. The 6 beta-hydroxycortisol/cortisol ratio did not provide a reliable index of CYP3A4 inhibition. Inhibition of CYP2D6-mediated O-demethylation was not observed in vivo. With use of carbamazepine, in vivo inhibition constants for CYP3A4 ranged between 12 and 35 mumol/L, whereas the corresponding in vitro value was 80 mumol/L. CONCLUSIONS: Stiripentol appears to inhibit several CYP450 enzymes in vitro and in vivo. In vivo inhibition constants show that stiripentol inhibition of CYP3A4 is linearly related to plasma concentration in patients with epilepsy.

Hypothesis: hyperstructures regulate bacterial structure and the cell cycle.

A myriad different constituents or elements (genes, proteins, lipids, ions, small molecules etc.) participate in numerous physico-chemical processes to create bacteria that can adapt to their environments to survive, grow and, via the cell cycle, reproduce. We explore the possibility that it is too difficult to explain cell cycle progression in terms of these elements and that an intermediate level of explanation is needed. This level is that of hyperstructures. A hyperstructure is large, has usually one particular function, and contains many elements. Non-equilibrium, or even dissipative, hyperstructures that, for example, assemble to transport and metabolize nutrients may comprise membrane domains of transporters plus cytoplasmic metabolons plus the genes that encode the hyperstructure's enzymes. The processes involved in the putative formation of hyperstructures include: metabolite-induced changes to protein affinities that result in metabolon formation, lipid-organizing forces that result in lateral and transverse asymmetries, post-translational modifications, equilibration of water structures that may alter distributions of other molecules, transertion, ion currents, emission of electromagnetic radiation and long range mechanical vibrations. Equilibrium hyperstructures may also exist such as topological arrays of DNA in the form of cholesteric liquid crystals. We present here the beginning of a picture of the bacterial cell in which hyperstructures form to maximize efficiency and in which the properties of hyperstructures drive the cell cycle.

Angiotensin-converting enzyme inhibition, anti-hypertensive activity and hemodynamic profile of trandolapril (RU 44570).

Trandolapril (RU 44570), a new non-sulfhydryl angiotensin-converting enzyme (ACE) inhibitor chemically related to enalapril, and its diacid (RU 44403), were investigated for their ability to inhibit angiotensin-converting enzyme. Trandolapril attenuated angiotensin I (Ang I)-induced pressor responses following i.v. administration to rats and dogs with ID50 values of 13.1 +/- 1.3 and 21.1 +/- 2.3 micrograms/kg. RU 44403 produced corresponding values of 9.9 +/- 0.7 and 7.2 +/- 2.3 micrograms/kg. Trandolapril (3-300 micrograms/kg) produced a dose-related attenuation of Ang I-induced pressor responses (ID50 30 micrograms/kg) following oral administration to rats. Oral administration of trandolapril (30-1000 micrograms/kg) to dogs inhibited Ang I pressor responses for over 6 h. The depressor action of bradykinin in the rat was potentiated by i.v. trandolapril and RU 44403 with ED50 values of 5.5 +/- 0.8 and 4.9 +/- 0.3 micrograms/kg respectively. Trandolapril was 2.3-10-fold more potent than enalapril in all experiments, depending on species or route of administration. RU 44403 and MK 422 were approximately equipotent, implying that trandolapril was more readily hydrolysed than enalapril. Trandolapril (0.3-30 mg/kg) produced dose-related, long-lasting (greater than 24 h) reductions in blood pressure (BP) in spontaneously hypertensive rats (SHR) following oral administration. The anti-hypertensive effect was potentiated significantly in hydrochlorothiazide-pretreated SHR when the plasma renin activity was increased. Enalapril was 10-fold less potent than trandolapril in reducing BP. The anti-hypertensive action of trandolapril (3 mg/kg) was abolished in SHR that were bilaterally nephrectomized 24 h beforehand, but was maintained in SHR pretreated by indomethacin (5 mg/kg p.o.). Trandolapril (1 mg/kg i.v.) produced a modest and transient reduction in BP in anesthetized dogs. Trandolapril produced dose-related (30-1000 micrograms/kg) reductions in BP, total peripheral resistance and heart work in dogs pretreated with hydrochlorothiazide to increase plasma renin activity.

Immobilized cycling enzyme active transport systems. pH feedback control.

Active transport can be induced by applying a pH gradient across a membrane containing a homogeneous mixture of two cycling enzymes. When the two reactions are inversely 'pH active', one producing protons and the other consuming them, a pH feedback control of the functional structure occurs and the active transport function of the membrane can be either stabilized or inhibited according to whether the endogenic pH modifications tend to enhance or reduce the exergonic pH gradient. When it is stabilized, the system looks like a thin active layer surrounded by two diffusive layers, leading to a fairly good model for biological transport systems. Under particular conditions, signals can be emitted.

pH feedback control of enzyme membranes.

pH feedback on immobilized enzymes is theoretically examined with respect to substrate and pH levels, strength of acids produced by the reaction, buffering and asymmetry of the system. All the productions of proton by the different reactions are taken into account by using a 'symbolic species' H. The system of differential diffusion-reaction equations is then integrated using numerical methods. The local 'effective enzyme activity' modulated by an acidity factor enables us to predict and quantify evolutions of the systems: NonMichaelian behavior of an immobilized Michaelis-Menten-type enzyme is shown, even when pH back-actions are excluded; the analysis of intramembrane pH profiles shows that the shift of the optimal pH is a complex function of the substrate and pH levels, the intrinsic pH dependence of the enzyme, and the membrane characteristics. This study may easily be transposed to other types of effector such as divalent cations and used in examining self-regulations of multienzyme systems where pH-active reactions are involved.

Monitoring of artificial enzyme membrane systems by electric currents. I. Analytical treatment with direct current and buffered conductivity.

Continuous electric fields (E) modify the transport flows and the intramembrane concentration profiles of protons or of ionic substrates or cofactors (inhibitors). These "mediators' induce variations in enzyme activity, quantifiable by a generalized Damköhler group II psi distinguishing electrotransport reactions from diffusion reactions. For three typical reaction schemas, using only one mediator, the steady-state equations have been established. Depending on boundary conditions, the direction of electric current (for asymmetrical systems) and the value of psi, activations, inhibitions or activations followed by inactivations have been found. With buffered conductivity (supporting electrolyte), the limiting concentration profiles (E leads to infinity) are uniformly equal to the boundary values; i.e., diffusion constraints are suppressed and the regime is controlled by the reaction. The calculations give the relative activity variations for partially suppressed transport controls.

Two-enzyme active transport in vitro with pH induced asymmetrical functional structures. III. Discussions based on numerical treatments of the time-dependent evolutions.

Three complementary models have been considered in which pH gradients (step function, linear pH or linear H+) impose asymmetry on a two-enzyme mixture. If the "combined pH dependences" of enzymes is pro-asymmetrical, the pH gradient induces an asymmetrical distribution of potential activities ("latent" asymmetry of functional structure). When substrate is added, "developed" asymmetry of effective activities appears which results in "substrate space wave" and pumping when the catalysed reaction couple is "inversible". It is shown that only one steady state exists for a given boundary condition and is attained when the "combined effective activity" of enzymes is nil; the stationary flux with symmetrical boundaries or the stationary load with moving boundaries is proportional to "effective global activities" of enzymes. "Equivalent square models" could be proposed that would be able to describe "functional" or "permanent" structure pumps as well. These models belong to the thermodynamic branch and the asymmetrical "space wave" substrate concentration profiles obtained must be distinguished from dissipative structures. It appears that such primary active transport pumps are chemical equivalents of heat pumps.