Multicentric investigations of the role in the disease severity of accelerated phospholipid changes in COVID-19 patient airway.

CONTEXT: The changes in host membrane phospholipids are crucial in airway infection pathogenesis. Phospholipase A2 hydrolyzes host cell membranes, producing lyso-phospholipids and free fatty acids, including arachidonic acid (AA), which contributes significantly to lung inflammation. AIM: Follow these changes and their evolution from day 1, day 3 to day 7 in airway aspirates of 89 patients with COVID-19-associated acute respiratory distress syndrome and examine whether they correlate with the severity of the disease. The patients were recruited in three French intensive care units. The analysis was conducted from admission to the intensive care unit until the end of the first week of mechanical ventilation. RESULTS: In the airway aspirates, we found significant increases in the levels of host cell phospholipids, including phosphatidyl-serine and phosphatidyl-ethanolamine, and their corresponding lyso-phospholipids. This was accompanied by increased levels of AA and its inflammatory metabolite prostaglandin E2 (PGE2). Additionally, enhanced levels of ceramides, sphingomyelin, and free cholesterol were observed in these aspirates. These lipids are known to be involved in cell death and/or apoptosis, whereas free cholesterol plays a role in virus entry and replication in host cells. However, there were no significant changes in the levels of dipalmitoyl-phosphatidylcholine, the major surfactant phospholipid. A correlation analysis revealed an association between mortality risk and levels of AA and PGE2, as well as host cell phospholipids. CONCLUSION: Our findings indicate a correlation between heightened cellular phospholipid modifications and variations in AA and PGE2 with the severity of the disease in patients. Nevertheless, there is no indication of surfactant alteration in the initial phases of the illness.

Effectiveness of exercise training on the dyspnoea of individuals with long COVID: A randomised controlled multicentre trial.

BACKGROUND: COVID-19-related acute respiratory distress syndrome (CARDS) is a severe evolution of the Sars-Cov-2 infection and necessitates intensive care. COVID-19 may subsequently be associated with long COVID, whose symptoms can include persistent respiratory symptoms up to 1 year later. Rehabilitation is currently recommended by most guidelines for people with this condition. OBJECTIVES: To evaluate the effects of exercise training rehabilitation (ETR) on dyspnoea and health-related quality of life measures in people with continuing respiratory discomfort following CARDS. METHODS: In this multicentre, two-arm, parallel, open, assessor-blinded, randomised controlled trial, we enroled adults previously admitted with CARDS to 3 French intensive care units who had been discharged at least 3 months earlier and who presented with an mMRC dyspnoea scale score > 1. Participants received either ETR or standard physiotherapy (SP) for 90 days. The primary outcome was dyspnoea, as measured by the Multidimensional Dyspnoea Profile (MDP), at day 0 (inclusion) and after 90 days of physiotherapy. Secondary outcomes were the mMRC and 12-item Short-Form Survey scores. RESULTS: Between August 7, 2020, and January 26, 2022, 487 participants with CARDS were screened for inclusion, of whom 60 were randomly assigned to receive either ETR (n = 27) or SP (n = 33). Mean MDP following ETR was 42% lower than after SP (26.15 vs. 44.76); a difference of -18.61 (95% CI -27.78 to -9.44; p<10-4). CONCLUSION: People who were still suffering from breathlessness three months after being discharged from hospital with CARDS had significantly improved dyspnoea scores when treated with ETR therapy for 90 days unlike those who only received SP. Study registered 29/09/2020 on Clinicaltrials.gov (NCT04569266).

[Impact of Home Return Assistance Service in Heart Failure (PRADO-IC) on the one year re-hospitalisation and mortality in a heart failure hospitalized population of patients]. / Impact du programme d'aide au retour à domicile-insuffisance cardiaque (PRADO-IC) sur la mortalité et la réhospitalisation à un an dans une population de patients insuffisants cardiaques.

INTRODUCTION: Congestive heart failure (CHF) is associated with prolonged and recurrent hospitalizations; the prognosis remains poor. Since 2013, the Caisse Primaire d'Assurance Maladie (CPAM) has set up a support program PRADO-IC (support program for returning home after hospitalisation for heart failure). The aim of this study was to evaluate the impact of PRADO-IC on the heart failure readmission rate and death rate at one year. METHODS: From September 2016 to September 2018, all patients hospitalized for heart failure at Saint-Joseph Hospital were included in an observational study. The inclusion in PRADO-IC program was at physician's discretion. Two groups were compared according to the inclusion in PRADO-IC or not (T). The primary endpoints were the comparison of one-year mortality and heart failure readmission rate between the two groups. RESULTS: Six hundred and thirty-three patients were included, 262 in the PRADO-IC group and 371 in the non-PRADO group. Patients in the PRADO-IC cohort more frequently present severity criteria (age, weight, BNP level, arrhythmia, anemia, renal failure). Mortality at one year (19.5% vs 16.2%, p = 0.28) are equivalent in both groups. There were no significant differences in one-year rehospitalization rate for heart failure (HF) (35.1% in PRADO cohort vs 28% in T group, p = 0.06), the time to first hospitalization (74.5 days in PRADO vs 54.5 days in T, p = 0.55) and the length of hospitalization (6.0 days in PRADO vs 7.0 days in T, p = 0.29) between the two groups. Age, hyponatremia, anemia, cancer, HF re-hospitalization were variables linked to a risk of mortality, in a multivariable analysis. CONCLUSION: Our study shows that the PRADO-IC program concerned to the most severe patients. Despite this, the one-year mortality and the HF readmission rate are similar between the two groups.

Electrochemical skin conductance by Sudoscan®: a new tool to predict intradialytic hypotension.

BACKGROUND: Intradialytic hypotension (IDH), a common complication in haemodialysis (HD) patients, is associated with multiple risk factors including cardiac dysfunction and alterations of the peripheral autonomic nervous system. To what extent dysautonomia may contribute to the occurrence of IDH remains elusive. We sought to investigate the clinical utility of Sudocan®, a device that quantifies dysautonomia, in the prediction of IDH. METHODS: We conducted a prospective monocentric study in adult HD patients from July 2019 to February 2020. Dysautonomia was assessed by the measurements of hand and foot electrochemical skin conductance (ESC) using Sudocan®, before HD. The primary endpoint was the incidence of IDH (The National Kidney Foundation/Kidney-Dialysis Outcome Quality Initiative definition), according to the presence of a pathological hand and/or foot ESC value, during the 3-month study period. RESULTS: A total of 176 HD patients (64 ± 14 years old) were enrolled. Mean pre-dialysis HD hand and foot ESC was 45 ± 20 and 54 ± 22 µS, respectively. About 35% and 40% of patients had a pathological ESC at the hand and foot, respectively. IDH occurred in 46 patients. Logistic regression showed that pathologic pre-dialysis HD hand ESC was associated with an increased risk of IDH [odds ratio = 2.56, 95% CI (1.04-6.67), P = 0.04]. The cumulative risk incidence of IHD during the study was 5.65 [95% CI (2.04-15.71), P = 0.001] and 3.71 [95% CI (1.41-9.76), P = 0.008], with a pathological hand and foot ESC, respectively. CONCLUSIONS: A pathological hand ESC, as assessed by a non-invasive Sudoscan® test, is associated with an increased risk of IDH.

A reappraisal of the presence of small or large fiber neuropathy in patients with erythromelalgia.

OBJECTIVE: To assess the contribution of large and small nerve fiber alteration in erythromelalgia (EM). METHODS: Thirty-three EM patients were included and underwent clinical evaluation based on EM severity score, DN4, and Utah Early Neuropathy Scale (UENS) score. Neurophysiological evaluation consisted in nerve conduction studies (NCS) for large nerve fibers and specific tests for small nerve fibers: electrochemical skin conductance, cold and warm detection thresholds, and laser evoked potentials. Finally, the evaluation of vascular changes was based on the presence of clinical feature of microvascular disorders and the measurement of the Toe Pressure Index (TPI). RESULTS: While 28 patients (85%) had vascular alteration on TPI or clinical features, 23 patients (70%) had small-fiber neuropathy on neurophysiological tests, and only 10 patients (30%) had large fiber neuropathy on NCS. Regarding clinical scores, there was no difference between groups (presence or absence of large- or small-fiber neuropathy or microvascular disorder) except for a higher UENS score in patients with large fiber neuropathy. CONCLUSION: Peripheral neuropathy, mostly involving small nerve fibers, is almost as common as microvascular changes in EM, but remains inconstant and not related to a specific neuropathic pattern or higher clinical severity. SIGNIFICANCE: The association of neuropathic and vascular factors is not systematic in EM, this syndrome being characterized by different pathophysiological mechanisms leading to a common clinical phenotype.

Low admission blood pressure as a marker of poor 1-year survival in patients with revascularized critical limb ischemia.

OBJECTIVE: To contrast the association between blood pressure (BP) level and antihypertensive medications at hospital admission with 1-year mortality in patients undergoing revascularization for critical limb ischemia (CLI). METHODS: From November 2013 to May 2019, 315 consecutive patients were retrospectively included. A median of seven (IQR 3-13) separate readings were recorded for each patient before revascularization procedure and the average represented patient's mean BP. BP-lowering medications, clinical and biological parameters were recorded at baseline. The main outcome was total 1-year mortality. RESULTS: The cohort included 172 men (55%) and 143 women (45%), with a mean age of 77.9 ±â€Š11.9 years. Treated hypertension was present in 245 (78%) patients; 288 (91%) patients had BP-lowering drug prescriptions (2.1 ±â€Š1.3 medications at baseline). Mean SBP, DBP, mean BP (MBP) and pulse pressure (PP) were 132 ±â€Š18, 70 ±â€Š8, 90 ±â€Š10 and 62 ±â€Š16mmHg. During 1-year follow-up, 80 (25.4%) patients died. In single-pressure multivariate analysis, SBP (hazard ratio 0.97; 95% CI 0.96-0.99; P = 0.005), MBP (hazard ratio 0.96; 95% CI 0.92-0.99; P = 0.01), PP (hazard ratio 0.97; 95% CI 0.95-0.99; P = 0.009), but not DBP, were inversely correlated with 1-year mortality, independently of age, coronary heart disease, left ventricular ejection fraction, brain natriuretic peptide, serum albumin, institutionalized status and antihypertensive drugs. Association between SBP, MBP and PP with 1-year mortality had a quite linear reverse pattern. CONCLUSION: Among patients undergoing revascularization for CLI, there is an inverse correlation between admission SBP, MBP and PP with 1-year mortality. BP may represent a modifiable therapeutic target to prevent poor outcome in CLI patients.

Anakinra for severe forms of COVID-19: a cohort study.

BACKGROUND: Coronaviruses can induce the production of interleukin (IL)-1ß, IL-6, tumour necrosis factor, and other cytokines implicated in autoinflammatory disorders. It has been postulated that anakinra, a recombinant IL-1 receptor antagonist, might help to neutralise the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related hyperinflammatory state, which is considered to be one cause of acute respiratory distress among patients with COVID-19. We aimed to assess the off-label use of anakinra in patients who were admitted to hospital for severe forms of COVID-19 with symptoms indicative of worsening respiratory function. METHODS: The Ana-COVID study included a prospective cohort from Groupe Hospitalier Paris Saint-Joseph (Paris, France) and a historical control cohort retrospectively selected from the Groupe Hospitalier Paris Saint-Joseph COVID cohort, which began on March 18, 2020. Patients were included in the prospective cohort if they were aged 18 years or older and admitted to Groupe Hospitalier Paris Saint-Joseph with severe COVID-19-related bilateral pneumonia on chest x-ray or lung CT scan. The other inclusion criteria were either laboratory-confirmed SARS-CoV-2 or typical lung infiltrates on a lung CT scan, and either an oxygen saturation of 93% or less under oxygen 6 L/min or more, or aggravation (saturation ≤93% under oxygen 3 L/min) with a loss of 3% of oxygen saturation in ambient air over the previous 24 h. The historical control group of patients had the same inclusion criteria. Patients in the anakinra group were treated with subcutaneous anakinra (100 mg twice a day for 72 h, then 100 mg daily for 7 days) as well as the standard treatments at the institution at the time. Patients in the historical group received standard treatments and supportive care. The main outcome was a composite of either admission to the intensive care unit (ICU) for invasive mechanical ventilation or death. The main analysis was done on an intention-to-treat basis (including all patients in the anakinra group who received at least one injection of anakinra). FINDINGS: From March 24 to April 6, 2020, 52 consecutive patients were included in the anakinra group and 44 historical patients were identified in the Groupe Hospitalier Paris Saint-Joseph COVID cohort study. Admission to the ICU for invasive mechanical ventilation or death occurred in 13 (25%) patients in the anakinra group and 32 (73%) patients in the historical group (hazard ratio [HR] 0·22 [95% CI 0·11-0·41; p<0·0001). The treatment effect of anakinra remained significant in the multivariate analysis (HR 0·22 [95% CI 0·10-0·49]; p=0·0002). An increase in liver aminotransferases occurred in seven (13%) patients in the anakinra group and four (9%) patients in the historical group. INTERPRETATION: Anakinra reduced both need for invasive mechanical ventilation in the ICU and mortality among patients with severe forms of COVID-19, without serious side-effects. Confirmation of efficacy will require controlled trials. FUNDING: Groupe Hospitalier Paris Saint-Joseph.

Predictive value of admission blood pressure for 3-month mortality in patients undergoing revascularization for critical limb ischemia.

OBJECTIVE: In patients with critical limb ischemia (CLI), blood pressure (BP) impact on mortality is unknown. We analyzed the predictive value of SBP, DBP and pulse pressure (PP) at hospital admission on 3-month mortality in patients with CLI undergoing revascularization procedure. METHODS: From November 2013 to December 2018, 297 consecutive patients were retrospectively included. Admission BP was recorded using automated brachial sphygmomanometer, before revascularization procedure. A median of seven (IQR3-13) separate readings were recorded for each patient and the average represented patient's mean BP (mBP). Clinical and biological parameters were recorded at baseline. RESULTS: The cohort included 163 men (55%) and 134 women (45%) with a mean age of 77.7 ±â€Š11.9 years. Treated hypertension and diabetes were present in, respectively, 62 and 48% of patients. Mean SBP, DBP and PP were 132 ±â€Š18, 70 ±â€Š8 and 62 ±â€Š16 mmHg. Thirty-four patients (11.4%) died during 3-month follow-up, mostly from cardiovascular causes. In univariate analysis, age, female sex, brain natriuretic peptide and C-reactive protein were positively correlated with mortality. BMI, mSBP, mDBP, mPP, hemoglobin, serum albumin and statin treatment were negatively correlated with mortality. In single-pressure multivariate analyses, mSBP (P = 0.024) and mPP (P = 0.030) were negatively correlated with mortality. Association between mSBP and mortality had an asymptotic curve pattern and SBP level 135 mmHg or less was significantly correlated with mortality. CONCLUSION: In patients undergoing revascularization for CLI, admission SBP is an independent predictor for short-term mortality with a negative relationship. SBP level 135 mmHg or less represents a warning sign to explore and correct associated comorbidities.

Mutation landscape of acquired cross-resistance to glycopeptide and ß-lactam antibiotics in Enterococcus faecium.

Bypass of the d,d-transpeptidase activity of penicillin-binding proteins by an l,d-transpeptidase (Ldtfm) results in resistance to ampicillin and glycopeptides in Enterococcus faecium M9, a mutant obtained by nine consecutive selection steps. Resistance requires activation of a cryptic locus for production of the essential tetrapeptide-containing substrate of Ldtfm and impaired activity of protein phosphatase StpA. Here, whole-genome sequencing revealed a high mutation rate for the entire selection procedure (79 mutations in 900 generations). Acquisition of a mutation in the mismatch repair gene mutL had little impact on the frequency of rifampin-resistant mutants although the mutation spectrum of M9 was typical of impaired MutL with high transversion to transition (40/11) and substitution to deletion (51/28) ratios. M9 did not mainly accumulate neutral mutations since base substitutions occurred more frequently in coding sequences than expected (χ(2) = 5.0; P < 0.05) and silent mutations were underrepresented (χ(2) = 5.72; P < 0.02). None of the mutations directly affected recognition of the tetrapeptide substrate of Ldtfm by peptidoglycan synthesis enzymes. Instead, mutations appear to remodel regulatory circuits involving two-component regulatory systems and sugar metabolism. The high number of mutations required for activation of the l,d-transpeptidase pathway may strongly limit emergence of cross-resistance to ampicillin and glycopeptides by this mechanism.

Serine/threonine protein phosphatase-mediated control of the peptidoglycan cross-linking L,D-transpeptidase pathway in Enterococcus faecium.

The last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of ß-lactam antibiotics. D,D-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, peptidoglycan cross-linking is performed mainly by active-site cysteine L,D-transpeptidases that use tetrapeptides in Mycobacterium tuberculosis, Clostridium difficile, and ß-lactam-resistant mutants of Enterococcus faecium. We have investigated reprogramming of the E. faecium peptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs by L,D-transpeptidase Ldtfm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of the L,D-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldtfm at the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of the D,D-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldtfm. Here we show that full bypass of PBPs by Ldtfm also requires increased Ser/Thr protein phosphorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of the L,D-transpeptidation pathway in the absence of mutational alteration of peptidoglycan synthesis enzymes. Importance: The mechanism of acquisition of high-level ampicillin resistance involving bypass of the penicillin-binding proteins (PBPs) by L,D-transpeptidase Ldtfm was incompletely understood, as production of tetrapeptide precursors following transcriptional activation of the ddc locus by the DdcRS two-component regulatory system was necessary but not sufficient for full activation of the L,D-transpeptidation pathway. Here, we identified the release of a negative control of Ser/Thr protein phosphorylation mediated by phosphatase StpA as the additional factor essential for ampicillin resistance. Thus, bypass of PBPs by Ldtfm requires the modification of signal transduction regulatory systems without any gain of function by mutational alteration of peptidoglycan biosynthetic enzymes. In contrast, previously characterized mechanisms of antibiotic resistance involve horizontal gene transfer and mutational alteration of drug targets. Activation of the L,D-transpeptidation pathway reported in this study is an unprecedented mechanism of emergence of a new metabolic pathway since it involved the recruitment of preexisting functions following modifications of regulatory circuits.

Colicin M, a peptidoglycan lipid-II-degrading enzyme: potential use for antibacterial means?

Colicins are proteins produced by some strains of Escherichia coli to kill competitors belonging to the same species. Among them, ColM (colicin M) is the only one that blocks the biosynthesis of peptidoglycan, a specific bacterial cell-wall polymer essential for cell integrity. ColM acts in the periplasm by hydrolysing the phosphoester bond of the peptidoglycan lipid intermediate (lipid II). ColM cytotoxicity is dependent on FkpA of the targeted cell, a chaperone with peptidylprolyl cis-trans isomerase activity. Dissection of ColM was used to delineate the catalytic domain and to identify the active-site residues. The in vitro activity of the isolated catalytic domain towards lipid II was 50-fold higher than that of the full-length bacteriocin. Moreover, this domain was bactericidal in the absence of FkpA under conditions that bypass the import mechanism (FhuA-TonB machinery). Thus ColM undergoes a maturation process driven by FkpA that is not required for the activity of the isolated catalytic domain. Genes encoding proteins with similarity to the catalytic domain of ColM were identified in pathogenic strains of Pseudomonas and other genera. ColM acts on several structures of lipid II representative of the diversity of peptidoglycan chemotypes. All together, these data open the way to the potential use of ColM-related bacteriocins as broad spectrum antibacterial agents.

Characterization of colicin M and its orthologs targeting bacterial cell wall peptidoglycan biosynthesis.

For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.

Revisiting the assignment of Rv0241c to fatty acid synthase type II of Mycobacterium tuberculosis.

The fatty acid synthase type II enzymatic complex of Mycobacterium tuberculosis (FAS-II(Mt)) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-II(Mt) cycles. Recently, an additional member of this family, Rv0241c, was proposed to have the same function, based on the heterologous complementation of a HAD mutant of the yeast mitochondrial FAS-II system. In the present work, Rv0241c was able to complement a HAD mutant in the Escherichia coli model but not a dehydratase-isomerase deficient mutant. However, an enzymatic study of the purified protein demonstrated that Rv0241c possesses a broad chain length specificity for the substrate, unlike FAS-II(Mt) enzymes. Most importantly, Rv0241c exhibited a strict dependence on the coenzyme A (CoA) as opposed to AcpM, the natural acyl carrier protein bearing the chains elongated by FAS-II(Mt). The deletion of Rv0241c showed that this gene is not essential to M. tuberculosis survival in vitro. The resulting mutant did not display any change in the mycolic acid profile. This demonstrates that Rv0241c is a trans-2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase that does not belong to FAS-II(Mt). The relevance of a heterologous complementation strategy to identifying proteins of such a system is questioned.

Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faecium.

Bypass of the penicillin-binding proteins by an L,D-transpeptidase (Ldt(fm)) confers cross-resistance to beta-lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt(fm) is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross-linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt(fm) is controlled by a two-component regulatory system (DdcRS) and a metallo-D,D-carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C-terminal D-Ala residue of the cytoplasmic peptidoglycan precursor UDP-MurNAc-pentapeptide. The T(161)A and T(161)M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP-MurNAc-pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E(127)K) and affected its interaction with the cell envelope (I(14)N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross-resistance to glycopeptides and beta-lactams in natural conditions.

The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis.

The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (>or=C(12)) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.

Rv3389C from Mycobacterium tuberculosis, a member of the (R)-specific hydratase/dehydratase family.

The (R)-specific 3-hydroxyacyl dehydratases/trans-enoyl hydratases are key proteins in the biosynthesis of fatty acids. In mycobacteria, such enzymes remain unknown, although they are involved in the biosynthesis of major and essential lipids like mycolic acids. First bioinformatic analyses allowed to identify a single candidate protein, namely Rv3389c, that belongs to the hydratases 2 family and is most likely made of a distinctive asymmetric double hot dog fold. The purified recombinant Rv3389c protein was shown to efficiently catalyze the hydration of (C(8)-C(16)) enoyl-CoA substrates. Furthermore, it catalyzed the dehydration of a 3-hydroxyacyl-CoA in coupled reactions with both reductases (MabA and InhA) of the acyl carrier protein (ACP)-dependent M. tuberculosis fatty acid synthase type II involved in mycolic acid biosynthesis. Yet, the facts that Rv3389c activity decreased in the presence of ACP, versus CoA, derivative and that Rv3389c knockout mutant had no visible variation of its fatty acid content suggested the occurrence of additional hydratase/dehydratase candidates. Accordingly, further and detailed bioinformatic analyses led to the identification of other members of the hydratases 2 family in M. tuberculosis.