The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP_Pks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP_Pks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.Mycolic acids, α-branched and β-hydroxylated fatty acids of unusual chain length (C30-C90), are the hallmark of the Corynebacterineae suborder that includes the causative agents of tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Members of each genus biosynthesize mycolic acids of specific chain lengths, a feature used in taxonomy. For example, Corynebacterium holds the simplest prototypes (C32-C36), called “corynomycolic acids,” which result from an enzymatic condensation between two regular size fatty acids (C16–C18). In contrast, the longest mycolates (C60-C90) are the products of condensation between a very long meromycolic chain (C40-C60) and a shorter α-chain (C22-C26) (1). These so-called “eumycolic acids” are found in mycobacteria and display various structural features present on the meromycolic chain. Eumycolic acids are major and essential components of the mycobacterial envelope where they contribute to the formation of the outer membrane (2, 3) that plays a crucial role in the permeability of the envelope. They also impact on the pathogenicity of some mycobacterial species (4).The first in vitro mycolate biosynthesis assays have been developed using Corynebacterium cell-wall extracts in the presence of a radioactive precursor (5, 6) and have brought key information about this pathway. Yet, any attempt to fractionate these extracts to identify the proteins involved has ended in failure. Later, enzymes catalyzing the formation of the meromycolic chain and the introduction of functions have been discovered with the help of novel molecular biology tools (for review, see Ref. 1), culminating with the identification of the putative operon fadD32-pks13-accD4 that encodes enzymes implicated in the mycolic condensation step in both corynebacteria and mycobacteria (see Fig. 1) (7–9). AccD4, a putative carboxyltransferase, associates at least with the AccA3 subunit to form an acyl-CoA carboxylase (ACC)³ complex that most likely activates, through a C₂-carboxylation step, the extender unit to be condensed with the meromycolic chain (see Fig. 1). In Corynebacterium glutamicum, the carboxylase would metabolize a C16 substrate (8, 10), whereas in M. tuberculosis the purified complex AccA3-AccD4 was shown to carboxylate C24-C26 acyl-CoAs (11). Furthermore, FadD32, predicted to belong to a new class of long-chain acyl-AMP ligases (FAAL) (12), is most likely required for the activation of the meromycolic chain prior to the condensation reaction. At last, the cmrA gene controls the reduction of the β-keto function to yield the final mycolic motif (13) (see Fig. 1).Open in a separate windowFIGURE 1.Proposed scheme for the biosynthesis of mycolic acids. The asymmetrical carbons of the mycolic motif have a R,R configuration. R₁-CO, meromycolic chain; R₂, branch chain. In mycobacteria, R₁-CO = C40-C60 and R₂ = C20-C24; in corynebacteria, R₁-CO = C16-C18 and R₂ = C14-C16; X₁, unknown acceptor of the mycolic α-alkyl β-ketoacyl chains; X₂, unknown acceptor of the mycolic acyl chains.Although the enzymatic properties of the ACC complex have been well characterized (9, 11), those of Pks13 and FadD32 are poorly or not described. Pks13 is a type I polyketide synthase (PKS) made of a minimal module holding ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and additional N-terminal ACP and C-terminal thioesterase domains (Fig. 1). Its ACP domains are naturally activated by the 4′-phosphopantetheinyl (P-pant) transferase PptT (14). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group. In the present article we report the purification of a soluble activated form of the large Pks13 protein. For the first time, the loading mechanisms of both types of substrates on specific domains of the PKS were investigated. We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of α-alkyl β-ketoacids, the precursors of mycolic acids.     

Prevalence and Spatial Distribution of Entamoeba histolytica/dispar and Giardia lamblia among Schoolchildren in Agboville Area (C?te d'Ivoire)

Background

New efforts are being made to improve understanding of the epidemiology of the helminths and intensifying the control efforts against these parasites. In contrast, relatively few studies are being carried out in this direction for the intestinal protozoa. To contribute to a better comprehension of the epidemiology of the intestinal protozoa, prevalence, and spatial distribution of Entamoeba histolytica/dispar and Giardia lamblia, and their association with drinking water supplies, were determined in the Agboville department in southeast Côte d''Ivoire.

Methods/Findings

Stool samples were taken from more than 1,300 schoolchildren in the third year of primary education (CE1) from 30 primary schools and preserved in SAF (sodium acetate-acetic acid-formalin). The samples were analyzed by formalin-ether concentration. Then, a survey questionnaire addressed to schoolchildren and school directors was used to collect data on water supplies. Prevalence of E. histolytica/dispar and G. lamblia were, respectively, 18.8% and 13.9%. No particular focus zone was observed in the spatial distribution of the two species. Significant negative association was observed between use of tap water and high prevalence of E. histolytica/dispar infection (OR = 0.83, p = 0.01). High prevalence of G. lamblia infection was positively associated with use of ponds as the source of drinking water (OR = 1.28, p = 0.009).

Conclusion

These two species of pathogenic protozoa are present with substantial prevalence in this area of Côte d''Ivoire. Although their spatial distribution is not focused in any one place, determination of the population segments with the highest levels of infection will help to target the chemotherapeutic fight. To reinforce treatment with chemotherapeutic agents, tap water should be made available in all the localities of this area.     

A human protein atlas for normal and cancer tissues based on antibody proteomics

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.     

Where Do We Go from Here? Prevalence of Trachoma Three Years after Stopping Mass Distribution of Antibiotics in the Regions of Kayes and Koulikoro,Mali

Objectives

A national survey in 1997 demonstrated that trachoma was endemic in Mali. Interventions to control trachoma including mass drug administration (MDA) with azithromycin were launched in the regions of Kayes and Koulikoro in 2003. MDA was discontinued after three annual rounds in 2006, and an impact survey conducted. We resurveyed all districts in Kayes and Koulikoro in 2009 to reassess trachoma prevalence and determine intervention objectives for the future. In this paper we present findings from both the 2006 and 2009 surveys.

Methods

Population-based cluster surveys were conducted in each of the nine districts in Koulikoro in 2006 and 2009, whilst in Kayes, four of seven districts in 2006 and all seven districts in 2009 were surveyed. Household members present were examined for clinical signs of trachoma.

Results

Overall, 29,179 persons from 2,528 compounds, in 260 clusters were examined in 2006 and 32,918 from 7,533 households in 320 clusters in 2009. The prevalence of TF in children aged 1–9 years in Kayes and Koulikoro was 3.9% (95%CI 2.9–5.0%, range by district 1.2–5.4%) and 2.7% (95%CI 2.3–3.1%, range by district 0.1–5.0%) respectively in 2006. In 2009 TF prevalence was 7.26% (95%CI 6.2–8.2%, range by district 2.5–15.4%) in Kayes and 8.19% (95%CI 7.3–9.1%, range by district 1.7–17.2%) in Koulikoro among children of the same age group. TT in adults 15 years of age and older was 2.37% (95%CI 1.66–3.07%, range by district 0.30–3.54%) in 2006 and 1.37% (95%CI 1.02–1.72%, range by district 0.37–1.87%) in 2009 in Kayes and 1.75% (95%CI 1.31–2.23%, range by district 1.06–2.49%) in 2006 and 1.08% (95%CI 0.86–1.30%, range by district 0.34–1.78%) in 2009 in Koulikoro.

Conclusions

Using WHO guidelines for decision making, four districts, Bafoulabe in Kayes Region; and Banamba, Kolokani and Koulikoro in Koulikoro Region, still meet criteria for district-wide implementation of the full SAFE strategy as TF in children exceeds 10%. A community-by-community approach to trachoma control may now be required in the other twelve districts. Trichiasis surgery provision remains a need in all districts and should be enhanced in six districts in Kayes and five in Koulikoro where the prevalence exceeded 1.0% in adults. Since 1997 great progress has been observed in the fight against blinding trachoma; however, greater effort is required to meet the elimination target of 2015.     

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.     

Trypanocidal and cytotoxic evaluation of synthesized thiosemicarbazones as potential drug leads against sleeping sickness

Thiosemicarbazones have become one of the promising compounds as new clinical candidates due to their wide spectrum of pharmaceutical activities. The wide range of their biological activities depends generally on their related aldehyde or ketone groups. Here, we report the pharmacological activities of some thiosemicarbazones synthesized in this work. Benzophenone and derivatives were used with N(4)-phenyl-3-thiosemicarbazide to synthesize corresponding five thiosemicarbazones (1–5). Their structures were characterized by spectrometrical methods analysis IR, NMR ¹H & ¹³C and MS. The compounds were then screened in vitro for their antiparasitic activity and toxicity on Trypanosoma brucei brucei and Artemia salina Leach respectively. The selectivity index of each compound was also determined. Four thiosemicarbazones such as 4, 2, 3 and 1 reveal interesting trypanocidal activities with their half inhibitory concentration (IC₅₀) equal to 2.76, 2.83, 3.86 and 8.48 μM respectively, while compound 5 (IC₅₀ = 12.16 μM) showed a moderate anti-trypanosomal activity on parasite. In toxicity test, except compound 1, which showed a half lethal concentration LC₅₀ >281 μM, the others exerted toxic effect on larvae with LC₅₀ of 5.56, 13.62, 14.55 and 42.50 μM respectively for thiosemicarbazones 4, 5, 3 and 2. In agreement to their selectivity index, which is greater than 1 (SI >1), these compounds clearly displayed significant selective pharmaceutical activities on the parasite tested. The thiosemicarbazones 2–5 that displayed significant anti-trypanosomal and cytoxicity activities are suggested to have anti-neoplastic and anti-cancer activities.     

Early growth and death from cardiovascular disease in women.

OBJECTIVE--To determine whether the link suggested between growth in utero and during infancy and death from cardiovascular disease in men is also present in women. DESIGN--Follow up study of women and men whose birth weight and weight at 1 year of age had been recorded. SETTING--Hertfordshire, England. SUBJECTS--5585 women and 10,141 men born during 1911-30. MAIN OUTCOME MEASURES--Standardised mortality ratios for cardiovascular disease. RESULTS--Among women and men death rates from cardiovascular disease fell progressively between the low and high birth weights groups (chi 2 = 4.3, p = 0.04 for women, chi 2 = 8.5, p < 0.005 for men). Cardiovascular deaths in men but not women were also strongly related to weight at 1 year, falling progressively between the low and high weight groups (chi 2 = 27.5, p < 0.0001). The highest cardiovascular death rates in women were among those with below average birth weight but above average weight at 1 year. In men the highest rates were among those with below average birth weight and below average weight at 1 year. CONCLUSION--Relations between cardiovascular disease and birth weight are similar in men and women. In men cardiovascular disease is also related to weight gain in infancy.