Expression of the Fab enzymes (Fab I and Fab Z) from Plasmodium falciparum after exposure to Artemisia afra plant extracts and drugs screening.

The emergence and spread of drug resistance of the malaria parasite to the main treatment emphasize the need to develop new antimalarial drugs. In this context, the fatty acid biosynthesis (FAS_II) pathway of the malaria parasite is one of the ideal targets due to its crucial role in parasite survival. In this study, we report the expression and the affinity binding of Fab_I and Fab_Z after exposure to the parasite with different extracts of the Artemisia afra. The parasites were exposed for 2 days to different extracts. Gene expression was done to determine the level of expression of the fab enzymes after treatments. A GCMS was run to determine the different compounds of the plant extracts, followed by a virtual screening between the fab enzymes and the active compounds using Pyrex. The results showed different expression patterns of the Fab enzymes. Fab_I expression was downregulated in the W2 and D6 strains by the ethanolic extract but was increased by Hexane and DCM extracts. A different expression pattern was observed for Fab_Z. It was all upregulated except in the D6 strain when exposed to the ethanolic and hexane extracts. Virtual screening showed an affinity with many compounds. Hits compounds with high binding energy were detected. 11alphaHydroxyprogesterone and Aspidospermidin-17-ol were found to have high binding energy with Fab_I respectively (- 10.7 kcal/mol; - 10.2 kcal/mol). Fab_Z shows also high affinity with 11alpha-Hydroxyprogesterone (- 10 kcal/mol) and Thiourea (- 8.4 kcal/mol). This study shows the potential of A. afra to be used as a new source of novel antimalarial compounds.

The Role of Type II Fatty Acid Synthesis Enzymes FabZ, ODSCI, and ODSCII in the Pathogenesis of Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which has a worldwide distribution and can infect a large number of warm-blooded animals and humans. T. gondii must colonize and proliferate inside the host cells in order to maintain its own survival by securing essential nutrients for the development of the newly generated tachyzoites. The type II fatty acid biosynthesis pathway (FASII) in the apicoplast is essential for the growth and survival of T. gondii. We investigated whether deletion of genes in the FASII pathway influences the in vitro growth and in vivo virulence of T. gondii. We focused on beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and oxidoreductase, short chain dehydrogenase/reductase family proteins ODSCI and ODSCII. We constructed T. gondii strains deficient in FabZ, ODSCI, and ODSCII using CRISPR-Cas9 gene editing technology. The results of immunofluorescence assay, plaque assay, proliferation assay and egress assay showed that in RHΔFabZ strain the apicoplast was partly lost and the growth ability of the parasite in vitro was significantly inhibited, while for RHΔODSCI and RHΔODSCII mutant strains no similar changes were detected. RHΔFabZ exhibited reduced virulence for mice compared with RHΔODSCI and RHΔODSCII, as shown by the improved survival rate. Deletion of FabZ in the PRU strain significantly decreased the brain cyst burden in mice compared with PRUΔODSCI and PRUΔODSCII. Collectively, these findings suggest that FabZ contributes to the growth and virulence of T. gondii, while ODSCI and ODSCII do not contribute to these traits.

Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease.

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of ΔlapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.

Interplay of Klebsiella pneumoniae fabZ and lpxC Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis.

Tight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging of Klebsiella pneumoniae in increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations in fabZ and lpxC occurring together (encoding either FabZR121L/LpxCV37G or FabZF51L/LpxCV37G). K. pneumoniae mutants having only LpxCV37G or LpxCV37A or various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37G accumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37G indicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37G was overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.IMPORTANCE Emergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation of K. pneumoniae resistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process in K. pneumoniae and how it may be linked to novel biosynthetic pathway inhibitors.

Antibacterial activity of flavonoid isolated from Trianthema decandra against Pseudomonas aeruginosa and molecular docking study of FabZ.

The natural product flavonoid demonstrates an extensive sort of pharmacological properties including antimicrobial activity. Although its Pseudomonas aeruginosa inhibition has been discovered, no target for action against flavonoid has been revealed to date. The anti - P. aeruginosa activity of the 2 - (3', 4' dihydroxy-phenyl) - 3, 5, 7-trihydroxy-chromen-4-one isolated from T. decandra was evaluated by disc diffusion and minimum inhibitory concentration methods. The molecular docking of the flavonoid isolated from T. decandra was carried out using CDOCKER (Discovery Studio 2.0). The flavonoid isolated from T. decandra was found to inhibit the growth of P. aeruginosa and the zone of inhibition was found to be 22 ±â€¯0.04 mm at 20 µg/ml while chloramphenicol showed 23 ±â€¯0.05 mm at 30 µg/ml. P. aeruginosa was found to be the most sensitive to both isolated flavonoid and standard control chloramphenicol with MIC values 39.05 µg/ml and 25 µg/ml respectively. Further, the FAS II ß-hydroxyacyl-ACP (FabZ) of P. aeruginosa was found to be a potential target of the flavonoid as it docked in silico effectively. Our work has demonstrated the anti - P. aeruginosa activity of flavonoid isolated from T. decandra and also resulted in the elucidation of a plausible mechanism of action of the isolated flavonoid by inhibiting the FabZ using in silico analysis.

Fused dimerization increases expression, solubility, and activity of bacterial dehydratase enzymes.

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ-FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.

Identification of a fabZ gene essential for flexirubin synthesis in Cytophaga hutchinsonii.

Cytophaga hutchinsonii, an aerobic soil bacterium which could degrade cellulose, produces yellow flexirubin pigments. In this study, fabZ, annotated as a putative ß-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase gene, was identified by insertional mutation and gene deletion as an essential gene for flexirubin pigment synthesis. The availability of a FabZ mutant that fails to produce flexirubin allowed us to investigate the biological role of the pigment in C. hutchinsonii. Loss of flexirubin made the FabZ mutant more sensitive to UV radiation, oxidative stress and alkaline stress than the wild type.

The initiation ketosynthase (FabH) is the sole rate-limiting enzyme of the fatty acid synthase of Synechococcus sp. PCC 7002.

Cyanobacteria are Gram-negative bacteria that are desirable hosts for biodiesel production, because they are photosynthetic, relatively fast growing, and can secrete products. We have reconstituted the fatty acid synthase (FAS) of the cyanobacterium Synechococcus sp. PCC 7002 and subjected it to in vitro kinetic analysis. Our data revealed that the overall rate of this metabolic pathway is exclusively limited by the FabH ketosynthase, which initiates product synthesis by condensing malonyl-ACP with acetyl-CoA to form acetoacetyl-ACP. This finding sharply contrasts with our previous findings that the Escherichia coli FAS is predominantly limited by its dehydratase (FabZ) and enoyl reductase (FabI) activities and that FabH activity is not limiting. We therefore reconstituted and analyzed a set of "hybrid" FASs. When the Synechococcus FabH was used to replace its counterpart in the reconstituted E. coli FAS, the resulting synthase was strongly limited by FabH activity. Conversely, replacement of the E. coli FabZ with its Synechococcus homolog dramatically alleviated the dependence of E. coli FAS activity on FabZ. In agreement with this finding, introduction of the E. coli FabH in the Synechococcus FAS virtually eliminated its dependence on this subunit, whereas substitution of the Synechococcus FabZ with its E. coli homolog shifted a substantial fraction of the overall flux control in the Synechococcus FAS to FabZ. Our findings demonstrate that the rate-limiting steps can differ dramatically between closely related bacterial fatty acid synthases, and that such regulatory behavior is fundamentally the property of the controlling enzyme(s).