Abel Baerga-Ortiz

Glutathione reductase-null malaria parasites have normal blood stage growth but arrest during development in the mosquito.

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.

Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

Background: Thioesterases are required for the release of polyunsaturated fatty acids in some bacteria. Results: The Orf6 protein from Photobacterium profundum has been characterized both functionally and structurally. Conclusion: Orf6 has a substrate preference for long-chain fatty acids. Significance: This is the first in vitro and structural characterization of a hotdog thioesterase from a deep-sea bacterium. Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg2+, which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg2+-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

Increasing the production of fatty acids by microbial fermentation remains an important step toward the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations toward accessible biodiesel precursors.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep‐sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl‐reduction. In this work, we have used the Udwary‐Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA‐homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl‐CoA to β‐hydroxybutyryl‐CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA‐homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA‐like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA‐homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.

Real-time detection of telomerase activity in cancer cells using a label-free electrochemical impedimetric biosensing microchip

The enzyme telomerase is present in about 85% of human cancers which makes it not only a good target for cancer treatment but also an excellent marker for cancer detection. Using a single stranded DNA probe specific for telomerase binding and reverse transcription tethered to an interdigital gold electrode array surface, the chromosome protection provided by the telomerase was replicated and followed by Electrochemical Impedance Spectroscopy as an unlabeled biosensor. Using this system designed in-house, easy and affordable, impedance measurements were taken while incubating at 37 °C and promoting the probe elongation. This resulted in up to 14-fold increase in the charge transfer resistance when testing a telomerase-positive nuclear extract from Jurkat cells compared to the heat-inactivated telomerase-negative nuclear extract. The electron transfer process at the Au electrodes was studied before the elongation, at different times after the elongation, and after desorption of non-specific binding.

Direct Detection and Quantification of Bacterial Genes Associated with Inflammation in DNA Isolated from Stool.

Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.

Fused dimerization increases expression, solubility, and activity of bacterial dehydratase enzymes: Fused Dimerization Improves Solubility and Activity

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.

The Presence of Genotoxic and/or Pro-inflammatory Bacterial Genes in Gut Metagenomic Databases and Their Possible Link With Inflammatory Bowel Diseases

Background: The human gut microbiota is a dynamic community of microorganisms that mediate important biochemical processes. Differences in the gut microbial composition have been associated with inflammatory bowel diseases (IBD) and other intestinal disorders. In this study, we quantified and compared the frequencies of eight genotoxic and/or pro-inflammatory bacterial genes found in metagenomic Whole Genome Sequences (mWGSs) of samples from individuals with IBD vs. a cohort of healthy human subjects. Methods: The eight selected gene sequences were clbN, clbB, cif, cnf-1, usp, tcpC from Escherichia coli, gelE from Enterococcus faecalis and murB from Akkermansia muciniphila. We also included the sequences for the conserved murB genes from E. coli and E. faecalis as markers for the presence of Enterobacteriaceae or Enterococci in the samples. The gene sequences were chosen based on their previously reported ability to disrupt normal cellular processes to either promote inflammation or to cause DNA damage in cultured cells or animal models, which could be linked to a role in IBD. The selected sequences were searched in three different mWGS datasets accessed through the Human Microbiome Project (HMP): a healthy cohort (N = 251), a Crohn’s disease cohort (N = 60) and an ulcerative colitis cohort (N = 17). Results: Firstly, the sequences for the murB housekeeping genes from Enterobacteriaceae and Enterococci were more frequently found in the IBD cohorts (32% E. coli in IBD vs. 12% in healthy; 13% E. faecalis in IBD vs. 3% in healthy) than in the healthy cohort, confirming earlier reports of a higher presence of both of these taxa in IBD. For some of the sequences in our study, especially usp and gelE, their frequency was even more sharply increased in the IBD cohorts than in the healthy cohort, suggesting an association with IBD that is not easily explained by the increased presence of E. coli or E. faecalis in those samples. Conclusion: Our results suggest a significant association between the presence of some of these genotoxic or pro-inflammatory gene sequences and IBDs. In addition, these results illustrate the power and limitations of the HMP database in the detection of possible clinical correlations for individual bacterial genes.

Abstract A09: Association of genotoxic and/or pro-inflammatory bacterial genes to colorectal neoplasia

Background: Although colorectal cancer (CRC) is a potentially preventable disease, it is still one of the most common and deadliest malignancies worldwide. Despite increasing adherence to routine screening and advances in therapeutic strategies, CRC is the 1st and 3rd leading cause of cancer-related death in Puerto Rico and the United States (US), respectively. Currently, CRC screening is the primary means for prevention; however, only 40% of CRC patients are diagnosed with localized-stage disease. The fact that in 60% of CRC patients are diagnosed at more advanced, less treatable stages emphasizes the need for CRC prevention and risk stratification strategies, that other than routine screening, are still unavailable. The etiology of CRC is complex and still incompletely understood. However, environmental factors including diet, the gut microbiota, and inflammation are accepted as major contributors to colorectal carcinogenesis. It has been shown that individuals with CRC have a distinct gut microbiota, but the mechanisms by which gut bacteria exert their CRC-promoting effects remains elusive. Certain pathogenic bacterial strains carry genes encoding toxins that promote DNA damage and perpetuate inflammation, yet the association of these toxins to CRC remains poorly understood. The aim of this study was to gain insight into the mechanisms by which a subset of the gut microbiota contribute to colorectal carcinogenesis by profiling six genes encoding genotoxic and/or proinflammatory bacterial toxins in stool from individuals with and without colorectal neoplasia (adenoma and CRC). Methods: The association between the genotoxic and/or pro-inflammatory bacterial genes and CRC was examined by characterizing the toxic colonic bacterial gene profile in stool samples from healthy individuals (controls) and individuals with colorectal neoplasia (cases). Stool samples from individuals in the mainland US (n= 20) and Caribbean Hispanics (n=33) living in Puerto Rico were provided by the NCI Early Detection Research Network (http://edrn.nci.nih.gov/) and the Puerto Rico Familial Colorectal Cancer Registry (http://purificar.rcm.upr.edu/index_eng.html), respectively. Detection pks, TcPC, GelE, cnf-1, murB, and usp in stool was performed by qPCR using gene-specific primers. Associations were assessed using odds ratios. Results: Four of the six toxic genes were detected more frequently in stool samples from individuals from the US with colorectal neoplasia (controls=10; adenoma=10). Results show borderline statistical significant associations (p=0.07) with the presence of usp and >2 genes and a higher odds of colorectal neoplasia (OR=5.44 and OR=9.33, respectively). In the cohort from our Caribbean Hispanic subjects (controls=13; adenomas=12; CRC=8), individuals with the presence GelE were 8.6-times more likely to have adenomas (p=.07) and individuals with ≥ 2 of the genes were 11.3-times more likely to have CRC than individuals without these genes (p=0.04). Conclusions: Analysis with a larger number of samples will be necessary to determine a more definite association between the presence of the toxic genes in this panel in stool and colorectal neoplasia. Additional mechanistic analysis will be required to fully understand how these bacterial toxins contribute to colorectal carcinogenesis. Citation Format: Maria Gonzalez-Pons, Ramon Gomez-Moreno, Abel Baerga-Ortiz, Marcia Cruz-Correa. Association of genotoxic and/or pro-inflammatory bacterial genes to colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A09.