Paul de Figueiredo

Microfabricated Microbial Fuel Cell Arrays Reveal Electrochemically Active Microbes

Microbial fuel cells (MFCs) are remarkable “green energy” devices that exploit microbes to generate electricity from organic compounds. MFC devices currently being used and studied do not generate sufficient power to support widespread and cost-effective applications. Hence, research has focused on strategies to enhance the power output of the MFC devices, including exploring more electrochemically active microbes to expand the few already known electricigen families. However, most of the MFC devices are not compatible with high throughput screening for finding microbes with higher electricity generation capabilities. Here, we describe the development of a microfabricated MFC array, a compact and user-friendly platform for the identification and characterization of electrochemically active microbes. The MFC array consists of 24 integrated anode and cathode chambers, which function as 24 independent miniature MFCs and support direct and parallel comparisons of microbial electrochemical activities. The electricity generation profiles of spatially distinct MFC chambers on the array loaded with Shewanella oneidensis MR-1 differed by less than 8%. A screen of environmental microbes using the array identified an isolate that was related to Shewanella putrefaciens IR-1 and Shewanella sp. MR-7, and displayed 2.3-fold higher power output than the S. oneidensis MR-1 reference strain. Therefore, the utility of the MFC array was demonstrated.

Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.

Pathogenesis and Immunobiology of Brucellosis: Review of Brucella–Host Interactions

This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics.

RNAi Screen of Endoplasmic Reticulum–Associated Host Factors Reveals a Role for IRE1α in Supporting Brucella Replication

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.

Air-cathode microbial fuel cell array: a device for identifying and characterizing electrochemically active microbes.

Microbial fuel cells (MFCs) have generated excitement in environmental and bioenergy communities due to their potential for coupling wastewater treatment with energy generation and powering diverse devices. The pursuit of strategies such as improving microbial cultivation practices and optimizing MFC devices has increased power generating capacities of MFCs. However, surprisingly few microbial species with electrochemical activity in MFCs have been identified because current devices do not support parallel analyses or high throughput screening. We have recently demonstrated the feasibility of using advanced microfabrication methods to fabricate an MFC microarray. Here, we extend these studies by demonstrating a microfabricated air-cathode MFC array system. The system contains 24 individual air-cathode MFCs integrated onto a single chip. The device enables the direct and parallel comparison of different microbes loaded onto the array. Environmental samples were used to validate the utility of the air-cathode MFC array system and two previously identified isolates, 7Ca (Shewanella sp.) and 3C (Arthrobacter sp.), were shown to display enhanced electrochemical activities of 2.69 mW/m(2) and 1.86 mW/m(2), respectively. Experiments using a large scale conventional air-cathode MFC validated these findings. The parallel air-cathode MFC array system demonstrated here is expected to promote and accelerate the discovery and characterization of electrochemically active microbes.

Microfabricated devices in microbial bioenergy sciences.

Microbes provide a platform for the synthesis of clean energy from renewable resources. Significant investments in discovering new microbial systems and capabilities, discerning the molecular mechanisms that mediate microbial bioenergy production, and optimizing existing microbial bioenergy systems have been made. However, further development is needed to achieve the economically feasible large-scale production of value-added energy products. Microfabricated lab-on-a-chip systems provide cost- and time-efficient opportunities for analyzing microbe-mediated bioenergy synthesis. Here, we review developments in the application of lab-on-a-chip systems to the bioenergy sciences. We focus on systems that support the analysis of microbial generation of bioelectricity, biogas, and liquid transportation fuels. We conclude by suggesting possible future directions.

A microfluidic microbial fuel cell array that supports long-term multiplexed analyses of electricigens

Microbial fuel cells (MFCs) are green energy technologies that exploit microbial metabolism to generate electricity. The widespread implementation of MFC technologies has been stymied by their high cost and limited power. MFC arrays in which device configurations or microbial consortia can be screened have generated significant interest because of their potential for defining aspects that will improve performance featuring high throughput characteristics. However, current miniature MFCs and MFC array systems do not support long-term studies that mimic field conditions, and hence, have limitations in fully characterizing and understanding MFC performances in varieties of conditions. Here, we describe an MFC array device that incorporates microfluidic technology to enable continuous long-term analysis of MFC performance at high throughput utilizing periodic anolyte/catholyte replenishment. The system showed 360% higher power output and 700% longer operating time when compared to MFC arrays without catholyte replenishment. We further demonstrate the utility of the system by reporting its successful use in screening microbial consortia collected from geographically diverse environments for communities that support enhanced MFC performance. Taken together, this work demonstrates that anolyte/catholyte replenishment can significantly improve the long-term performance of microfabricated MFC arrays, and support the characterization of diverse microbial consortia.

Fifty shades of immune defense.

1 Comparative Immunogenetics Laboratory, Texas A&M University, College Station, Texas, United States of America, 2 Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas, United States of America, 3 Borlaug Center, Texas A&M University, College Station, Texas, United States of America, 4 Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, Texas, United States of America, 5 Department of Plant Pathology & Microbiology, College Station, Texas, United States of America

DNA watermarking of infectious agents: progress and prospects.

Following the 2001 anthrax attacks, infectious disease research laboratories and personnel were subjected to increased scrutiny amid concerns that the released agent originated from within such facilities. Since then, enhanced regulatory controls have been implemented to thwart the possibility of future releases. However, improved microbial forensics technologies have not been employed to facilitate fault attribution or to control and track agent inventories. We believe that novel systems employing enhanced identity protection will instill new public confidence in scientists and avoid erroneous assignment of liability in the case of a release. We propose a DNA watermarking system that includes institution-, laboratory-, and/or investigator-specific watermarks in the genomes of organisms, especially Select Agents. The system will achieve five key goals critical to any watermarking system, phrased in general information theoretic terms: message fidelity, error tolerance, ease of interpretation, availability of signatures, and resistance to attack (Table 1). Table 1 Goals and features of watermarking system. A DNA watermark is a unique synthetic DNA sequence embedded into the genome of a genetically tractable organism. The watermark provides a means for agent, isolate, or strain identification and tracking by PCR amplification and sequencing of the embedded tag. The power of watermarking for agent control emerges when the technology is linked to the activities of a trusted authorizing entity (Figure 1). This entity could, for example, be charged with distributing organisms containing unique watermark sequences to individual laboratories and/or investigators. These watermarks would distinguish their organisms from those of others in the research community. Laboratories would be encouraged, permitted, or required to use only strains that contain their approved, and confidential, watermark. In the event of release, the offending pathogen would be interrogated for the presence of an approved watermark. If such a watermark were present, then information about the possible source would become immediately available. Of course, pathogen-specific standard operating procedures (SOPs) that ensure the integrity of the watermarking system (to prevent cross-contamination, manage the sharing of strains, and prevent accidental or intentional misuse) would be a necessary component of any watermarking strategy. Figure 1 Proposed watermark implementation strategies. Previously developed watermarking technologies include approaches for embedding watermarks in microbial genomes [1]–[4] and strategies for encryption [2], [5], [6]. Each method seeks to develop a genetic cipher that is 1) robust to mutation, 2) easy for intended users to decipher, and 3) difficult for third parties to decipher or alter. While these strategies for manipulating watermarks have been successful at watermark encoding, placement in a genome, retrieval from a genome, and decoding, none of the techniques achieves all of the five goals (outlined in Table 1) that are necessary for a watermarking system for Select Agent tracking. In our opinion, however, these techniques are worthy of further investigation, as regards their utility for the research and biosecurity communities. We propose investigation proceed on three discrete but interconnected fronts. First, the theoretical mathematical and information aspects of watermarking systems must be examined and rigorously tested in silico. Second, insertion and removal of watermarks from microbial genomes must be assessed, and the phenotypic invisibility of the watermarks tested. Finally, pathogen-specific SOPs must be developed, keeping in mind the need for transparency and collaboration in research, and tested in a “role playing” scenario. Our initial work indicates that the model is mathematically plausible. Previous work in the use of watermarks suggests that appropriately placed watermarks can be phenotypically neutral [1], [3]. The technologies to introduce watermarks into several of the highest risk Select Agent genomes are currently available, using site-specific insertion tools such as Targetron (intron-based homing) and Lambda red mutagenesis. Adaptation of these or comparable genetic tools for less tractable Select Agents would require technological advances that would also broadly benefit research of each agent. Adoption of a watermarking strategy by research groups would need to be justified by a cost-benefit analysis, from an institutional liability perspective, and from the perspective of the research community. Several salient concerns can be readily identified. Our proposed system does not protect against covert usage of naturally occurring wild-type strains or remediate existing stocks, but instead provides a forward-looking strategy. To address cost concerns, funding agencies that require enhanced inventory control could be encouraged or required to support the cost of implementing watermarking systems. Similarly, these agencies could support or collaborate with private or public authorizing entities to develop SOPs for strain management. Finally, convincingly establishing phenotypic neutrality of genomic modifications will be non-trivial, and thus, will constitute an important area for future research. Despite these potential impediments, watermarking would nearly eliminate the potential for mistaken assignment of source for a suspected agent release. Moreover, the development and implementation costs may prove to be much less than other proposed measures for enhancing laboratory security, including around-the-clock security patrols. We considered two variations on the operational infrastructure required (Figure 1). An authorizing entity could (Figure 1A) design, insert, and distribute, or (Figure 1B) simply distribute, the secured watermark to requesting laboratory. In the latter scenario, the requesting laboratory would be responsible for adapting genetic technology to deliver the watermark. We do not propose that previously generated modified strains (mutant collections, etc.) would be modified and restocked. The transition to marked strains would be incremental but stable. We speculate that an efficient approach to this scenario would be to provide funding opportunities to establish and validate agent-specific systems. While there are several potential impediments to implementing the proposed watermarking systems, the combination of positive impact on lay perception of responsible scientific activity and an increased confidence in control of liability by investigators and institutions provides a rationale to investigate the development of watermarking tools for Select Agent research.

A PLA1-2 punch regulates the Golgi complex

The mammalian Golgi complex, trans Golgi network (TGN) and ER-Golgi intermediate compartment (ERGIC) are comprised of membrane cisternae, coated vesicles and membrane tubules, all of which contribute to membrane trafficking and maintenance of their unique architectures. Recently, a new cast of players was discovered to regulate the Golgi and ERGIC: four unrelated cytoplasmic phospholipase A (PLA) enzymes, cPLA(2)α (GIVA cPLA(2)), PAFAH Ib (GVIII PLA(2)), iPLA(2)-β (GVIA-2 iPLA(2)) and iPLA(1)γ. These ubiquitously expressed enzymes regulate membrane trafficking from specific Golgi subcompartments, although there is evidence for some functional redundancy between PAFAH Ib and cPLA(2)α. Three of these enzymes, PAFAH Ib, cPLA(2)α and iPLA(2)-β, exert effects on Golgi structure and function by inducing the formation of membrane tubules. We review our current understanding of how PLA enzymes regulate Golgi and ERGIC morphology and function.