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Unlikely allies

Viruses that attack bacteria and genetically engineered microorganisms help to fight diseases and produce drugs and other chemical substances

Léo Ramos Chaves Lysis halos: layer of bacteria over a culture medium is ruptured by viral activity that destroys themLéo Ramos Chaves

Viruses to combat human infections, bacteria that fight cancer, DNA programmed like a computer code–solutions sought by science that may have been developed by evolution in single-cell organisms.  One such solution arises from the existence of viruses capable of identifying and destroying specific bacteria.  They are what are known as bacteriophages, or simply phages, and they are increasingly recognized as potential alternatives to antibiotics and a source for innovative applications in biotechnology.  Another line of research, synthetic biology, involves manipulating the genetic material of microorganisms and directing bacteria and yeast to produce new treatments, function as sensors and open the door to other huge—albeit microscopic—possibilities.

Many of these projects are the result of basic research, which tries to understand organisms and mechanisms without initially aiming for applications or products.  Such is the case of the microbiological research study of compost heaps at the São Paulo Zoo (see Pesquisa FAPESP Issue No.181), whose objective was to catalogue bacterial diversity and investigate processes involved in decomposition (see the report on page 20). But “where there are many bacteria, there are many phages,” says biochemist Aline Maria da Silva of the University of São Paulo Chemistry Institute (IQ-USP), who is working on the project together with institute colleague and bioinformatician João Carlos Setubal. “Phages modulate the populations of bacteria,” explains Silva, who at first was only interested in the viruses because they interfered with her subject of study. But her master’s advisee, Deyvid Amgarten, an ardent fan of phages, went further and began studying the viral genetic material found in the compost heaps that contain the food and animal waste from the zoo.

Just as one idea leads to the next and sparks curiosity, the group decided to look for phages that would be useful in fighting disease-causing pathogenic bacteria.  The compost heap held bacteria of the genus Pseudomonas, a relative of the dangerous P. aeruginosa, which causes hospital infections.  In a report published in early 2017, the World Health Organization classified that bacteria as the second most important in terms of the urgent need for research to find new antibiotics to fight it because it is highly resistant to existing drugs.  The advantage offered by the phages is that they evolve together with their targets, maintaining the special ability to attack one type of bacteria without harming the human body.

Victor Balcão / UNISO Lighter phages already injected DNA into a fragment of bacteriaVictor Balcão / UNISO

The IQ group was able to isolate–and genetically characterize–three distinct phages, as described in a paper published in the journal BMC Genomics in May 2017. “The phages degrade the bacterial biofilm within 24 hours,” says Silva.  To find out how this virus behaves in the environment and inside the body of mammals, other research groups would need to become interested in conducting those tests.  Other phages have already been successfully tested against P. aeruginosa in mice, as shown in a study led by immunologist Aras Kadioglu at the U.K.s University of Liverpool, published in the July 2017 issue of the journal Thorax. The treatment eliminated bacteria from the lungs of 70% of mice that had an established infection–the condition that in humans is one of the principal causes of hospital deaths.

The method used to isolate the phages at IQ-USP is the same used in medical applications in Eastern Europe.  Since 1923, the Eliava Institute in Tbilisi, Georgia, has been using viral agents to fight all sorts of bacterial infections, including those that no other treatment was able to eliminate (see infographic). A review published in 2001 in the journal Antimicrobial Agents and Chemotherapy by researchers from Eliava and the University of Maryland compares the treatment success using antibiotics (64%) to phages (82%), and claims that the viral treatment is even more effective (95%) when applied intravenously.  For this reason, it is sought by patients all over the world. The story is that the discovery of use of the virus as a possible treatment occurred at the same time as the discovery of antibiotics.  Since their action is more broad-ranging, and they are more readily produced on an industrial scale, that strategy gained global prominence–everywhere but in the former Soviet bloc, where isolation imposed by the Iron Curtain required creative solutions, independent from the West.

Over decades of using antibiotics, evolution has tried to select resistant bacteria and this has given rise to lineages responsible for severe cases of hospital infections.  In late April 2017, there was a report of a case involving American psychiatrist Tom Patterson at the School of Medicine at University of California, San Diego, who in early 2016 had gone into a coma as a result of an infection that failed to respond to antibiotics.  His wife, Steffanie Strathdee, and physician Robert Schooley, both professors at the same school, made a last-ditch appeal and obtained authorization to try phage therapy.  “Patient samples were sent to two research groups that selected the phages,” says Schooley, who argues that phages are widely recognized as a weapon in the arsenal for fighting bacterial infections.  “Companies from several countries are beginning to work on developing this type of therapy,” he says.

Patterson came out of the coma and his case, the subject of a study for publication in a scientific journal, was presented on April 27, 2017 at the Pasteur Institute in Paris during the event commemorating a century of research on bacteriophages. The date celebrates the discovery of phages by French-Canadian microbiologist Felix d’Herelle (1873-1949) of the Pasteur Institute at the same time as Englishman Frederick Twort (1877-1950), and this is why it is known as Human Phage Therapy Day. D’Herelle actually had a part in establishing the Eliava Institute but did not return to Tbilisi after bacteriologist George Eliava (1892-1937) was executed by members of the Stalinist regime.

In most countries, as long as the treatment possibility is restricted to cases considered hopeless, researchers are limited to describing these abundant and ubiquitous viruses.  Microbiologist Sylvia Cardoso Leão of the Federal University of São Paulo (Unifesp), an expert on mycobacteria–which cause tuberculosis and hard-to-treat infections–recently became interested in phages.  Working together with microbiologist Cristina Viana Niero of the Diadema campus of Unifesp, a participant in the zoo project, Leão and her then-master’s student James Daltro Lima-Junior looked for specialized phages in the mycobacteria of the composting heaps.  They found them, but not the bacteria with which they interact.  In collaboration with Silva and Setubal, the group characterized those viruses from the genetic standpoint, which molecular biologist Graham Hatfull of the University of Pittsburgh, in the United States, then compared to the sequences in the collection he maintains.  “It is the world’s largest collection of bacteriophages that infect mycobacteria, an important tool for better understanding these species,” Leão says.  The surprise was in determining that one of the São Paulo phages studied is very similar to another one isolated from a sample collected in South Africa.  Perhaps it comes from some African animal that lives at the Zoo.

Leão is monitoring mycobacterial infections and has helped establish protocols to fight outbreaks and detect the cause of the problem, such as resistance to disinfectants used in hospitals.  She argues that the phages would be good allies in this ongoing battle.  “They are what is most common, nature’s own weapon.” Today, it is possible to buy products made with phages to eliminate bacteria from vegetables and reduce the level of Salmonella in chickens, for example, but not to fight human diseases.

Phages’ capacity to recognize their corresponding bacteria could also result in the development of sensors, such as that which Portuguese food engineer Victor Balcão, at the University of Sorocaba (UNISO), in inland São Paulo, is trying to devise in partnership with biochemist Marta Vila of the same institution. “The idea is to create an adhesive strip that you can hang on hospital walls to detect Pseudomonas aeruginosa,” he explains.  The project, named PneumoPhageColor, involves coating these adhesives with a gel containing phages captured in hospital sewage water. Any bacteria in the air will penetrate the hydrogel, and their interaction with the virus and subsequent rupture (through a process known as lysis) will produce a glow, like fireflies and some mushrooms (see Pesquisa FAPESP Issue No. 255) through a chemical reaction between substances contained in the hydrogel.  The warning could indicate the need to disinfect the environment.  “You have a 30% chance of entering an emergency room and leaving with an infection,” the researcher warns.

These are steps that he hopes to complete by the end of next year, now that he has assembled the laboratory and can build upon the success achieved in a previous project. “We were able to stabilize the phage in the gel via entrapment followed by a chemical reaction,” he says, as published in 2013 in the journal Enzyme and Microbial Technology. The Sorocaba group then demonstrated that it takes the bacteria six minutes to penetrate 3 millimeters into the hydrogel, enough time to come in contact with the phages, as shown in 2014 in the journal Applied Biochemistry and Biotechnology.

Soonhee Moon / Columbia University Bacterial cultures dyed for a morphology study become art in Tal Danino’s laboratorySoonhee Moon / Columbia University

Genetics under control
The outlook for biotechnology applications that use phages is broad and may go beyond taking advantage of their natural abilities.  These viruses can be genetically altered to act as sensors for pathogens or as weapons against them, or to transport desirable genetic sequences into cells.  Another property of the interaction between viruses and bacteria is the CRISPR-Cas9 system, which is like an immune system for bacteria that maintains a record of the viruses they have come in contact with.  The system has given rise to a tool that allows genetic material to be precisely edited to act as a sensor for specific strands of DNA that several Brazilian laboratories have already started using (see Pesquisa FAPESP Issue No. 240). These are some of the tools of synthetic biology, which can dispense with phages and set up DNA completely independently from organisms and thus build bacteria with prearranged genomes, a possibility launched in 2010 by American biochemist Craig Venter (see Pesquisa FAPESP Issue No. 172).

This type of approach is in its infancy in Brazil, but projects to modify microorganisms to produce substances have already appeared.  “The application potential is already on the radar of those who conduct molecular biology for diagnostic purposes and the treatment of diseases,” says physician Roger Chammas of the USP School of Medicine (FMUSP).

Biologist Aparecida Maria Fontes at the Ribeirão Preto School of Medicine of USP (FMRP) had contact with synthetic biology through a partnership with Venter.  Together with colleagues, she is seeking an alternative for the treatment of Gaucher’s disease, whose carriers lack an enzyme that degrades a particular type of fat.  The accumulation of this fat in cells causes a series of problems and even death. “There are close to 600 carriers in Brazil and the Ministry of Health spends R$200,000 a year to treat each of them,” she explains. In a collaboration with American Ron Weiss of the Massachusetts Institute of Technology (MIT), she selected two molecules to be inserted in a virus, which in turn will carry the gene of interest to the nucleus of human cells.  Preliminary results with in vitro cell lineages have been encouraging.  “We have been able to produce stable viruses, the molecule is functional, the enzyme is able to degrade the substrate and the cells are maintaining ongoing production of the enzyme,” she affirms. Fontes plans to file a patent application by the end of this year.

Soonhee Moon / Columbia UniversityOther organisms can also be transformed into micro-factories using synthetic biology.  The group led by biochemist Cleslei Zanelli of the School of Pharmaceutical Sciences at São Paulo State University (Unesp) in Araraquara is attempting to miniaturize and promote efficiency in the production of the active substance of the espinheira-santa (Maytenus ilicifolia), used in popular medicine for the treatment of stomach problems.  To do so, the researchers identified the gene responsible for producing the enzyme that synthesizes the drug, friedelin, and transferred it to Saccharomyces cerevisae yeast, as described in a 2016 article published in the journal Scientific Reports. “We have a project underway to increase levels of production to the same as or greater than we see in the plant,” Zanelli says.  The advantage of using yeast in production is that aside from requiring much less space than plants, production can be constant–it is independent of growth cycles or seasons.  “You just need to add sugar, vitamins and a source of nitrogen to the culture medium for them to constantly synthesize the substance,” he says.  Once production is optimized, the group from Araraquara, which includes chemist Maysa Furlan, plans to use the modified yeast system to produce other substances with antitumoral and anti-inflammatory properties that are already in clinical trials.

Some bacteria are already being used in industry to produce compounds such as vitamins and amino acids.  The group led by biologist Danielle Pedrolli, also from Unesp in Araraquara, is building synthetic RNAs to improve the capacity of Bacillus subtilis bacteria in producing vitamin B2 and its precursors, purines, by activating five targets in the genetic material. “We’re going to link the RNA tool to a system that causes it to enter into action when the bacteria achieve an adequate density,” she explains.  It has already worked in vitro, and now needs only to be tested in the bacteria.

Bacteria’s’ ability to detect the density of companions has important uses, such as that in the synthetic biology laboratory of American Tal Danino, of Columbia University in New York, who was in São Paulo in March 2017 for a scientific event.  “Our laboratory is looking for ways to program bacteria to detect and treat cancer,” he says.  “We’ve designed these bacteria to communicate with each other, synchronizing their attacks on the tumor as they produce a drug.”  The group is programming DNA sequences like computer codes and is thus able to build circuits that allow the bacteria to produce substances that are toxic to the cells. “We were able to determine that the bacteria express a gene only when they find a tumor,” says biomedical engineer Tetsuhiro Harimoto, a doctoral candidate in Danino’s laboratory.  That targeted action would be possible because the environment inside a tumor, in terms of oxygen and pH content, for example, is specific and can be programmed to be ideal for the action of the synthetic bacteria.

When they achieve a clinical density inside the tumor, the bacteria can rupture and release the drug they produce through genetic engineering.  In addition to its targeted precision, the medication feeds off itself. “Some bacteria survive and multiply, forming a cycle of growth and lysis,” Harimoto says.  The group has already achieved positive results with in vitro human cells and was able to reduce tumors in mice when used in combination with chemotherapy, as described in a 2016 paper published in the journal Nature.

The promising results explain Danino’s optimism, which anticipates positive therapeutic effects before long.  “There are several companies and laboratories, including mine, that are moving forward with the idea of using bacteria or specially designed cells for diseases such as cancer, diabetes and intestinal disorders,” he says.  “It’s just a matter of time before we improve our engineering of these organisms so that they have a huge impact on society.”

In the meantime, he is also applying bacteria manipulation to the arts, including a project with Brazilian artist Vik Muniz. For the researcher, the aesthetic exercise could help communicate complex scientific concepts.  “Not only are the visual arts universal, but they transcend the barriers of language and scientific jargon, inviting a wider audience to see science and ask questions.”

Live computing
Biologist Rafael Silva Rocha of the FMRP-USP is studying the gene function of bacteria through the lens of computing rather than molecular biology.  “We have conducted research in reprogramming bacteria, engineering fungi for bioenergy, and developing biosensors and computational tools for engineering gene circuits,” he says.  He works together with his wife, biologist María Eugenia Guazzaroni, a professor in the Biology Department of the School of Philosophy, Sciences and Languages and Literature at the same university.

Soonhee Moon / Columbia UniversityHe exemplifies his study by referring to the genetic structure of the Escherichia coli bacteria: it has 4,500 genes whose function is orchestrated by 200 regulators acting in unison. One gene may be controlled by 20 different regulators while one regulator may affect the function of hundreds of genes.  By studying these networks of interaction, it is possible to select the regulators that have the broadest action and use them to modulate and alter specific aspects of the bacterial behavior.  The group has already produced initial studies, such as that published in 2015 in the journal ACS Synthetic Biology in which they were able to insert two strands of synthetic DNA, which regulate gene activity, into live bacteria.  The idea is to integrate information about how the genome of bacteria is regulated to implement biotechnological innovations.

That integration of how the different genetic scales of an organism function – genomic, transcriptomic, proteomic and enzymatic–is also one of the focuses of a group led by botanist Marcos Buckeridge at the USP Biosciences Institute.  Bioinformatician Amanda Rusiska Piovezani, the doctoral candidate Buckeridge advises, has developed a computational tool that enables exploration of the relationships between elements of the complex network that make up the functioning of a specific part of sugarcane plant roots.  Although seemingly a minor detail, its purpose is to understand how air cavities (a channel known as aerenchyma) form in the roots through degradation of the cell walls, an essential property for optimizing biofuel production.  The software will be added to a statistical testing tool now under development by master’s candidate Vinícius Carvalho and may be employed in a variety of other systems.  “Even the running of a city,” muses Buckeridge. He is involved in creating a research center called Biomass and Synthetic Biology Systems, to be headquartered at USP, which will focus precisely on this type of approach to resolving problems connected to bioenergy, under the BIOEN Program of FAPESP.

Secret community

Researchers have spent the past eight years studying a rather unappealing object: decomposing animal waste in a compost heap at the São Paulo Zoo. Aside from the obvious, the invisible life of compost heaps has revealed itself as a promising source.  “We want to establish a collection of the bacterial consortium and identify those that may have promising biotechnology applications,” says bioinformatician João Setubal at the USP Chemistry Institute (IQ-USP).

His group is extracting the DNA from these compost samples in search of the microorganisms that live there and he recently obtained the nearly complete genome of six bacteria–four of them new to scientists–according to an April 2017 paper in the journal Frontiers in Microbiology. The compost heap was also monitored for 100 days, with genetic material collected every 10 days.  “We wanted to see how microbial populations change over time,” Setubal explains.  A paper published in the December 2016 journal Scientific Reports shows how the bacterial composition changes according to the availability of nutrients.  Bacteria that live off of cellulose are more abundant at first, degrading that substance.   Lignin, which makes plant tissues rigid, is the last to be degraded by specialized microorganisms.  The enzymes they produce, which are stable in the high temperatures of the compost heap, are promising sources for industrial applications.

Scientific articles
LEMOS, L. N. et al. Genome-centric analysis of a thermophilic and cellulolytic bacterial consortium derived from composting. Frontiers in Microbiology. V. 8, 644. 19 April 2017.
ANTUNES, L. P. et al. Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatransciptomics. Scientific Reports. V. 6, 38915. December 12, 2016.

Competition drives synthetic biology

Brazil is gaining a name for itself in international competitions looking to advance synthetic biology using projects developed by undergraduate students, through the International Genetically Engineered Machine Competition (iGEM) (see Pesquisa FAPESP Issue No. 247). This year, four Brazilian groups are competing: from Unesp Araraquara campus, the Federal University of Amazonas and two divisions of USP–the main campus in São Paulo and the Luiz de Queiroz School of Agriculture (ESALQ) in Piracicaba. “It’s as if there were a catalogue of 2,000 Lego pieces that can be combined to form a circuit,” says Rafael Silva Rocha, one of the coordinators of the São Paulo team. “The goal is not to get to the final product, but to get as far as possible.” They are limited by a one-year deadline, but the same group or others can continue to develop the projects after the competition.

In early 2017, the Synthetic Biology Club at USP–SynbioBrasil–organized a meeting with participants from all over Brazil.  “We’re showing students from other places that organizing clubs is a worthwhile endeavor and that iGEM opens doors,” says chemist Otto Heringer, who recently earned his undergraduate degree in chemistry at USP and opened a company that plans to use synthetic biology tools to fight agricultural pests. He is referring as much to what he learned in developing a project in this field as to the social and scientific network created as a result.

Biologist Cauã Westmann, a master’s degree candidate in Rocha’s laboratory, is collecting silver medals from iGEM. He has won four of them by taking part in competitions from 2012 to 2016, one as a member of the team from the University of York in the United Kingdom.  This year he is helping organize the work of the São Paulo team of USP. “Brazil has a unique situation, with a community that is growing and becoming very collaborative,” he says.  He is organizing a synthetic biology club on the Ribeirão Preto campus of USP, patterned after the others: a group of students organizes themselves, without the help of professors, and meets regularly to discuss science. “It’s quite gratifying to be able to combine mathematics, chemistry, biology, engineering and even the social sciences,” he says.  One social scientist is writing her master’s thesis about the science meeting held by Synbio. “The club has even become a topic of study!”

1. Studies of microbial diversity in the State of São Paulo Zoo Park (No. 11/50870-6); Grant Mechanism Thematic Project; Biota Program; Principal Investigator João Carlos Setubal (USP); Investment R$2,355,648.35.
2. Mycobacteria and extrachromosomal elements: molecular characterization and biotechnological applications (No. 11/18326-4); Grant Mechanism Thematic project; Principal Investigator Sylvia Luisa Pincherle Cardoso Leão (Unifesp); Investment R$1,181,733.92.
3. PneumoPhageColor – development of a colorimetric biodetection kit for Pseudomonas aeruginosa based on phage particles (No. 16/08884-3); Grant Mechanism Regular Research Project; Principal Investigator Vitor Manuel Cardoso Figueiredo Balcão (UNISO); Investment R$138,199.67.
4. Synthetic biology approaches for deciphering the logic of signal integration in complex bacterial promoters (No. 12/22921-8); Grant Mechanism Young Investigator; Principal Investigator Rafael Silva Rocha (USP); Investment  R$1,370,671.34.
5. Cloning and functional characterization of oxidoesqualene cyclases of Maytenus ilicifolia (No. 14/03819-3); Grant Mechanism Regular Research Project; Principal Investigator Cleslei Fernando Zanelli (Unesp); Investment  R$235,403.07.
6. Synthetic biology, use of humanized codons and microRNAs for the production of a biopharmaceutical for Gaucher’s disease (No. 13/50450-2); Grant Mechanism Regular Research Grant – Research Partnership for Technological Innovation (PITE); Agreement Agilent; Principal Investigator Aparecida Maria Fontes (USP); Investment R$948,505.23.
7. Reprogramming the purine metabolism of Bacillus subtilis employing sRNA-technology (No.14/17564-7); Grant Mechanism Young Investigator; Principal Investigator Danielle Biscaro Pedrolli (Unesp); Investment R$854,252.72.
8. Using systems biology approach to develop a model for whole plant functioning (No.11/52065-3); Grant Mechanism Regular Research Grant – Research Partnership for Technological Innovation (PITE); Agreement Microsoft; Principal Investigator Marcos Silveira Buckeridge (USP); Investment  R$228,500.00 and $12,000.00.

Artigos científicos
AMGARTEN, D. et al. Three novel Pseudomonas phages isolated from composting provide insights into the evolution and diversity of tailed phagesBMC Genomics. v. 18, n. 1, 346. 4 mai. 2017.
WATERS, E. M. et al. Phage therapy is highly effective against chronic lung infections with Pseudomonas aeruginosaThorax. v. 72, n. 7, p. 666-7. jul. 2017.
LIMA-JUNIOR, J. D. et al. Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U. BMC Microbiology. v. 16, n. 1, 111. 17 jun. 2016.
BALCÃO, V. M. et al. Structural and functional stabilization of phage particles in carbohydrate matrices for bacterial biosensingEnzyme and Microbial Technology. v. 53, n. 1, p. 55-69. 10 jun. 2013.
BALCÃO, V. M. et alCarbohydrate hydrogels with stabilized phage particles for Bacterial biosensing: bacterium diffusion studiesApplied Biochemistry and Biotechnology. v. 172, n. 3, p. 1194-214. fev. 2014.
AMORES, G. R. et alEngineering synthetic cis-regulatory elements for simultaneous recognition of three transcriptional factors in bacteriaACS Synthetic Biology. v. 4, n. 12, p. 1287-94. 18 dez. 2015.
DIN, M. O. et alSynchronized cycles of bacterial lysis for in vivo deliveryNature. v. 536, n. 7614, p. 81-5. 4 ago. 2016.
SOUZA-MOREIRA, T. M. et al. Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an essential role in the production of the most rearranged pentacyclic triterpeneScientific Reports. v. 6, 36858. 22 nov. 2016.