New antibiotics and their use in citrus canker control

Citrus farming is considered one of the main Brazilian economic activities, generating revenue in the order of US$ 2 billion/year in juice exports (Neves et al, 2010). Brazilian juice represents more than 80% of the product consumed in the world, and the state of São Paulo is responsible for approximately 80% of Brazilian orange production.

This notable success could be expanded if Brazilian citrus farming were able to resolve serious problems relating to irrigation, soil poverty, pests and diseases that cause production fluctuations. Of the diseases that affect citrus, two have caused great concern: citrus canker and HLB (huanglongbing/greening), caused by the bacteria Xanthomonas citri subsp. citri (X. citri) and Candidatus Liberibacter asiaticus/Americans, respectively. HLB is undoubtedly a serious disease at the moment, due to its very high incidence in orchards and the lack of resistant plants or cure. Citrus canker, however, has strength. It has been with us since 1957, when it was first detected in the Presidente Prudente region. It is a severe disease that affects all citrus species and varieties of economic interest (Gottwald et al, 2002).

X. citri is not spread by insect vectors, as in HLB, and the main form of contagion occurs plant-to-plant, through the combined action of rain and wind and human-mediated contact. Infection occurs when the bacteria enter through natural openings and wounds caused by broken branches or when insect larvae feed on the leaves (e.g.: citrus leafminer; Phyllocnistis citrella). Once inside the plant tissue, the bacteria induce typical symptoms that culminate in the appearance of brownish eruptive lesions. Depending on the severity of the disease, it promotes defoliation, premature fruit drop and even death of the plant.

There is no cure for citrus canker and the most effective form of control is the elimination of the symptomatic plant and its neighbors (eradication). This practice was the norm in São Paulo between 1999-2009 and maintained the health of São Paulo's orchards with disease rates well below 1% (Fundecitrus). During this period, if the incidence of disease in the orchard reached 0.5%, the entire plot would have to be eliminated. However, from 2009 onwards, various issues led to the relaxation of legislation and, as a consequence, cancer experienced an unprecedented rise in the following years. In March 2017, Regulation 37 of the Ministry of Agriculture, Livestock and Supply (MAPA) came into force, allowing producers to create a plan to mitigate the disease. Now, the largest orange production area in the world, the State of São Paulo, will have the bacteria in an endemic condition, as is already the case in the southernmost states of Brazil, Argentina and Florida. As a control in a mitigation system, it is recommended to plant orange trees that are more resistant to canker (remembering that total resistance does not exist), the use of green barriers to minimize the spread of the bacteria due to rain and wind and the massive use of cupric formulations.

Copper, in association with other management practices, has been effective in controlling cancer. However, its excessive use presents the risk of environmental contamination at different levels, thus creating the need to search for alternative antimicrobials capable of reaching X. citri and which necessarily present a lower environmental impact with their use.

At the beginning of 2010, Professor Henrique Ferreira, from the Laboratory of Bacterial Genetics (LGB), shortly after the changes in legislation that led to an increase in the incidence of the disease, started a project with his doctoral student Isabel Cristiane da Silva, seeking alternatives to use of copper, with support from FAPESP.

As one of the research focuses at LGB (Faculdade de Ciências Farmacêuticas de Araraquara/Unesp, 2010-2012; Instituto de Biociências de Rio Claro, IBRC/Unesp, 2013-present) was cell division of X. citri, we thought: “it would be possible control X. citri with compounds that disrupt its division?” Armed with various chemical structures of compounds that inhibit bacterial cell division found in the literature, tested against other bacteria and none in commercial use, Prof. Ferreira and Isabel looked for Pharmacist Dr. Luis Octavio Regasini. We asked if he had something similar to those molecules that we could test against X. citri.

Dr. Regasini, today Professor and coordinator of the Green and Medicinal Chemistry Laboratory (LQVM) at IBILCE, Unesp, São José do Rio Preto Campus, returned dozens of compounds that were tested by Prof.'s group. Ferreira and, to our surprise, 40% were able to kill the bacteria. The series of gallic acid esters (alkyl gallates) especially caught our attention due to their great activity, which was reported in Silva et al (2013).

Preparation of Alkyl Galates
In 2013 Prof. Regasini began planning new esters using the latency tool, more common in the development of pharmaceutical products than agrochemicals. This molecular modification tool produces compounds that are more capable of overcoming plant or animal cellular barriers, concentrating the compound inside the cells. The task of synthesizing alkyl gallate esters was carried out by chemist Carlos Roberto Polaquini, a postgraduate student in Chemistry at IBILCE/Unesp, and was carried out at LQVM, in São José do Rio Preto, which has the infrastructure for the synthesis , purification and analysis of bioactive compounds.

The compounds were prepared in a few steps, with satisfactory yields, and using some sustainability principles in Chemistry, the so-called “12 Principles of Green Chemistry”. Preparation was sought on a laboratory scale with reduced waste production, which could be more harmful than the action of the compounds on the environment.

Approximately 100 new esters were synthesized on the milligram scale, which required a lot of efforts from LQVM. After selecting the most active ones, today the group focuses on synthesis on a gram scale, which will allow advances in field trials, facilitating patenting and interest in the fine chemicals industry in the agrochemicals area. Fortunately, one of the most active esters was produced on a scale greater than 50 grams.

Mechanisms of action of Alkyl Galates: The cell division machinery as a target for antibacterials
In Prof.'s laboratory. Ferreira, LGB, the first tests were carried out to identify antibacterials capable of killing X. citri, through metabolic assays. After exposure to the compounds, the bacteria's ability to breathe was evaluated and those that had proven to cause cell death were selected to have their mechanism of action investigated.

In combating diseases, it is desirable to use compounds, called antibiotics, that selectively kill bacteria and are of little harm to humans, animals and plants. The ideal antibiotic should target molecules or biochemical pathways that are exclusive to bacteria, as is the case with penicillin, the first antibiotic to be discovered. Penicillin acts on the bacterial cell wall, a structure made up of molecules that do not occur in plants and animals. Another target of interest is the 'cell division machinery'. The machinery used by bacteria is fundamentally different from that used by plants and animals. If division is stopped, the infection is contained and the bacteria die.

At LGB we constructed, through genetic manipulation, X. citri cells used to unveil the mechanisms and
targets of antimicrobial action. In optical and fluorescence microscopy it is possible to identify, for example, membrane impairment, which results in extravasation of cellular contents and death. The increase in size of the bacteria, a common characteristic of cells growing and unable to divide, indicates action in division. Finally, it is possible to directly identify the interference of compounds in intracellular structures or proteins labeled with fluorophores: if a compound acts on cell division, it may destroy the divisional septum necessary for the cell division process to occur.

Among the proteins essential for cell division and which are exclusive to bacteria, the most abundant is FtsZ, which forms a ring around the bacteria at the site of division before it occurs. The FtsZ ring is the structure in which the division machinery is assembled and which, at the end of the process, forms a constriction separating the two daughter cells. Bacterial cells treated with alkyl gallates have difficulty dividing and so we thought that FtsZ would be a likely target for the compounds. To prove this, we contacted Professor Dirk-Jan Scheffers from the University of Groningen, Netherlands.

Prof. Scheffers, a current member of our research group funded by FAPESP and the Dutch agency NWO, hosted Isabel for a period of one year, where we studied the interaction of alkyl gallates with the FtsZ protein. We studied the effects of gallates on isolated FtsZ molecules from two different types of bacteria, Bacillus subtilis (model bacteria for studying Gram-positive bacteria) and X. citri. In the Groningen laboratory, we use a set of experiments that allow us to study whether FtsZ can still form rings and whether these rings are still capable of forming constrictions. We found that gallates strongly affect FtsZ activity, but not enough to explain all the effects observed. We identified that other mechanisms are also used by gallates to kill bacteria. They create small holes in the bacteria's membrane and the resulting leakage of nutrients and energy effectively leads to cell death (Krol et al, 2015).

This dual function can be very useful in the prevention and treatment of plant diseases. Plant treatment results in the release of the compound directly into the environment, posing the risk of bacteria developing resistance against it – so far, resistant bacteria have emerged for every antibiotic used in the clinic. However, when hitting more than one target at the same time, it becomes more difficult for the bacteria to develop resistance through mutations.

Safety of use of alkyl gallates
Before we could move forward with the idea of ​​using alkyl gallates as agricultural pesticides, we needed to study their effects and understand more about their toxicological safety. On this front, we have the support of collaborator Professor Fernando Pavan from the Faculty of Pharmaceutical Sciences of Araraquara/UNESP, who has the infrastructure to carry out pre-clinical trials to discover new molecules.

In 2014, Prof. Pavan welcomed Dr. Isabel, now a postdoc within the FAPESP/Brazil-NWO/Netherlands bilateral collaboration project coordinated by Prof. Ferreira, to study the toxicological effects of alkyl gallates in vitro. Cytotoxicity, apoptosis/necrosis, mutagenicity and chemoprevention studies were carried out and the results were more than satisfactory. It was possible to conclude that in addition to these molecules guaranteeing toxicological safety for humans, they also presented a chemopreventive effect, that is, they reversed the mutagenic effects induced in the laboratory (Silva et al, 2017).

Initial plant protection tests were very promising, as gallates, when sprayed on orange tree leaves, blocked infection by X. citri (Fig. 8), with the advantage of being a compound with less environmental impact and presenting positive characteristics. for human/animal health. We are currently working to ensure that the joint efforts of these collaborations materialize into solutions for Brazilian citrus farming and a return to society in the form of environmental 'preservation'.