University of Groningen Antibacterial activity of monoacetylated alkyl gallates against Xanthomonas citri subsp citri Savietto,

22 Asiatic Citrus Canker (ACC) is an incurable disease of citrus plants caused by the Gram-negative 23 bacterium Xanthomonas citri subsp. citri (X. citri). It affects all the commercially important citrus 24 varieties in the major orange producing areas around the world. Control of the pathogen requires recurrent 25 sprays of copper formulations that accumulate in soil and water reservoirs. Here, we describe the 26 improvement of the alkyl gallates, which are potent anti-X. citri compounds, intended to be used as 27 alternatives to copper in the control of ACC. Acetylation of alkyl gallates increased their lipophilicity, 28 which resulted in potentiation of the antibacterial activity. X. citri exposed to the acetylated compounds 29 exhibited increased cell length that is consistent with the disruption of the cell division apparatus. Finally, 30 we show that inhibition of cell division is an indirect effect that seemed to be caused by membrane 31 permeabilization, which is apparently the primary target of the acetylated alkyl gallates. 32 33 Introduction 34 Xanthomonas citri subsp. citri is the etiological agent of Asiatic Citrus Canker, a severe disease that 35 affects orange trees, and for which no healing process is known (Brunings and Gabriel 2003). The host 36 range of this pathogen consists of a wide diversity of Citrus spp. of economic importance around the 37 world. Symptomatic plants exhibit brownish eruptive lesions on their aerial parts, which may be 38 surrounded by chlorotic halos. Untreated infections may lead to premature fruit-drop, stem dieback and 39 defoliation, which is responsible for major economic losses to citriculture (Gottwald et al. 2002). X. citri 40 can be introduced to new areas by the movement of infected citrus fruits and seedlings. Upon infection, 41 the bacterium is rapidly disseminated by rainwater and wind passing over the surfaces of lesions and 42 splashing onto uninfected nearby trees (Bock et al. 2005; Gottwald et al. 2002). 43 The control of citrus canker in the major orange producer area in the world, the state of São 44 Paulo, Brazil, was satisfactorily achieved by the plant eradication program that took place between the 45 years 1999-2009 (Belasque Jr and Behlau 2011; Belasque Jr. et al. 2009). During that period, 46 symptomatic plants and the neighboring ones had to be eliminated to refrain the spread of the bacterium. 47 The drawback of eradication was the high cost of visual inspections, and the enormous number of plants 48 that had to be eliminated over the course of the years. Pressures from different sectors of the orange 49 producing chain culminated in the current scenario in which control is exerted by the plantation of less 50 susceptible cultivars of citrus, the use of wind-breaks to avoid bacterial lateral spreading by the combined 51 action of wind and rain, and the use of cupric formulations as bactericides. According to the current 52 legislation, the state of São Paulo was declared as an area of Risk Mitigation System from 2017, and the 53 control of citrus canker is now similar to what is already performed in the Southern states of Brazil 54 (Behlau et al. 2008). 55 Concerns have now been raised about the massive use of copper as the only bactericide to 56 control the spread of citrus canker. Copper sprays have to be applied repeatedly for effectiveness, 57 especially after a new leaf flush, thereby control by mitigation will increase the chemical residuals left on 58 fruits, soil, and water reservoirs. Copper can be bio-cumulative and it is a toxic metal (Brunetto et al. 59 2016; Cornu et al. 2017; Fones and Preston 2013). Besides, the emergence of bacterial strains resistant to 60 copper is a fact (Behlau et al. 2012; Canteros 1999). Altogether, citriculture requires new formulations as 61 alternatives to copper in order to combat bacterial and fungal infections. 62 Our research team is focused on the development of environmental friendly compounds able to 63 combat X. citri. We described the use of esters of gallic acid, the alkyl gallates, as potent cell division 64 inhibitors of X. citri (Król et al. 2015; Silva et al. 2013). Moreover, alkyl gallates were able to preclude 65 the ability of X. citri to infect citrus plants. Finally, alkyl gallates are safer than copper, and even exhibit 66 chemo-preventive action reducing the mutagenicity caused by agents that induce chromosomal damage 67 (e.g. compounds that generate Reactive Oxygen Species) (Silva et al. 2017). A downside of their 68 application in the field would be the possible broad anti-bacterial spectrum of the compounds, which may 69 be circumvented, at least in part, by the preparation of formulations able to attach specifically to citrus 70 leaves. In addition to this, compounds can be modified for increased potency, therefore minimizing the 71 dose necessary for effectiveness and the need for recurrent applications in the field. 72 One of the strategies used to modify and perhaps improve the action of lead compounds is the 73 optimization of physicochemical properties by the conversion of some of their functional groups. The 74 ester group is the main alternative to the carboxyl and hydroxyl polar groups, due to the increase of 75 lipophilicity and thus the biomembrane permeability (Beaumont et al. 2003; Rautio et al. 2008). Previous 76 studies performed by Sardi et al. (2017) demonstrated that an acetylated derivative of curcumin, a natural 77 polyphenolic compound, was more potent than its natural prototype against Staphylococcus aureus 78 strains, showing the importance of converting hydroxyl to ester groups for antibacterial activity. Here we 79 demonstrate that acetylation of some of the previously described alkyl gallates increased 100% their 80 potency against X. citri. Compounds stimulated morphological alterations in X. citri, which is consistent 81 with a disruption of the bacterial cell division process. However, our data support the view that the action 82 on division is indirect and a consequence of breakage of the cell transmembrane potential, which is 83 required for the correct assembly/positioning of the divisome. 84 85 Materials and methods 86 Synthesis and 1H NMR spectrum data of monoacetylated alkyl gallates 87 Monoacetylated alkyl gallates were synthesized by the acetylation of alkyl gallates according to 88 Changtam et al. (2010) with minor modifications. First, alkyl gallates with side chains varying from five 89 to eight carbons were synthetized as described in Silva et al. (2013). Next, acetic anhydride (10 mL) was 90 added to the solutions containing the alkyl gallates (1 mmol) in pyridine (10 mL), mixtures were stirred at 91 100 °C for 7 days, and monitored by successive TLC analyses. When reactions were finished, residues 92 were poured into crushed ice. The resulting solutions were partitioned with ethyl acetate and the organic 93 phase dried at room temperature. The crude products were purified over silica gel column eluted with 94 mixtures of hexane and ethyl acetate, furnishing monoacetylated alkyl gallates (8a ̶ 11a). Compound 95 numbers were chosen to keep in line with our previous reports (Krol et al. 2015; Silva et al. 2013). NMR 96 spectra were recorded at 600 MHz for 1H nucleus on a Bruker Avance III spectrometer at 25 °C. 97