Global Expression Profile of Biofilm Resistance to Antimicrobial Compounds in the Plant-Pathogenic Bacterium Xylella fastidiosa Reveals Evidence of Persister Cells

Abstract

Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.

INTRODUCTION

Xylella fastidiosa is a bacterium naturally transmitted by xylem sap feeding leafhoppers. It colonizes the xylem vessels of several host plants causing diseases in economically important crops, including plum, almond, peach, coffee, grapevine, and citrus. In citrus plants it causes citrus variegated chlorosis (CVC), which is responsible for millions of dollars in damages to the Brazilian citrus industry (6). It has been proposed that the water and nutrient stress symptoms in CVC are the consequence of the bacterial cell multiplication inside the vessel resulting in the blockage of the xylem by biofilm formation (2, 12, 26). Cells in biofilm have adaptation advantages, such as increased resistance to a broad range of antimicrobial agents (24). These resistance mechanisms can be due to the presence of exopolymer matrices, changes in gene expression, and metabolic alterations, which make the microbiological population difficult to control (9, 36). Moreover, another mechanism associated with antimicrobial compound tolerance is the formation of persister cells, which are cells that neither grow nor die in the presence of bactericidal agents and thus exhibit multidrug tolerance (20). It has been shown for different bacterial species that persister cells constitute ca. 1% of cells in stationary phase and biofilm culture (17) and, in contrast to resistant cells, the tolerance of persister cells to antibiotics might function by preventing target corruption by a bactericidal agent through the blockage of the antimicrobial target(s) (21). In microarray analysis, it was shown that toxin-antitoxin (TA) systems are expressed only in persister cells and are likely contributors to this condition (34). It has been also reported that persister cells are largely responsible for the recalcitrant infections caused by bacterial biofilms (20, 21). The resistance of biofilm cells to antimicrobial compounds is known for many different bacteria, but little information is available for plant pathogens. For X. fastidiosa, this information would be extremely relevant considering the role of biofilm for its pathogenicity (2, 12, 14).

In agriculture, copper-containing compounds are among the main antimicrobial substances used to limit the spread of plant-pathogenic bacteria and fungi on vegetable and fruit crops (38). Since copper is widely used in agriculture against phytopathogens and since X. fastidiosa grows inside the xylem vessels of the host plant and vector's foregut, forming biofilm, it became important to monitor the responses of X. fastidiosa biofilm to this antimicrobial compound. Recently, the expression of some genes related to multidrug resistance was investigated in X. fastidiosa biofilm under different copper concentrations (28). However, the focus of the analyses was restricted to only few genes. In addition, it was reported that with 7 mM copper, which was the inhibitory concentration for cell growth in culture, high-quality RNA was obtained and the expression of genes associated with copper resistance was verified. These results suggest that copper was toxic to the cells but fails to kill the whole population. One of the hypotheses to explain this finding was that the cells could be in a resistant physiological state (28). To better understand the genetic mechanisms used by X. fastidiosa biofilm in response to copper, we investigated the global transcription profile of biofilm cells in response to inhibitory and subinhibitory concentrations of this compound. To broaden our knowledge on the response of this bacterium to antimicrobials, we used another compound, tetracycline, that unveils common and specific mechanisms associated with these stress responses. This comparison is relevant because most of the knowledge about genetic responses to antimicrobial compounds comes from studies with antibiotics (1, 5, 23, 24, 27).

Our results showed that tolerance in X. fastidiosa may occur via a shutdown of target functions and inhibition of movement for both compounds, whereas keeping cells in biofilm seems to be a copper-specific response. An increase in the expression of genes encoding the TA system suggests that the formation of persister cells may constitute a mechanism for the survival of X. fastidiosa under stress conditions.

MATERIALS AND METHODS

Bacterial strain and growth conditions.

X. fastidiosa strain 9a5c was isolated from CVC symptomatic sweet orange plants (Citrus sinensis [L.] Osbeck) in periwinkle wilt (PW) medium (10). To obtain cells attached to a glass surface, several individual colonies were transferred to a polypropylene tube containing 3 ml of PW broth. When the A₆₀₀ reached 0.3, the tubes were vortexed, and the cells were transferred to a 250-ml flask containing 50 ml of PW broth. The flasks were kept at 28°C with rotary agitation at 120 rpm. After 15 days of growth in these conditions, different concentrations of the antimicrobial compounds were added to the flasks. The subinhibitory and inhibitory concentrations of copper to X. fastidiosa biofilm (3 and 7 mM CuSO₄, respectively) were established according to the method of Rodrigues et al. (28). To determine the subinhibitory and inhibitory concentrations of tetracycline for biofilm cells, we used the MIC for planktonic cells as the initial concentration (100 μg/ml) for the tests and increased from that eight different tetracycline concentrations (ranging from 100 to 800 μg/ml) until growth inhibition. After 48 h of incubation with the compound, the biofilms were scraped, dislodged, and washed twice phosphate-buffered saline (PBS) buffer. For each concentration tested, cells were collected from three different flasks. The percentage of viable cells was estimated after growth at 28°C in PW plates by comparison with untreated culture.

For the microarray experiments, cultures growing in subinhibitory concentrations were obtained by inoculating 3 mM CuSO₄ and 100 μg of tetracycline/ml. Cultures exposed to inhibitory concentrations of copper and tetracycline were obtained by adding 7 mM and 800-μg/ml concentrations of these compounds, respectively. Biofilm cells were scraped from the flask 24 h after the addition of copper and 48 h after the addition of tetracycline (due to its bacteriostatic characteristic) and washed three times by centrifugation at 8,000 × g for 5 min at 4°C with RNase-free water. The pellets were stored at −80°C until required.

Total RNA extraction and cDNA synthesis.

Total RNA was extracted according to Rhodious (www.microarrays.org/pdfs/total_RNA_from_ecoli.pdf) and treated with an RNase-free DNase set (Qiagen). The RNA concentration and integrity were analyzed in denaturing electrophoresis gel and spectrophotometer (A₂₆₀/A₂₈₀ and A₂₃₀/A₂₈₀ ratios). Portions (30 μg) of total RNA of each biological sample from all of the experiments were used for microarray experiments (cDNA synthesis, labeling with Cy3, hybridizations, and data capture; Roche-NimbleGen, Inc., Madison, WI). The cDNAs for real-time reverse transcription-PCR (RT-PCR) analysis were synthesized using 1 μg of total RNA with RevertAid (Fermentas) according to the manufacturer's instructions and diluted 1:3 with RNase-free water.

DNA microarrays and analysis.

DNA microarrays were designed with 13 60-mer oligonucleotide probes per gene, based on the available X. fastidiosa genome sequence (www.lbi.ic.unicamp.br/xf/). Probes were synthesized in situ by photolithography on glass slides using a computer-generated randomized pattern on the array determined by Roche-NimbleGen. All gene probes set were repeated five times in each chip, and we used three biological replicates per treatment where each replicate comprised a pool of three biological repetitions. The raw normalized data were processed by a robust multichip average (RMA) convolution model applied for background correction. The corrected probe intensities were then normalized using a quantile-based normalization procedure (4). Differentially expressed genes were identified using the ArrayStar software with Student t test (P < 0.05), and the multiple test correction of raw P values was performed using the false discovery rate. We also used a fold change cutoff of 2.0. The differentially expressed genes were divided into functional categories as proposed by Simpson et al. (35; see also www.lbi.ic.unicamp.br/xf/). After that, the open reading frames classified as hypothetical or hypothetical conserved proteins were analyzed by BLASTP (3), and some of them were re-annotated here. A detailed description of the array results can be found under reference codes GPL10380 and GSE21645 at the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) database. We also identified common induced or repressed genes expressed in both copper and tetracycline treatments using graphics of Venn (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Cluster analysis of some genes, which belong to different functional categories was done using the MultiExperiment Viewer software (29, 30).

Real-time RT-PCR.

RT-PCR was performed for 23 genes chosen after the microarray analysis to confirm the gene expression (see Table S1 in the supplemental material; note that additional supplemental material for this article may be found at http://powerswingle.centrodecitricultura.br/microarray_xylella/suplemental.html). Biological experiments were carried out to obtain cells in biofilm treated with inhibitory and subinhibitory concentrations of copper and tetracycline. Biofilm cells were collected after 3, 5, 10, 20, and 30 days of growth. The cells were washed with RNase-free water, harvested by centrifugation (4,000 × g, 3 min, 4°C), and immediately subjected to RNA extraction as described above.

The primers were designed using Prime Express software v2.0 (Applied Biosystems). The specificity of the primers was checked by sequencing the amplicons with the BigDye terminator kit v3.0 in an ABI 3730 automatic sequencer (Applied Biosystems). The nucleotide sequences were evaluated by aligning them with the original sequences using the BLASTN tool (3). In addition, melting-curve analysis was performed from 60 to 95°C for each primer at the end of the RT-PCR to confirm the amplification of a unique product for each gene. To evaluate the efficiency of the primers, standard curves were constructed by plotting the threshold cycle (C_T) values against the logarithm of cDNA copies of a set of standard solutions. The slopes of these standard curves were used to calculate the efficiency (E) according to the following formula: E = 10^(−1/slope) − 1. Primers that presented efficiency values between 0.9 and 1 were considered satisfactory for the experiments (see Table S1 in the supplemental material).

RT-PCR was performed in an ABI Prism 7500 sequence detection system (Applied Biosystems) using default parameters. Reactions were prepared with 12.5 μl of SYBR green PCR master mix (Applied Biosystems), 50 ng of each primer, 2 μl of cDNA, and water to 25 μl. No template controls were included to detect any spurious signals. The fluorescence threshold was set automatically to 0.2.

Assays were performed in triplicate using XF1353 (parC) and XF0656 (gltT) as endogenous controls to normalize the amount of cDNA per sample. These genes encode topoisomerase and a glutamate symport protein, respectively (see Table S1 in the supplemental material). They were determined by the Normfinder and Genorm softwares to be the best genes for endogenous controls in our experiments (data not shown). This was the result of an assay with four genes (XF2421, XF1353, XF0656, and XF0204) whose transcript levels remained constant during all of the stress conditions tested in microarray experiments, and they are also X. fastidiosa housekeeping genes according to Scally et al. (31).

Scanning electron microscopy (SEM).

In order to obtain X. fastidiosa biofilms, we used an experimental protocol developed by de Souza et al. (12). X. fastidiosa cells were incubated at 28°C on 12-mm-diameter cover glass slips immersed in PW medium in Nunclon delta SI Multidish 24 wells (Nunc A/S, Roskilde, Denmark). To this end, individual colonies were transferred to microcentrifuge tubes containing 1 ml of PW broth. The tubes were vortexed, and the cell suspensions were transferred to the wells. Copper and tetracycline at concentrations of 7 mM and 800 μg/ml, respectively, were added to X. fastidiosa biofilms after 15 days of growth. Three glass coverslips were analyzed for each concentration. The samples were rinsed, dried and coated with an ∼50-nm-thick sputtered gold film. After processing, the samples were observed in a scanning electron microscope (Leo 435 VP) in high-vacuum mode at 25 kV. The images were processed for display using Photoshop software (Adobe, Mountain View, CA).

Immunolocalization of X. fastidiosa hemagglutinin in biofilms with subinhibitory and inhibitory concentrations of copper and tetracycline.

For the immunofluorescence analysis, biofilms were obtained using glass-bottom microwell dishes (a 35-mm petri dish and a 14-mm microwell MatTek). The experiment was performed as defined above for SEM. The biofilm adhering to cover glasses was washed as described by Caserta et al. (7). The antibody against hemagglutinin was provided by B. C. Kirkpatrick, University of California at Davis (37). The antibody was used in a 1:400 dilution. For localization of proteins in the biofilms, we used 1 ml of goat anti-rabbit rhodamine-conjugated IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:10,000 dilution in PBS. The biofilm cells were stained with Syto 9 diluted 1:600 in autoclaved Milli-Q H₂O. The cells were visualized in an Olympus UIS2 fluorescence microscope, using for the Syto 9 an excitation wavelength between 460 and 490 nm and an emission wavelength between 500 and 520 nm and for rhodamine an excitation wavelength between 510 and 550 nm and emission wavelength between 570 and 590 nm.

Determination of the number of persister cells of X. fastidiosa biofilm in the presence of inhibitory concentration of copper and tetracycline.

X. fastidiosa biofilm cells were growth as described above. Cells attached to the glass surface were exposed to inhibitory concentrations of copper (7 mM) and tetracycline (800 μg/ml) at 15 days after inoculation. Biofilm cells were scraped from the flask 24 h after addition of copper and 48 h for tetracycline. Controls without antimicrobial compounds were collected at the same time. The cells were washed three times by centrifugation at 8,000 × g for 5 min at 4°C in PBS buffer. The pellets were resuspended in 1 ml of PBS for serial dilutions and plated on PWG media. All samples were conducted in triplicate, and each experiment was performed twice.

Number of persister cells after pretreatment with subinhibitory concentration of copper and tetracycline.

To verify the effect of pretreating X. fastidiosa biofilm cells with subinhibitory concentrations of copper and tetracycline in the induction of persistence, we conducted an experiment using three different conditions, with three biological replicates. In condition 1 we inoculated the cells in medium containing subinhibitory concentrations of copper and tetracycline (separately) and, after 15 days of growth, the supernatant (containing planktonic cells) was discarded and replaced by medium with inhibitory concentrations of copper or tetracycline. After 24 h (copper) or 48 h (tetracycline), the planktonic cells were discarded, and the biofilm was scraped and plated after serial dilution. For condition 2, the cells were grown for 10 days to form biofilm on the glass surface, and then subinhibitory concentrations of copper or tetracycline were added. At day 15 of growth, we added the inhibitory concentrations and left the cultures for 24 h (copper) or 48 h (tetracycline); the planktonic cells were then discarded, the biofilm was scraped, and the cells were plated as described above. Condition 3 was the control without pretreatment with the subinhibitory concentrations of copper or tetracycline.

RESULTS

Effects of inhibitory and subinhibitory concentrations of copper on global gene expression profile in X. fastidiosa biofilm.

The global effects of subinhibitory and inhibitory concentrations of two antimicrobial compounds on transcription in X. fastidiosa biofilm were examined by microarray. The differentially expressed genes were identified and classified into functional categories (Fig. 1). A total of 23 genes were chosen for further investigation by RT-PCR analysis. Some of these genes were differentially expressed in different conditions therefore a total of 39 RT-PCR analyses were performed (see Fig. S1 in the supplemental material). The overall Spearman's rank correlation coefficient was 0.978, indicating a very good correlation between the log ratio values from microarray experiments and RT-PCR and thus validating the expression profiles.

Fig 1.

Categorization of differentially expressed genes. Genes identified as differentially expressed in microarray analyses after exposure of X. fastidiosa biofilm cells to subinhibitory/inhibitory concentrations of copper and tetracycline were grouped according to their functions according to the X. fastidiosa genome database. The bars represent the absolute number of genes significantly expressed for each category in the different treatments, and the values correspond to the percentages of these genes in relation to the total number of genes in the category.

The microarray data analysis of the treatment with the subinhibitory concentration of copper (3 mM) showed 223 genes induced and 150 repressed. Among the induced genes the most remarkable feature was the induction of phage genes, as well as pathogenicity, virulence, and adaptation, including hemagglutinins, hemolysin secretion/activation protein, copper homeostasis protein, and drug efflux among others (Fig. 1 and see Fig. S2 and Table S2 in the supplemental material). The main group of repressed genes was involved in RNA and protein synthesis (functional categories I and III), as well as type IV pilus components (functional category IV, cell structure), suggesting a probable reduction in metabolism and movement (Fig. 1 and see Fig. S2 and Table S2 in the supplemental material).

The analysis of biofilm treatment with the inhibitory concentration of copper (7 mM) revealed 461 induced genes and 407 repressed. The higher number of genes differentially expressed observed at 7 mM copper suggests a dose-response mechanism (Fig. 2A and see Table S2 in the supplemental material). The increase corresponds to more genes being modulated inside the same functional categories, such as those involved with induction of phage and adaptation-related genes, and the repression of general metabolic function and movement, which indicates the activation of a common resistance/tolerance mechanism.

Fig 2.

Venn diagrams of gene expression responses of X. fastidiosa under different conditions. Common and unique expression patterns of X. fastidiosa exposed to subinhibitory and inhibitory concentrations of copper (A), subinhibitory concentrations of copper and tetracycline (B), and inhibitory concentrations of both compounds (C) are compared.

Comparative expression analysis of copper and tetracycline responses.

To verify the similarity in the mechanisms of resistance of X. fastidiosa biofilm to different antimicrobial compounds, microarrays were also performed for subinhibitory and inhibitory concentrations of tetracycline. The antibiotic concentrations used in these experiments were 100 and 800 μg/ml, respectively (see Fig. S3 in the supplemental material). The comparison between the subinhibitory and inhibitory treatments with two antimicrobial compounds (copper and tetracycline) shows different responses to each compound and concentration, even though some are common for both, the majority of them seems to be specific (Fig. 2B and C). In relation to the subinhibitory treatments, it was verified that 252 genes were modulated only in the presence of copper and 742 with tetracycline (Fig. 2B and see Table S3 in the supplemental material). With the inhibitory concentrations, we observed an opposite behavior regarding the gene modulation since 808 genes were differentially expressed in the presence of copper, whereas only 100 genes were differentially expressed in the presence of tetracycline (Fig. 2C and see Table S4 in the supplemental material). In contrast to our observations using copper, where a higher number of genes modulated in the inhibitory concentration indicated a dose-response mechanism, with tetracycline this response was not observed. These results suggest that X. fastidiosa biofilm cells may respond more efficiently to copper than to tetracycline.

For the set modulated just in the copper treatment, the most remarkable feature was the induction of phage-related genes, as well as genes related to pathogenicity, virulence, and adaptation such as the ones encoding hemagglutinins. Since these latter proteins have been shown to be involved in cell-cell adhesion in X. fastidiosa biofilm (14), this result suggests that biofilm maintenance could be a strategy to survive under copper stress conditions. Copper and tetracycline repressed several genes encoding type IV pilus proteins, which have been associated with X. fastidiosa movement (22). To better understand the transcriptional behavior of the genes involved in biofilm formation (pspA [hemagglutinin]) and movement (pilP and pilT) in the presence of copper and tetracycline, we used RT-PCR to investigate their expression patterns at different stages of biofilm development. As shown in Fig. S4 in the supplemental material, the gene expression profile at 15 days agrees with the microarray data, with a reduction in the expression of pili in the presence of both compounds and an induction of hemagglutinin-encoding gene with the copper treatment. Moreover, gene expression modulation clearly depends on the biofilm developmental phase in which the cells are treated with the antimicrobial compounds. We also analyzed the differences in biofilm morphology between treatments with inhibitory concentrations of copper and tetracycline by SEM and verified hemagglutinin expression by immunofluorescence analysis. Small biofilms were observed in the presence of both antimicrobial compounds, but with tetracycline the biofilms were smaller or the remaining cells were individually dispersed on the glass surface (Fig. 3, left). In addition, greater expression of hemagglutinin was observed in the biofilms in the presence of copper compared to the presence of tetracycline (Fig. 3, right). These results support the idea that keeping cells in biofilm may be a strategy for copper resistance in X. fastidiosa.

Fig 3.

(Left) Scanning electron micrographs of X. fastidiosa biofilm. Cells were evaluated after growing for 15 days on a glass surface (A and B) and exposure to inhibitory concentrations of copper (C and D) and tetracycline (E and F). The biofilms were visualized 24 h after the addition of the antimicrobial compounds. Bars correspond to 1 μm (A, C, and E) and 5 μm (B, D, and F). (Right) Fluorescence labeling of hemagglutinin in the X. fastidiosa biofilm. Biofilm cells without (A) or with 3 mM copper (B), 7 mM copper (C), 100 μg of tetracycline (D), and 800 μg of tetracycline (E) were marked in green, and the red spots correspond to the antibody recognition of hemagglutinin (secondary antibody labeled with rhodamine). In the first column are images representing hemagglutinin labeling, with X. fastidiosa cells depicted in red. In the second column are images representing Syto 9 staining, with X. fastidiosa cells depicted in green. In the third column are superposed images of the two channels (green and red). The presence of hemagglutinin is depicted in orange in the first column. Note that there is no or very low fluorescence associated with the tetracycline treatment. Scale bars, 20 μm.

In addition to the repression of movement, common repressed genes also include some encoding ribosomal proteins and general metabolic proteins (Fig. 2B and C). These results reinforce the idea that a decrease in metabolic functions, translation, and cell movement is a common response of the X. fastidiosa biofilm to copper and tetracycline. The repression of all of these genes in the presence of these antibacterial compounds suggests that the cells may reduce their metabolism when entering into a resistant physiological state.

The response to tetracycline and copper involves the production of persister cells.

One of the most interesting observations was the number of differentially expressed genes that encode components of TA systems in the treatments. Because of their reduced size, they are sometimes either misannotated or missed in sequenced genomes. We therefore performed a search for TA systems in X. fastidiosa 9a5c using the rapid automated search for toxins and antitoxins in bacteria (RASTA-Bacteria) tool (33). Using a likelihood score of 60%, we identified 65 genes in this genome that belong to the TA family of genes (see Table S5 in the supplemental material). The genes were carefully analyzed, and those that lay in operons were considered for further analyses. Among these, 12 TA systems were differentially expressed in our experiments (Table 1). Most of the TA systems were induced in treatments with both antimicrobial compounds, but we also observed specific induction according to the compound and concentration, such as one plasmodial TA system that was induced only with tetracycline treatments. The expression of TA systems has been associated with persister cells in different organisms. These cells neither grow nor die in the presence of bactericidal agents, representing a particular physiological state (20). Interestingly, treatments with 800 μg of tetracycline/ml and 7 mM copper led to a drastic reduction in cell growth, even though it was possible to purify RNA from cells collected before plating. To verify the percentages of persister cells in an X. fastidiosa population, we carried out an experiment where we treated the cells with inhibitory concentrations of copper and tetracycline and plated a serial dilution of the population (Fig. 4). In this experiment, the percentage of the population that survived was ca. 0.05%. This result is in accordance with the amount of persister cells (17), suggesting that X. fastidiosa biofilm contains persister cells that could play an important role in multidrug tolerance.

Table 1.

Probable TA loci differentially expressed in X. fastidiosa treated with a subinhibitory or inhibitory concentration of copper or tetracycline^a

LBI gene IDb	TA system Information	Fold changec				Localization	Description by BLASTP similarity
		3 mM CuSO4	7 mM CuSO4	Tetracycline
				100 μg/ml	800 μg/ml
XF1695	Toxin	2.182	2.357*	0.486*	0.499*	Chromosome (phage Xfp4)	SpoVT/AbrB-like domain
XF1696	Putative antitoxin	0.810	1.359	0.577*	0.455*	Chromosome (phage Xfp4)	Predicted DNA-binding protein with an HTH domain
XFa0045	Toxin (Gp49 superfamily)	1.514	0.673	2.026*	1.946*	Plasmid	Molecular function unknown
XFa0046	Antitoxin	2.219	0.438	4.712*	3.178*	Plasmid	DNA binding proteins belonging to the xenobiotic response element family of transcriptional regulators
XF2080	Toxin (RelE family)	1.098	0.865	3.370*	1.228	Chromosome	Addiction module toxin, RelE/StbE family
XF2081 (dinJ)	Antitoxin	2.543*	2.749*	2.299*	1.087	Chromosome	Addiction module antitoxin, RelB/DinJ family
XF1589 (Y4JK)	Toxin	1.713*	1.363	1.410	1.000	Chromosome (phage Xfp3)	Addiction module (protein contained PIN domain)
XF1590 (Y4JJ)	Antitoxin (probable)	1.301	0.972	2.041*	1.177	Chromosome (phage Xfp3)	Addiction module plasmid stabilization protein (StbC superfamily)
XF1596	Antitoxin (HigA)	2.298*	2.087*	1.540*	1.007	Chromosome (phage Xfp3)	Helix-turn-helix XRE family-like proteins
XF1597	Toxin (HigB)	2.778*	3.129*	2.208*	1.371	Chromosome (phage Xfp3)	Addiction module killer protein
XF1709	Toxin	0.939	0.866	2.529*	1.071	Chromosome (phage Xfp4)	Plasmid maintenance system killer
XF1710	Antitoxin	0.461*	0.699*	1.037	0.591	Chromosome (phage Xfp4)	Helix-turn-helix XRE family-like proteins
XF2490	Toxin (MqsR)	11.459*	8.473*	3.969*	1.023	Chromosome (phage Xfp2)	GCU-specific mRNA interferase toxin of the MqsR-MqsA TA system and biofilm/motility regulator
XF2491	Antitoxin	14.591*	8.445*	2.178*	0.931	Chromosome (phage Xfp2)	Sequence-specific DNA binding
XF2032 (parE)	Toxin	0.888	1.111	3.020*	0.828	Chromosome	Plasmid stabilization protein (ParE)
XF2031 (parD)	Antitoxin	1.158	1.040	2.532*	0.724	Chromosome	Plasmid stabilization protein (ParD)
XF2066 (yacB)	Toxin	1.436*	1.865*	5.717*	1.402	Chromosome	Addiction module toxin, RelE/StbE
XF2067	Hypothetical protein	2.388*	1.735*	2.803*	1.044	Chromosome	Conserved hypothetical protein
XF2068	Antitoxin	3.805*	5.068*	4.553*	1.347	Chromosome	RelB/DinJ family addiction module antitoxin
XF2074	Toxin	1.303	1.058	3.242	0.870	Chromosome	Addiction module toxin, RelE/StbE family
XF2075	Antitoxin	1.948	2.528*	3.657*	0.900	Chromosome	Helix-turn-helix protein, CopG
XF2126	Antitoxin	1.244	1.636*	2.118*	1.389	Chromosome	DNA binding proteins belonging to the xenobiotic response element family of transcriptional regulator
XF2125	Toxin (probable)	2.424*	4.556*	1.415	1.119	Chromosome	Hypothetical protein from X. fastidiosa
XF2763	Antitoxin	2.252*	1.531*	1.415	1.119	Chromosome	Putative addiction module antidote protein
XF2764	Toxin	2.558*	2.333*	1.293*	1.067	Chromosome	Addiction module killer protein (phage-derived protein Gp49-like)

^aDifferential expression was defined for values below 0.5 and above 2.0, which were considered repressed or induced respectively. Statistical analyses were also used to evaluate the data where a P value of 0.05 was the cutoff for significance.

^bSee http://www.lbi.ic.unicamp.br/xf/.

^c*, Genes differentially expressed using statistical analyses as specified in Materials and Methods.

Fig 4.

Survival of X. fastidiosa biofilm cells treated with inhibitory concentrations of copper and tetracycline. Cells were grown for 15 days, forming a mature biofilm, and then copper and tetracycline were added to final concentrations of 7 mM and 800 μg/ml, respectively. After 24 h (copper) and 48 h (tetracycline) of exposure, the cells were scraped, washed, diluted, and spot plated. The data are averages of the log percent survival of two independent experiments. Error bars indicate the standard errors of the means.

Persistence increases when cells are pretreated with copper.

Since the expression of TA systems directly correlates to the persistence phenotype in other bacteria (16, 17, 39), and since we observed the induction of expression of TA genes even in subinhibitory concentrations of copper and tetracycline, we hypothesize that a pretreatment with antimicrobial compounds could lead to an increase in the number of persister cells. To test this hypothesis, we performed an experiment using three different conditions. In condition 1, the cells were exposed to subinhibitory concentrations of copper or tetracycline for 15 days, and after that the cells were incubated with inhibitory concentrations. This treatment led to a decrease in biofilm development in both antimicrobial compounds. In addition, probably because of the low number of total cells, we were not able to recover persister cells from these experiments. Upon comparing conditions 2 (pretreated mature biofilm) and 3 (nonpretreated mature biofilm), we did not see any significant increase in the number of persister cells in the population pretreated with subinhibitory concentrations of tetracycline (0.9-fold). On the other hand, pretreatment with a subinhibitory concentration of copper induced a 26-fold increase in the number of persister cells.

DISCUSSION

Globally, the responses to copper and tetracycline present some similar mechanisms, but most of them are rather specific, which is expected since the compounds present very different modes of action. A model shown in Fig. 5 is presented for a better visualization of the common and specific responses that occur when X. fastidiosa biofilm is exposed to copper and tetracycline inhibitory and subinhibitory concentrations. Tolerance to antimicrobial compounds may occur in X. fastidiosa via a shutdown of target functions (e.g., protein synthesis and DNA replication), as well as an inhibition of movement. Modulation of bacterial metabolism, by altering transcription patterns or inhibiting growth by the inhibition of specific target functions, is a common response observed for bacteria treated with different antimicrobial compounds (1, 13, 17). Biofilm formation and phage activity seem to be copper-specific responses. Keeping the cells in biofilms in response to copper comprises the expression of hemagglutinins, as verified by gene expression (microarray and real-time PCR) and microscopy. Multilayer biofilm formation in X. fastidiosa depends on hemagglutinin functions of cell-cell adhesion (14). Thus, the expression of these proteins in the presence of copper could prevent biofilm disruption promoting a higher protection of the cells in biofilm.

Fig 5.

Schematic representation of X. fastidiosa genetic responses to copper and tetracycline. On the left are shown the gene-specific responses to copper (yellow oval), and on the right are shown the gene-specific responses to tetracycline (blue oval). The shared region represents common responses to both antimicrobial compounds. Upward-pointing arrows indicate induced genes, and downward-pointing arrows represent repressed ones. Small arrows correspond to 3 mM copper (in red) or 100 μg of tetracycline/ml (in blue), and large arrows represent 7 mM copper (in red) or 800 μg of tetracycline/ml (in blue). For more details, see the Discussion.

We also suggest that formation of persister cells is a mechanism of population survival in X. fastidiosa under both stress conditions. It has been demonstrated that, if the cells do not eliminate the compound effectively by multidrug resistance (MDR), the population starts producing persister cells that are multidrug tolerant (20, 21). Therefore, bacteria have evolved two complementary and highly redundant strategies to protect themselves from antimicrobials: multidrug efflux and, when this fails, tolerance of persister cells (20, 21). Few genes related to MDR were induced in our experimental conditions and, moreover, subinhibitory and inhibitory concentrations of both antimicrobial compounds led to induction of TA genes, which has been associated with the persistence phenotype (8, 16, 32). The majority of induced TA systems were found in phage regions, but their roles in bacteriophages have not been confirmed. In addition to the suggested role in maintaining the phage in the bacterial chromosome, phage TA systems may have a physiological function since the expression of phage toxins in Escherichia coli leads to growth arrest and a decrease in cell viability in a reversible manner by targeting the protein synthesis machinery (11, 19). Based on that, these phage TA systems in X. fastidiosa could have a role in bacteriostasis for an attempted survival of a small subpopulation of cells during stressful conditions similar to that recently described for the Treponema denticola biofilm (25).

There seems to be a concentration issue for the expression of TA systems. For tetracycline, modulation in expression was observed mostly for the subinhibitory concentration that correlates with the shutdown of metabolic functions. For copper, on the other hand, induction of the TA systems was observed in both concentrations but with an increase in the number of genes modulated in the presence of higher concentrations. Interestingly, the percentage of persister cells in treatments with both antimicrobial agents and inhibitory concentrations does not significantly differ. Since it is known that TA systems are related to the persistence phenotype, we suggest that the inhibitory tetracycline treatment leads to an expression of these TA systems in some early stage in the response to this compound, as seen for the subinhibitory concentration. On the other hand, pretreatment with the subinhibitory concentration of copper induced an increase of 26-fold in the amount of persister cells in the population. Since the modulation of these TA systems occurs in a broader range of concentrations with copper compared to tetracycline, we speculate that there could be an adaptation behavior of cell responses to this compound. An increase in the number of persister cells was also observed by Hong et al. (15) when E. coli cells were pretreated with oxidative and acid stresses. According to these authors, the decision whether the cell actively combats the stress or becomes persistent may depend on intensity and type of stresses. Thus, our results indicate that the induction of persister cells in pretreatment with copper, but not with tetracycline, could reflect the natural condition to which this bacterium is exposed since this compound is commonly used in agriculture. Therefore, this bacterium may be more adapted to the presence of copper with a probable activation of genes to improve the cell fitness to survive at higher copper concentrations.

Some TA systems found in X. fastidiosa are similar to other TA systems described for different bacteria such as relBE, higBA, and mqsRA. The expression of the toxin reduces metabolic functions, leading the cells to enter in a dormant, multidrug-tolerant state (21), which could be reversible (16). In addition, TA systems influence biofilm formation and the general stress response in different bacterial species (39). All of these physiological changes were observed in our experiments, reinforcing the participation of TA systems in X. fastidiosa stress response. Even though there is one report for functional characterization of a TA system in X. fastidiosa, which is similar to the R100 pemI pemK TA system of E. coli (18), there is no information about the function of TA systems in induction of persister cells for this bacterium. Nevertheless, the induction of these genes in X. fastidiosa under stress conditions, as observed for other microorganisms, suggests a similar function related to the protection of the cell population against death. The formation of persister cells seems to be a typical response in a population that decreases under unfavorable conditions: after the stress condition is reduced, these particular cells can repopulate, and the infection relapses (21). This is the first evidence of expression of TA systems associated with a survival strategy for a plant-pathogenic bacterium and reveals possible implications of the frequent use of antimicrobial agents, such as copper-containing compounds, in agriculture.