The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

Bacterial strains and growth conditions

Escherichia coli strain BL21.DE3 and Xanthomonas oryzae pv. oryzae (Xoo) Philippines race 6 PXO99^A (here after called PXO99) were used in this study. Transformed BL21.DE3 cultures were grown in lysogenic broth (LB) with 50 µg/mL kanamycin at 37°C and 230 rpm. PXO99 was cultured on peptone sucrose (PS) agar containing 20 µg/mL cephalexin. The mutant strains PXO99ΔraxST, PXO99ΔraxA, PXO99ΔraxB, PXO99ΔraxC, and PXO99Δax21 were described previously (Da Silva et al., 2004; Lee et al., 2009) and cultured in the presence of kanamycin at 50 µg/mL. Xanthomonas campestris pv. campestris (Xcc) strain 33913 and Xanthomonas euvesicatoria (Xe) strain 85–10 were cultured similarly to PXO99.

For enrichment of Ax21 in the supernatant, cells were grown in 10 mL of yeast extract broth (YEB) media (5 g/L yeast extract, 10 g/L tryptone, 5 g/L NaCl, 5 g/L sucrose, 0.5 g/L MgSO₄, pH 7.3) to an OD₆₀₀ of ∼1.5, spun down and resuspended in 2 mL of M9 minimal media containing 1.5% glucose and 0.3% casamino acids. Cultures were further incubated at 28°C for 48 h. Then, cells were spun down and the supernatant was passed through a 0.22 µM-filtering unit. The resulting supernatant was enriched for Ax21.

Rice inoculation, lesion measurements and in planta bacterial growth curve analysis

Rice (Oryza sativa) TP309 and a transgenic TP309 line carrying the Xa21 gene driven by its native promoter [TP309-XA21, derivative of line 106-17 (Song et al., 1995)], were germinated in distilled water at 28°C for one week, and then transplanted into soil and grown in 4.5-L pots (4 seedlings/pot) in a greenhouse. Five-to-six-week old plants were transferred to a growth chamber at least two days prior to inoculation. The growth chamber conditions were set to 28°C, 85% humidity and 14/10 day/night cycle. Xoo cells were prepared by culturing on PS agar plates with appropriate antibiotics. The cultures were incubated at 28°C for three days. Rice leaves were inoculated with the scissors-clipping method (Kauffman et al., 1973), using cells suspended in distilled water at a density of 10⁸ CFU (colony forming unit)/mL. Lesion lengths were measured 13–14 days after inoculation. The results represent the averages of measurements taken from more than 15 inoculated leaves per strain/plant genotype combination.

For in planta bacterial growth analysis, plants were inoculated as described above. At each time point, 6 leaves (from 3 different tillers) were harvested for each bacterial strain/plant genotype combinations. Two leaves from each tiller, representing one biological replicate, were cut to about 2 mm pieces and incubated in 10 mL of water for 2 h at 230 rpm. This suspension was then serially diluted and plated on PSA plates with appropriate antibiotics. Three days later the colonies were counted and the cell number per leaf calculated.

Statistical analysis was carried out using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software (SAS Institute Inc., Cary, NC, U.S.A.)

Plasmid construction

The DNA sequence encoding the entire ax21 gene was amplified from PXO99 genomic DNA and ligated into a modified pET27 vector (Merck KGaA, Darmstadt, Germany) such that the encoded protein has a C-terminal 8x-histidine tag. The sequence cloned included the entire protein with the longer signal peptide MKTSLLALGLLAALPFAASA (see Results and Discussion). Amino acids 18-20, Alanine-Serine-Alanine (the AXA motif), were mutated to Aspartate-Aspartate-Aspartate (DDD) by QuikChange site-directed mutagenesis.

Western blot

Western blots were carried out following standard procedures (Sambrook Fritsch & Maniatis, 1989). Ax21 protein was detected using an anti-Ax21 antibody. The generation of this antibody was first described in Han et al. (2011), and is described here again. Ax21 monoclonal antibodies were raised in rabbit against the 17 aa Ax21 synthetic peptide (axY ^S22). Peptides and antibodies were generated by Pacific Immunology (http://www.pacificimmunology.com/). For Western blots, the Ax21 antibody and a secondary anti-rabbit IgG antibody were used at dilutions of 1:3,000 and 1:5,000, respectively. Western blots detecting the His tag were performed using anti-His (Qiagen 34660) and anti-mouse IgG antibodies at dilutions of 1:2,000.

PXO99 supernatant fractionation and proteinase K treatment

To separate the outer membrane vesicle (OMVs) fraction from cell-free supernatants ultracentrifugation was applied at ∼180,000 g for 2 h. The resulting membrane pellet was resuspended in 200 µL of water. Proteinase K treatment was carried out by incubating 40 µL of the OMVs pellet with 2 µg of proteinase K at 37°C for 30 min.

Ax21 secretion does not require raxA, raxB, or raxC

In earlier experiments, we assayed for the presence of Ax21 in supernatants of wild type and the raxA and raxC mutant strains using silver staining of PAGE gels (Lee et al., 2009). To more carefully assess Ax21 secretion in these mutant strains, we used antibodies generated against Ax21. We carried out Western blot analysis to validate the presence of secreted Ax21 in the extracellular milieu. PXO99 cultures were grown similarly to the previously described methods for enriching for Ax21 in the supernatant (Han et al., 2011), and probed with an anti-Ax21 antibody (Fig. 1). As reported previously, Ax21 is detected in the supernatants of PXO99 (Lee et al., 2009). However, using this more sensitive and specific detection method, we found that Ax21 is also present in the supernatants of PXO99ΔraxA, PXO99ΔraxB and PXO99ΔraxC strains (Fig. 1). These results indicate that RaxA, RaxB and RaxC are not required for Ax21 secretion. These experiments also show that the Ax21 protein is of the mature size in all the samples, including PXO99ΔraxB, indicating that Ax21 does not require the presence of RaxB for processing, as was previously hypothesized (Lee et al., 2009; Lee et al., 2013).

We next tested if Ax21 is present in supernatants of Xcc. The rationale for this experiment is that Xcc lacks predicted orthologs of RaxA and RaxB. Thus, if Ax21 is secreted in Xcc, it would indicate that these genes are not required for its secretion. We found that Ax21 is present in Xcc supernatants grown under enriching conditions (Fig. S1). Together, these experiments indicate that Ax21 is not processed by RaxB and does not require the predicted type I secretion components (RaxA, RaxB and RaxC) for secretion as previously suggested (Lee et al., 2009; Lee et al., 2013; Han et al., 2011; Han et al., 2013).

These results conflict with publications by two independent research groups. The first, by McCarthy, Dow & Ryan (2011), showed that Ax21 activity from supernatants of S. maltophilia strain K279a carrying a mutation in a presumed raxB ortholog (Smlt2001) is reduced compared with that of supernatants from the wild type strain. From this experiment the authors concluded that RaxB is partially required for Ax21 secretion. However, our analysis of Smlt2001 reveals that it lacks the unique N-terminal peptidase domain (C39, residues 17-143 in the PXO99A genome annotation), characteristic of the RaxB ABC-transporter. Instead, Smlt2001 shows a much higher similarity to PXO_01193, a PXO99 ABC-transporter that lacks the N-terminal C39 peptidase domain. This analysis suggests that Smlt2001 is not an ortholog of RaxB. We were not able to identify a predicted RaxB ortholog in the S. maltophilia K279a genome. The second publication, by Wang et al. (2013), reported that Ax21 is not secreted in the absence of RaxA in Xoc. We have not tested the role of RaxA in Xoc.

Ax21 is processed by the general secretory (Sec) system

Many secreted proteins have N-terminal leader sequences, which are cleaved during export. We have shown through silver staining and Western blot analyses of Xoo supernatants that the size of Ax21 secreted from PX099 corresponds to the size of the mature, processed protein. MS/MS analysis of the excised band reveals that peptides corresponding to Ax21 lack the N-terminal region. These results indicate that the N-terminal region of Ax21 is cleaved before or during secretion (Lee et al., 2009; Lee et al., 2013). The involvement of RaxB, an ABC-transporter containing a peptidase domain, in triggering XA21-mediated immunity, led us to the hypothesis that RaxB processes Ax21 before or during secretion. RaxB carries an N-terminal C39 peptidase domain, which is predicted to recognize and cleave GG-leader peptides (Da Silva et al., 2004). Such leader sequences typically have the motif LSX₂ELX₂IXGG (where X can be any amino acid) with cleavage occurring immediately after the double glycine (Dirix et al., 2004). These are called GG-type or double glycine leader peptides. Ax21 lacks this motif.

Because RaxB does not process Ax21 (Fig. 1), and Ax21 lacks the GG-leader motif, we investigated other mechanisms of processing. Reanalysis of the Ax21 leader sequence revealed a signal peptide that typically targets proteins to the general secretory (Sec) pathway. The Sec system is the primary mechanism by which proteins are delivered across the microbial cytoplasmic membrane (Natale, Brüser & Driessen, 2008). Sec-targeted proteins are delivered to the primary Sec machinery by the secretion specific chaperone SecB (Natale, Brüser & Driessen, 2008). The protein is translocated across the membrane through the heterotrimeric integral membrane complex SecYEG driven by the associated molecular motor SecA. The signal peptide is cleaved from the protein during or shortly after translocation by a membrane bound signal peptidase.

We previously reported that secreted Ax21 is missing the first 15 amino acids MLALGLLAALPFAASA (Lee et al., 2009). This sequence is based on the annotation of the PXO99 genome (Salzberg et al., 2008). This annotation of the ax21 gene begins translation with the alternate start codon TTG. However, examination of the genomic DNA sequence suggests that translation likely begins at a typical ATG start codon 12 base pairs upstream. Moreover, a putative Shine-Dalgarno Sequence (AGG) is positioned 10 bp upstream of the ATG codon. Based on this analysis, we predict that the correct leader sequence is MKTSLLALGLLAALPFAASA. Most of the Ax21 homologs from other strains and species are annotated with similar longer leader sequences.

The program SignalP 4.1, which predicts the presence of classical, but not GG-type signal peptides, indicates that both the annotated shorter sequence (16 amino acids) and the longer sequence (20 amino acids) are predicted signal peptides (Fig. 2A) (Petersen et al., 2011). The Ax21 leader sequence has the hallmarks of a typical Sec signal peptide as shown in Fig. 2A. These include a positively charged N-terminus (n-region), a hydrophobic core (h-region), and a short C-terminal region containing the motif AXA (Natale, Brüser & Driessen, 2008). The leader peptidase cleaves the protein immediately after the AXA motif. This corresponds with the region that was found to be absent from extracellular Ax21 (Lee et al., 2009).

To test if Ax21 is processed by the Sec system and not by RaxB, we expressed recombinant C-terminal His-tagged Ax21 in E. coli (BL21.DE3), a strain that has no homologs of RaxB. We analyzed Ax21 processing by Western blot analysis, probing for the C-terminal His-tag. We observed that in total cell cultures and in cell pellets the N-terminus of Ax21 was processed (Fig. 2B). When the predicted ASA cleavage motif was mutated, Ax21 was no longer processed (Fig. 2B). Ax21 was not detected in E. coli supernatants probably since the conditions used for expression were not set for enriching Ax21 in the supernatant (see below and Fig. S4). Altogether, these results indicate that Ax21 possesses a typical Sec signal peptide, which is processed by the Sec machinery.

Ax21 secretion is associated with outer membrane vesicles

Not all proteins targeted to the Sec pathway are secreted. In Gram-negative bacteria, proteins processed by the Sec system can be destined to the inner membrane, periplasmic space, outer membrane, or the extracellular space. We previously showed that Ax21 was found in the supernatant of Xoo cultures, and culturing methods were developed for Ax21 enrichment in the supernatant (Lee et al., 2009; Han et al., 2011).

PFAM searches of the Ax21 sequence suggest that it falls into the family of β-barrel outer membrane proteins (OMP) (e-value = 9.1e−08) (Altschul et al., 1990; Sonnhammer, Eddy & Durbin, 1997). The β-barrel topology prediction servers PRED-TMBB (Bagos et al., 2004) and BOCTOPUS (Hayat & Elofsson, 2012) predict that Ax21 forms a 10-stranded β-barrel (Fig. S2). There are several known 10-stranded beta barrel proteins with solved three-dimensional structure (Vandeputte-Rutten et al., 2001; Prince, Achtman & Derrick, 2002; Eren et al., 2010). However, these have very low amino acid sequence similarity to Ax21. Using the automated homology-modeling program I-TASSER, Ax21 is predicted to form an 8-stranded β-barrel structure. Despite the variation between these prediction tools, all three show that Ax21 has clear features of an outer-membrane protein with a β-barrel structure (Koebnik, Locher & Van Gelder, 2000).

Because Ax21 is found in supernatants and due to its predicted β-barrel structure, we hypothesized that secreted Ax21 is embedded in outer membrane vesicles (OMVs). OMVs are spherical structures derived from the budding of sections of the outer membrane (McBroom & Kuehn, 2007). To test this hypothesis, we generated Xoo supernatants using the previously described method for enrichment of Ax21. The enrichment method used in our previous studies to purify Ax21 required growing Xoo to a high concentration, spinning cells down, removing the supernatant and resuspending the cell pellet in a small volume of minimal media. The concentrated culture was further incubated for 24–48 h. The cells were pelleted by centrifugation, and the supernatant was filtered through 0.22 µM filter. To separate the OMVs from soluble, secreted proteins, the cell-free supernatant was ultracentrifuged at ∼180,000 g for 2 h. The resulting supernatant and OMV fractions were probed for the presence of Ax21 by Western blot. As shown in Fig. 3, Ax21 can be detected in the enriched supernatant; however, it was absent from the supernatant following ultracentrifugation. In contrast, the OMVs pellet had a strong band representing Ax21, indicating that Ax21 in the cell-free supernatant was associated with OMVs. These results further suggest that Ax21 is not a soluble secreted protein, but rather an outer membrane protein secreted via the OMVs secretory pathway.

To test whether the secretion of Ax21 via the OMVs pathway is conserved in different Xanthomonas stains, we examined OMVs of Xcc and Xanthomonas euvesicatoria (Xe). These strains were chosen as representatives of strains lacking or containing RaxA/B orthologs, respectively. Similar to Xoo, Ax21 was associated exclusively with the OMV pellets of both strains (Fig. S1), suggesting that the secretion of Ax21 via the OMV secretory pathway is conserved among at least 3 different Xanthomonas species and is independent of the RaxA/B components.

To test whether Ax21 is embedded in membrane vesicles, or merely associated with them, isolated OMVs were treated with proteinase K and then analyzed by Western blot. While the majority of the OMV proteins appeared to be degraded, Ax21 was not (Fig. S3). Adding the Triton X-100 detergent caused a slight decrease in the intensity of the Ax21 band. These results indicate that Ax21 is embedded in the outer membrane and are consistent with the homology and structural prediction, which suggest that Ax21 is an OMP.

OMVs production is increased as a response to cellular stress (McBroom & Kuehn, 2007). This may explain why more Ax21 is found in the supernatant when Xoo is grown using the enrichment method as described above (Fig. S4). The high cell concentration and lack of nutrients may induce stress responses in the cells leading to elevated levels of OMVs production. Ax21 is not detected in supernatants from Xoo grown normally in rich media unless the supernatants are highly concentrated (Fig. S4).

Ax21 is not required for activation of XA21-mediated immunity

The findings that Ax21 is an OMP, processed by the Sec system and released in OMVs, does not fall in line with our previous findings and proposed model that Ax21 is a RaxA/B secreted activator of XA21 mediated immunity. We had previously reported that a PXO99Δax21 insertion mutant strain could overcome XA21-mediated immunity (Lee et al., 2009; Lee et al., 2013). However, a careful examination of our collection of bacterial mutant strains revealed that several strains were mixed up or had the incorrect genetic profile (Fig. S5) (Ronald, 2013).

We identified a correct PXO99Δax21 mutant strain in the collection, which we validated by PCR, Southern blot and Western blot (Fig. S5). The correct mutant had a kanamycin resistance cassette inserted near the C-terminus of the gene by double homologous recombination. We inoculated TP309 and TP309-XA21 with the validated mutant using the scissor clipping method. As shown in Fig. 4, PXO99Δax21 forms short lesions on TP309-XA21 rice, similarly to the wild type strain, while PXO99ΔraxST forms long water-soaked lesions as previously reported (Da Silva et al., 2004). These results were further supported by an in planta growth curve analysis, where no statistical differences in bacterial burden could be observed between the wild type strain PXO99 and the PXO99Δax21 throughout the time course (Fig. S6). To confirm this finding, we generated two additional ax21 mutant strains, using two different knockout plasmids. These mutants had identical in planta phenotype to the PXO99Δax21 strain (Fig. 4). These results indicate that the PXO99Δax21 mutant does not evade XA21-mediated immunity as previously reported (Lee et al., 2009; Lee et al., 2013).