The Ax 21 protein influences virulence and biofilm formation in

Ax21 is an outer membrane protein that is extensively conserved in plant pathogenic Xanthomonas and the associated genera Xylella and Stenotrophomonas, including S. maltophilia, some strains of which are hospital-acquired human pathogens (Ryan et al. 2009). Studies on Ax21 in Xanthomonas oryzae pv. oryzae (Xoo) have shown that the protein can be sulphated and that it is secreted into the bacterial medium in association with the outer membrane vesicles (Han et al. 2012; Bahar et al. 2014). Although a sulphated peptide derived from the N-terminus of Ax21 was originally thought to be a specific trigger for XA21-dependent innate immunity in rice, subsequent work has shown that this is not the case. Nevertheless, this peptide does induce defence-related responses in plants (Danna et al. 2011). A second proposed role for Ax21 is as a diffusible signal that controls the gene expression in Xoo as a response to bacterial cell density (Bahar et al. 2014). These observations led us to examine the potential role(s) of Ax21 in S. maltophilia. In 2011, it was reported that the mutation of ax21 had effects on different phenotypes in S. maltophilia (McCarthy et al. 2011). However, this paper was recently retracted due to errors in data presentation of Fig. 2 (McCarthy et al. 2017). Here, we report on the outcomes of repeated key experiments that indicate the pleiotropic nature of ax21 mutation and the effects of addition of the Ax21 protein on the restoration of the wild-type phenotype. Here, our aim was to repeat key experiments that indicate the pleiotropic nature of ax21 mutation and the effects of the addition of the Ax21 protein on the restoration of the wild-type phenotype. Abstract Stenotrophomonas maltophilia is an antibioticresistant Gram-negative pathogen, which is associated with hospital-acquired infection. The genome encodes a protein highly related to the Ax21 protein of Xanthomonas oryzae that is implicated in interactions of this plant pathogen with rice. Here, we report on the pleiotropic nature of ax21 mutation in S. maltophilia and the effects of addition of the Ax21 protein on the restoration of the wild-type phenotype. We show that loss by mutation of Ax21 leads to reduced motility, reduced biofilm formation, reduced tolerance to the antibiotic tobramycin and reduced virulence to larvae of Galleria mellonella, as well as alteration in the expression of specific genes associated with virulence or antibiotic resistance. Addition of the Ax21protein restored motility and the level of gene expression towards wild type. These findings are consistent with the notion that the Ax21 protein is involved in intraspecies communication, although other interpretations cannot be discounted.


Introduction 24
Ax21 is an outer membrane protein that is extensively conserved in plant pathogenic 25 Xanthomonas and the associated genera Xylella and Stenotrophomonas, including S. 26 maltophilia, some strains of which are hospital acquired human pathogens (Ryan et al., 27 2009). Studies on Ax21 in Xanthomonas oryzae pv. oryzae (Xoo) have shown that the 28 protein can be sulfated and that it is secreted into the bacterial medium in association with 29 outer membrane vesicles (Han et al., 2012;Bahar et al., 2014). Although a sulfated 30 peptide derived from the N-terminus of Ax21 was originally thought to be a specific 31 trigger of XA21-dependent innate immunity in rice, subsequent work has shown that this 1 is not the case. Nevertheless, this peptide does induce defense-related responses in plants 2 (Danna et al., 2011). A second proposed role for Ax21 is as a diffusible signal that 3 controls gene expression in Xoo as a response to bacterial cell density (Bahar et al., 4 2014). These observations led us to examine the potential role(s) of Ax21 in S. 5 maltophilia. In 2011, it was reported that mutation of ax21 had effects on different 6 phenotypes in S. maltophilia (McCarthy et al., 2011). However, this paper was recently 7 retracted due to errors in data presentation of Figure 2 (McCarthy et al., 2017). Here we 8 report on the outcomes of repeated key experiments that indicate the pleiotropic nature of 9 ax21 mutation and the effects of addition of the Ax21 protein on restoration of the wild-10 type phenotype. 11 12

Bacterial Strains and Growth Conditions 14
The wild-type S. maltophilia was strain K279a (Crossman et al., 2008). A mutant with a 15 deletion of smlt0387 (designed as ax21) was generated using pEX18Gm (Hoang et al., 16 1998). For complementation studies, the smlt0387 gene was cloned into pBBR1MCS 17 (Kovach et al., 1995). Strains and plasmids used during this study are detailed in Table 1. 18 For the majority of experiments, NYGB medium was used as growth media for S. 19 maltophilia strains, which comprises 20 g/L glycerol, 3 g/L yeast extract (Difco) and 20 5 g/L bacteriological peptone (Oxoid). While the assessment bacterial clumping or 21 biofilm formation was carried out in L medium, which comprises of; sodium chloride, 5 22 g/L; yeast extract, 5 g/L; Bactotryptone (Difco), 10 g/L and D-glucose, 1 g/L. Peptides 23 Ax21 (Smlt0387) and Ax21Y (Smlt0387 with Y altered to A) used in experiments were 24 generated by Cambridge Peptides and used at 500 nM unless otherwise stated. 25

RNA extraction and qRT-PCR. 27
For RNA extractions, S. maltophilia strains were cultivated at 30°C in NYGB broth 28 (without antibiotic) to logarithmic phase (OD 600 ≈ 0.8). A volume of 800 µl of RNA 29 protect (Qiagen) was added to 400 µl of culture and incubated at room temperature for 30 5 min. These suspensions were centrifuged and the resulting pellets were stored at −80°C 31 after removal of the supernatant. Following the manufacturer's instructions total RNA 1 was isolated from cells after thawing, using the RNeasy Mini Kit (Qiagen) and then 2 treated with DNase (Ambion). PCR was used to confirm removal of DNA contamination. 3 Specific RT-PCR primers were used to amplify central fragments of approximately 4 200 bp in length from smlt1112, smlt1390, smlt2175 and smlt3949 are described in Table  5 2. Quantification of gene expression was assessed using a Rotor-Gene Q (Qiagen) and 6 QuantiFast SYBR Green PCR Kit (Qiagen). 7 8

Motility assays 9
Bacterial motility was assayed on NYGB that was solidified using 0.6% Eiken agar 10 (Eiken Chemical, Tokyo). A sterile 200-µl tip was used to inoculate S. maltophilia strains 11 to the centre of the plate. Plates are visualized after incubated at 30°C for 48 h. Killing curves were carried out at 30°C as previously described by Macfarlane et al. 24 (1999). S. maltophilia strains were grown to mid-log phase on NYGB and then diluted to 25 10 6 in 100 mL of pre-warmed PBS containing antibiotic as indicated. Similarly, an 26 antibiotic-free control was inoculated. At 0, 10, 20, 30, 50, 100, 120 and 180 min after 27 antibiotic exposure, 0.1 mL volumes were removed, diluted in PBS and inoculated onto 28 NYG agar plates. In order to determine viable CFU, these plates were incubated for 24 h 29 at 30°C. 30

Virulence assay 1
Galleria mellonella larvae were stored at 4°C in wood shavings. For experiments, live 2 versus dead larvae were observed after 24 h post-infection. G. mellonella were injected 3 with 10µl of successively diluted bacteria (1 × 10 6 CFU). Infected G. mellonella were 4 placed on Whatman paper lined Petri dishes and incubated at 37°C. The G. mellonella 5 were monitored for their survival after a 24-h period. Three separate tests were conducted 6 consisting of 10 larvae for each strain. The control groups for each experiment consisted 7 G. mellonella injected with PBS alone and a group of uninfected G. mellonella. Addition of the protein at 500 nM restored wild type motility (Fig. 3A). A variant Ax21 4 protein (Ax21Y) in which the tyrosine residue that is sulfated in Ax21 of Xoo was 5 replaced by an alanine residue also restored motility (Fig. 3A). This is intriguing since S. 6 maltophilia lacks homologs of RaxST believed to be responsible for the sulfation of 7 Ax21 in Xoo. through an influence on DSF signaling. 30

Ax21 influences a diverse range of functions in the nosocomial pathogen 2
Stenotrophomonas maltophilia leading to altered virulence, tobramycin tolerance and 3 biofilm formation. Further work is needed to establish whether Ax21 is truly a cell-cell 4 signal. 5 6 Acknowledgements 7 We thank Robert Ryan, Max Dow and Delphine Caly for initial data, helpful discussions 8 and critical reading of the manuscript. 9

References 10
Bahar O, Pruitt R, Luu DD, Schwessinger B, Daudi A, Liu F, Ruan R, Fontaine-Bodin L, 11 Koebnik R, Ronald P. 2014. The Xanthomonas Ax21 protein is processed by the general 12 secretory system and is secreted in association with outer membrane vesicles.