Twitching motility of Stenotrophomonas maltophilia under iron limitation: In-silico, phenotypic and proteomic approaches

Figures & data

Table 1. S. maltophiia strains used in this study and their average zone of twitching under normal and iron-depleted conditions

				Average zone of twitching in mm (SD)
S. maltophilia isolates	Biological source	Geographical source	Isolates information	Normal	Iron-depleted
In-silico
K279a	Clinical	Bristol, UK	Blood infection
R551-3	Environmental	Washington, USA	Poplar tree endophyte
D457	Clinical	Mostoles, Spain	-
JV3	Environmental	Brazil	Rhizosphere
Clinical strains
ATCC13637	Pleural fluid	Stafford, England	Oral carcinoma	0	0
CS17	Blood	Malaysia	-	15 (0.58)	20 (1.15)
CS24	Wound swab	Malaysia	-	0	0
SM5	Pus	Malaysia	Surgery	14 (0.58)	20 (2.31)
SM6	Pus	Malaysia	Surgery	24 (2.89)	25 (2.31)
SM7	CSF	Malaysia	Neurosurgery	14 (0.58)	17 (1.73)
SM8	CSF	Malaysia	Neurosurgery	0	0
SM9	Tracheal aspirate	Malaysia	Pediatric	0	0
SM10	Tracheal aspirate	Malaysia	Anesthesiology	0	0
SM11	CSF	Malaysia	Neurosurgery	26 (1.15)	26 (1.15)
SM20	Pus	Malaysia	Pediatric	13 (1.53)	18 (3.46)
SM22	Tracheal aspirate	Malaysia	Pediatric	16 (0.58)	16 (1.63)
SM23	Sputum	Malaysia	Respiratory	14 (0.58)	14 (1.00)
SM24	Blood	Malaysia	Urology and Nephrology	20 (1.53)	25 (1.53)
SM25	Tracheal aspirate	Malaysia	Pediatric	14 (0.58)	17 (1.15)
SM27	Blood	Malaysia	Urology and Nephrology	29 (1.73)	33 (2.08)
SM28	Tracheal aspirate	Malaysia	Pediatric	16 (1.73)	18 (1.73)
SM30	Tracheal aspirate	Malaysia	Anesthesiology	0	0
SM39	Tracheal aspirate	Malaysia	Anesthesiology	34 (1.00)	38 (2.89)
SM40	Blood	Malaysia	Surgery	33 (2.89)	36 (0.58)
SM41	Blood	Malaysia	Pediatric	29 (1.53)	35 (2.52)
SM42	Sputum	Malaysia	Urology and Nephrology	36 (2.31)	40 (4.17)
SM43	Sputum	Malaysia	Medical	0	0
SM44	Pus	Malaysia	Pediatric	22 (1.00)	27 (2.00)
SM45	Tracheal aspirate	Malaysia	Pediatric	45 (0)	50 (2.52)
SM46	Tracheal aspirate	Malaysia	Anesthesiology	0	0
SM47	Urine	Malaysia	Neurosurgery	0	0
SM48	Tracheal aspirate	Malaysia	Medical	34 (3.21)	34 (3.79)
Environmental strains
LMG959	Rice paddy	Japan	-	0	0
LMG10871	Soil	Japan	-	0	0
LMG10879	Rice paddy	Japan	-	23 (0.58)	23 (0)
LMG11104	Roots	Unknown	Cichorium intybus, rhizosphere tuberous roots	0	0
LMG11108	Roots	Unknown	Triticum, roots	0	0

0 indicate absence of hazy zone (no twitching motility observed).

Table 2. Functional roles of type IV pilus associated with twitching motility and their abbreviations obtained from RAST server

Targets^a	Functional Role	Locus tag^b
DQS	3-dehydroquinate synthase	SMLT_RS17230
pilD	Leader peptidase (Prepilin peptidase)	SMLT_RS17910
MTT	Multimodular transpeptidase-transglycosylase	SMLT_RS17525
pilDm	N-methyltransferase	SMLT_RS17910
pilT	Twitching motility protein PilT	SMLT_RS05270
pilSe	Two-component sensor PilS	SMLT_RS17880
pilR	Type IV fimbriae expression regulatory protein PilR	SMLT_RS17885
pilC	Type IV fimbrial assembly protein PilC	SMLT_RS17905
pilB	Type IV fimbrial assembly, ATPase PilB	SMLT_RS17890
fimT	Type IV fimbrial biogenesis protein FimT	SMLT_RS07795
pilV	Type IV fimbrial biogenesis protein PilV	SMLT_RS07795
pilW	Type IV fimbrial biogenesis protein PilW	SMLT_RS07800
pilX	Type IV fimbrial biogenesis protein PilX	SMLT_RS07810
pilY1	Type IV fimbrial biogenesis protein PilY1	SMLT_RS07815
pilA	Type IV pilin PilA	SMLT_RS17895
pilE	Type IV pilus biogenesis protein PilE	SMLT_RS07820
pilM	Type IV pilus biogenesis protein PilM	SMLT_RS18210
pilN	Type IV pilus biogenesis protein PilN	SMLT_RS18205
pilO	Type IV pilus biogenesis protein PilO	SMLT_RS18200
pilP	Type IV pilus biogenesis protein PilP	SMLT_RS18200
pilQ	Type IV pilus biogenesis protein PilQ	SMLT_RS18195

^aThe abbreviations of the targets and name of the functional roles are derived from RAST server; ^bCorresponding locus tag respective to S. maltophilia K279a from GenBank, NCBI.

Figure 1. (a) Typical subsurface twitching motility agar interpretation. A colony on the surface of agar around the inoculation point (top colony) and a visible halo or hazy zone of bacteria that have twitched across the plate between the bottom of the agar and the Petri plate (interstitial colony). (b) A representative of the zone of twitching motility on twitching agar plates on normal and iron-depleted conditions. P. aeruginosa ATCC27853 (positive control), SM45 (clinical isolate) and LMG10879 (environmental isolate). The plates were flooded with TM developer solution. The zone of twitching motility was marked, and 1 cm line was drawn onto the plates. (c) Box-and-whisker plot for clinical isolates under normal and iron-depleted conditions. The twitching zone under normal condition (min: 13 mm, median: 22 mm, max: 45 mm), while iron-depleted condition (min:14 mm, median: 25 mm, max: 50 mm)

Table 3. Differentially expressed motility-associated proteins identified via ITRAQ assay for CS17 and LMG959 isolates under iron-depleted condition

Protein name	Accession number^a	Percentage coverage^b	Fold change^c	p-value^d
CS17
Flagellar biosynthesis protein FliC	A0A0X3QSM8	73.5	20.14	0.0065
Flagellar biosynthesis protein FliC	A0A2Y9U6E5	75.0	1.54	0.0046
Pili assembly chaperone: bacterial pili assembly chaperone	A0A0U5I3M8	27.7	3.66	0.5431
Fimbrial protein	A0A0U5I9J4	33.5	0.70	0.0147
Twitching motility protein PilT	A0A501PPD4	13.6	99.08	0.0156
LMG959
Flagellar biosynthesis protein FliC	A0A0X3QSM8	75.3	17.70	0.2522
Flagellin	A0A2W6J2T0	50.0	1.43	0.6261
Fimbrial protein	A0A064BLK2	7.4	0.64	0.2778
Flagellar biosynthesis protein FliC	A0A0M0NGJ6	33.7	2.19	0.9019
Pili chaperone protein	A0A2T1I8L3	19.0	18.37	0.1309

^aUniProt protein accession number, ^b Percentage of identified peptides over the whole protein sequence, ^c Fold change comparing protein level for the strain growing under iron-depleted versus normal iron conditions, ^d the fold changes in bold are consider statistically significant, set at (p < 0.05).

Figure 2. Structure of type IV pili and mechanisms of bacterial twitching motility. (a) Structurally, T4P is composed of: (1) Major pilin PilA/PilE; (2) Secretin PilQ; (3) Alignment proteins PilM, PilN, PilO, PilP; (4) Retraction ATPase PilT; (5) Assembly ATPase PilB; and (6) Minor pilins PilX, PilW, PilV. (b) Pilus retraction results in the forward movement of the cell across the surface. Arrows indicate the direction of pilus retraction/extension

Data availability statement

The datasets generated and/or analysed during the current study are available in the ProteomeXchange Consortium database (http://proteomecentral.proteomexchange.org) via PRoteomics IDEntifications (PRIDE) with the dataset identifier PXD004370.