In silico detection of virulence gene homologues in the human pathogen sphingomonas spp.

There is an ongoing debate about the clinical significance of Sphingomonas paucimobilis as a virulent bacterial pathogen. In the present study, we investigated the presence of different virulence factors and genes in Sphingomonas bacteria. We utilized phylogenetic, comparative genomics and bioinformatics analysis to investigate the potentiality of Sphingomonas bacteria as virulent pathogenic bacteria. The 16S ribosomal RNA gene (16S rDNA) phylogenetic tree showed that the closest bacterial taxon to Sphingomonas is Brucella with a bootstrap value of 87 followed by Helicobacter, Campylobacter, Pseudomonas, and then Legionella. Sphingomonas shared no virulence factors with Helicobacter or Campylobacter, despite their close phylogenic relationship. In spite of the phylogenetic divergence between Sphingomonas and Pseudomonas, they shared many major virulence factors, such as adherence, antiphagocytosis, iron uptake, proteases, and quorum sensing. In conclusion, Sphingomonas spp. contains several major virulence factors resembling Pseudomonas sp., Legionella sp., Brucella sp., and Bordetella sp. virulence factors. Similarity of virulence factors did not match phylogenetic relationships. These findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor. Sphingomonas spp. is potential virulent bacterial pathogen.

Introduction

Sphingomonas is a group of Gram-negative, rod-shaped, non-spore-forming, chemoheterotrophic, strictly aerobic bacterium that produces yellow or off-white pigmented colonies. The distinctiveness of Sphingomonas lies in its possession of ubiquinone 10 as its major respiratory quinone, presence of glycosphingolipids (GDLs) in their cell envelopes, and its metabolic versatility.1 The genome of Sphingomonas is approximately 3,948 kB and contains 70 structural RNAs. It encodes for approximately 3,914 proteins.2 Sphingomonas utilize glucose as its primary source of carbon. However, it can also utilize a wide variety of other sugars such as arabinose, fucose, glactose, lactose, mannose, melibiose, sucrose, trehalose, and xylose. Moreover, Sphingomonas can also degrade polysaccharides, and many of the strains were also able to utilize one or more of the contaminants as their source of carbon.2 The capability of Sphingomonas to utilize a wide range of organic compounds, and to grow and survive under low-nutrient conditions has resulted in its widespread distribution in various environments, including drinking water, soil, air, sinks, shower curtains, and corroding copper pipes. In addition, Sphingomonas paucimobilis has also been found in several clinical specimens, which include hospital water supplies; temperature probe respirators; stocked distilled water; blood; removes; hospital dialysis equipment; patients with meningitis, septicemia, bacteremia, and peritonitis; and wound infections.3 Reported cases of nosocomial infections caused by S. paucimobilis are rarely serious and could be effectively treated with antibiotics. On the contrary, some other reports have concluded that S. paucimobilis nosocomial infections have the ability to severely threaten immune-compromised or ill patients causing health problems with consistent exposure to the source of infection such as shower curtains.4 For example, in 2007, reports concluded that S. paucimobilis was the cause of bacteremia outbreak in the hemato/oncology units in Gülhane Military Hospital in Ankara, Turkey. The reports also showed that the clinical isolates were traced back neither to the health care workers nor to the environmental isolates.5 Moreover, it was strongly documented that S. paucimobilis created significant problems in various clinical settings, being the most widespread cause of nosocomial infections including bacteremia/septicemia caused by contaminated solutions such as distilled water, hemodialysis fluid, and sterile drug solutions. Cases of pseudo-bacteremia have been recorded in association with S. paucimobilis, as have many cases of unusual infections both invasive and severe, eg, septic arthritis and osteomyelitis.6 Moreover, S. paucimobilis caused bloodstream infection in a patient with Down syndrome. It was thereby concluded that S. paucimobilis should be recognized as a nosocomial infectious agent in patients with Down syndrome and immunosuppressive disorders.7 In addition, it was also reported that S. paucimobilis has the ability to cause infections in both previously healthy and immune-compromised children8 and can act as a causal agent of osteomyelitis in an immune-competent patient.9 Frequent S. paucimobilis infections were observed among our diabetic foot ulcer patients (23% of observed Gram-negative infections). S. paucimobilis general infection rate was 9.5%, falling just behind Staphylococcus aureus (unpublished data). There is still an ongoing debate about the clinical virulence of S. paucimobilis with a possible conclusion that its clinical importance cannot be neglected. Henceforth, this study employs comparative genomics and bioinformatics techniques in order to investigate the pathogenic potentials of Sphingomonas spp.

Materials and Methods

Phylogenetic relationships reconstruction

Partial 16S rDNA sequences of selected pathogenic bacteria, namely, S. paucimobilis (D16144.1), Bacillus anthracis (GQ280034.1), Bartonella bacilliformis (AF442955.1), Brucella sp. (DQ413258.1), Burkholderia sp. (AB379686.1), Campylobacter jejuni (AY621112.1), Clostridium difficile (HM245939.1), Corynebacterium sp. (D83375.1), Escherichia coli (KC504012.1), Haemophilus sp. (AB004027.1), Helicobacter sp. (AY034821.1), Legionella pneumophila (NR_041742.1), Neisseria meningitidis (AF059671.1), Pseudomonas sp. (AB379690.1), Salmonella typhimurium (DQ153191.1), Shigella spp. (JN626189.1), Vibrio cholerae (Z21856.1), Yersinia pestis (AJ232235.1), were all acquired from the GenBank. These sequences were then aligned using the Bioedit built-in clustal W program (gap opening penalty = 10, gap extension penalty = 5, delay divergent sequences = 40%). The resulting alignments were compared, and the final alignments were improved manually and prepared in FASTA and MEGA formats using format converter tool v2.2.5 available online at http://www.hiv.lanl.gov/content/sequence/FORMAT_CONVERSION/form.html.

In order to establish the phylogenetic relationships among taxa, tree was constructed using the maximum likelihood (ML) method based on the Tamura–Nei model.10 The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search was(were) obtained automatically by applying Neighbor-Joining and BIONJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then the topology was selected with superior log-likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 18 nucleotide sequences. Codon positions included were first + second + third + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 253 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.11

Comparative genomics and bioinformatics analysis

Virulence genes sequences and functions, corresponding to different major bacterial virulence factors of selected pathogens, were collected from GenBank and validated in virulence factors of pathogenic bacteria database at http://www.mgc.ac.cn/VFs/. Supplementary Table 1 shows the tested major pathogenic virulence factors. Selected gene sequences were tested against available Sphingomonas gene information using Sphingomonas nucleotide BLAST tool available at http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=MicrobialGenomes_13687&DB_GROUP=AllMG. The search set all Sphingomonas complete genomes, and selected organism was Sphingomonas (tax Id: 13687). The program selected for the search was blastn, optimizes for fairly similar sequences because of evolutionary divergence of the tested and query taxa.

Results and Discussion

In this study, the presence of the major known bacterial virulence factors in Sphingomonas spp. was examined. In order to decide on the accurate common pathogenic bacterial species for comparison with Sphingomonas spp., a phylogenetic tree using the ML method was constructed using partial 16S rDNA sequences of selected pathogenic bacteria as mentioned above (Fig. 1). The phylogenetic tree showed that the selected bacterial species are divided into two major clades (groups), namely, Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacterial group contained Clostridium spp., Corynebacterium sp., and Bacillus spp., whereas the Gram-negative bacterial group contained the remaining bacterial species. These results also agree with the known taxonomic arrangement of the tested bacterial species with the exception of Bartonella spp. that accrued in an independent clad outside the Gram-negative bacteria. More importantly, the 16S rDNA phylogenetic tree showed Brucella sp. to be the closest bacterial taxon to Sphingomonas with a bootstrap value of 87 followed by Helicobacter spp., Campylobacter sp., Pseudomonas sp., and then Legionella sp. Based on the suggested phylogenetic relationships, the following bacterial species which include Brucella sp., Helicobacter spp., Campylobacter sp., Pseudomonas sp., and Legionella sp. were selected for further comparative genomic and bioinformatics analyses. Table 1 presents the five selected bacterial genera with its corresponding species, the selected hosts, and the diseases caused by these pathogens. The selected pathogens were mainly human pathogens having also the ability to infect mammals, protozoa (Legionella sp.), and plants (Pseudomonas syringae). All the virulent factors acquired by these pathogens (Table 2) were tested for their presence in Sphingomonas genomic information. The major categories of bacterial virulence factors such as adherence, endotoxin, mobility, secretion systems, quorum sensing, and many others are shown in (Table 2).

Figure 1

Partial 16SrDNA-based Maximum Likelihood (ML) phylogenetic tree for a major pathogenic bacterial taxa utilizing base substitution Tamura–Nei model.

Table 1

Major pathogenic taxa used in the comparative analysis against Sphingomonas spp.

GENUS	SPECIES	HOST	DISEASE
Brucella	B. abortus	Human and cattle	Brucellosis, Osteoarthritis, endocarditis and several neurological disorders.
	B. canis	Human and dogs
	B. melitensis	Human goats and sheep
	B. ovis	Sheep
	B. suis	Human and pigs
Helicobacter	H. acinonychis	Humans and other mammals	Bacterial carcinogen, Gastroduodenal diseases
	H. hepaticus
	H. pylori
Campylobacter	C. fetus	Humans	Bacterial gastroenteritis Guillain-Barre syndrome (GBS)
	C. jejuni
Legionella	L. pneumophila	Humans and protozoa	Legionnaires’ disease
Pseudomonas	P. aeruginosa	Human	Eye, burn and wound infections
	P. syringae	Plant	Bacterial speck and bacterial blight

Table 2

Major pathogenic virulence factors and its corresponding genes and functions used in the comparative analysis against Sphingomonas spp.

GENUS	MAJOR TESTED VIRULENCE FACTORS	CORRESPONDING TESTED GENES OR FUNCTION
Brucella	Immune-evasion	Btp1/TcpB (Toll-interleukin-1 receptor (TIR) domain)
	Intracellular survival	Cyclic β-1,2-glucan synthase (Cgs), gmd; manA; manC; per; pgm; pmm/manB; wbkA; wbkB; wbkC; wzm; wzt; and RicA (Rab2 interacting conserved protein A)
	Regulation	BvrR-BvrS two-component system
	Secretion system	BMEII0025; BMEII0026; BMEII0027; BMEII0028; BMEII0029; BMEII0030; BMEII0031; BMEII0032; BMEII0033; BMEII0034; BMEII0035; type IV secretion system
Helicobacter	Adherence	babA; babB; hopZ; sabA;
	Endotoxin	gluE; gluP; kdtB; lpxB; rfaC; rfaJ; rfbD; rfbM; wbcJ; wbpB;
	Enzyme	ureA; ureB; ureE; ureF; ureG; ureH; ureI;
	Molecular mimicry	fucT; fucU; neuA; neuB;
	Motility	flaA; flaB; flgG
	Proinflammatory effect	napA; oipA;
	Secretion system	cag1; cag10; cag11; cag12; cag13; cag14; cag15; cag16; cag17; cag18/cagL; cag19; cag2; cag20; cag21; cag22; cag23 (cagE/picB); cag24; cag25; cag3; cag4; cag5 (virD4); cag6; cag7; cag8; cag9; virB11; type IV secretion system.
	Toxin	vacA
	Type IV secretory protein	cagA
	Pathogenicity islands	cag-PAI
Campylobacter	Adherence	cadF; Cj1415c; Cj1416c; Cj1421c; Cj1422c; Cj1423c; Cj1425c; Cj1426c; Cj1427c; Cj1429c; Cj1430c; Cj1431c; Cj1432c; Cj1433c; Cj1434c; Cj1435c; Cj1436c; Cj1437c; Cj1438c; Cj1440c; Cj1442c; fcl; glf; gmhA2; kfiD; kpsC; kpsD; kpsE; kpsF; kpsM; kpsS; kpsT; Cj0983; Cj1135; Cj1136; Cj1137c; Cj1138; Cj1139c; Cj1140; Cj1144c; Cj1145c; gmhA; htrB; neuA1; neuB1; neuC1; waaC; waaD; waaE; waaF; waaV; porA; peb1 A.
	Invasion	ciaB, ciaC (invasion antigens)
	Motility	Cj0371; Cj1312; Cj1313; flaA; flaB; flaC; flaD; flaG; flgB; flgC; flgD; flgE; flgE2; flgG; flgG2; flgH; flgI; flgK; flhA; flhB; flhF; fliA; fliD; fliE; fliF; fliG; fliH; fliI; fliL; fliM; fliN; fliP; fliQ; fliR; fliS; fliY; motA; motB; pflA; ptmA; ptmB.
	Secretion system	Cjp54; virB10; virB11; virB4; virB8; virB9; virD4; type IV secretion system.
	Toxin	cdtA; cdtB; cdtC;
Legionella	Adherence	htpB; omp28; pilB; pilC; pilD;
	Enzyme	mip;
	Iron uptake	ccmC; iraaB; frgA; feoA; feoB;
	Motility	flaA; flgA; flgB; flgC; flgD; flgE; flgF; flgG; flgH; flgI; flgK; flgL; flhA; flhB; flhF; fliA; fliD; fliE; fliF; fliG; fliH; fliJ; fliM; fliN; fliO; fliP; fliQ; fliR; fliS; motA; motB;
	Nutrient acquisition	phtA;
	Regulation	csrA; letA; letS; relA; rpoS;
	Secretion system	lspD; lspE; lspF; lspG; lspH; lspI; lspJ; lspK; lspL; lspM; pilD; (Type II secretion system). dotA; dotB; dotC; dotD; icmB; icmC; icmD; icmE; icmF; icmG; icmH; icmJ; icmK; icmL; icmM; icmN; icmO; icmP; icmQ; icmR; icmS; icmT; icmV; icmW; icmX; lepA; lepB; lidA; lvgA; ralF; sdeA/laiA; sdeB; sdeC; sdeD; sidA; sidB; sidC; sidE; sidF; sidG; sidH; vipA; vipD; vipE; vipF; wipA; wipB; wipC; ylfA; ylfB (type IV secretion system)
	Stress protein	gspA (global stress gene) katA; katB; sodB; sodC;
	Toxin	RtxA
	Unclassified	enhA; enhB; enhC; ligA;
Pseudomonas	Adherence	fleN; fleQ; fleR; flgC; flgD; flgE; flgF; flgG; flgH; flgI; flgJ; flgK; flgL; flhA; flhB; flhF; fliC; fliD; fliE; fliF; fliG; fliH; fliI; fliJ; fliM; fliN; fliO; fliP; fliQ; fliR; waaA; waaC; waaF; waaG; waaP; wzy; wzz; chpA; chpB; chpC; chpD; chpE; fimT; fimU; fimV; pilA; pilB; pilC; pilD; pilE; pilF; pilG; pilH; pilI; pilJ; pilK; pilM; pilN; pilO; pilP; pilQ; pilR; pilS; pilT; pilU; pilV; pilW; pilX; pilY1; pilY2;
	Antiphagocytosis	Alg44; Alg8; algA; algB; algD; algE; algF; algG; algI; algJ; algK; algL; algP; algQ; algR; algU; algX; algZ; mucA; mucB; mucC;
	Biosurfactant	rhlA; rhlB;
	Iron uptake	fptA; pchA; pchB; pchC; pchD; pchE; pchF; pchG; pchH; pchI; pchR; fpvA; pvdA; pvdD; pvdE; pvdS;
	Pigment	phzM; phzS (Pyocyanin)
	Protease	aprA; lasA; lasB.
	Regulation	lasI; lasR; rhlL; rhlR;
	Secretion system	xcpP; xcpQ; xcpR; xcpS; xcpT; xcpU; xcpV; xcpW; xcpX; xcpY; xcpZ (Type II secretion system)
	Toxin	toxA; exoS; exoT; exoU; exoY; plcH;

Table 3 and Figure 2 present the shared virulence factors among Sphingomonas and the selected five bacterial pathogens. Results in Table 3 showed that Sphingomonas spp. shares the genes responsible for intracellular survival ability (Cgs, manC, and pgm) with Brucella sp. with e-values ranging from 0 to 3.00E − 09.12–14 In addition, Sphingomonas spp. shares the genes encoding for Type IV secretion system such as BMEII0026 with Brucella sp. with e-value of 6.00E − 04 and identity similarity of 90%. On the contrary, Sphingomonas spp. shared no virulence factors with Helicobacter spp. or Campylobacter sp., despite their close phylogenic relationship when compared to Pseudomonas sp. and Legionella sp. Sphingomonas spp. shared Legionella sp. genes responsible for adherence and motility, namely, htpB and flip. Moreover, they also shared the gene responsible for stress tolerance and sodB. The sodB encodes for superoxide dismutase, which is a cytoplasmic iron superoxide dismutase important for intracellular survival and transmission.15

Table 3

Comparative analysis against Sphingomonas spp. against major bacterial virulence factors and functions from different pathogenic bacteria.

BACTERIAL TAXA	MAJOR BACTERIAL VIRULENCE FACTORS (VFS)	SUB VFS	RELATED GENE	E VALUE	IDENT	Sphingomonas GENBANK ACCESSION
Brucella	Intracellular survival and Immuno-modulatory activity	CβG (cyclic β-1,2 glucan)	cgs	6.00E-59	66%	CP006644.1
		Mannose-1-phosphate guanylyltransferase	manC	3.00E-09	77%	NC_009511.1
		Phosphoglucomutase	pgm	0	74%	NC_009511.1
	Secretion system	VirB type IV secretion system	BMEII 0025	4.00E-04	83%	CP006644.1
		VirB type IV secretion system	BMEII 0026	6.00E-04	90%	NC_020561.1
		VirB type IV secretion system	BMEII 0035	4.00E-06	72%	NC_020561.1
Legionella	Adherence	Hsp60	htpB	2.00E-25	64%	NC_020561.1
	Motility	Flagella	fliP	4.00E-11	68%	NC_020561.1
	Stress protein	SodB	sodB	3.00E-05	82%	NC_020561.1
Pseudomonas	Adherence	Flagella	fleQ	4.00E-65	72%	NC_009511.1
			flgE	5.00E-19	67%	NC_009511.1
			flgF	7.00E-14	71%	NC_009511.1
			flgG	4.00E-55	68%	NC_020561.1
			flgH	4.00E-29	72%	NC_020561.1
			flgI	6.00E-93	68%	NC_020561.1
			flgJ	6.00E-05	89%	NC_020561.1
			flgK	1.00E-04	94%	NC_009511.
			flhA	2.00E-141	69%	NC_020561.1
			flhB	1.00E-18	68%	NC_020561.1
			flhF	6.00E-05	89%	NC_020561.1
			fliC	1.00E-32	72%	NC_009511.1
			fliE	6.00E-04	74%	NC_020561.1
			fliF	3.00E-04	66%	NC_020561.1
			fliG	8.00E-15	69%	NC_020561.1
			fliH	4.00E-05	85%	CP006644.1
			fliI	1.00E-115	71%	NC_009511.
			fliN	3.00E-16	70%	NC_009511.1
			fliP	9.00E-102	75%	NC_020561.
			fliQ	2.00E-08	84%	NC_020561.1
			fliR	7.00E-08	72%	NC_020561.1
GENUS	MAJOR VFS	SUB VFS	RELATED GENE	E VALUE	IDENT	ACCESSION
Pseudomonas	Adherence	LPS (lipopolysaccharide)	waaA	2.00E-04	89%	CP006644.1
			waaG	4.00E-07	76%	NC_020561.1
			waaP	1.00E-04	83%	NC_020561.1
		Type IV pili	chpA	7.00E-26	67%	NC_020561.1
			chpC	8.00E-05	82%	NC_020561.1
			chpE	3.00E-05	86%	CP006644.1
			fimU	3.00E-04	93%	NC_009511.1
			pilB	3.00E-04	93%	NC_009511.1
			pilD	4.00E-05	76%	CP006644.1
			pilF	4.00E-05	97%	CP006644.1
			pilG	2.00E-04	80%	NC_020561.1
			pilH	2.00E-04	77%	NC_009511.1
			pilI	7.00E-06	84%	CP006644.1
			pilJ	5.00E-21	76%	NC_020561.1
			pilK	3.00E-06	83%	NC_020561.1
			pilN	8.00E-04	82%	CP006644.1
			pilQ	1.00E-09	73%	NC_009511.1
			pilR	9.00E-54	70%	NC_009511.1
			pilS	6.00E-06	74%	NC_020561.1
			pilT	1.00E-07	83%	NC_009511.1
			pilU	1.00E-12	75%	NC_009511.1
			pilV	2.00E-06	72%	CP006644.1
			pilW	1.00E-05	88%	NC_009511.1
	Antiphagocytosis	Alginate	Alg 44	7.00E-04	85%	NC_020561.1
			algA	1.00E-21	65%	NC_009511.1
			algB	2.00E-36	69%	NC_009511.1
			algD	2.00E-23	72%	CP006644.1
			algF	3.00E-05	91%	CP006644.1
			algG	8.00E-05	85%	CP006644.
			algI	1.00E-59	67%	NC_009511.1
			algJ	7.00E-04	94%	NC_009511.1
			algP	1.00E-07	80%	CP006644.1
			algZ	5.00E-05	91%	CP006644.1
Pseudomonas	Biosurfactant	Rhamnolipid	rhlA	5.00E-04	89%	NC_009511.1
	Iron uptake	Pyochelin	fptA	6.00E-08	86%	NC_009511.1
			pchA	8.00E-04	93%	NC_009511.1
			pchB	6.00E-04	67%	NC_020561.1
			pchC	1.00E-04	80%	NC_009511.1
			pchD	1.00E-09	67%	NC_009511.1
			pchE	5.00E-12	94%	CP006644.1
			pchF	9.00E-17	79%	CP006644.1
			pchG	1.00E-05	79%	NC_009511.1
			pchH	2.00E-12	75%	CP006644.1
			pchI	3.00E-16	74%	NC_020561.1
		Pyoverdine	fpvA	3.00E-12	68%	NC_020561.1
			pvdA	6.00E-05	85%	NC_020561.1
			pvdD	6.00E-33	67%	CP006644.1
			pvdE	7.00E-06	82%	NC_009511.1
	Pigment	Pyocyanin	phzM	5.00E-05	77%	NC_009511.1
		Pyocyanin	phzS	6.00E-05	68%	NC_009511.1
	Protease	Alkaline protease	aprA	2.00E-12	79%	CP006644.1
		LasA	lasA	2.00E-04	86%	NC_020561.1
		LasB (Elastase)	lasB	9.00E-04	86%	CP006644.1
	Regulation	Quorum sensing	rhlL	3.00E-04	93%	NC_009511.1
			rhlR	5.00E-09	72%	NC_009511.1
	Secretion system	xcp secretion system	xcpQ	2.00E-46	70%	NC_009511.1
			xcpR	3.00E-174	72%	NC_009511.1
			xcpS	1.00E-44	66%	NC_009511.1
			xcpT	3.00E-23	69%	NC_009511.1
			xcpU	2.00E-07	78%	NC_020561.1
			xcpW	4.00E-04	73%	NC_009511.1
			xcpX	1.00E-05	88%	NC_020561.1
	Toxin	PLC (Phospholipase C)	plcH	6.00E-33	68%	CP006644.1

Figure 2

Percentage of different virulence factors associated with Sphingomonas spp.

Regardless of the phylogenetic divergence between Sphingomonas spp. and Pseudomonas sp., it was observed from our results that they shared several major virulence factors such as adherence, antiphagocytosis, iron uptake, proteases, quorum sensing, and others. With regard to adherence, they shared 20 genes, including flgK with e-value of 1.00E − 04 and identity similarity of 94%, and flgJ with e-value of 6.00E − 05 and identity similarity of 89%. Flagella plays an important role as a virulence factor such as in swimming motility toward the infection site and a role in biofilm formation and other pathogenic adaptations.16–20 Type IV pili play an important role in adherence by assisting the pathogens to attach with their host cells and causing a twitching motility that allows the bacteria to move along the cell surface, and in biofilm formation.17,21–25 Both Sphingomonas spp. and Pseudomonas sp. shared many genes implicated in Type IV pili biogenesis and mechanical function of pili, such as pilF with e-value of 4.00E − 05 and identity similarity of 97%, and pilB with e-value of 3.00E − 04 and identity similarity of 93%.

Furthermore, Sphingomonas spp. and Pseudomonas sp. shared many genes implicated in antiphagocytosis through the production of alginate. They shared 10 alginate genes, including algJ with e-value of 7.00E − 04 and identity similarity of 94%, and algZ with e-value of 5.00E − 05 and identity similarity of 91%. The production of alginate permits pathogenic bacteria to form biofilm and contributes to the persistence of bacteria in the lung by acting as an adhesin, which prevents the bacteria from being expelled from the infection site, and the alginate slime layer makes it more difficult for phagocytes to ingest and kill the bacteria.26–30 Another important bacterial virulence factor shared between Sphingomonas spp. and Pseudomonas sp. is quorum sensing. Sphingomonas spp. showed acquiring of both rhlL and rhlR with e-values of 3.00E − 4 and 5.00E − 9, respectively. These results show that Sphingomonas spp. possesses only rhl system of quorum sensing. While in Pseudomonas sp., quorum sensing consists of two separate but interrelated systems, namely, las and rhl, which are found to regulate the production of multiple virulence factors and are also crucial for proper biofilm formation.31–33

It is also worth mentioning that both Sphingomonas spp. and Pseudomonas sp. share seven genes encoding for xcp secretion system (Type II secretion system), including xcpX and xcpR with e-values of 1.00E − 5 and 3.00E − 174, respectively. The xcp secretion system is found to be responsible for secretion of toxins and enzymes into the extracellular fluid.34,35 It was also observed that both Sphingomonas spp. and Pseudomonas sp. shared many genes involved in iron uptake using both pyochelin (10 genes) and pyoverdine (4 genes). The pyochelin is effective at promoting iron uptake in Pseudomonas aeruginosa, catalyzes the formation of tissue-damaging free radicals, and also binds other transition metals (eg, Mo(IV), Co(II)) with appreciable affinity, and is also implicated in the delivery of both Co(II) and Mo(IV) to P. aeruginosa cells.36,37 The pyoverdine is effective at acquiring iron from transferrin and lactoferrin. Moreover, pyoverdine is cytotoxic because of its ability to stimulate the production of reactive oxygen species.38,39 Interestingly, Sphingomonas spp. and Pseudomonas sp. shared only one gene responsible for toxin production, namely, plcH that is responsible for degrading the phospholipids surfactant, which functions to reduce the surface tension so that the alveoli do not collapse completely when air leaves them during breathing. It is noteworthy that sphingomyelin (PlcH is a multifunctional enzyme) has been isolated from Pseudomonas aeruginosa in 2003.40,41

Our comparative analysis was further extended to search for other bacterial toxins that Sphingomonas spp. may acquire. Table 4 shows other toxins that were found to be shared between Sphingomonas spp. and Bordetella pertussis, which is a Gram-negative species and strictly aerobic coccobacilli. B. pertussis is a strict human pathogen causing whooping cough, a highly contagious respiratory disease marked by severe, spasmodic coughing episodes.42 It was also observed that Sphingomonas spp. contains genes for invasive adenylate cyclase/hemolysin, cyclolysin secretion protein, which is a bifunctional toxin harboring both adenylate cyclase and hemolytic activities, and functions primarily as an anti-inflammatory factor.43–45 Moreover, Sphingomonas spp. contains genes responsible for pertussis toxin and its secretion system, which assists in the attachment of B. pertussis to ciliated respiratory cells, important immunogen and activate cyclic adenosine phosphate (cAMP), histamine sensitizing factor (HSF), lymphocytosis promoting factor (LPF), islet-activating protein (IAP); interferes with leucocyte function; and is hemolytic.46,47

Table 4

Suggested Sphingomonas spp. toxin information in relation to Bordetella pertussis toxins.

BORDETELLA TOXIN	RELATED GENE	PRODUCT NAME	BORDETELLA PERTUSSIS TOHAMA I	Sphingomonas SPP.
			PROTIEN GENBANK ID	E VALUE	IDENT	ACCESSION
Cya (Invasive Adenylate cyclase/haemolysin)	cyaA	Bifunctional hemolysin-adenylate cyclase precursor	33591934	1.00E-21	79%	CP006644.1
	cyaD	Cyclolysin secretion protein	33591936	8.00E-04	81%	NC_009511.1
	cyaE	Cyclolysin secretion protein	33591937	2.00E-04	89%	NC_009511.1
Ptx (Pertussis toxin)	ptlA	Pertussis toxin transport protein	33594643	6.00E-04	84%	CP006644.1
	ptlC	Putative bacterial secretion system protein	33594645	3.00E-06	88%	NC_009511.1
	ptlD	Putative membrane protein	33594646	8.00E-04	78%	CP006644.
	ptlF	Putative bacterial secretion system protein	33594649	5.00E-04	76%	NC_009511.1
	ptlH	Putative bacterial secretion system protein	33594651	6.00E-10	71%	CP006644.1
	ptxA	Pertussis toxin subunit 1 precursor	33594638	1.00E-04	83%	CP006644.1

Conclusion

Results of this study showed that Sphingomonas spp. contains several major virulence factors, mainly resembling those of Pseudomonas sp. Other virulence factors from Legionella sp., Brucella sp., and Bordetella sp. have also been observed. Moreover, the similarity of virulence factors did not correspond to the phylogenetic relationships. These findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor.

We selected Sphingomonas spp. to test its potentiality for being potential virulent pathogen.

We tested the phylogenetic relationship of Sphingomonas spp. against known virulent pathogens.

We screened the presence of various virulent factors from different virulent pathogens in Sphingomonas spp.

Sphingomonas spp. contains several major virulence factors, mainly resembling those of Pseudomonas sp.

Supplementary Table 1. This table shows the tested major pathogenic virulence factors.