Identification of conditionally essential genes for Streptococcus suis infection in pigs

Figures & data

Table 1. Essential genes required for growth of S. suis in pigs.

			Tn-Seq Fold change^3
Locus tag^1	Gene	Product^2	Blood	Brain	CSF
Carbon metabolism
SSU0479	pep	Phosphoenolpyruvate carboxylase	−8.6	−7.4	−5.0
SSU1636	pdhB	Pyruvate dehydrogenase E1 component, Beta sub	−3.4	−2.1	−2.9
SSU1025	zwf	Glucose-6-phosphate 1-dehydrogenase	−4	−2.7	−4.3
SSU1839	tkt	transketolase	−3.3		−3.7
SSU1315		Lactonase	−9.9
SSU0936	deoC	Deoxyribose-phosphate aldolase	−7.8	−6.9
SSU1269	deoB	Phosphopentomutase		−7.6
SSU1270	rpiA	Ribose-5-phosphate isomerase A	−8.7	−7.6	−4.7
SSU0021	prsA1	Ribose-phosphate pyrophosphokinase	−4.3	−2.9	−4.1
SSU0648	fhs	Formate-tetrahydrofolate ligase	−5.6	−4.4	−6.1
Purine and pyrimidine metabolism
SSU0122	rpoB	DNA-directed RNA polymerase subunit Beta	−5.0	−3.6	−5.1
SSU0123	rpoC	DNA-directed RNA polymerase subunit Beta	−4.6	−3.2	−4.7
SSU0816	guaA	GMP synthase	−9.8	−8.6	−2.5
SSU0014	hpt	Hypoxanthine-guanine Phosphoribosyltransferase	−7.6	−6.5	−4.1
SSU1758	purA	Adenylosuccinate synthetase	−3.1	−9.4	−3.6
SSU0037	purB	Adenylosuccinate lyase	−4.7	−9.1	−4.0
SSU0378	gmk	Guanylate kinase	−3.5
Biosynthesis of amino acids
SSU0621	serC	Phosphoserine aminotransferase	−10.2	−8.9	−3.2
SSU1325		Haloacid dehalogenase	−4.11	−7.8	−5.3
SSU0319		Aminotransferase	−3.1	−5.6
SSU1720	cysE	Serine acetyltransferase	−4.3	−3.0	−4.7
SSU0748	thrB	Homoserine kinase	−8.7	−7.6
SSU0671	asD	Aspartate-semialdehyde dehydrogenase	−8.6	−7.4	−3.4
SSU0157	glnA	Glutamine synthetase	−4.5
SSU0262		Threonine synthase	−9.4	−8.3	−3.6
SSU1634	acoL	Dihydrolipoamide dehydrogenase	−4.9	−3.6
Transporters
SSU0114		Metal cation ABC transporter (Permease)	−7.4	−6.3	−8.1
SSU1865		Metal cation ABC transporter membrane protein	−4.2	−2.8	−4.5
SSU0883		Glutamine ABC transporter, glutamine-binding protein/permease protein	−2.2
SSU1675		Glutamine ABC transporter, glutamine-binding protein/permease protein	−2.3
SSU1852		Amino acid ABC transporter permease	−3.8	−2.5	−3.7
SSU1662	oppF	Oligopeptide transport ATP-binding protein		−5.1
SSU1583	manL	Mannose-specific phosphotransferase system	−7.4	−6.2
SSU0751	potA	Part of permidine/putrescine ABC transporter	−7.8
SSU0249	glA	Putative aquaporin	−9.2
SSU1869		Metal cation ABC transporter TroA	−5.2	−3.7	−5.5
Cell envelope biosynthesis
SSU0516	cps2B	Chain length determinant protein	−7,0	−5.9	−7.7
SSU0519	cps2E	Galactosyl transferase	−8.8	−5.6	−4.3
SSU1186	penA	Penicillin-binding protein 2b	−8.3	−4.8	−9.4
SSU0018	merC	Rod shape-determining protein MreC	−7.8	−6.7	−8.5
SSU0373	gpsB	Cell division protein GpsB	−3.4	−2.1	−4.0
SSU1028	ftsY	Cell division protein FtsY	−6.2	−5.1	−6.2
SSU1775	secE	Preprotein translocase subunit SecE	−3.4	−2.1	−3.7
SSU1615	secA	Preprotein translocase subunit SecA	−3.7
Membrane and secreted structures
SSU1128		Surface anchored protein	−3.9
SSU0375		Membrane protein	−9.8
SSU0386		Membrane protein	−7.9	−6.8	−8.6
SSU0473		Membrane protein	−8.6	−7.5	−3.2
Stress conditions
SSU0279	grpE	Heat shock protein GrpE	−3.3	−2.0	−3.6
SSU0280	dnaK	Molecular chaperone DnaK	−6.2	−4.9	−6.8
SSU0306	tig	Trigger factor	−3.8	−7.0
SSU0023	recO	DNA repair protein RecO	−4.2
Transcriptional regulators
SSU0511		TetR family transcriptional regulator	−5.9
SSU1326	ezrA	Septation ring formation regulator EzrA	−7.7		−4.7
SSU1608		MarR family transcriptional regulator	−4.6	−4.0
SSU0063	spxA	Spx family transcriptional regulator	−7.5	−6.4
SSU1202	ccpA	Catabolite control protein A CcpA	−7.5	−4.5
SSU0320	codY	CodY family transcriptional regulator	−7.5	−6.3
SSU0869		LysR family transcriptional regulator	−10.1
Unknown function
SSU0469		Hypothetical protein	−3.6	−2.3
SSU1501		CsbD-like protein	−5.5
SSU1940		c-di-AMP phosphodiesterase GdpP	−9.0

¹The Locus tag according to S. suis P1/7 genome is provided. ² Putative gene products according to genome annotations. ³ Log₂ fold change decrease found in different sites of infection as compared with inoculum in Tn-Seq. Only fold changes differences lower than 2 are shown. spec, spectinomycin-resistance cassette. MTDC, methylene-tetrahydrofolate dehydrogenase/cyclohydrolase; Glu/Gln, Glutamic/Glutamine.

Figure 1. Bacterial loads in (a) blood and (b) CSF after intravenous and intrathecal infection of pigs with S. suis strain 10 and (c) histological changes in central nervous tissues. Bacterial loads were calculated by CFU determination each hour post infection (hpi). Each symbol and line represent one individual animal of four. Histological examinations show typical suppurative meningitis (arrows) in various degrees in the spinal cord (left panel), cerebellum (central panel), and cerebrum (right panel) of inoculated pigs. Star symbol indicates the location of nervous tissue.

Figure 2. Global overview of Tn-Seq screen. (a) Hierarchical clustering of the complete gene set obtained for each infection site. The level of essentiality for each gene is color-coded as indicated. Genes with an increased log₂ fold change lower than 2 were considered essential in vivo, higher than 2 were considered as beneficial; otherwise, they were considered as no required. (b) VENN diagram indicating the number of unique or shared essential genes within infection sites. (c) Categorization of gene ontologies of the essential gene set for blood, brain, and CSF according to genome annotations.

Figure 3. Survival of S. suis strain 10 and derivative mutants in ex vivo (a) porcine blood, (b) porcine serum, and (c) porcine CSF. Bacteria were cultured in THB medium, diluted, and then incubated in the different body fluids. The number of bacteria was determined by CFU counting at time 0 and every 2 h over a time course of 6 h. Data are expressed as the total area under the curve obtained in three independent experiments and relative to that of the wild type (left) representative growth curves for selected strains are shown (right). One or two asterisks indicate statistically significant differences compared to the wild type at P < 0.05 and P < 0.001, respectively.

Figure 4. Growth curve of S. suis strain 10 and mutant derivatives under different environmental conditions. Bacteria were cultured in THB medium (a, d, e, f) at 37̊C, (b) at 42̊C and (c) at 34̊C in presence of (d) 5 mM NaF, (e) 2 mM H₂O₂ and (f) 1 mM of Bipyridyl, and the optical density at 600 nm (OD₆₀₀) was measured every 20 minutes over a time course of 18 hours.

Figure 5. Bacterial load in blood and organs after intravenous infection of pigs with strain 10 and mutant derivatives (a) 10∆penA, (b) 10∆SSU1869, and (c) 10∆SSU1940. Cells of strain 10 and mutant derivatives carrying a spectinomycin-resistance marker, were mixed 1:1 and the resulting suspensions were used to infect animals. Bacterial loads of the wildtype and mutants in blood, one- and two-day post-infection (dpi), in internal organs and brain, four dpi, were determined by plating on solid media containing or not spectinomycin and counting colony-forming units after overnight incubation. The ratio wild type/mutant is shown and the number of animals that resulted positive for each strain is depicted at the top. The ratio wildtype/mutant in blood remained similar to the three dpi.