In vivo negative selection screen identifies genes required for Francisella virulence

Weiss et al. 10.1073/pnas.0609675104.

Supporting Information

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SI Figure 5
SI Table 1
SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Material and Methods
SI Table 2

SI Figure 5

Fig. 5. Francisella encounters a bottleneck in the passage from the skin to the spleen after s.c. infection. Five mice were s.c. infected with 2 ´ 10⁵ cfu of the transposon insertion library. Bacteria were collected from the input and from the spleen of each mouse 48 h postinfection. The transposon mutant clones present in each sample were determined by microarray analysis. Input/input samples are shown as controls. Blue represents overabundance of signal compared to the input, yellow signal represents absence relative to the input.

SI Figure 6

Fig. 6. Verification of targeted mutant and complemented strains used in this study. Genomic DNA was prepared from the indicated bacterial strains. PCR reactions used the appropriate checkF and checkR primers (see Table S1) to amplify the given region of DNA from the wild-type (W), mutant (M) or complemented (C) strain. (A-D) The difference in size of the PCR products from reactions using wild-type DNA as a template or DNA from the mutant or complemented strains is due to the insertion of the antibiotic resistance cassette as well as the deletion or insertion of the gene of interest. For complemented strains, the deleted gene was re-introduced into the appropriate region, followed by a chloramphenicol resistance cassette downstream of the gene, resulting in a region of DNA that is larger than that present in the wild-type strain. The FTT0584 and FPI regions were not amplified from wild-type DNA due to their large sizes, ~6.4 kb and ~29 kb, respectively. (E) PCRs for portions of genes within the FPI were performed; the wild-type strain encodes the indicated genes while the FPI deletion mutant does not, confirming the deletion of the FPI.

SI Figure 7

Fig. 7. Targeted mutants are attenuated after i.p. inoculation in mice. Groups of four or five mice were infected i.p. with a 1:1 mixture of wild-type bacteria and the indicated bacterial strain. The data represent the competitive index (CI) value for cfu of mutant/wild-type in the spleen 48 h after infection. Bars represent the geometric mean CI value for each group of mice. P < 0.005 (**), P < 0.0005 (***).

SI Figure 8

Fig. 8. FTT0584 and FTT0748 mutants induce faster macrophage cell death than wild-type bacteria. Bone marrow-derived macrophages were activated with heat-killed F. novicida for 12 h before infection with 10 bacteria per macrophage of the indicated bacterial strains. Cell death was quantified by staining with DAPI and ethidium homodimer-2 (EthD-2) at 5 h postinfection and A) visualization by fluorescence microscopy and B) calculation of the % EthD-2 positive cells. P < 0.05 (*), P < 0.0005 (***).

SI Figure 9

Fig. 9. FTT0584 and FTT0748 mutants escape the Francisella-containing vacuole with similar kinetics as the wild-type strain. Bone marrow-derived macrophages were infected with the indicated GFP-expressing bacterial strains at 100 bacteria per macrophage. Samples were fixed at 30 and 60 min postinfection and stained for LAMP-1. Co-localization with LAMP-1 was determined for 100-200 bacteria per sample. The results are representative of three independent experiments.

SI Figure 10

Fig. 10. FTT0584 and FTT0748 mutants induce increased IL-1b release from peritoneal macrophages. Thioglycollate-elicited peritoneal macrophages were infected with the indicated Francisella strains. Supernatants were collected 3 h postinfection and IL-1b was quantified by ELISA. P < 0.05 (*), P < 0.005 (**).

Table 1. Complete list of genes negatively selected in the spleen 48 hr after intraperitoneal infection

Schu4 designation	Gene name	Protein function	Attenuated		Reference
			in vitro	in vivo
FTT0027c	lysA	Diaminopimelate decarboxylase
FTT0028c	NA	Conserved membrane protein
FTT0029c	NA	Conserved hypothetical protein
FTT0051	rbfA	Ribosome-binding factor A
FTT0052	hisS	Histidyl-tRNA synthetase
FTT0053	NA	Major facilitator superfamily (MFS) transport protein
FTT0054	NA	ATPase
FTT0055	rluC	Ribosomal large subunit pseudouridine synthase C
FTT0056c	NA	Major facilitator superfamily (MFS) transport protein	Yes	NA	(1)
FTT0057	NA	Hypothetical membrane protein
FTT0072	sdhC	Succinate dehydrogenase, cytochrome b556
FTT0077	sucB	Dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex
FTT0078	apt	Adenine phosphoribosyltransferase
FTT0094c	qseC	Sensor histidine kinase
FTT0107c	dsbB	Disulfide bond formation protein	Yes	Yes	(1, 2)
FTT0108c	cca	tRNA nucleotidyltransferase
FTT0113	deoB	Phosphopentomutase
FTT0118	prfC	Peptide chain release factor 3
FTT0134	NA	Hypothetical membrane protein
FTT0154	xerD	Integrase/recombinase
FTT0162	ampD	N-acetylmuramoyl-L-alanine amidase
FTT0165c	NA	Conserved hypothetical lipoprotein
FTT0203c	purH	Bifunctional purine biosynthesis protein	Yes	NA	(1, 2)
FTT0256c	NA	Lipopolysaccharide protein
FTT0282	cyoB	Cytochrome O ubiquinol oxidase subunit I
FTT0283	cyoC	Cytochrome O ubiquinol oxidase, subunit III
FTT0284	cyoD	Cytochrome O ubiquinol oxidase subunit IV
FTT0288c	pdxY	Pyridoxal/pyridoxine/pyridoxamine kinase
FTT0295	NA	Conserved hypothetical membrane protein
FTT0296	pcp	Pyrrolidone-carboxylate peptidase
FTT0315	pyrH	Uridylate kinase
FTT0326	rplD	50S ribosomal protein L4
FTT0327	rplW	50S ribosomal protein L23
FTT0351	rplQ	50S ribosomal protein L17
FTT0356	htpG	Chaperone Hsp90, heat-shock protein HtpG	Yes	Yes	(2)
FTT0397	mtn	5'-methylthioadenosine\S-adenosylhomocysteine nucleosidase
FTT0398c	NA	Hypothetical membrane protein
FTT0399c	NA	BNR/Asp-box repeat protein
Intergenic (FTT0399c-400)
FTT0405	lolD	Lipoprotein releasing system, subunit D, ABC transporter, ATP-binding protein
FTT0406	cadA	Lysine decarboxylase, inducible
FTT0407	gcvT	Glycine cleavage complex protein T (aminomethyltransferase)
FTT0408	gcvH	Glycine cleavage system H protein
FTT0409	gcvP1	Glycine cleavage system P protein, subunit 1
FTT0414	pgm	Phosphoglucomutase
FTT0453c	NA	Conserved hypothetical protein
FTT0454	yfdH	Glycosyl transferase, group 2 family protein
Intergenic (FTT0454--55c)
FTT0455c	NA	Dolichyl-phosphate-mannose-protein mannosyltransferase family protein
FTT0468	surA	Peptidyl-prolyl cis-trans isomerase (PPIase)
FTT0469	ksgA	Dimethyladenosine transferase , kasugamycin resistance
FTT0480c	xasA	Glutamate:g-aminobutyric acid antiporter family protein (APC family protein)
FTT0509c	NA	Conserved hypothetical protein
Intergenic (FTT0509c-10)
FTT0584	NA	Hypothetical protein
FTT0589	NA	Hypothetical protein
FTT0615c	NA	Metal ion transporter protein
FTT0617c	phoH	phoH-like protein
FTT0645c	NA	Conserved hypothetical membrane protein
FTT0667	NA	Hypothetical protein
FTT0685c	NA	Potassium channel protein
FTT0712c	sdaC1	Serine transporter
Intergenic (FTT0747c-48)
FTT0748	NA	Conserved hypothetical protein
FTT0756	NA	Cation-efflux family protein
FTT0766	deoD	Purine nucleoside phosphorylase
FTT0790	NA	Bacterial sugar transferase family protein
FTT0791	galE	UDP-glucose 4-epimerase
FTT0792	NA	Glycosyl transferases group 1 family protein
FTT0793	NA	ABC transporter, ATP-binding and membrane protein
FTT0797	NA	Glycosyl transferase family protein
FTT0798	NA	Glycosyl transferase family protein
FTT0799	NA	Glycosyl transferases group 1 family protein
FTT0800	NA	Haloacid dehalogenase-like hydrolase family protein
FTT0805	capB	Capsule biosynthesis protein capB
FTT0806	capC	Capsule biosynthesis protein CapC
FTT0891	NA	Conserved hypothetical membrane protein
FTT0892	folD	Methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase putative bifunctional protein
FTT0893	purM	Phosphoribosylaminoimidazol (AIR) synthetase	Yes	Yes	(2, 3)
FTT0895	purN	Phosphoribosylglycinamide formyltransferase
FTT0896	purE	Phosphoribosylaminoimidazole carboxylase,catalyic subunit
FTT0903	NA	Hypothetical protein
FTT0904	lpnB	Conserved hypothetical lipoprotein
FTT0905	NA	Type IV pili glycosylation protein
FTT0910	NA	Hypothetical protein
FTT0928c	NA	b-glucosidase-related glycosidase
FTT0936c	bioF	8-amino-7-oxononanoate synthase
FTT0937c	bioB	Biotin synthase
FTT0938	bioA	Adenosylmethionine-8-amino-7-oxononanoate aminotransferase
FTT0940c	NA	Conserved hypothetical protein
FTT0941c	NA	Lipase/esterase
FTT0944	NA	AMP-binding family protein
FTT0945	NA	Chorismate binding family protein
FTT0946	trpG	Anthranilate synthase component II
FTT0968c	NA	Amino acid antiporter
FTT1013c	ruvB	Holliday junction DNA helicase, subunit B
FTT1047c	NA	Hypothetical protein
FTT1048c	NA	Hypothetical protein
FTT1129c	NA	Conserved hypothetical protein
FTT1130c	cphA	Cyanophycin synthetase
FTT1150c	putA	Multifunctional protein, transcriptional repressor of proline utilization, proline dehydrogenase,pyrroline-5-carboxylate dehydrogenase
FTT1151c	NA	Conserved hypothetical protein
FTT1223	dedA1	DedA family protein
FTT1236	NA	Hypothetical protein	Yes	NA	(1)
FTT1237	NA	Glycosyl transferase family 8 protein
FTT1238c	NA	Hypothetical membrane protein
FTT1239	NA	Hypothetical membrane protein
FTT1254	fadD1	Acyl-CoA synthetase (long-chain-fatty-acid--CoA ligase)
FTT1257	emrA1	HlyD family secretion protein
FTT1258	NA	Outer membrane efflux protein
FTT1327	cdd	Cytidine deaminase
FTT1334c	NA	Hypothetical protein
FTT1344; FTT1699	pdpA; pdpA1	Conserved hypothetical protein	Yes	Yes	(4)
FTT1345; FTT1700	pdpB; pdpB1	Conserved hypothetical protein	Yes	Yes	(2, 5)
FTT1346; FTT1701	NA; NA	Hypothetical protein
FTT1347; FTT1702	NA; NA	Conserved hypothetical protein