Molecular bases of proliferation of Francisella tularensis in Arthropod vectors

Rexford Asare; Christine Akimana; Snake Jones; Yousef Abu Kwaik

doi:10.1111/j.1462-2920.2010.02230.x

. Author manuscript; available in PMC: 2011 Sep 1.

Published in final edited form as: Environ Microbiol. 2010 May 7;12(9):2587–2612. doi: 10.1111/j.1462-2920.2010.02230.x

Molecular bases of proliferation of Francisella tularensis in Arthropod vectors

Rexford Asare ^1,^†, Christine Akimana ^1,^†, Snake Jones ¹, Yousef Abu Kwaik ^1,^*

PMCID: PMC2957557 NIHMSID: NIHMS215232 PMID: 20482589

Summary

Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis-arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes-derived macrophages and D. melanogaster-derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella-containing phagosome matures to a late endosome-like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod-derived cells, we screened a comprehensive library of mutants of F. tularensis subsp novicida for their defect in intracellular proliferation within D. melanogaster-derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster-derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), Type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis-arthropod vector interaction and its patho-adaptation to infect mammals.

Keywords: S2 cells, intracellular, tularemia, Drosophila, macrophages

Introduction

Francisella tularensis is a Gram-negative intracellular bacterium that causes tularemia in small mammals and humans (Ellis et al., 2002; Hazlett and Cirillo, 2009; Santic et al., 2010). Because of low infectivity, ease of dissemination, and high morbidity and mortality, F. tularensis is classified by the CDC as a category A select bioterrorism agent (Dennis et al., 2001). The bacteria are maintained in nature primarily through infection of rodents and lagomorphs, and are transmitted to humans through insects bites, skin abrasions, inhalation, or ingestion (Ellis et al., 2002; Hazlett and Cirillo, 2009; Santic et al., 2010).

There are four subspecies of F. tularensis, which are subspecies tularensis, holarctica, mediasiatica and novicida (Keim et al., 2007; Nigrovic and Wingerter, 2008). All the four subspecies share about 97% genomic identities (Champion et al., 2009; Larsson et al., 2009). Human infections are mostly caused by subspecies tularensis and holarctica. F. tularensis subspecies tularensis is restricted to North America while the other species are distributed across the northern hemisphere (Keim et al., 2007; Nigrovic and Wingerter, 2008). Unlike the other subspecies, F. tularensis subsp. novicida is attenuated in humans, but it causes disease in animal models similar to the virulent subspecies (Santic et al., 2006; Santic et al., 2010). In addition, it replicates intracellularly within human and mouse macrophages, which is an important step in the disease process in mammals (Oyston et al., 2004).

Vector-borne transmission of tularemia to mammalian hosts has an important role in pathogenesis of the disease, and F. tularensis-arthropod vector interaction has likely played a major role in bacterial ecology and maintenance in the environment (Keim et al., 2007). Deer flies, horse flies, ticks and mosquitoes are common arthropod vectors of transmission between mammals (Keim et al., 2007). Although transmission via arthropod vectors may play an important role in the infectious life cycle of F. tularensis and subsequent pathogenesis to mammalian host, very little is known about the interaction of F. tularensis with the arthropod vector at the molecular, cellular, and organism level.

The well-studied and genetically tractable arthropod Drosophila melanogaster has been developed as an arthropod host model for the study of host-pathogen interactions at the organism level for fungi, gram positive and gram negative bacteria (Apidianakis and Rahme, 2009). Recent studies have shown that adult flies or Drosophila cell line could be used as a model system to study Francisella pathogenesis (Vonkavaara et al., 2008; Santic et al., 2009). D. melanogaster is an attractive model system for a number of reasons: 1) Studies of the signal transduction cascade underlying the innate immune system in D. melanogaster have revealed striking similarities to the mammalian innate immune response (Hoffmann et al., 1999; Anderson, 2000); 2) In insects and mammals, Toll family receptors (Hoffmann et al., 1999; Anderson, 2000) trigger host innate immune responses that are highly conserved; 3) Amenability for genetic manipulations; and most importantly 4) Arthropods are vectors for transmission of tularemia between mammals.

The genetic tractability of Drosophila has enabled unbiased approaches to the identification of host-encoded factors that impact the pathogen-host interface both at the cellular and molecular levels (D’Argenio et al., 2001; Dionne et al., 2003; Kurz et al., 2003; Needham et al., 2004; Kim et al., 2008). Although many studies using Drosophila S2 cells as a model system has focused on RNAi screening of host factors required for the pathogen-host interaction (Cherry, 2008), no comprehensive screen has been performed to identify genes of Francisella or any other intracellular pathogens required for proliferation in arthropod-derived cells.

The sualB cell line from Anopheles gambiae has also been used as a model to study intracellular replication of F. tularensis (Read et al., 2008). Interestingly, trafficking of F. tularensis subsp novicida in D. melanogaster-derived S2 cells is similar to mammalian macrophages (Santic et al., 2009). Within both host cells, F. tularensis transiently occupies a late endosome-like phagosome followed by rapid bacterial escape into the cytosol, where the bacteria proliferate robustly (Golovliov et al., 2003; Clemens et al., 2004; Santic et al., 2005a; Santic et al., 2005b; Checroun et al., 2006; Santic et al., 2007; Bonquist et al., 2008; Chong et al., 2008; Santic et al., 2008; Qin et al., 2009; Wehrly et al., 2009). This may suggest that some common mechanisms are utilized by F. tularensis to modulate phagosome biogenesis, escape into the cytosol, and to proliferate within mammalian and arthropod-derived cells.

Intracellular trafficking and robust intracellular proliferation of F. tularensis subsp novicida within mammalian macrophages and S2 cells is very similar. In addition, the ability of F. tularensis subsp novicida to infect D. melanogaster and mice has made F. tularensis subsp novicida a very useful model to dissect molecular basis of the intracellular infection by F. tularensis under BSL2 containment (Santic et al., 2006; Santic et al., 2010). Several genes within the 30-Kb pathogenicity island have been shown to be required for intracellular replication of F. tularensis within macrophages (Baron and Nano, 1998; Santic et al., 2005b; Bonquist et al., 2008; Schmerk et al., 2009). Similar to macrophages, intracellular replication of F. tularensis in S2 and SualB cells has been shown to be dependent on MglA, MglB, IglA, IglC, IglD as well as PdpA and PdpB (Read et al., 2008; Vonkavaara et al., 2008; Santic et al., 2009). We have shown that replication of F. tularensis subsp novicida within human macrophages involve a large percentage of the bacterial genome (see accompanying manuscript).

Since the molecular bases of intracellular proliferation of F. tularensis in arthropod-derived cells are not known, we screened a comprehensive sequence-defined mutant library of F. tularensis subsp novicida containing 3,050 alleles corresponding to 1448 non-essential genes for mutants defective in intracellular proliferation (Gallagher et al., 2007). Our data show that 394 genes, representing 22% of the genome, are required for replication within D. melanogaster S2 cells. Among 168 most defective mutants in S2 cells, 80 are required for replication and lethality to D. melanogaster adult flies. Interestingly, 135 of the 394 mutants that are defective in S2 cells are also defective in macrophages (see accompanying manuscript). Therefore, F. tularensis might have acquired some of the mechanisms to proliferate within mammalian cells through patho-adaptation to the arthropod host. However, additional distinct molecular mechanisms are also required for proliferate within both evolutionarily distant hosts.

Results and Discussion

Replication of F. tularensis mutants in S2 Cells

Arthropod vectors are important vehicles for transmission of. F. tularensis between mammals, but knowledge on the interaction between the bacteria and the arthropod host is very limited. The well-studied and genetically tractable arthropod model D. melanogaster has been recently explored as an arthropod vector model for F. tularensis subsp holarctica-derived LVS strain and F. tularensis subsp novicida with similar findings for both subsp (Vonkavaara et al., 2008; Santic et al., 2009). There are approximately 1800 genes in the genome of F. tularensis subsp novicida, of which 312 genes are essential. To identify the repertoire of genes essential for intracellular replication of F. tularensis in S2 cells, we screened F. tularensis subsp novicida mutant library containing 3050 sequence-defined multiple-allele insertion mutants corresponding to the 1448 non-essential genes of the genome (Gallagher et al., 2007). The S2 cells were seeded at 1 × 10⁶ cells ml⁻¹ in 96 well plates. Infections were performed at MOI of 10 for 1 h followed by 1 h of gentamicin treatment to kill extracellular bacteria, which resulted in infection of ~25% of the cells with an average of 1 bacterium/cell. At 24 h post-infection, cells were lysed and serial dilutions were plated on agar plates for colony enumeration. The CFU for the wild type strain at 24h post-infection was ~ 1×10⁸ with slight variations between multiple experiments.

Our primary screen showed that 501 mutant alleles were defective in S2 cells, but the observed defect for some of the mutants may be to a defect in uptake, which was not accounted for in our large and comprehensive screen. To confirm findings of the primary screen, we re-screened the 501 mutants twice. For these infections, the OD of the bacteria for all the mutants was measured and adjusted to a similar OD for all infections, to ensure equivalent input. Our data confirmed that 476 alleles, corresponding to 394 genes, were consistently and reproducibly required for proliferation within S2 cells (Table 1 and 2). Remarkably, this represented about 22% of the genome. The defect in growth ranged from 10 fold reductions in growth for the less attenuated mutants to 10⁷ fold reductions in growth for the severely attenuated mutants, compared to the wild type strain (Table 1). This indicates that while some of the mutants were completely attenuated for growth in S2 cells others exhibited only modest reduction in intracellular growth. About 38% of the mutants that were defective had insertions in either hypothetical proteins or proteins of unknown function (Fig. 1). Other functional gene classes that showed growth defect included metabolic (25%), FPI (2%), Type IV pili (1%), transport (16%), and DNA modification (5%) (Table 1 and Fig. 1). Clearly, there were a large number of genes that belong to proteins of unknown function or hypothetical proteins that are required for intracellular proliferation. It will be interesting to identify the functions of these proteins. This will facilitate deciphering the molecular mechanisms utilized by F. tularensis to proliferate in arthropod-derived cells. Many of the FPI genes were identified in our screen including iglC, iglD, pdpC and pdpD (Table 1 and 2).

Table 1.

List of growth-defective mutants of F. tularensis within S2 and U937 cells according to their functions.

List of growth defective mutants in both U937 and S2 Cells
Strain Name	Locus Tag	Gene	Description	Log reduction in Growth relative to WT
Controls
Wild type				0	0
Intracellular growth locus C		IglC		5	5
Proteins of unknown Function
tnfn1_pw060323p08q148	FTN_0027		conserved protein of unknown function	4	6^*
tnfn1_pw060510p03q161	FTN_0027		conserved protein of unknown function	2	2^*
tnfn1_pw060323p03q103	FTN_0041		protein of unknown function	5	2^#
tnfn1_pw060420p01q149	FTN_0041		protein of unknown function	2	3^#
tnfn1_pw060420p04q143	FTN_0149		conserved protein of unknown function	5	5
tnfn1_pw060323p02q193	FTN_0275		conserved protein of unknown function	2	2^#
tnfn1_pw060419p03q124	FTN_0275		conserved protein of unknown function	2	2^#
tnfn1_pw060510p02q121	FTN_0275		conserved protein of unknown function	3	2^#
tnfn1_pw060420p04q134	FTN_0297		conserved protein of unknown function	7	7
tnfn1_pw060328p05q119	FTN_0444		membrane protein of unknown function	6	6^#
tnfn1_pw060420p03q175	FTN_0444		membrane protein of unknown function	5	5^#
tnfn1_pw060323p07q141	FTN_0788		conserved protein of unknown function	5	5
tnfn1_pw060420p04q176	FTN_0855		protein of unknown function	5	2
tnfn1_pw060323p03q147	FTN_0930		protein of unknown function	6	3^#
tnfn1_pw060323p05q150	FTN_0930		protein of unknown function	6	3^#
tnfn1_pw060510p01q108	FTN_0977		conserved protein of unknown function	7	7
tnfn1_pw060510p01q128	FTN_1170		conserved protein of unknown function	3	3^*
tnfn1_pw060418p02q157	FTN_1170		conserved protein of unknown function	5	6^*
tnfn1_pw060420p04q196	FTN_1256		membrane protein of unknown function	4	5
tnfn1_pw060323p01q113	FTN_1343		conserved protein of unknown function	4	4^#
tnfn1_pw060418p02q105	FTN_1343		conserved protein of unknown function	4	4^#
tnfn1_pw060328p02q110	FTN_1457		protein of unknown function	5	5^#
tnfn1_pw060420p02q183	FTN_1457		protein of unknown function	6	6^#
tnfn1_pw060328p01q172	FTN_1542		conserved protein of unknown function	2	2^#
tnfn1_pw060328p02q177	FTN_1713		protein of unknown function	3	3
tnfn1_pw060328p06q155	FTN_1764		protein of unknown function	6	7^#
Hypothetical Proteins
tnfn1_pw060323p03q142	FTN_0030		hypothetical membrane protein	4	3^#
tnfn1_pw060420p02q155	FTN_0030		hypothetical membrane protein	3	3^#
tnfn1_pw060328p06q180	FTN_0038		hypothetical protein	4	4^#
tnfn1_pw060419p02q127	FTN_0038		hypothetical protein	2	2^#
tnfn1_pw060420p02q173	FTN_0169		conserved hypothetical membrane protein	6	6^*
tnfn1_pw060510p01q193	FTN_0169		conserved hypothetical membrane protein	5	5^*
tnfn1_pw060328p05q136	FTN_0384		conserved hypothetical protein	4	7
tnfn1_pw060328p05q130	FTN_0534		conserved hypothetical membrane protein	3	8
tnfn1_pw060418p01q143	FTN_0556		hypothetical protein	7	7
tnfn1_pw060419p03q188	FTN_0696		hypothetical membrane protein	2	2^#
tnfn1_pw060323p01q155	FTN_0696		hypothetical membrane protein	5	3^#
tnfn1_pw060328p06q185	FTN_0709		hypothetical protein	3	7
tnfn1_pw060323p07q129	FTN_0759		conserved hypothetical protein	4	2
tnfn1_pw060419p02q102	FTN_0792		hypothetical protein	5	6^#
tnfn1_pw060420p01q167	FTN_0792		hypothetical protein	2	2^#
tnfn1_pw060323p02q140	FTN_0895		hypothetical protein	2	2^*
tnfn1_pw060323p07q105	FTN_0895		hypothetical protein	4	2^*
tnfn1_pw060328p08q188	FTN_1098		conserved hypothetical membrane protein	2	2^#
tnfn1_pw060510p03q192	FTN_1098		conserved hypothetical membrane protein	2	2^#
tnfn1_pw060510p04q192	FTN_1098		conserved hypothetical membrane protein	7	6^#
tnfn1_pw060419p04q117	FTN_1156		hypothetical protein	2	4
tnfn1_pw060328p02q129	FTN_1612		hypothetical protein	2	2
Metabolic Proteins
tnfn1_pw060323p08q120	FTN_0020	carB	carbamoyl-phosphate synthase large chain	5	7
tnfn1_pw060419p01q106	FTN_0111	ribH	riboflavin synthase beta-chain	4	5
tnfn1_pw060328p06q174	FTN_0125	ackA	propionate kinase 2/acetate kinase A	4	4^#
tnfn1_pw060418p03q133	FTN_0199	cyoE	heme O synthase	2	4
tnfn1_pw060323p04q102	FTN_0211	pcp	pyrrolidone carboxylylate peptidase	1	1^#
tnfn1_pw060418p03q177	FTN_0211	pcp	pyrrolidone carboxylylate peptidase	3	4^#
tnfn1_pw060418p01q187	FTN_0319		amino acid-polyamine-organocation family protein	6	7
tnfn1_pw060323p06q113	FTN_0420		SAICAR synthetase/phosphoribosylamine-glycine ligase	7	5
tnfn1_pw060323p05q182	FTN_0504		lysine decarboxylase	4	4
tnfn1_pw060510p01q124	FTN_0507	gcvP1	glycine cleavage system P protein, subunit 1	5	7
tnfn1_pw060510p02q154	FTN_0511		shikimate 5-dehydrogenase	2	2^#
tnfn1_pw060510p02q157	FTN_0511		shikimate 5-dehydrogenase	6	6^#
tnfn1_pw060510p04q157	FTN_0511		shikimate 5-dehydrogenase	3	2^#
tnfn1_pw060323p06q194	FTN_0527	thrC	threonine synthase	7	7^#
tnfn1_pw060510p01q172	FTN_0527	thrC	threonine synthase	5	5^#
tnfn1_pw060510p03q172	FTN_0527	thrC	threonine synthase	2	2^#
tnfn1_pw060323p03q127	FTN_0567		tRNA synthetase class II (D, K and N)	6	2
tnfn1_pw060510p03q171	FTN_0588		asparaginase	2	2
tnfn1_pw060419p03q116	FTN_0593	sucD	succinyl-CoA synthetase, alpha subunit	2	2
tnfn1_pw060418p02q128	FTN_0633	katG	peroxidase/catalase	7	7
tnfn1_pw060328p06q130	FTN_0692	nadA	quinolinate sythetase A	3	2^#
tnfn1_pw060419p04q164	FTN_0692	nadA	quinolinate sythetase A	2	2^#
tnfn1_pw060510p01q159	FTN_0695	add	deoxyadenosine deaminase/adenosine deaminase	3	7
tnfn1_pw060328p06q156	FTN_0811	birA	biotin--acetyl-CoA-carboxylase ligase	6	7
tnfn1_pw060328p01q128	FTN_0840	mdaB	NADPH-quinone reductase (modulator of drug activity B)	5	5
tnfn1_pw060420p02q175	FTN_0877	cls	cardiolipin synthetase	7	5
tnfn1_pw060328p06q142	FTN_0954		histidine acid phosphatase	4	4
tnfn1_pw060420p01q130	FTN_0965		metal-dependent exopeptidase	3	3
tnfn1_pw060328p01q151	FTN_0983		bifunctional protein: glutaredoxin 3/ribonucleotide reductase beta subunit	5	3^#
tnfn1_pw060328p06q189	FTN_0995	hslV	ATP-dependent protease HslVU, peptidase subunit	2	2^#
tnfn1_pw060420p04q195	FTN_0995	hslV	ATP-dependent protease HslVU, peptidase subunit	4	4^#
tnfn1_pw060510p02q187	FTN_1018		aldolase/adducin class II family protein	3	3
tnfn1_pw060323p02q168	FTN_1046	wzb	low molecular weight (LMW) phosphotyrosine protein phosphatase	3	2
tnfn1_pw060328p06q184	FTN_1061		acid phosphatase, HAD superfamily protein	2	2^#
tnfn1_pw060420p02q103	FTN_1061		acid phosphatase, HAD superfamily protein	3	3^#
tnfn1_pw060510p04q113	FTN_1121	phrB	deoxyribodipyrimidine photolyase	5	7
tnfn1_pw060328p02q175	FTN_1131	putA	bifunctional proline dehydrogenase, pyrroline-5- carboxylate dehydrogenase	6	6
tnfn1_pw060328p02q174	FTN_1135	aroB	3-dehydroquinate synthetase	3	4^#
tnfn1_pw060328p03q107	FTN_1222	kpsF	phosphosugar isomerase	4	3
tnfn1_pw060510p02q164	FTN_1231	gloA	lactoylglutathione lyase	4	4^*
tnfn1_pw060420p04q194	FTN_1231	gloA	lactoylglutathione lyase	3	5^*
tnfn1_pw060510p04q146	FTN_1231	gloA	lactoylglutathione lyase	2	2^*
tnfn1_pw060510p01q142	FTN_1333	tktA	transketolase I	5	5
tnfn1_pw060418p02q109	FTN_1376		disulfide bond formation protein, DsbB family	4	4
tnfn1_pw060328p06q150	FTN_1494	aceE	pyruvate dehydrogenase complex, E1 component, pyruvate dehydrogenase	4	7
tnfn1_pw060419p01q104	FTN_1523		amino acid-polyamine-organocation family protein	4	4
tnfn1_pw060328p02q165	FTN_1523		amino acid-polyamine-organocation family protein	4	5^#
tnfn1_pw060419p02q191	FTN_1523		amino acid-polyamine-organocation family protein	2	2^#
tnfn1_pw060510p01q118	FTN_1553	nudH	dGTP pyrophosphohydrolase	5	5^#
tnfn1_pw060418p01q131	FTN_1557		oxidoreductase iron/ascorbate family protein	7	7
tnfn1_pw060420p04q105	FTN_1584	glpD	glycerol-3-phosphate dehydrogenase	3	5
tnfn1_pw060419p04q130	FTN_1585	glpK	glycerol kinase	3	3
tnfn1_pw060510p01q146	FTN_1597	prfC	peptide chain release factor 3	5	5
tnfn1_pw060419p02q112	FTN_1619	appC	cytochrome bd-II terminal oxidase subunit I	5	7
tnfn1_pw060328p02q105	FTN_1620	appB	cytochrome bd-II terminal oxidase subunit II	6	3
tnfn1_pw060418p04q111	FTN_1621		predicted NAD/FAD-dependent oxidoreductase	3	3^#
tnfn1_pw060418p04q112	FTN_1621		predicted NAD/FAD-dependent oxidoreductase	2	2^#
tnfn1_pw060420p04q169	FTN_1621		predicted NAD/FAD-dependent oxidoreductase	4	4^#
tnfn1_pw060323p04q160	FTN_1655	rluC	ribosomal large subunit pseudouridine synthase C	7	7^#
tnfn1_pw060510p02q165	FTN_1655	rluC	ribosomal large subunit pseudouridine synthase C	2	2^#
Transporter Proteins
tnfn1_pw060420p04q149	FTN_0008		10 TMS drug/metabolite exporter protein	4	4^#
tnfn1_pw060420p02q151	FTN_0018	sdaC	serine permease	2	4
tnfn1_pw060418p04q168	FTN_0141		ABC transporter, ATP-binding protein	5	6^#
tnfn1_pw060418p03q147	FTN_0299	putP	proline:Na+ symporter	2	2^#
tnfn1_pw060510p02q139	FTN_0299	putP	proline:Na+ symporter	2	2^#
tnfn1_pw060323p03q141	FTN_0619		pseudogene: nicotinamide ribonucleoside (NR) uptake permease (PnuC) family protein	3	3^*
tnfn1_pw060328p06q129	FTN_0619		pseudogene: nicotinamide ribonucleoside (NR) uptake permease (PnuC) family protein	2	5^*
tnfn1_pw060510p02q156	FTN_0624		serine permease	2	2^*
tnfn1_pw060323p06q164	FTN_0624		serine permease	2	2^*
tnfn1_pw060418p01q161	FTN_0636	glpT	glycerol-3-phosphate transporter	7	7
tnfn1_pw060419p04q142	FTN_0687	galP1	galactose-proton symporter, major facilitator superfamily (MFS) transport protein	2	3^*
tnfn1_pw060510p04q158	FTN_0687	galP1	galactose-proton symporter, major facilitator superfamily (MFS) transport protein	2	2^*
tnfn1_pw060328p06q132	FTN_0728		predicted Co/Zn/Cd cation transporter	2	5
tnfn1_pw060418p03q103	FTN_0739	potG	ATP-binding cassette putrescine uptake system, ATP-binding protein	2	2^#
tnfn1_pw060328p08q153	FTN_0739	potG	ATP-binding cassette putrescine uptake system, ATP-binding protein	2	5^#
tnfn1_pw060510p04q103	FTN_0799	emrE	putative membrane transporter of cations and cationic drugs, multidrug resistance protein	2	2
tnfn1_pw060323p01q177	FTN_0799	emrE	putative membrane transporter of cations and cationic drugs, multidrug resistance protein	4	3
tnfn1_pw060328p04q109	FTN_0885		proton-dependent oligopeptide transporter (POT) family protein, di- or tripeptide:H+ symporter	5	2
tnfn1_pw060328p04q167	FTN_0997		proton-dependent oligopeptide transporter (POT) family protein, di- or tripeptide:H+ symporter	5	3
tnfn1_pw060323p05q110	FTN_1215	kpsC	capsule polysaccharide export protein KpsC	2	5
tnfn1_pw060323p07q172	FTN_1344		major facilitator superfamily (MFS) transport protein	4	4^*
tnfn1_pw060420p04q148	FTN_1344		major facilitator superfamily (MFS) transport protein	5	5^*
tnfn1_pw060323p01q175	FTN_1441		sugar porter (SP) family protein	4	4^#
tnfn1_pw060420p02q182	FTN_1441		sugar porter (SP) family protein	6	6^#
tnfn1_pw060419p02q126	FTN_1581		small conductance mechanosensitive ion channel (MscS) family protein	3	3
tnfn1_pw060323p03q106	FTN_1593	oppA	ABC-type oligopeptide transport system, periplasmic component	2	2^*
tnfn1_pw060420p03q104	FTN_1593	oppA	ABC-type oligopeptide transport system, periplasmic component	4	6^*
tnfn1_pw060420p01q189	FTN_1611		major facilitator superfamily (MFS) transport protein	7	5
tnfn1_pw060328p02q121	FTN_1716	kdpC	potassium-transporting ATPase C chain	2	1^*
tnfn1_pw060420p02q159	FTN_1716	kdpC	potassium-transporting ATPase C chain	2	2^*
tnfn1_pw060418p03q187	FTN_1733		nicotinamide ribonucleoside (NR) uptake permease (PnuC) family protein	2	4
Transferases
tnfn1_pw060323p02q177	FTN_0019	pyrB	aspartate carbamoyltransferase	2	2^#
tnfn1_pw060323p03q119	FTN_0019	pyrB	aspartate carbamoyltransferase	2	2^#
tnfn1_pw060510p01q103	FTN_0063	ilvE	branched-chain amino acid aminotransferase protein (class IV)	3	5
tnfn1_pw060323p03q121	FTN_0343		aminotransferase	7	2
tnfn1_pw060328p03q179	FTN_0358		tRNA-methylthiotransferase MiaB protein	4	4^*
tnfn1_pw060419p01q169	FTN_0358		tRNA-methylthiotransferase MiaB protein	2	2^*
tnfn1_pw060323p06q168	FTN_0545		glycosyl transferase, group 2	4	4^#
tnfn1_pw060419p01q187	FTN_0545		glycosyl transferase, group 2	5	5^#
tnfn1_pw060328p01q142	FTN_0928	cysD	sulfate adenylyltransferase subunit 2	3	3^#
tnfn1_pw060323p03q182	FTN_1428	wbtO	transferase	3	2^#
tnfn1_pw060510p01q119	FTN_1428	wbtO	transferase	2	6^#
DNA modifying
tnfn1_pw060323p03q125	FTN_0133		ribonuclease II family protein	2	2
tnfn1_pw060510p02q141	FTN_0133		ribonuclease II family protein	5	5
tnfn1_pw060323p03q122	FTN_0577	mutL	DNA mismatch repair enzyme with ATPase activity	7	6^#
tnfn1_pw060510p01q148	FTN_0577	mutL	DNA mismatch repair enzyme with ATPase activity	5	5^#
tnfn1_pw060510p04q193	FTN_0680	uvrC	excinuclease ABC, subunit C	6	3
tnfn1_pw060328p04q156	FTN_1027	ruvC	holliday junction endodeoxyribonuclease	3	4^#
tnfn1_pw060510p01q132	FTN_1027		holliday junction endodeoxyribonuclease	3	3^#
tnfn1_pw060510p01q114	FTN_1073		DNA/RNA endonuclease G	5	6^*
tnfn1_pw060510p02q114	FTN_1073		DNA/RNA endonuclease G	2	2^*
tnfn1_pw060510p01q153	FTN_1154		type I restriction-modification system, subunit S	5	6
tnfn1_pw060323p03q167	FTN_1197	recR	RecFOR complex, RecR component	2	4^#
tnfn1_pw060510p02q106	FTN_1197	recR	RecFOR complex, RecR component	3	3^#
tnfn1_pw060328p06q158	FTN_1293	rnhB	ribonuclease HII	2	5
tnfn1_pw060323p07q175	FTN_1487		restriction endonuclease	3	6
Cell Division
tnfn1_pw060328p03q149	FTN_0162	ftsQ	cell division protein FtsQ	2	2^#
tnfn1_pw060328p01q167	FTN_0330	minD	septum formation inhibitor-activating ATPase	2	2
FPI Proteins
Type IV Pilin
tnfn1_pw060323p03q109	FTN_1137	pilQ	Type IV pili secretin component	2	2
tnfn1_pw060418p02q167	FTN_1137	pilQ	Type IV pili secretin component	4	4
tnfn1_pw060323p06q157	FTN_1139	pilO	Type IV pili glycosylation protein	2	2
Others
tnfn1_pw060323p06q138	FTN_0107	lepA	GTP-binding protein LepA	2	4^#
tnfn1_pw060418p02q123	FTN_0107	lepA	GTP-binding protein LepA	2	4^#
tnfn1_pw060420p04q150	FTN_0155		competence protein	2	7^*
tnfn1_pw060510p04q189	FTN_0155		competence protein	6	3^*
tnfn1_pw060418p04q181	FTN_0338		MutT/nudix family protein	2	2
tnfn1_pw060328p06q137	FTN_0465		Sua5/YciO/YrdC family protein	2	2^#
tnfn1_pw060323p03q111	FTN_0465		Sua5/YciO/YrdC family protein	2	2^#
tnfn1_pw060323p06q115	FTN_0768	tspO	tryptophan-rich sensory protein	3	3^#
tnfn1_pw060420p03q193	FTN_0768	tspO	tryptophan-rich sensory protein	3	3^#
tnfn1_pw060510p01q120	FTN_0768	tspO	tryptophan-rich sensory protein	3	3^#
tnfn1_pw060328p06q167	FTN_0985		DJ-1/PfpI family protein	6	6^#
tnfn1_pw060328p06q167	FTN_0985		DJ-1/PfpI family protein	5	5^#
tnfn1_pw060420p04q127	FTN_1031	ftnA	ferric iron binding protein, ferritin-like	2	6
tnfn1_pw060419p02q137	FTN_1034	rnfB	iron-sulfur cluster-binding protein	2	3
tnfn1_pw060420p03q121	FTN_1064		PhoH family protein, putative ATPase	2	4
tnfn1_pw060328p06q178	FTN_1241		DedA family protein	4	5
tnfn1_pw060418p01q185	FTN_1355		regulatory factor, Bvg accessory factor family	6	7
tnfn1_pw060328p03q154	FTN_1453		two-component regulator, sensor histidine kinase	2	2
tnfn1_pw060323p06q110	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase	2	2^*
tnfn1_pw060323p07q167	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase	4	4^*
Intergenic
tnfn1_pw060323p03q164	intergenic			3	2
tnfn1_pw060328p06q190	intergenic			3	3
tnfn1_pw060419p03q131	intergenic			2	2
tnfn1_pw060419p04q189	intergenic			5	3
tnfn1_pw060323p08q139	intergenic			4	4
List of growth defective mutants in only S2 cells according to their functions
Proteins of unknown Function
tnfn1_pw060419p01q176	FTN_0043		conserved protein of unknown function		2
tnfn1_pw060418p01q155	FTN_0044		protein of unknown function		3
tnfn1_pw060418p02q158	FTN_0050		protein of unknown function		4
tnfn1_pw060328p08q104	FTN_0051		conserved protein of unknown function		3
tnfn1_pw060420p01q142	FTN_0052		protein of unknown function		2
tnfn1_pw060419p04q191	FTN_0077		protein of unknown function		3^#
tnfn1_pw060323p06q122	FTN_0077		protein of unknown function		2^#
tnfn1_pw060510p04q143	FTN_0099		conserved protein of unknown function		2
tnfn1_pw060418p04q193	FTN_0109		protein of unknown function		4
tnfn1_pw060418p04q117	FTN_0207		protein of unknown function containing a von Willebrand factor type A (vWA) domain		2
tnfn1_pw060328p04q119	FTN_0325		membrane protein of unknown function		2
tnfn1_pw060328p08q156	FTN_0340		protein of unknown function		2
tnfn1_pw060323p03q157	FTN_0364		conserved protein of unknown function		2^*
tnfn1_pw060418p04q136	FTN_0364		conserved protein of unknown function		3^*
tnfn1_pw060328p08q149	FTN_0439		protein of unknown function		4^#
tnfn1_pw060418p01q142	FTN_0482		protein of unknown function		6
tnfn1_pw060328p04q110	FTN_0573		protein of unknown function		2
tnfn1_pw060418p02q126	FTN_0573		protein of unknown function		4
tnfn1_pw060328p08q173	FTN_0584	araJ	conserved inner membrane protein of unknown function		5
tnfn1_pw060510p04q147	FTN_0599		protein of unknown function		2^#
tnfn1_pw060328p06q173	FTN_0599		protein of unknown function		2^#
tnfn1_pw060510p01q183	FTN_0782		protein of unknown function		5
tnfn1_pw060323p03q129	FTN_0786		protein of unknown function		7^#
tnfn1_pw060323p05q127	FTN_0791		protein of unknown function		3^#
tnfn1_pw060419p03q107	FTN_0791		protein of unknown function		2^#
tnfn1_pw060418p01q141	FTN_0817		conserved protein of unknown function		2
tnfn1_pw060328p05q126	FTN_0828		protein of unknown function		5
tnfn1_pw060510p04q111	FTN_0861		conserved protein of unknown function		4
tnfn1_pw060418p04q148	FTN_0878		protein of unknown function		2
tnfn1_pw060328p02q106	FTN_0884		drug/metabolite transporter superfamily protein		2^#
tnfn1_pw060328p03q163	FTN_0884		drug/metabolite transporter superfamily protein		4^#
tnfn1_pw060323p03q150	FTN_0900		protein of unknown function with predicted hydrolase and phosphorylase activity		2^#
tnfn1_pw060418p03q108	FTN_0900		protein of unknown function with predicted hydrolase and phosphorylase activity		6^#
tnfn1_pw060323p04q104	FTN_0918		conserved protein of unknown function		2^#
tnfn1_pw060418p02q131	FTN_0918		conserved protein of unknown function		3^#
tnfn1_pw060419p04q188	FTN_0925		protein of unknown function		5
tnfn1_pw060419p04q179	FTN_1001		protein of unknown function		2^#
tnfn1_pw060323p07q181	FTN_1001		protein of unknown function		3^#
tnfn1_pw060418p02q145	FTN_1020		conserved protein of unknown function		5
tnfn1_pw060419p01q172	FTN_1044		conserved protein of unknown function		3
tnfn1_pw060420p01q111	FTN_1053		outer membrane protein of unknown function		3
tnfn1_pw060420p02q158	FTN_1071		protein of unknown function		5
tnfn1_pw060418p02q133	FTN_1093		protein of unknown function		5
tnfn1_pw060420p01q134	FTN_1103		protein of unknown function		2^#
tnfn1_pw060328p01q140	FTN_1103		protein of unknown function		3^#
tnfn1_pw060323p08q143	FTN_1235		protein of unknown function		2
tnfn1_pw060510p03q135	FTN_1254		protein of unknown function		4
tnfn1_pw060323p03q102	FTN_1257		membrane protein of unknown function		3^#
tnfn1_pw060419p03q150	FTN_1257		membrane protein of unknown function		4^#
tnfn1_pw060418p04q121	FTN_1261		protein of unknown function		2
tnfn1_pw060323p08q134	FTN_1270		conserved membrane protein of unknown function		2
tnfn1_pw060418p01q149	FTN_1298		GTPase of unknown function		7
tnfn1_pw060419p01q143	FTN_1334		conserved protein of unknown function		2^#
tnfn1_pw060328p08q108	FTN_1334		conserved protein of unknown function		3^#
tnfn1_pw060328p05q124	FTN_1372		protein of unknown function		5
tnfn1_pw060323p04q183	FTN_1386		protein of unknown function		3
tnfn1_pw060328p01q156	FTN_1442		conserved protein of unknown function		2^#
tnfn1_pw060420p01q165	FTN_1442		conserved protein of unknown function		4^#
tnfn1_pw060418p02q186	FTN_1448		protein of unknown function		3
tnfn1_pw060328p02q116	FTN_1449		conserved protein of unknown function		3^#
tnfn1_pw060419p03q173	FTN_1449		conserved protein of unknown function		2^#
tnfn1_pw060323p07q176	FTN_1534		conserved protein of unknown function		3
tnfn1_pw060328p02q177	FTN_1713		protein of unknown function		3
tnfn1_pw060328p05q185	FTN_1734		protein of unknown function		5
tnfn1_pw060328p08q107	FTN_1774		protein of unknown function		3
Hypothetical Protein
tnfn1_pw060418p04q139	FTN_0011		hypothetical protein		2^#
tnfn1_pw060420p02q108	FTN_0012		hypothetical protein		2
tnfn1_pw060420p02q139	FTN_0013		hypothetical protein		3
tnfn1_pw060328p01q141	FTN_0014		conserved hypothetical protein		3
tnfn1_pw060419p04q178	FTN_0028		conserved hypothetical membrane protein		3^#
tnfn1_pw060323p04q145	FTN_0028		conserved hypothetical membrane protein		2^#
tnfn1_pw060418p04q143	FTN_0053		hypothetical protein		2
tnfn1_pw060328p06q157	FTN_0170		conserved hypothetical membrane protein		5
tnfn1_pw060418p03q151	FTN_0212		hypothetical membrane protein		3
tnfn1_pw060323p08q114	FTN_0326		conserved hypothetical protein		3
tnfn1_pw060328p05q165	FTN_0360		hypothetical protein		5
tnfn1_pw060419p01q145	FTN_0368		hypothetical protein		2
tnfn1_pw060419p03q186	FTN_0375		hypothetical protein		3
tnfn1_pw060420p02q163	FTN_0398		hypothetical membrane protein		3
tnfn1_pw060420p04q104	FTN_0466		conserved hypothetical protein		4
tnfn1_pw060328p08q148	FTN_0548		conserved hypothetical protein		2^#
tnfn1_pw060418p04q176	FTN_0548		conserved hypothetical protein		2^#
tnfn1_pw060328p06q164	FTN_0630		hypothetical protein		5
tnfn1_pw060328p05q141	FTN_0701		conserved hypothetical protein		5
tnfn1_pw060418p02q152	FTN_0706		hypothetical membrane protein		3
tnfn1_pw060418p02q175	FTN_0717		conserved hypothetical membrane protein		5
tnfn1_pw060328p06q126	FTN_0732		hypothetical protein		5
tnfn1_pw060323p07q129	FTN_0759		conserved hypothetical protein		2
tnfn1_pw060323p04q134	FTN_0938		hypothetical protein		2^#
tnfn1_pw060418p02q170	FTN_0938		hypothetical protein		4^#
tnfn1_pw060419p03q187	FTN_1123		conserved hypothetical protein		3
tnfn1_pw060418p04q105	FTN_1180		hypothetical membrane protein		3
tnfn1_pw060420p04q159	FTN_1223		conserved hypothetical membrane protein		7
tnfn1_pw060323p08q166	FTN_1232		conserved hypothetical membrane protein		2
tnfn1_pw060328p03q180	FTN_1260		hypothetical membrane protein		2
tnfn1_pw060510p01q184	FTN_1299		hypothetical protein		5
tnfn1_pw060419p04q127	FTN_1342		conserved hypothetical protein		3
tnfn1_pw060328p05q157	FTN_1379		pseudogene: hypothetical membrane protein, fragment		5
tnfn1_pw060323p06q178	FTN_1389		conserved hypothetical membrane protein		3^#
tnfn1_pw060420p01q172	FTN_1389		conserved hypothetical membrane protein		2^#
tnfn1_pw060420p01q153	FTN_1458		conserved hypothetical protein		2
tnfn1_pw060323p04q147	FTN_1761		pseudogene: hypothetical protein, fragment		3
tnfn1_pw060418p04q149	FTN_1765		conserved hypothetical protein		2
Metabolic
tnfn1_pw060510p02q160	FTN_0021	carA	carbamoyl-phosphate synthase small chain		2
tnfn1_pw060418p04q115	FTN_0095		nitroreductase		7
tnfn1_pw060420p02q191	FTN_0113	ribC	riboflavin synthase alpha chain		6
tnfn1_pw060328p05q159	FTN_0118		serine peptidase, S49 family		3^#
tnfn1_pw060420p02q187	FTN_0118		serine peptidase, S49 family		5^#
tnfn1_pw060328p06q139	FTN_0127	gabD	succinate semialdehyde dehydrogenase (NAD(P)+ dependent)		5
tnfn1_pw060510p01q130	FTN_0154	rimK	glutathione synthase/ribosomal protein S6 modification enzyme		3
tnfn1_pw060328p01q150	FTN_0168	lysU	lysyl-tRNA synthetase		2^#
tnfn1_pw060510p02q178	FTN_0217		L-lactate dehydrogenase		2
tnfn1_pw060323p07q113	FTN_0362		deoxyribodipyrimidine photolyase-related protein		4
tnfn1_pw060323p04q144	FTN_0406		sterol desaturase		3^#
tnfn1_pw060418p01q189	FTN_0406		sterol desaturase		6^#
tnfn1_pw060328p06q134	FTN_0443	maeA	NAD-dependent malic enzyme		5^#
tnfn1_pw060328p06q125	FTN_0496	slt	soluble lytic murein transglycosylase		3
tnfn1_pw060418p04q116	FTN_0512	glgX	pullulanase		4
tnfn1_pw060510p03q154	FTN_0516	glgA	glycogen synthase		7
tnfn1_pw060420p01q135	FTN_0540	pckA	phosphoenolpyruvate carboxykinase		2
tnfn1_pw060419p04q153	FTN_0597		protein-disulfide isomerase		2
tnfn1_pw060510p02q110	FTN_0603	mutM	formamidopyrimidine-DNA glycosylase		2
tnfn1_pw060328p02q139	FTN_0621	eno	enolase (2-phosphoglycerate dehydratase)		2^#
tnfn1_pw060510p03q188	FTN_0627	chiA	chitinase, glycosyl hydrolase family 18		6
tnfn1_pw060418p01q120	FTN_0651	cdd	cytidine deaminase		5^#
tnfn1_pw060419p01q168	FTN_0651	cdd	cytidine deaminase		2^#
tnfn1_pw060328p04q151	FTN_0661	guaB	IMP dehydrogenase/GMP reductase		6^#
tnfn1_pw060328p06q131	FTN_0674	glxK	glycerate kinase		3
tnfn1_pw060420p01q148	FTN_0694	nadB	L-aspartate oxidase		4
tnfn1_pw060323p06q103	FTN_0711		predicted metal-dependent hydrolase		2
tnfn1_pw060328p04q116	FTN_0765		choloylglycine hydrolase family protein		2
tnfn1_pw060510p03q119	FTN_0806		glycosyl hydrolase family 3		3
tnfn1_pw060323p07q185	FTN_0814	bioF	8-amino-7-oxononanoate synthase		3^#
tnfn1_pw060419p02q138	FTN_0814	bioF	8-amino-7-oxononanoate synthase		3^#
tnfn1_pw060328p04q175	FTN_0818		lipase/esterase		5
tnfn1_pw060418p02q142	FTN_0826		aldo/keto reductase family protein		3
tnfn1_pw060328p08q145	FTN_0907		D-alanyl-D-alanine carboxypeptidase		4^#
tnfn1_pw060418p04q131	FTN_0907		D-alanyl-D-alanine carboxypeptidase		4^#
tnfn1_pw060418p04q167	FTN_0935	asnB	asparagine synthase		2
tnfn1_pw060510p02q145	FTN_0945	rsuA	16S rRNA pseudouridine synthase		4
tnfn1_pw060328p08q120	FTN_0987		tRNA-dihydrouridine synthase		3^#
tnfn1_pw060323p08q141	FTN_1015		isochorismatase family protein		3^#
tnfn1_pw060420p01q129	FTN_1015		isochorismatase family protein		2^#
tnfn1_pw060323p05q141	FTN_1033	grxB	glutaredoxin 2		3^#
tnfn1_pw060420p01q193	FTN_1033	grxB	glutaredoxin 2		4^#
tnfn1_pw060418p01q153	FTN_1055	lon	DNA-binding, ATP-dependent protease La		2
tnfn1_pw060328p06q184	FTN_1061		acid phosphatase, HAD superfamily protein		3^#
tnfn1_pw060420p02q103	FTN_1061		acid phosphatase, HAD superfamily protein		7^#
tnfn1_pw060510p04q113	FTN_1121	phrB	deoxyribodipyrimidine photolyase		6
tnfn1_pw060328p02q175	FTN_1131	putA	bifunctional proline dehydrogenase, pyrroline-5- carboxylate dehydrogenase		4
tnfn1_pw060328p02q174	FTN_1135	aroB	3-dehydroquinate synthetase		5^#
tnfn1_pw060328p08q131	FTN_1174	murI	glutamate racemase		2^#
tnfn1_pw060419p03q164	FTN_1186	pepO	M13 family metallopeptidase		7
tnfn1_pw060418p01q124	FTN_1245	iscS	cysteine desulfurase		7^#
tnfn1_pw060323p04q139	FTN_1264	rluD	ribosomal large subunit pseudouridine synthase D		2^#
tnfn1_pw060510p03q183	FTN_1264	rluD	ribosomal large subunit pseudouridine synthase D		6^#
tnfn1_pw060328p06q166	FTN_1273		long chain fatty acid CoA ligase		2
tnfn1_pw060419p03q126	FTN_1278	nadE	NAD synthase		5
tnfn1_pw060328p05q128	FTN_1329	fbaA	fructose bisphosphate aldolase Class II		3
tnfn1_pw060323p06q195	FTN_1390		Zn-dependent hydrolase		5
tnfn1_pw060510p04q137	FTN_1425	wbtF	NAD dependent epimerase		2
tnfn1_pw060419p03q166	FTN_1431	wbtA	dTDP-glucose 4,6-dehydratase		2
tnfn1_pw060323p07q169	FTN_1438		bifunctional protein: 3-hydroxacyl-CoA dehydrogenase/acyl-CoA-binding protein		4^#
tnfn1_pw060418p02q122	FTN_1438		bifunctional protein: 3-hydroxacyl-CoA dehydrogenase/acyl-CoA-binding protein		3^#
tnfn1_pw060328p08q196	FTN_1459		short chain dehydrogenase		5
tnfn1_pw060328p06q128	FTN_1530	lysA	diaminopimelate decarboxylase		6
tnfn1_pw060328p05q101	FTN_1532	gdhA	glutamate dehydrogenase (NADP+)		2^#
tnfn1_pw060419p04q163	FTN_1532	gdhA	glutamate dehydrogenase (NADP+)		6^#
tnfn1_pw060418p02q178	FTN_1536		amino acid-polyamine-organocation (APC) superfamily protein		4
tnfn1_pw060323p06q106	FTN_1552		acid phosphatase, PAP2 family		5
tnfn1_pw060510p01q118	FTN_1553	nudH	dGTP pyrophosphohydrolase		2
tnfn1_pw060323p04q110	FTN_1678	nuoC	NADH dehydrogenase I, C subunit		5^#
tnfn1_pw060328p05q160	FTN_1729	dapB	dihydrodipicolinate reductase		4^#
tnfn1_pw060510p01q178	FTN_1729	dapB	dihydrodipicolinate reductase		3^#
tnfn1_pw060328p04q104	FTN_1730	lysC	aspartate kinase III		2
tnfn1_pw060328p03q174	FTN_1768	pepN	aminopeptidase N		3
Transporter proteins
tnfn1_pw060323p03q117	FTN_0005	corA	divalent inorganic cation transporter		2^#
tnfn1_pw060420p01q131	FTN_0005	corA	divalent inorganic cation transporter		3^#
tnfn1_pw060420p01q180	FTN_0097		hydroxy/aromatic amino acid permease (HAAAP) family protein		4^#
tnfn1_pw060419p03q162	FTN_0115		Na+/H+ antiporter		4
tnfn1_pw060323p08q162	FTN_0151		ABC-type nitrate/sulfonate/bicarbonate transport system, ATPase component		2
tnfn1_pw060419p01q165	FTN_0183		manganese/Zinc/Iron chelate uptake transporter family protein		3^#
tnfn1_pw060419p04q103	FTN_0183		manganese/Zinc/Iron chelate uptake transporter family protein		2^#
tnfn1_pw060510p02q174	FTN_0184		major facilitator superfamily (MFS) transport protein		2
tnfn1_pw060323p03q161	FTN_0276	mviN	multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) transporter		2^*
tnfn1_pw060510p02q151	FTN_0276	mviN	multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) transporter		3^*
tnfn1_pw060323p08q118	FTN_0345		DNA uptake protein, SMF family		2
tnfn1_pw060419p03q195	FTN_0363		sodium bile acid symporter family protein		4
tnfn1_pw060420p03q115	FTN_0566		mechanosensitive ion channel protein		3
tnfn1_pw060328p08q167	FTN_0579		major facilitator superfamily (MFS) transport protein		2
tnfn1_pw060419p04q167	FTN_0620		major facilitator superfamily (MFS) transport protein		5
tnfn1_pw060328p06q114	FTN_0631		metabolite:H+ symporter (MHS) family protein		2^#
tnfn1_pw060510p02q115	FTN_0631		metabolite:H+ symporter (MHS) family protein		5^#
tnfn1_pw060510p02q167	FTN_0631		metabolite:H+ symporter (MHS) family protein		5^#
tnfn1_pw060418p02q189	FTN_0640	dctA	C4-dicarboxylate transport protein		3
tnfn1_pw060510p02q159	FTN_0688	galP2	galactose-proton symporter, major facilitator superfamily (MFS) transport protein		3
tnfn1_pw060510p03q140	FTN_0741		proton-dependent oligopeptide transporter (POT) family protein, di- or tripeptide:H+ symporter		5
tnfn1_pw060328p05q107	FTN_0767	betT	betaine/carnitine/choline transporter (BCCT) family protein		4
tnfn1_pw060420p03q116	FTN_0824		major facilitator superfamily (MFS) transport protein		2
tnfn1_pw060510p04q173	FTN_0872		small conductance mechanosensitive ion channel (MscS) family protein		5
tnfn1_pw060328p06q175	FTN_0875		metabolite:H+ symporter (MHS) family		2
tnfn1_pw060328p02q106	FTN_0884		drug/metabolite transporter superfamily protein		1^#
tnfn1_pw060328p03q163	FTN_0884		drug/metabolite transporter superfamily protein		4^#
tnfn1_pw060328p01q188	FTN_0910		sugar:cation symporter family protein		2^#
tnfn1_pw060419p04q109	FTN_0910		sugar:cation symporter family protein		2^#
tnfn1_pw060419p01q175	FTN_0984		ABC transporter, ATP-binding protein		2
tnfn1_pw060419p01q170	FTN_1006		transporter-associated protein, HlyC/CorC family		4
tnfn1_pw060418p02q160	FTN_1010		major facilitator superfamily (MFS) transport protein		2
tnfn1_pw060419p01q133	FTN_1014		nicotinamide ribonucleoside (NR) uptake permease (PnuC) family protein		2
tnfn1_pw060328p02q109	FTN_1107	metlQ	methionine uptake transporter (MUT) family protein, membrane and periplasmic protein		2
tnfn1_pw060323p05q139	FTN_1166		metabolite:H+ symporter (MHS) family protein		7
tnfn1_pw060419p02q107	FTN_1267		ATP-binding Cassette (ABC) superfamily protein		4
tnfn1_pw060418p02q182	FTN_1275		drug:H+ antiporter-1 (DHA2) family protein		5
tnfn1_pw060420p04q186	FTN_1404		ATP-binding cassette (ABC) superfamily protein		2
tnfn1_pw060510p02q118	FTN_1409		major facilitator superfamily (MFS) transport protein		6
tnfn1_pw060328p06q119	FTN_1549		drug:H+ antiporter-1 (DHA1) family protein		3
tnfn1_pw060419p02q126	FTN_1581		small conductance mechanosensitive ion channel (MscS) family protein		2
tnfn1_pw060418p01q150	FTN_1586		sugar transporter, MFS superfamily		2
tnfn1_pw060420p01q146	FTN_1681	fur	ferric uptake regulation protein		2^*
tnfn1_pw060510p04q167	FTN_1681	fur	ferric uptake regulation protein		2^*
tnfn1_pw060323p03q163	FTN_1683		drug:H+ antiporter-1 (DHA1) family protein		3^*
tnfn1_pw060328p02q192	FTN_1683		drug:H+ antiporter-1 (DHA1) family protein		4^*
tnfn1_pw060323p06q117	FTN_1685		drug:H+ antiporter-1 (DHA1) family protein		3
tnfn1_pw060418p02q140	FTN_1685		drug:H+ antiporter-1 (DHA1) family protein		5
tnfn1_pw060328p05q182	FTN_1707	nhaD	Na+:H+ antiporter		5
tnfn1_pw060328p02q121	FTN_1716	kdpC	potassium-transporting ATPase C chain		2^*
tnfn1_pw060420p02q159	FTN_1716	kdpC	potassium-transporting ATPase C chain		2^*
tnfn1_pw060510p03q118	FTN_1717	kdpB	potassium-transporting ATPase B chain		3
tnfn1_pw060420p01q113	FTN_1752	nhaA	Na+:H+ antiporter		3
Transferase
tnfn1_pw060510p04q127	FTN_0071		LPS fatty acid acyltransferase		2^#
tnfn1_pw060419p03q160	FTN_0080		SAM-dependent methyltransferase		4
tnfn1_pw060328p08q125	FTN_0120		rhodanese-related sulfurtransferase		4
tnfn1_pw060328p01q137	FTN_0153		RimI-like acetyltransferase		3
tnfn1_pw060418p01q110	FTN_0200		UDP-3-O-[3-fatty acid] glucosamine N- acyltransferase		2^#
tnfn1_pw060510p02q131	FTN_0200		UDP-3-O-[3-fatty acid] glucosamine N- acyltransferase		2^#
tnfn1_pw060420p02q146	FTN_0300		glycosyl transferase, group 2		5
tnfn1_pw060328p03q179	FTN_0358		tRNA-methylthiotransferase MiaB protein		4^*
tnfn1_pw060419p01q169	FTN_0358		tRNA-methylthiotransferase MiaB protein		2^*
tnfn1_pw060420p01q152	FTN_0453		glycosyl transferase		5
tnfn1_pw060419p02q135	FTN_0560	ksgA	dimethyladenosine transferase		3
tnfn1_pw060419p04q168	FTN_1091	aroA	3-phosphoshikimate 1-carboxyvinyltransferase		2
tnfn1_pw060418p03q185	FTN_1400		S-adenosylmethionine-dependent methyltransferase		5
tnfn1_pw060418p04q172	FTN_1418	manC	mannose-1-phosphate guanylyltransferase		4
DNA Modification
tnfn1_pw060510p04q169	FTN_0122	recA	recombinase A protein		2
tnfn1_pw060328p06q179	FTN_0492	parC	DNA topoisomerase IV subunit A		2
tnfn1_pw060510p04q168	FTN_0666	uvrA	excinuclease ABC, subunit A		2
tnfn1_pw060328p06q140	FTN_0704		type I restriction-modification system, subunit M (methyltransferase)		5
tnfn1_pw060510p02q180	FTN_0704		type I restriction-modification system, subunit M (methyltransferase)		2
tnfn1_pw060510p03q158	FTN_1294		rRNA methylase, SpoU family		2
tnfn1_pw060510p02q176	FTN_1413		ATPase, AAA family, related to the helicase subunit of the Holliday junction resolvase		2
tnfn1_pw060328p08q179	FTN_1491		adenine specific DNA methylase		2
tnfn1_pw060328p06q176	FTN_1544	hemK	modification methylase, HemK family		5
tnfn1_pw060328p05q164	FTN_1594	uvrD	DNA helicase II		6
*Cell Division*
tnfn1_pw060328p01q167	FTN_0330	minD	septum formation inhibitor-activating ATPase		2
tnfn1_pw060323p08q146	FTN_0331	minC	septum formation inhibitor		4^#
tnfn1_pw060420p02q170	FTN_0331	minC	septum formation inhibitor		4^#
Transcription/Translation
tnfn1_pw060328p06q196	FTN_0552	yhbY	RNA-binding protein		5^#
tnfn1_pw060510p03q150	FTN_0949	rplI	50S ribosomal protein L9		2
tnfn1_pw060328p06q170	FTN_1099		transcriptional regulator, LysR family		7
tnfn1_pw060419p03q165	FTN_1300		transcriptional regulator, LysR family		2
tnfn1_pw060328p02q148	FTN_1393		transcriptional regulator, ArsR family		3^#
tnfn1_pw060418p01q138	FTN_1393		transcriptional regulator, ArsR family		2^#
tnfn1_pw060419p02q151	FTN_1628		transcriptional regulator, LysR family		2^#
tnfn1_pw060510p03q194	FTN_1628		transcriptional regulator, LysR family		2^#
FPI
tnfn1_pw060328p01q144	FTN_1313		hypothetical protein		3
tnfn1_pw060323p03q179	FTN_1314		conserved hypothetical protein		1
tnfn1_pw060328p06q163	FTN_1315		protein of unknown function		5
tnfn1_pw060328p06q115	FTN_1322	iglC	intracellular growth locus protein C		5
tnfn1_pw060419p04q108	FTN_1325	pdpD	protein of unknown function		2
Type IV Pili
tnfn1_pw060418p04q123	FTN_0070	pilE	Type IV pili, pilus assembly protein		3
tnfn1_pw060510p03q129	FTN_0070	pilE	Type IV pili, pilus assembly protein		3
tnfn1_pw060323p06q179	FTN_0305		pilus assembly protein		4
tnfn1_pw060419p01q196	FTN_0414		Type IV pili, pilus assembly protein		2
tnfn1_pw060419p03q141	FTN_0664	fimT	Type IV pili, pilus assembly protein		2
tnfn1_pw060328p05q146	FTN_0946	pilF	Type IV pili, pilus assembly protein		5
tnfn1_pw060418p02q167	FTN_1137	pilQ	Type IV pili secretin component		4
Others
tnfn1_pw060328p08q161		isftu1	isftu1		2
tnfn1_pw060323p03q115		isftu3	isftu3		1
tnfn1_pw060328p04q157		isftu2	isftu2		1
tnfn1_pw060510p02q150		isftu6	isftu6		2
tnfn1_pw060328p01q179	FTN_0010		phage terminase, small subunit		3
tnfn1_pw060328p08q114	FTN_0266	htpG	chaperone Hsp90, heat shock protein HtpG		2
tnfn1_pw060328p04q152	FTN_0322		VacJ like lipoprotein		3^#
tnfn1_pw060418p01q140	FTN_0322		VacJ like lipoprotein		2^#
tnfn1_pw060328p08q155	FTN_0357	pal	peptidoglycan-associated lipoprotein, OmpA family		4^*
tnfn1_pw060419p01q158	FTN_0357	pal	peptidoglycan-associated lipoprotein, OmpA family		2^*
tnfn1_pw060510p02q122	FTN_0367		phage integrase		4
tnfn1_pw060328p08q132	FTN_0372		regulatory protein, AlpA family		4
tnfn1_pw060323p07q171	FTN_0585	cutC	copper homeostasis protein CutC family protein		2
tnfn1_pw060328p06q127	FTN_0713	ostA2	organic solvent tolerance protein OstA		5^#
tnfn1_pw060419p01q180	FTN_0713	ostA2	organic solvent tolerance protein OstA		4^#
tnfn1_pw060323p06q105	FTN_0810		ROK family protein		4
tnfn1_pw060419p01q139	FTN_0836		kinase-like protein		2
tnfn1_pw060418p04q134	FTN_1051	hfq	host factor I for bacteriophage Q beta replication		2
tnfn1_pw060420p03q121	FTN_1064		PhoH family protein, putative ATPase		4
tnfn1_pw060328p05q177	FTN_1192		chitin-binding protein		6
tnfn1_pw060419p04q183	FTN_1240		BolA family protein		4
tnfn1_pw060418p02q190	FTN_1242		DedA family protein		5
tnfn1_pw060419p01q120	FTN_1488		prophage maintenance system killer protein (DOC)		6
tnfn1_pw060419p01q135	FTN_1665		magnesium chelatase		2
tnfn1_pw060419p04q180	FTN_1682	frgA	siderophore biosynthesis protein		5
tnfn1_pw060510p04q122	FTN_1698		Dam-replacing family protein		2
Intergenic
tnfn1_pw060418p01q125	intergenic				7
tnfn1_pw060323p06q165	intergenic				5
tnfn1_pw060323p08q117	intergenic				3
tnfn1_pw060328p05q195	intergenic				6
tnfn1_pw060419p01q148	intergenic				3
tnfn1_pw060420p03q148	intergenic				3
tnfn1_pw060420p01q164	intergenic				2
tnfn1_pw060510p02q127	intergenic				2
tnfn1_pw060328p08q109	intergenic				2
tnfn1_pw060510p04q116	intergenic				2
List of growth defective mutants in only U937 Cells
Proteins of unknown function
tnfn1_pw060328p06q147	FTN_0109		protein of unknown function	3^#
tnfn1_pw060418p04q193	FTN_0109		protein of unknown function	4^#
tnfn1_pw060510p01q123	FTN_0132		protein of unknown function	3
tnfn1_pw060323p07q115	FTN_0290		protein of unknown function	5
tnfn1_pw060328p04q122	FTN_0428		protein of unknown function	2^*
tnfn1_pw060510p04q109	FTN_0428		protein of unknown function	2^*
tnfn1_pw060419p03q140	FTN_0477		conserved protein of unknown function	2
tnfn1_pw060420p02q178	FTN_0915		conserved protein of unknown function	7
tnfn1_pw060419p04q188	FTN_0925		protein of unknown function	4
tnfn1_pw060420p02q181	FTN_0933		protein of unknown function	7
tnfn1_pw060419p04q118	FTN_1172		conserved protein of unknown function	2
tnfn1_pw060420p01q127	FTN_1175		membrane protein of unknown function	4
tnfn1_pw060420p01q109	FTN_1367		protein of unknown function	2
tnfn1_pw060420p01q132	FTN_1624		conserved protein of unknown function	4
tnfn1_pw060420p02q184	FTN_1696		protein of unknown function	7
Hypothetical Proteins
tnfn1_pw060323p01q181	FTN_0336		hypothetical protein	3
tnfn1_pw060510p01q147	FTN_0403		hypothetical membrane protein	4
tnfn1_pw060323p01q163	FTN_0727		hypothetical membrane protein	3
tnfn1_pw060418p03q110	FTN_0847		conserved hypothetical protein	2^#
tnfn1_pw060510p02q108	FTN_0847		conserved hypothetical protein	4^#
tnfn1_pw060419p02q152	FTN_0888		hypothetical membrane protein	2
tnfn1_pw060418p01q191	FTN_1349		hypothetical protein	4^#
tnfn1_pw060328p06q182	FTN_1395		conserved hypothetical protein	4
tnfn1_pw060328p04q136	FTN_1406		conserved hypothetical membrane protein	4
tnfn1_pw060420p02q127	FTN_1656		conserved hypothetical protein	2
tnfn1_pw060420p02q176	FTN_1686		hypothetical membrane protein	5
tnfn1_pw060418p03q159	FTN_1736		hypothetical protein	2
Metabolic Proteins
tnfn1_pw060419p02q150	FTN_0090	acpA	acid phosphatase	5
tnfn1_pw060419p03q169	FTN_0218	nfnB	dihydropteridine reductase	2
tnfn1_pw060420p01q123	FTN_0524	asd	aspartate semialdehyde dehydrogenase	5
tnfn1_pw060323p06q168	FTN_0545		glycosyl transferase, group 2	5^#
tnfn1_pw060419p01q187	FTN_0545		glycosyl transferase, group 2	5^#
tnfn1_pw060510p03q168	FTN_0598		tRNA-dihydrouridine synthase	3
tnfn1_pw060328p04q196	FTN_0746	alr	alanine racemase	6^#
tnfn1_pw060420p04q108	FTN_0822		para-aminobenzoate synthase component I	5
tnfn1_pw060420p04q140	FTN_0957		short chain dehydrogenase	4
tnfn1_pw060420p02q174	FTN_1233		haloacid dehalogenase-like hydrolase	6
tnfn1_pw060420p04q116	FTN_1421	wbtH	glutamine amidotransferase/asparagine synthase	3
tnfn1_pw060419p04q135	FTN_1415		thioredoxin	6
tnfn1_pw060510p04q185	FTN_1701		glutamate decarboxylase	3
tnfn1_pw060510p04q136	FTN_1767	rbsK	ribokinase, pfkB family	3
tnfn1_pw060328p05q154	FTN_1777	trpG	anthranilate synthase component II	2^#
Transporter Proteins
tnfn1_pw060420p04q158	FTN_0800		ArsB arsenite/antimonite exporter	2
tnfn1_pw060510p01q152	FTN_1711	tyrP	tyrosine permease	6
DNA Modification
tnfn1_pw060419p04q116	FTN_0287		type I restriction-modification system, subunit R (restriction)	2
tnfn1_pw060420p03q134	FTN_0710		type I restriction-modification system, subunit R (restriction)	4
tnfn1_pw060510p04q179	FTN_0838	xthA	exodeoxyribonuclease III	3
tnfn1_pw060419p04q152	FTN_1017		pseudogene: DNA-3-methyladenine glycosylase	5
tnfn1_pw060323p04q111	FTN_1176	uvrB	excinuclease ABC, subunit B	2
Transferases
tnfn1_pw060420p02q180	FTN_0483		bifunctional NMN adenylyltransferase/Nudix hydrolase	7
tnfn1_pw060510p01q158	FTN_0988	prmA	50S ribosomal protein L11, methyltransferase	7
tnfn1_pw060510p02q144	FTN_1234	queA	S-adenosylmethionine:tRNA ribosyltransferase- isomerase	6
Transcription/Translation
tnfn1_pw060323p03q127	FTN_0567		tRNA synthetase class II (D, K and N)	2
tnfn1_pw060510p03q168	FTN_0598		tRNA-dihydrouridine synthase	3
tnfn1_pw060419p04q129	FTN_1290	mglA	macrophage growth locus, protein A	3^#
Others
tnfn1_pw060328p08q161	-	isftu1	isftu1	2
tnfn1_pw060510p04q176	FTN_0182		ATP-binding cassette (ABC) superfamily protein	2
tnfn1_pw060323p08q110	FTN_0286		transposase	3
tnfn1_pw060420p01q168	FTN_0646	cscK	ROK family protein	5
tnfn1_pw060328p04q123	FTN_0672	secA	preprotein translocase, subunit A (ATPase, RNA helicase)	2
tnfn1_pw060328p04q112	FTN_1002	blaA	beta-lactamase class A	2^#
tnfn1_pw060419p02q192	FTN_1002	blaA	beta-lactamase class A	2^#
tnfn1_pw060420p02q177	FTN_1145	era	GTP-binding protein	6
tnfn1_pw060418p03q107	FTN_1217		ATP-binding cassette (ABC) superfamily protein	2
tnfn1_pw060328p06q171	FTN_1263	comL	competence lipoprotein ComL	2^#
tnfn1_pw060420p02q179	FTN_1263	comL	competence lipoprotein ComL	7^#
tnfn1_pw060323p06q110	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase	2^*
tnfn1_pw060323p07q167	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase	4^*
Intergenic
tnfn1_pw060328p03q108	intergenic			2
tnfn1_pw060419p04q165	intergenic			5
tnfn1_pw060510p01q102	intergenic			5
tnfn1_pw060510p01q112	intergenic			4
tnfn1_pw060510p01q135	intergenic			4

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- Mutants for which all the alleles showed growth defect

- Mutants for which two out of three or three out of four alleles showed growth defect

Table 2.

List of growth defective or dissemination defective mutants in S2 and U937 cells identified in previous screens

Strain Name	Locus Tag	Gene	Description
tnfn1_pw060323p03q172^α	FTN_0008		10 TMS drug/metabolite exporter protein
tnfn1_pw060420p02q151^β ^γ ^δ	FTN_0018	sdaC	serine permease
tnfn1_pw060323p02q177 ^β ^γ ^δ	FTN_0019	pyrB	aspartate carbamoyltransferase
tnfn1_pw060323p08q120 ^β ^γ ^δ	FTN_0020	carB	carbamoyl-phosphate synthase large chain
tnfn1_pw060510p02q160 ^β ^γ ^δ	FTN_0021	carA	carbamoyl-phosphate synthase small chain
tnfn1_pw060419p04q178 ^α	FTN_0028		conserved hypothetical membrane protein
tnfn1_pw060418p04q123^β	FTN_0070	pilE	Type IV pili, pilus assembly protein
tnfn1_pw060420p01q180 ^β ^γ ^δ ^π	FTN_0097		hydroxy/aromatic amino acid permease (HAAAP) family protein
tnfn1_pw060419p01q106 ^α	FTN_0111	ribH	riboflavin synthase beta-chain
tnfn1_pw060510p04q169^δ	FTN_0122	recA	recombinase A protein
tnfn1_pw060510p01q123 ^α	FTN_0132	lpsA	protein of unknown function
tnfn1_pw060323p03q125 ^α	FTN_0133		ribonuclease II family protein
tnfn1_pw060420p02q173 ^α	FTN_0169		conserved hypothetical membrane protein
tnfn1_pw060418p03q133 ^α	FTN_0199	cyoE	heme O synthase
tnfn1_pw060323p04q102 ^β ^γ ^δ	FTN_0211	pcp	pyrrolidone carboxylylate peptidase
tnfn1_pw060510p02q178 ^α	FTN_0217		L-lactate dehydrogenase
tnfn1_pw060420p04q134 ^α	FTN_0297		conserved protein of unknown function
tnfn1_pw060418p03q147 ^α	FTN_0299	putP	proline:Na+ symporter
tnfn1_pw060420p02q146 ^β ^γ ^δ	FTN_0300		glycosyl transferase, group 2
tnfn1_pw060328p01q167 ^β ^γ ^δ	FTN_0330	minD	septum formation inhibitor-activating ATPase
tnfn1_pw060323p08q146^δ	FTN_0331	minC	septum formation inhibitor
tnfn1_pw060328p08q156 ^α	FTN_0340		protein of unknown function
tnfn1_pw060323p06q113 ^β ^γ ^δ	FTN_0420	purCD	SAICAR synthetase/phosphoribosylamine- glycine ligase
tnfn1_pw060328p06q134 ^β ^γ ^δ	FTN_0443	maeA	NAD-dependent malic enzyme
tnfn1_pw060328p05q119 ^β ^γ ^δ ^π	FTN_0444		membrane protein of unknown function
tnfn1_pw060323p05q182 ^β ^γ ^δ	FTN_0504	cadC	lysine decarboxylase
tnfn1_pw060510p01q124 ^β ^γ ^δ	FTN_0507	gcvP1	glycine cleavage system P protein, subunit 1
tnfn1_pw060323p06q168 ^β ^γ ^δ	FTN_0545		glycosyl transferase, group 2
tnfn1_pw060419p02q135^β	FTN_0560	ksgA	dimethyladenosine transferase
tnfn1_pw060419p03q116 ^β ^γ ^δ	FTN_0593	sucD	succinyl-CoA synthetase, alpha subunit
tnfn1_pw060510p04q147 ^β ^γ ^δ	FTN_0599		protein of unknown function
tnfn1_pw060323p06q164 ^β ^γ ^δ	FTN_0624		serine permease
tnfn1_pw060418p02q128^δ	FTN_0633	katG	peroxidase/catalase
tnfn1_pw060420p01q168 ^α	FTN_0646	cscK	ROK family protein
tnfn1_pw060419p01q168 ^β ^γ ^δ	FTN_0651	cdd	cytidine deaminase
tnfn1_pw060510p04q168^δ	FTN_0666	uvrA	excinuclease ABC, subunit A
tnfn1_pw060328p04q123 ^β ^γ ^δ	FTN_0672	secA	preprotein translocase, subunit A (ATPase, RNA helicase)
tnfn1_pw060420p03q134 ^α	FTN_0710		type I restriction-modification system, subunit R (restriction)
tnfn1_pw060328p06q127 ^α	FTN_0713	ostA2	organic solvent tolerance protein OstA
tnfn1_pw060328p06q132 ^β ^γ ^δ	FTN_0728		predicted Co/Zn/Cd cation transporter
tnfn1_pw060323p06q115 ^α	FTN_0768	tspO	tryptophan-rich sensory protein
tnfn1_pw060323p06q105 ^α	FTN_0810		ROK family protein
tnfn1_pw060323p07q185 ^β ^γ ^δ	FTN_0814	bioF	8-amino-7-oxononanoate synthase
tnfn1_pw060418p01q141^β	FTN_0817		conserved protein of unknown function
tnfn1_pw060420p04q108 ^β ^γ ^δ ^π	FTN_0822		para-aminobenzoate synthase component I
tnfn1_pw060420p03q116 ^α	FTN_0824		major facilitator superfamily (MFS) transport protein
tnfn1_pw060328p01q128 ^α	FTN_0840	mdaB	NADPH-quinone reductase (modulator of drug activity B)
tnfn1_pw060420p04q176 ^β ^γ ^δ	FTN_0855		protein of unknown function
tnfn1_pw060420p02q175 ^α	FTN_0877	cls	cardiolipin synthetase
tnfn1_pw060323p04q104 ^α	FTN_0918		conserved protein of unknown function
tnfn1_pw060419p04q188 ^β ^γ ^δ	FTN_0925		protein of unknown function
tnfn1_pw060420p02q181 ^α	FTN_0933		protein of unknown function
tnfn1_pw060323p04q134 ^α	FTN_0938		hypothetical protein
tnfn1_pw060419p01q170 ^α	FTN_1006		transporter-associated protein, HlyC/CorC family
tnfn1_pw060323p08q141 ^α	FTN_1015		isochorismatase family protein
tnfn1_pw060418p01q153 ^α	FTN_1055	lon	DNA-binding, ATP-dependent protease La
tnfn1_pw060510p01q114^π	FTN_1073		DNA/RNA endonuclease G
tnfn1_pw060419p04q168 ^β ^γ ^δ	FTN_1091	aroA	3-phosphoshikimate 1- carboxyvinyltransferase
tnfn1_pw060328p08q188 ^α	FTN_1098		conserved hypothetical membrane protein
tnfn1_pw060328p02q109 ^α	FTN_1107	metlQ	methionine uptake transporter (MUT) family protein, membrane and periplasmic protein
tnfn1_pw060328p02q175 ^β ^γ ^δ	FTN_1131	putA	bifunctional proline dehydrogenase, pyrroline-5-carboxylate dehydrogenase
tnfn1_pw060418p03q107 ^β ^γ ^δ	FTN_1217		ATP-binding cassette (ABC) superfamily protein
tnfn1_pw060323p08q166 ^α	FTN_1232		conserved hypothetical membrane protein
tnfn1_pw060328p06q178 ^β ^γ ^δ	FTN_1241		DedA family protein
tnfn1_pw060510p03q135 ^β	FTN_1254		protein of unknown function
tnfn1_pw060420p04q196 ^β ^γ ^δ ^π	FTN_1256		membrane protein of unknown function
tnfn1_pw060323p03q102 ^β ^γ ^δ	FTN_1257		membrane protein of unknown function
tnfn1_pw060420p02q179 ^β ^γ ^δ	FTN_1263	comL	competence lipoprotein ComL
tnfn1_pw060418p01q149 ^β ^γ ^δ	FTN_1298		GTPase of unknown function
tnfn1_pw060328p01q144 ^β ^γ ^δ	FTN_1313		hypothetical protein
tnfn1_pw060328p06q163^β	FTN_1315		protein of unknown function
tnfn1_pw060510p01q110 ^α ^β ^γ ^δ	FTN_1321	iglD	intracellular growth locus protein D
tnfn1_pw060328p06q115 ^β ^γ ^δ	FTN_1322	iglC	intracellular growth locus protein C
tnfn1_pw060419p04q108 ^β ^γ ^δ	FTN_1325	pdpD	protein of unknown function
tnfn1_pw060510p01q142 ^β ^γ ^δ	FTN_1333	tktA	transketolase I
tnfn1_pw060418p01q191 ^α	FTN_1349		hypothetical protein
tnfn1_pw060418p01q185 ^α	FTN_1355		regulatory factor, Bvg accessory factor family
tnfn1_pw060418p02q109^π	FTN_1376		disulfide bond formation protein, DsbB family
tnfn1_pw060418p03q185 ^α	FTN_1400		S-adenosylmethionine-dependent methyltransferase
tnfn1_pw060419p04q135 ^α	FTN_1415		thioredoxin
tnfn1_pw060420p04q116 ^β ^γ ^δ	FTN_1421	wbtH	glutamine amidotransferase/asparagine synthase
tnfn1_pw060510p04q137 ^β ^γ ^δ	FTN_1425	wbtF	NAD dependent epimerase
tnfn1_pw060419p03q166 ^β ^γ ^δ ^π	FTN_1431	wbtA	dTDP-glucose 4,6-dehydratase
tnfn1_pw060418p02q122 ^β ^γ ^δ	FTN_1438		bifunctional protein: 3-hydroxacyl-CoA dehydrogenase/acyl-CoA-binding protein
tnfn1_pw060328p08q196 ^α	FTN_1459		short chain dehydrogenase
tnfn1_pw060323p06q110 ^β ^γ ^δ	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase
tnfn1_pw060328p06q128^β	FTN_1530	lysA	diaminopimelate decarboxylase
tnfn1_pw060323p07q176 ^α	FTN_1534		conserved protein of unknown function
tnfn1_pw060418p02q178 ^α	FTN_1536		amino acid-polyamine-organocation (APC) superfamily protein
tnfn1_pw060418p01q150^γ	FTN_1586		sugar transporter, MFS superfamily
tnfn1_pw060510p01q146 ^β ^γ ^δ	FTN_1597	prfC	peptide chain release factor 3
tnfn1_pw060420p01q189 ^α	FTN_1611		major facilitator superfamily (MFS) transport protein
tnfn1_pw060323p04q160 ^β ^γ ^δ	FTN_1655	rluC	ribosomal large subunit pseudouridine synthase C
tnfn1_pw060419p04q180^δ	FTN_1682	frgA	siderophore biosynthesis protein
tnfn1_pw060323p03q163 ^β ^γ ^δ	FTN_1683		drug:H+ antiporter-1 (DHA1) family protein
tnfn1_pw060328p05q154 ^β ^γ ^δ	FTN_1777	trpG	anthranilate synthase component II

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^α

- (Kraemer et al., 2009)

^β

- (Weiss et al., 2007)

^γ

- (Qin and Mann, 2006)

^δ

- (Su et al., 2007)

^π

- (Maier et al., 2007)

Fig 1 — The S2 cells were infected with mutants of *F. tularensis* subsp *novicida* at MOI of 10 for 1 h followed by 1 h of gentamicin treatment. Growth of the mutants was compared to the wild type strain at 24 h post-infection and the relative reduction in growth relative to the wild type strain was determined. Mutants were considered defective if they exhibited ≥10 fold reduction in growth. The growth defective mutants were grouped according to the function of the genes.

The metabolic genes were grouped according to the putative biochemical pathway. Our analysis showed that a large number of the metabolic genes that were required for replication in S2 cells were involved in both amino acid and carbohydrate metabolism or involved in the synthesis of co-enzymes or cofactors required for carbohydrate and amino acid metabolic pathways (Table 3). At least 11 genes required for nucleotide metabolism were required for intracellular proliferation (Table 3). Therefore, inability of F. tularensis to efficiently metabolize amino acids, carbohydrates, and nucleotides affected its ability to replicate within S2 cells. This indicates that the intracellular environment does not have sufficient nutrients required for intracellular bacterial proliferation and the bacteria require de novo synthesis of metabolic intermediates to support intracellular proliferation. Remarkably, the distribution of the metabolic genes across the various functional groups is similar for S2 cells and human macrophages (see accompanying manuscript), indicating similar metabolic requirements for F. tularensis to proliferate within evolutionarily distant host cells.

Table 3.

Metabolic genes required for intracellular proliferation of F. tularensis within S2 cells grouped according to metabolic pathways

Amino acid metabolism
tnfn1_pw060323p08q120	FTN_0020	carB	carbamoyl-phosphate synthase large chain
tnfn1_pw060510p02q160	FTN_0021	carA	carbamoyl-phosphate synthase small chain
tnfn1_pw060328p05q159	FTN_0118		serine peptidase, S49 family
tnfn1_pw060420p02q187	FTN_0118		serine peptidase, S49 family
tnfn1_pw060328p06q174	FTN_0125	ackA	propionate kinase 2/acetate kinase A
tnfn1_pw060323p05q182	FTN_0504		lysine decarboxylase
tnfn1_pw060510p01q124	FTN_0507	gcvP1	glycine cleavage system P protein, subunit 1
tnfn1_pw060510p02q154	FTN_0511		shikimate 5-dehydrogenase
tnfn1_pw060510p02q157	FTN_0511		shikimate 5-dehydrogenase
tnfn1_pw060510p04q157	FTN_0511		shikimate 5-dehydrogenase
tnfn1_pw060323p06q194	FTN_0527	thrC	threonine synthase
tnfn1_pw060510p01q172	FTN_0527	thrC	threonine synthase
tnfn1_pw060510p03q172	FTN_0527	thrC	threonine synthase
tnfn1_pw060420p01q135	FTN_0540	pckA	phosphoenolpyruvate carboxykinase
tnfn1_pw060510p03q171	FTN_0588		asparaginase
tnfn1_pw060418p04q167	FTN_0935	asnB	asparagine synthase
tnfn1_pw060328p06q142	FTN_0954		histidine acid phosphatase
tnfn1_pw060328p02q175	FTN_1131	putA	bifunctional proline dehydrogenase, pyrroline-5- carboxylate dehydrogenase
tnfn1_pw060328p08q131	FTN_1174	murI	glutamate racemase
tnfn1_pw060328p06q128	FTN_1530	lysA	diaminopimelate decarboxylase
tnfn1_pw060328p05q101	FTN_1532	gdhA	glutamate dehydrogenase (NADP+)
tnfn1_pw060419p04q163	FTN_1532	gdhA	glutamate dehydrogenase (NADP+)
tnfn1_pw060328p05q160	FTN_1729	dapB	dihydrodipicolinate reductase
tnfn1_pw060510p01q178	FTN_1729	dapB	dihydrodipicolinate reductase
tnfn1_pw060328p04q104	FTN_1730	lysC	aspartate kinase III
tnfn1_pw060328p04q116	FTN_9765		choloylglycine hydrolase family protein
Carbohydrate metabolism
tnfn1_pw060328p06q139	FTN_0127	gabD	succinate semialdehyde dehydrogenase (NAD(P)+ dependent)
tnfn1_pw060510p02q178	FTN_0217		L-lactate dehydrogenase
tnfn1_pw060328p06q134	FTN_0443	maeA	NAD-dependent malic enzyme
tnfn1_pw060418p04q116	FTN_0512	glgX	pullulanase
tnfn1_pw060510p03q154	FTN_0516	glgA	glycogen synthase
tnfn1_pw060419p03q116	FTN_0593	sucD	succinyl-CoA synthetase, alpha subunit
tnfn1_pw060328p02q139	FTN_0621	eno	enolase (2-phosphoglycerate dehydratase)
tnfn1_pw060510p03q188	FTN_0627	chiA	chitinase, glycosyl hydrolase family 18
tnfn1_pw060328p06q131	FTN_0674	glxK	glycerate kinase
tnfn1_pw060510p03q119	FTN_0806		glycosyl hydrolase family 3
tnfn1_pw060510p02q187	FTN_1018		aldolase/adducin class II family protein
tnfn1_pw060328p03q107	FTN_1222	kpsF	phosphosugar isomerase
tnfn1_pw060328p05q128	FTN_1329	fbaA	fructose bisphosphate aldolase Class II
tnfn1_pw060510p04q137	FTN_1425	wbtF	NAD dependent epimerase
tnfn1_pw060419p03q166	FTN_1431	wbtA	dTDP-glucose 4,6-dehydratase
tnfn1_pw060323p07q169	FTN_1438		bifunctional protein: 3-hydroxacyl-CoA dehydrogenase/acyl-CoA-binding protein
tnfn1_pw060418p02q122	FTN_1438		bifunctional protein: 3-hydroxacyl-CoA dehydrogenase/acyl-CoA-binding protein
tnfn1_pw060328p06q150	FTN_1494	aceE	pyruvate dehydrogenase complex, E1 component, pyruvate dehydrogenase
tnfn1_pw060420p04q105	FTN_1584	glpD	glycerol-3-phosphate dehydrogenase
tnfn1_pw060419p04q130	FTN_1585	glpK	glycerol kinase
tnfn1_pw060419p02q112	FTN_1619	appC	cytochrome bd-II terminal oxidase subunit I
tnfn1_pw060328p02q105	FTN_1620	appB	cytochrome bd-II terminal oxidase subunit II
tnfn1_pw060510p04q136	FTN_1767	rbsK	ribokinase, pfkB family
Nucleotide metabolism
tnfn1_pw060323p08q120	FTN_0020	carB	carbamoyl-phosphate synthase large chain
tnfn1_pw060510p02q160	FTN_0021	carA	carbamoyl-phosphate synthase small chain
tnfn1_pw060418p03q133	FTN_0199	cyoE	heme O synthase
tnfn1_pw060323p06q113	FTN_0420		SAICAR synthetase/phosphoribosylamine-glycine ligase
tnfn1_pw060419p01q168	FTN_0651	cdd	cytidine deaminase
tnfn1_pw060328p04q151	FTN_0661	guaB	IMP dehydrogenase/GMP reductase
tnfn1_pw060510p01q159	FTN_0695	add	deoxyadenosine deaminase/adenosine deaminase
tnfn1_pw060328p01q151	FTN_0983		bifunctional protein: glutaredoxin 3/ribonucleotide reductase beta subunit
tnfn1_pw060419p04q135	FTN_1415		thioredoxin
tnfn1_pw060419p04q181	FTN_1415		thioredoxin
tnfn1_pw060510p01q118	FTN_1553	nudH	dGTP pyrophosphohydrolase
Reductive Metabolism
tnfn1_pw060418p04q115	FTN_0095		nitroreductase
tnfn1_pw060510p01q130	FTN_0154	rimK	glutathione synthase/ribosomal protein S6 modification enzyme
tnfn1_pw060419p04q153	FTN_0597		protein-disulfide isomerase
tnfn1_pw060418p02q128	FTN_0633	katG	peroxidase/catalase
tnfn1_pw060418p02q142	FTN_0826		aldo/keto reductase family protein
tnfn1_pw060328p01q128	FTN_0840	mdaB	NADPH-quinone reductase (modulator of drug activity B)
tnfn1_pw060420p03q153	FTN_0840	mdaB	NADPH-quinone reductase (modulator of drug activity B)
tnfn1_pw060323p05q141	FTN_1033	grxB	glutaredoxin 2
tnfn1_pw060420p01q193	FTN_1033	grxB	glutaredoxin 2
tnfn1_pw060420p04q194	FTN_1231	gloA	lactoylglutathione lyase
tnfn1_pw060510p02q164	FTN_1231	gloA	lactoylglutathione lyase
tnfn1_pw060510p04q146	FTN_1231	gloA	lactoylglutathione lyase
tnfn1_pw060328p08q196	FTN_1459		short chain dehydrogenase
tnfn1_pw060418p01q131	FTN_1557		oxidoreductase iron/ascorbate family protein
tnfn1_pw060418p04q112	FTN_1621		predicted NAD/FAD-dependent oxidoreductase
tnfn1_pw060420p04q169	FTN_1621		predicted NAD/FAD-dependent oxidoreductase
cholesterol Metabolism
tnfn1_pw060323p04q144	FTN_0406		sterol desaturase
tnfn1_pw060418p01q189	FTN_0406		sterol desaturase
Lipid Metabolism
tnfn1_pw060328p04q175	FTN_0818		lipase/esterase
tnfn1_pw060420p02q175	FTN_0877	cls	cardiolipin synthetase
tnfn1_pw060328p06q166	FTN_1273		long chain fatty acid CoA ligase
conenzyme synthesis
tnfn1_pw060328p06q130	FTN_0692	nadA	quinolinate sythetase A
tnfn1_pw060419p04q164	FTN_0692	nadA	quinolinate sythetase A
tnfn1_pw060420p01q148	FTN_0694	nadB	L-aspartate oxidase
tnfn1_pw060328p06q156	FTN_0811	birA	biotin--acetyl-CoA-carboxylase ligase
tnfn1_pw060323p07q185	FTN_0814	bioF	8-amino-7-oxononanoate synthase
tnfn1_pw060419p02q138	FTN_0814	bioF	8-amino-7-oxononanoate synthase
tnfn1_pw060418p01q124	FTN_1245	iscS	cysteine desulfurase
tnfn1_pw060419p03q126	FTN_1278	nadE	NAD synthase
tnfn1_pw060323p04q110	FTN_1678	nuoC	NADH dehydrogenase I, C subunit
tnfn1_pw060419p01q106	FTN_0111	ribH	riboflavin synthase beta-chain
tnfn1_pw060420p02q191	FTN_0113	ribC	riboflavin synthase alpha chain
Peptidoglycan biosynthesis
tnfn1_pw060328p06q125	FTN_0496	slt	soluble lytic murein transglycosylase
tnfn1_pw060323p04q102	FTN_0211	pcp	pyrrolidone carboxylylate peptidase
tnfn1_pw060418p03q177	FTN_0211	pcp	pyrrolidone carboxylylate peptidase
Aromatic compound biosynthesis
tnfn1_pw060323p08q141	FTN_1015		isochorismatase family protein
tnfn1_pw060420p01q129	FTN_1015		isochorismatase family protein
tnfn1_pw060328p02q174	FTN_1135	aroB	3-dehydroquinate synthetase
ppGpp biosynthesis
tnfn1_pw060323p06q110	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase
tnfn1_pw060323p07q167	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase

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For some mutants defective in intracellular proliferation, the two or more mutant alleles resulted in different defective phenotype (Table 1). The lack of consistent phenotype for the two mutant alleles for some of the mutants may be due to the site of the insertion, which may generate a functional or partially functional protein. The intracellular growth defect for most of the mutant alleles that exhibited growth defect was not due to a general defect in growth, since more than 98% of the mutants exhibited normal growth in vitro, compared to the wild type strain. The in vitro growth was analyzed by measuring the OD of all the 501 mutants identified in the primary screen and the wild type strain after overnight growth in broth. It is possible the defect in some of the mutants may be due to a defect in attachment and/or invasion. It is likely that the reduction in intracellular growth for some of the mutants was due to a polar effect of the transposon insertion on downstream genes. However, the possible polar effect would implicate the identified disrupted operon in intracellular proliferation.

Interestingly, 75 of the 394 mutants that showed defect in intracellular proliferation within S2 cells, have been shown in previous screens to exhibit growth defect for various aspects of virulence of F. tularensis in mammals (Table 2) (Qin and Mann, 2006; Maier et al., 2007; Su et al., 2007; Weiss et al., 2007; Kraemer et al., 2009). Importantly, ~90% of F. tularensis subsp novicida genes that affect intracellular proliferation in S2 cells are conserved in the virulent F. tularensis subsp tularensis, which validates the use of subsp novicida as a model system.

Comparison of growth-defective mutants in S2 cells to the phenotype in human macrophages

A concurrent study from our lab has identified mutants that exhibit growth defect in human macrophages (see accompanying manuscript). Infections of U937 human macrophages were performed similar to S2 cells. To assess whether similar molecular mechanisms are utilized by F. tularensis to infect arthropod-derived cells and human macrophages, we compared the mutants identified to be defective in S2 cells to the mutants identified to be defective in human macrophages. Among the 394 mutants defective in replication in D. melanogaster S2 cells 135 of them were also required for replication in human macrophages (Fig. 2). This large number (259) of loci indicates that some common mechanisms are utilized by F. tularensis to proliferate within both insect-derived cells and human macrophages.

Fig 2 — Growth of the mutants was compared to the wild type strain and the relative reduction in cfu relative to the wild type strain at 24 h post-infection was determined. The mutants were divided into two groups depending on whether they showed growth defect in S2 cells only or both S2 cells and U937 macrophages. The defective mutants in each group were categorized according to the function of the genes.

However, there is a significant difference in the molecular mechanisms required for replication in the two evolutionarily distinct host cells, since 259 of the genes identified to be required for intracellular replication are specific to S2 cells. Similarly, among 202 mutants defective in replication in human macrophages more than 143 were specific to U937 cells, indicating common as well as distinct mechanisms required for the infection of the two evolutionarily distant host cells. However, we can’t exclude the possibility that some of the differences in the phenotypes of the mutants between the two host cells may be due to the fact that simply different cell lines were used and the differences might not be host-specific for some of the mutants. We also can’t exclude the possibility that some of the differences of the phenotypes of the mutants between U937 and S2 cells may be due to the fact that the infection of both host cells was done at different temperatures where the S2 cells must be incubated below 30°C. The U937 cells are PMA-differentiated human macrophage-like cells, while S2 are embryonic macrophage-like cells of an insect. The distribution of the mutants across the different functional categories in the S2 cells was similar to mutants that were defective in both S2 cells and human macrophages and mutants defective in S2 cells only (Fig. 2). Among 70% of the mutants that were attenuated in both S2 cells and human macrophages, the level of attenuation of growth was similar in both S2 cells and human macrophages. However, the other 30% of the mutants showed up to 10³ fold difference in the levels of attenuation between S2 cells and human macrophages (Table 1).

Previous studies have shown that in both arthropod cells and human macrophages, F. tularensis transiently occupies a late endosome-like phagosome followed by rapid bacterial escape into the cytosol, where the bacteria proliferate robustly (Golovliov et al., 2003; Clemens et al., 2004; Santic et al., 2005a; Santic et al., 2005b; Checroun et al., 2006; Santic et al., 2007; Bonquist et al., 2008; Chong et al., 2008; Santic et al., 2008; Qin et al., 2009; Wehrly et al., 2009). In our concurrent study, we showed that of the 135 genes required for intracellular proliferation in both macrophages and S2 cells, 59 loci were required for modulation of phagosome biogenesis and bacterial escape into the cytosol of macrophages (see accompanying manuscript). Since we have shown that modulation of phagosome biogenesis and escape into the cytosol by F. tularensis is similar in both arthropod cells and macrophages (Vonkavaara et al., 2008; Santic et al., 2009), it is most likely that these 59 loci are also required for modulation of phagosome biogenesis and phagosomal escape in S2 cells, similar to their role in macrophages. This indicates that conserved as well as distinct molecular mechanisms are utilized by F. tularensis to escape from the phagosome of evolutionarily distant host cells. It is likely that interaction of F. tularensis with arthropods has played a role in its ecology and patho-adaptation to infect mammals. This may not be surprising, since there are numerous evolutionary conserved pathways between the two species, such as innate immune responses and intracellular trafficking events.

Lethality of F. tularensis mutants to D. melanogaster adult flies

Previous in vivo studies on replication of F. tularensis in adult D. melanogaster has shown that some mutants, which are attenuated in human cells or in other mammalian models of infection, are also attenuated in growth and are less lethal to D. melanogaster adult flies (Vonkavaara et al., 2008; Santic et al., 2009). Thus, we determined whether mutants that were attenuated in S2 cells were also attenuated in virulence in adult flies. A total of 168 most defective mutants in intracellular proliferation in S2 cells were selected to be tested in the adult flies for lethality first, then for proliferation (Table 4). To obtain the survival ratio of infected flies, the number of flies that survived the infection at day 15 was divided by the number of flies immediately after infection. We calculated from 13 independent experiments that the average percent survival of wild type strain-infected flies by day 15 was ~22% whereas the percentage of survival of flies infected with the iglC mutant was 85.5% (p < 0.001, unpaired student t-test). We considered a mutant to be attenuated if flies infected with that mutant showed ≥50% survival rate at 15 days post-infection, which is statistically different from the wild type strain-infected flies for the same period (p< 0.0012, unpaired student t-test). A 50–74% survival rate for infected flies was considered moderate attenuation for the mutant and 75–100% survival rate for the flies represented highly attenuated mutant strain. From the 168 mutants tested, a total of 80 mutants were attenuated in lethality to D. melanogaster (Table 4). Twenty two of 80 mutant strains were highly attenuated for growth in D. melanogaster and 58 mutant strains exhibited moderate attenuation (Table 4 and Fig 3). We classified the mutants that showed attenuation in D. melanogaster to groups of functional categories. Higher percentages (60%) of mutants from the “Others” and “unknown function” functional categories were found to be attenuated in lethality to adult D. melanogaster (Table 4), which indicates that further studies of these novel genes might reveal unique mechanism that F. tularensis utilizes for interaction with the arthropod host. For the rest of the functional categories, the percentage of mutants attenuated in lethality to D. melanogaster was 35–45% (Table 4). Among mutants defective in intracellular growth in S2 cells that were tested, 21 of them were also found in other screens to be attenuated in vivo or in-vitro growth of mammalian models of tularemia (Qin and Mann, 2006; Maier et al., 2007; Su et al., 2007; Weiss et al., 2007; Kraemer et al., 2009). We observed that 10 of these loci were also associated with less lethality to D. melanogaster in our screen. Mutants that had differential effects in D. melanogaster and the mammalian host may perhaps highlight the differences of these hosts during F. tularensis infection and warrant further studies.

Table 4.

List of F. tularensis mutants defective in growth and lethality to D. melanogaster grouped according to function.

Strain Name	Locus Tag	Gene	Description	Log Reduction in Growth Relative to WT		Percent survival	Mut/WT cfu ratio lls
Strain Name	Locus Tag	Gene	Description	S2	U937	Percent survival	Mut/WT cfu ratio lls
Controls
Wild type strain		WT
Intracellular growth locus		IglC
Cell Division
tnfn1_pw060328p01q167	FTN_0330	minD	septum formation inhibitor-activating ATPase	2	2	13%	24.6E-03
DNA Modification
tnfn1_pw060510p01q114	FTN_1073		DNA/RNA endonuclease G	6	5	90%	1.2E-02
tnfn1_pw060328p04q156	FTN_1027	ruvC	holliday junction endodeoxyribonuclease	4	3	60%	4.7E-01
tnfn1_pw060328p08q179	FTN_1491		adenine specific DNA methylase	2		50%	6.5E-01
tnfn1_pw060323p03q167	FTN_1197	recR	RecFOR complex, RecR component	4	2	44%	6.7E-04
tnfn1_pw060328p06q158	FTN_1293	rnhB	ribonuclease HII	5	3	40%	1.4E+00
tnfn1_pw060328p05q121	FTN_1487		restriction endonuclease	1	1	40%	9.0E+00
tnfn1_pw060510p04q193	FTN_0680	uvrC	excinuclease ABC, subunit C	3	6	20%	1.0E-01
tnfn1_pw060510p01q153	FTN_1154		type I restriction-modification system, subunit S	6	5	20%	3.2E-01
tnfn1_pw060323p03q122	FTN_0577	mutL	DNA mismatch repair enzyme with ATPase activity	5	5	0%	2.5E-02
FPI
tnfn1_pw060418p04q106	FTN_1319	pdpC	hypothetical protein	5	3	90%	1.2E-04
tnfn1_pw060510p01q110	FTN_1321	iglD	intracellular growth locus protein D	6	4	60%	2.1E-01
tnfn1_pw060328p06q115	FTN_1322	iglC	intracellular growth locus protein C	5		33%
Hypothetical Protein
tnfn1_pw060420p04q159	FTN_1223		conserved hypothetical membrane protein	7		90%	3.1E-02
tnfn1_pw060419p03q188	FTN_0696		hypothetical membrane protein	2	2	78%	2.1E-02
tnfn1_pw060323p07q129	FTN_0759		conserved hypothetical protein	2	4	71%	2.1E+00
tnfn1_pw060328p06q180	FTN_0038		hypothetical protein	4	4	70%	3.5E-04
tnfn1_pw060323p03q142	FTN_0030		hypothetical membrane protein	3	4	63%	2.2E-03
tnfn1_pw060328p05q136	FTN_0384		conserved hypothetical protein	7	4	62%	2.3E-02
tnfn1_pw060328p06q126	FTN_0732		hypothetical protein	5		60%	1.6E+01
tnfn1_pw060328p02q129	FTN_1612		hypothetical protein	2	2	50%	2.3E-02
tnfn1_pw060328p04q136	FTN_1406		conserved hypothetical membrane protein	3	5	50%	9.0E-02
tnfn1_pw060328p08q101	FTN_0054		hypothetical protein	1	1	40%	3.1E-07
tnfn1_pw060418p01q191	FTN_1349		hypothetical protein	6	6	40%	2.0E-03
tnfn1_pw060323p07q105	FTN_0895		hypothetical protein	2	4	38%	4.4E-02
tnfn1_pw060420p02q139	FTN_0013		hypothetical protein	3		33%	2.3E+02
tnfn1_pw060418p02q175	FTN_0717		conserved hypothetical membrane protein	5		30%	2.3E-02
tnfn1_pw060420p02q173	FTN_0169		conserved hypothetical membrane protein	6	6	30%	1.9E+03
tnfn1_pw060323p06q178	FTN_1389		conserved hypothetical membrane protein	3		25%	9.6E-05
tnfn1_pw060419p04q117	FTN_1156		hypothetical protein	4	4	25%	3.0E+01
tnfn1_pw060419p02q102	FTN_0792		hypothetical protein	6	5	22%	2.3E+01
tnfn1_pw060419p03q187	FTN_1123		conserved hypothetical protein	3		20%	1.0E-01
tnfn1_pw060420p04q104	FTN_0466		conserved hypothetical protein	4		20%	1.3E+00
tnfn1_pw060328p06q185	FTN_0709		hypothetical protein	7	3	20%	1.3E+00
tnfn1_pw060510p04q192	FTN_1098		conserved hypothetical membrane protein	6	7	11%	2.7E-02
tnfn1_pw060510p01q184	FTN_1299		hypothetical protein	5		10%	1.3E-01
tnfn1_pw060418p03q151	FTN_0212		hypothetical membrane protein	3		10%	3.3E+00
Intergenic
tnfn1_pw060328p06q190	intergenic			3	3	89%	9.9E-03
tnfn1_pw060420p03q148	intergenic			5		80%	5.0E-02
tnfn1_pw060323p08q139	intergenic			4	4	44%	1.6E-02
tnfn1_pw060419p03q131	intergenic			2	2	40%	5.5E-01
tnfn1_pw060419p04q189	intergenic			3	5	22%	9.8E-02
Metabolic
tnfn1_pw060420p04q105	FTN_1584	glpD	glycerol-3-phosphate dehydrogenase	5	3	100%	5.6E-04
tnfn1_pw060323p05q182	FTN_0504		lysine decarboxylase	4	4	100%	6.7E-04
tnfn1_pw060510p03q154	FTN_0516	glgA	glycogen synthase	4		90%	3.6E-04
tnfn1_pw060328p06q142	FTN_0954		histidine acid phosphatase	4	4	90%	9.8E-03
tnfn1_pw060328p06q130	FTN_0692	nadA	quinolinate sythetase A	2	3	90%	2.3E-01
tnfn1_pw060328p02q174	FTN_1135	aroB	3-dehydroquinate synthetase	4	3	80%	3.5E-04
tnfn1_pw060328p06q150	FTN_1494	aceE	pyruvate dehydrogenase complex, E1 component, pyruvate dehydrogenase	1	4	70%	4.7E-01
tnfn1_pw060418p02q109	FTN_1376		disulfide bond formation protein, DsbB family	4	4	70%	6.1E+02
tnfn1_pw060420p04q169	FTN_1621		predicted NAD/FAD-dependent oxidoreductase	4	4	67%	4.2E-01
tnfn1_pw060510p01q124	FTN_0507	gcvP1	glycine cleavage system P protein, subunit 1	7	5	67%	4.0E+00
tnfn1_pw060323p06q106	FTN_1552		acid phosphatase, PAP2 family	4		60%	2.8E-04
tnfn1_pw060419p01q106	FTN_0111	ribH	riboflavin synthase beta-chain	5	4	60%	5.6E-03
tnfn1_pw060328p08q131	FTN_1174	murI	glutamate racemase	5		60%	2.3E-01
tnfn1_pw060419p03q116	FTN_0593	sucD	succinyl-CoA synthetase, alpha subunit	2	2	60%	2.6E+01
tnfn1_pw060323p04q110	FTN_1678	nuoC	NADH dehydrogenase I, C subunit	2		55%	7.9E-04
tnfn1_pw060323p03q127	FTN_0567		tRNA synthetase class II (D, K and N)	2	6	55%	5.6E-03
tnfn1_pw060510p03q171	FTN_0588		asparaginase	2	1	50%	3.0E-02
tnfn1_pw060510p03q188	FTN_0627	chiA	chitinase, glycosyl hydrolase family 18	2		50%	1.4E+00
tnfn1_pw060510p01q142	FTN_1333	tktA	transketolase I	5	5	45%	1.8E-06
tnfn1_pw060510p02q178	FTN_0217		L-lactate dehydrogenase	2		44%	4.1E+01
tnfn1_pw060323p06q195	FTN_1390		Zn-dependent hydrolase	3		40%	1.2E-01
tnfn1_pw060419p01q168	FTN_0651	cdd	cytidine deaminase	6		38%	3.5E+00
tnfn1_pw060510p02q187	FTN_1018		aldolase/adducin class II family protein	3	3	33%	3.0E-03
tnfn1_pw060328p02q105	FTN_1620	appB	cytochrome bd-II terminal oxidase subunit II	3	6	33%	4.4E-02
tnfn1_pw060323p06q103	FTN_0711		predicted metal-dependent hydrolase	4		30%	2.4E-04
tnfn1_pw060328p08q145	FTN_0907		D-alanyl-D-alanine carboxypeptidase	3		30%	1.7E-01
tnfn1_pw060419p04q130	FTN_1585	glpK	glycerol kinase	3	3	29%	4.4E-01
tnfn1_pw060328p01q151	FTN_0983		bifunctional protein: glutaredoxin 3/ribonucleotide reductase beta subunit	3	5	25%	1.9E-03
tnfn1_pw060419p02q112	FTN_1619	appC	cytochrome bd-II terminal oxidase subunit I	7	5	22%	4.9E-02
tnfn1_pw060323p07q167	FTN_1518	relA	GDP pyrophosphokinase/GTP pyrophosphokinase	4	4	22%	3.6E-01
tnfn1_pw060420p02q175	FTN_0877	cls	cardiolipin synthetase	5	7	20%	6.8E-02
tnfn1_pw060328p03q174	FTN_1768	pepN	aminopeptidase N	2		20%	8.0E+00
tnfn1_pw060418p02q122	FTN_1438		bifunctional protein: 3-hydroxacyl- CoA dehydrogenase/acyl-CoA- binding protein	4		20%	8.8E+01
tnfn1_pw060323p06q113	FTN_0420		SAICAR synthetase/phosphoribosylamine- glycine ligase	5	7	13%	2.0E-01
tnfn1_pw060510p01q146	FTN_1597	prfC	peptide chain release factor 3	5	5	13%	6.8E+02
tnfn1_pw060510p01q118	FTN_1553	nudH	dGTP pyrophosphohydrolase	5	5	11%	1.3E-01
tnfn1_pw060328p06q156	FTN_0811	birA	biotin--acetyl-CoA-carboxylase ligase	7	6	11%	4.5E-02
tnfn1_pw060328p08q120	FTN_0987		tRNA-dihydrouridine synthase	4		11%	2.7E+00
tnfn1_pw060420p03q153	FTN_0840	mdaB	NADPH-quinone reductase (modulator of drug activity B)	5	5	10%	2.9E-01
tnfn1_pw060420p01q130	FTN_0965		metal-dependent exopeptidase	3	3	9%	1.2E+00
tnfn1_pw060419p03q164	FTN_1186	pepO	M13 family metallopeptidase	2		0%	4.0E-01
Others
tnfn1_pw060418p02q123	FTN_0107	lepA	GTP-binding protein LepA	4	4	90%	9.4E-02
tnfn1_pw060323p06q105	FTN_0810		ROK family protein	4		70%	2.4E-04
tnfn1_pw060328p08q114	FTN_0266	htpG	chaperone Hsp90, heat shock protein HtpG	2		67%	5.8E-01
tnfn1_pw060419p01q120	FTN_1488		prophage maintenance system killer protein (DOC)	6		60%	7.2E-01
tnfn1_pw060328p06q178	FTN_1241		DedA family protein	5	4	55%	2.8E-04
tnfn1_pw060418p04q181	FTN_0338		MutT/nudix family protein	2	1	50%	1.3E-01
tnfn1_pw060420p03q193	FTN_0768	tspO	tryptophan-rich sensory protein	3	3	50%	1.4E-01
tnfn1_pw060419p02q137	FTN_1034	rnfB	iron-sulfur cluster-binding protein	3	2	50%	1.2E+00
tnfn1_pw060323p03q111	FTN_0465		Sua5/YciO/YrdC family protein	2	2	33%	2.8E+00
tnfn1_pw060328p06q167	FTN_0985		DJ-1/PfpI family protein	6	2	20%	5.2E-01
tnfn1_pw060420p03q121	FTN_1064		PhoH family protein, putative ATPase	4	1	11%	1.2E-01
tnfn1_pw060328p03q154	FTN_1453		two-component regulator, sensor histidine kinase	2	2	11%	1.0E-02
tnfn1_pw060420p04q127	FTN_1031	ftnA	ferric iron binding protein, ferritin- like	6	2	0%	3.0E+00
Proteins of unknown function
tnfn1_pw060420p01q142	FTN_0052		protein of unknown function	2		100%	1.6E-04
tnfn1_pw060420p04q176	FTN_0855		protein of unknown function	2	5	100%	6.9E-01
tnfn1_pw060323p06q122	FTN_0077		protein of unknown function	2		91%	3.2E-05
tnfn1_pw060418p02q186	FTN_1448		protein of unknown function	6	6	90%	8.8E-01
tnfn1_pw060418p03q108	FTN_0900		protein of unknown function with predicted hydrolase and phosphorylase activity	6		78%	1.3E-03
tnfn1_pw060418p04q193	FTN_0109		protein of unknown function	4		70%	2.9E-01
tnfn1_pw060328p06q173	FTN_0599		protein of unknown function	2		67%	3.3E-03
tnfn1_pw060328p08q104	FTN_0051		conserved protein of unknown function	3		67%	2.4E-02
tnfn1_pw060328p08q149	FTN_0439		protein of unknown function	4		60%	1.2E-03
tnfn1_pw060328p01q172	FTN_1542		conserved protein of unknown function	2	2	60%	5.5E-03
tnfn1_pw060323p05q127	FTN_0791		protein of unknown function	1		60%	8.0E-03
tnfn1_pw060418p04q121	FTN_1261		protein of unknown function	2		60%	7.7E-01
tnfn1_pw060418p02q157	FTN_1170		conserved protein of unknown function	6	5	60%	2.6E+01
tnfn1_pw060418p04q117	FTN_0207		protein of unknown function containing a von Willebrand factor type A (vWA) domain	2		56%	5.0E+00
tnfn1_pw060510p04q111	FTN_0861		conserved protein of unknown function	4		55%	4.2E+02
tnfn1_pw060323p04q183	FTN_1386		protein of unknown function	3		50%	1.2E-03
tnfn1_pw060418p01q142	FTN_0482		protein of unknown function	6		50%	4.8E-03
tnfn1_pw060328p05q124	FTN_1372		protein of unknown function	5		50%	1.5E+00
tnfn1_pw060323p07q176	FTN_1534		conserved protein of unknown function	3		50%	2.7E+00
tnfn1_pw060418p02q105	FTN_1343		conserved protein of unknown function	4	4	50%	6.9E+00
tnfn1_pw060323p03q157	FTN_0364		conserved protein of unknown function	2		45%	6.2E-01
tnfn1_pw060328p06q155	FTN_1764		protein of unknown function	7	6	40%	3.5E-06
tnfn1_pw060323p08q148	FTN_0027		conserved protein of unknown function	6	4	40%	2.7E-02
tnfn1_pw060418p02q145	FTN_1020		conserved protein of unknown function	5		40%	6.0E+00
tnfn1_pw060420p02q158	FTN_1071		protein of unknown function	5		33%	2.6E+02
tnfn1_pw060328p08q107	FTN_1774		protein of unknown function	3		33%	3.6E-01
tnfn1_pw060419p04q110	FTN_0048		conserved protein of unknown function	2	2	29%	4.2E+01
tnfn1_pw060419p04q188	FTN_0925		protein of unknown function	5		22%	5.4E+00
tnfn1_pw060323p07q141	FTN_0788		conserved protein of unknown function	5	5	22%	5.4E+00
tnfn1_pw060328p02q110	FTN_1457		protein of unknown function	5	5	20%	1.5E+00
tnfn1_pw060420p04q143	FTN_0149		conserved protein of unknown function	5	5	20%	3.2E+00
tnfn1_pw060418p01q155	FTN_0044		protein of unknown function	3		10%	3.5E+01
tnfn1_pw060328p02q177	FTN_1713		protein of unknown function	3	3	0%	7.0E-04
Transcription/Translation
tnfn1_pw060510p03q150	FTN_0949	rplI	50S ribosomal protein L9	2		75%	2.8E-05
tnfn1_pw060328p02q148	FTN_1393		transcriptional regulator, ArsR family	3		30%	2.3E-01
Transferase
tnfn1_pw060323p06q168	FTN_0545		glycosyl transferase, group 2	4	4	87%	2.7E-03
tnfn1_pw060328p01q142	FTN_0928	cysD	sulfate adenylyltransferase subunit 2	3	3	57%	9.6E-02
tnfn1_pw060510p01q119	FTN_1428	wbtO	transferase	6	2	50%	4.1E-01
tnfn1_pw060420p02q146	FTN_0300		glycosyl transferase, group 2	5		30%	6.8E-02
tnfn1_pw060323p03q121	FTN_0343		aminotransferase	2	7	20%	1.9E+01
tnfn1_pw060418p01q110	FTN_0200		UDP-3-O-[3-fatty acid] glucosamine N-acyltransferase	2	2	20%	2.4E+02
tnfn1_pw060510p01q103	FTN_0063	ilvE	branched-chain amino acid aminotransferase protein (class IV)	5	3	13%	3.1E-01
tnfn1_pw060418p04q172	FTN_1418	manC	mannose-1-phosphate guanylyltransferase	4		10%	8.3E-01
Transporter proteins
tnfn1_pw060420p01q180	FTN_0097		hydroxy/aromatic amino acid permease (HAAAP) family protein	4	4	89%	3.0E-05
tnfn1_pw060420p01q189	FTN_1611	-	major facilitator superfamily (MFS) transport protein	5	7	89%	9.2E-02
tnfn1_pw060420p03q104	FTN_1593	oppA	ABC-type oligopeptide transport system, periplasmic component	6	4	70%	8.4E-04
tnfn1_pw060323p06q117	FTN_1685		drug:H+ antiporter-1 (DHA1) family protein	4		70%	9.5E-02
tnfn1_pw060418p04q168	FTN_0141		ABC transporter, ATP-binding protein	6	5	70%	1.1E-01
tnfn1_pw060328p06q119	FTN_1549		drug:H+ antiporter-1 (DHA1) family protein	6		62%	1.3E-01
tnfn1_pw060510p03q140	FTN_0741		proton-dependent oligopeptide transporter (POT) family protein, di- or tripeptide:H+ symporter	3		60%	3.7E-02
tnfn1_pw060419p01q175	FTN_0984		ABC transporter, ATP-binding protein	2		56%	5.0E-01
tnfn1_pw060323p05q110	FTN_1215	kpsC	capsule polysaccharide export protein KpsC	5	2	55%	8.7E-07
tnfn1_pw060323p01q175	FTN_1441		sugar porter (SP) family protein	4	4	50%	9.5E-05
tnfn1_pw060328p04q167	FTN_0997		proton-dependent oligopeptide transporter (POT) family protein, di- or tripeptide:H+ symporter	3	5	50%	2.4E-03
tnfn1_pw060419p01q170	FTN_1006		transporter-associated protein, HlyC/CorC family	2		50%	6.7E-01
tnfn1_pw060328p06q132	FTN_0728		predicted Co/Zn/Cd cation transporter	5	2	50%	2.1E+00
tnfn1_pw060328p08q167	FTN_0579		major facilitator superfamily (MFS) transport protein	3		44%	1.6E+00
tnfn1_pw060419p03q162	FTN_0115		Na+/H+ antiporter	4	4	40%	4.4E-02
tnfn1_pw060419p02q126	FTN_1581		small conductance mechanosensitive ion channel (MscS) family protein	3	3	40%	1.6E-01
tnfn1_pw060418p03q187	FTN_1733	-	nicotinamide ribonucleoside (NR) uptake permease (PnuC) family protein	4	2	40%	3.4E+00
tnfn1_pw060418p02q182	FTN_1275		drug:H+ antiporter-1 (DHA2) family protein	4		40%	4.4E+00
tnfn1_pw060510p02q159	FTN_0688	galP2	galactose-proton symporter, major facilitator superfamily (MFS) transport protein	3		40%	2.2E+04
tnfn1_pw060323p07q172	FTN_1344		major facilitator superfamily (MFS) transport protein	4	4	37%	4.3E-05
tnfn1_pw060420p02q159	FTN_1716	kdpC	potassium-transporting ATPase C chain	2	2	33%	2.0E+03
tnfn1_pw060419p01q133	FTN_1014		nicotinamide ribonucleoside (NR) uptake permease (PnuC) family protein	2		30%	3.8E+00
tnfn1_pw060510p02q118	FTN_1409		major facilitator superfamily (MFS) transport protein	2		20%	4.8E-01
tnfn1_pw060323p05q139	FTN_1166		metabolite:H+ symporter (MHS) family protein	1		20%	1.4E+00
tnfn1_pw060323p03q117	FTN_0005	corA	divalent inorganic cation transporter	3		12%	6.9E+03
tnfn1_pw060328p03q163	FTN_0884		drug/metabolite transporter superfamily protein	4	4	11%	1.6E-03
tnfn1_pw060510p04q167	FTN_1681	fur	ferric uptake regulation protein	2		10%	3.5E-01
tnfn1_pw060420p02q151	FTN_0018	sdaC	serine permease	4	1	10%	9.9E+02
Type IV pili
tnfn1_pw060418p02q167	FTN_1137	pilQ	Type IV pili secretin component	4		60%	1.5E+03

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Fig 3 — *D. melanogaster* was infected and survival curves of representative *F. tularensis* subsp *novicida* mutants are shown. Wild type *F. tularensis* subsp *novicida* U112 and its isogenic *iglC mutant* are positive and negative controls, respectively. Data are represented as a daily mean % survival of 20 flies divided into two groups of 10. Error bar are standard deviation (SD) of the two groups.

Proliferation of F. tularensis mutants within D. melanogaster adult flies

We examined proliferation of the attenuated mutants within adult flies to determine if the defect in S2 cells correlated with reduced proliferation within D. melanogaster. To examine bacterial growth within the adult fly, 15 flies were infected with each mutant strain. A total of five flies were used for determination of average CFU at day 0 post infection and 10 flies used for the day 3 post-infection. The average CFU on day 15 was divided by the average cfu on day 0 to obtain the relative bacterial growth rate of each mutant in the fly. Furthermore, to normalize bacterial growth rate of all mutants in order to compare them to each other, the results are expressed as the ratio of mutant bacterial load to wild type (control) bacterial load. The averages cfu within the same test group were calculated using a trimmean, which excluded outliers (~20%) from each end of a data set. The ratio of the wild type strain of 1 was considered a reference. A mutant was considered to be attenuated if it showed 10 or more fold reduction in growth compared to the wild type bacteria. The average cfu of inoculums recovered from flies immediately after pricking considered day 0, was ~30 cfu per fly. By day 3 post infection, the wild type bacterial growth increased by ~10⁸ cfu, whereas the iglC mutant (control) increased by ~10³ cfu. Some mutants such as FTN_0054 (hypothetical), FTN_0097 (transporter), and FTN_0516 (metabolic) were severely attenuated in proliferation within D. melanogaster (Table 4). Other mutants such as FTN_0984 (transport) exhibited only a slight reduction in growth whereas some mutants such as FTN_0688 (transport) showed higher levels of growth compared to the wild type strain (Table 4). Thirteen out of 24 mutants from the “hypothetical proteins” functional category were found to be attenuated in replication within D. melanogaster adults. Mutants in the small category “intergenic” are interesting, 4 out 5 of these mutants, showed less proliferation within D. melanogaster. For the remaining categories, a fair percentage (~40%) of mutants tested showed defect in growth within D. melanogaster (Table 4).

The difference observed between intracellular growth in S2 cells and proliferation of F. tularensis within the fly might indicate that other organs, tissues or cells other than the macrophage-like cells might have a crucial role in replication of F. tularensis in arthropods. Importantly, previous findings have shown that, albeit hemocytes that are macrophage-like cells being important for infection, F. tularensis also multiply within extracellular spaces in the head, legs and wings of D. melanogaster (Vonkavaara et al., 2008). Other pathogens such as Listeria monocytogenes also spread through D. melanogaster tissues such as the fat body and epithelium (Mansfield et al., 2003). Mutants with the lowest bacterial load (more than 10³ fold less than the wild type strain) were not necessarily the least lethal mutants to D. melanogaster, as only 13/40 of these caused ≤25% lethality to infected flies (Table 4). This finding indicates that the bacterial factors defective in these mutants have additional roles that modulate virulence within D. melanogaster. Among mutants defective in intracellular growth in S2 cells, 21 of them were involved in other aspects of virulence or dissemination in animal models of tularemia (Table 2) (Qin and Mann, 2006; Maier et al., 2007; Su et al., 2007; Weiss et al., 2007; Kraemer et al., 2009), 7 of these mutated genes were also associated with less lethality to D. melanogaster in our screen. Mutants that have differential effects in D. melanogaster and the mammalian host may highlight host-specific differences during infection by F. tularensis of evolutionarily distant hosts

When we classified our data according to lethality effects on D. melanogaster, we uncovered a correlation between the number of bacteria in the fly and the survival of the fly (Table 4). Mutants that exhibited low CFU resulted in higher survival rate for the infected flies. In addition, D. melanogaster infected with mutants that proliferated to high rates were less likely to survive (Table 4). Conversely, the least lethal a mutant was to D. melanogaster, the more likely it was to be attenuated in intracellular proliferation (Table 4). Approximately 64% of the mutants that caused up to 25% lethality in D. melanogaster exhibited at least 10³ fold less CFU than wild type strain. Overall, 86% of these mutants had at least 10 fold less bacterial load than the wild type strain.

Combined, these observations indicate that decreased bacterial load offers survival advantage in most cases. Thus, screening F. tularensis strains for lethality to D. melanogaster might be a reasonable approach to identify important bacterial factors involved in arthropod-Francisella interaction. Although maintenance of F. tularensis in mammals is thought to be associated mainly with cottontail rabbits (Farlow et al., 2005; Keim et al., 2007; Goethert et al., 2009), recent cases of tularemia are mainly associated with arthropod vectors (2002; Goethert et al., 2009). In addition, many different kinds of hematophagous arthropods are vectors for transmission of tularemia, including tabanid flies and mosquitoes (Goethert et al., 2009). The mode of perpetuation seems to involve both horizontal (feeding of an arthropod on an infected mammal) and vertical (inheritance of infection by arthropod progeny) transmission (Hopla, 1974).

The association between F. tularensis and arthropod has most likely resulted in patho-adaptation that has enabled the bacteria to survive within the natural environment, and has likely equipped F. tularensis with molecular mechanisms for survival in mammalian cells. This is supported by our observations that a large number of the F. tularensis genes that are required for intracellular proliferation within human macrophages are also required for proliferation within S2 cells, for lethality to and proliferation within adult flies. Importantly, we have recently shown that modulation of phagosome biogenesis and phagosomal escape of F. tularensis into the cytosol are similar in both arthropod cells and macrophages (Santic et al., 2009). Since 59 bacterial loci are required for phagosomal escape in macrophages (see accompanying manuscript), it is most likely that these loci are also required for phagosomal escape in S2 cells. This indicates conserved as well as distinct molecular mechanisms utilized by F. tularensis to escape from the phagosome of evolutionarily distant host cells. Taken together, it is likely that interaction of F. tularensis with arthropods has played a factor in its ecology and patho-adaptation to infect mammals, since there are numerous evolutionary conserved pathways between the two species, such as innate immune responses and intracellular trafficking events. This is the first comprehensive molecular identification of the genetic loci of an intracellular pathogen required for intracellular proliferation within arthropod-derived cells and adult arthropod model system. Our studies will facilitate deciphering the molecular aspects of F. tularensis-arthropod vector interaction and its role in bacterial ecology and patho-adaptation to infect mammals.

Experimental Procedure

Bacterial strains, U937 macrophages, S2 cells and Media

F. tularensis subsp. novicida strain U112 and its isogenic mutants mglA and iglC have been described previously (Lauriano et al., 2004). The construction of the F. tularensis subsp. novicida mutants library which was obtained from Biodefence and Emerging Infections Resource Repository (http://www.beiresources.org) has been described previously (Gallagher et al., 2007). F. tularensis subsp. novicida strain U112 and its isogenic iglC mutant harboring the plasmid pKK214, which encodes gfp, were used in fly experiments as positive and negative controls, respectively, and have been described elsewhere (Abd et al., 2003; Lauriano et al., 2004; Santic et al., 2005b; Santic et al., 2007). All F. tularensis subsp. novicida strains were grown on tryptic soy agar (TSA) plates or in tryptic soy broth (TSB) supplemented with 0.1% cysteine and 10 mg/ml of kanamycin for 2 days or overnight respectively. Drosophila S2 cells has been described previously (Santic et al., 2009). For recovering bacteria from flies, tryptic soy agar (TSA) plates supplemented with 0.1 % cysteine, 10 mg ml⁻¹ tetracycline or 10 mg ml⁻¹ kanamycin were used as described previously (Santic et al., 2009).

Intracellular survival assays in S2 cells

Intracellular growth kinetics of F. tularensis in S2 cells was performed as previously described (Santic et al., 2009) with minor modifications. Briefly, S2 cells were seeded at 1 × 10⁶ cells ml⁻¹ in 96 well plates pre-coated with 0.5 mg ml⁻¹ concavalin A (ConA) and incubated at 28°C with SDM containing 10% FBS (SDM-10) overnight. Cells were washed 3 X with SDM-10 and infected in duplicates with all the alleles of each mutant as well as wild type F. tularensis subsp. novicida and its isogenic iglC mutant at a MOI of 10. MOI was determined for each of the mutant strains. Infected cells were incubated for 1h at 28°C followed by treatment with 50μg ml⁻¹ gentamicin for 1 h to kill extracellular bacteria. Infected cells were subsequently washed three times and incubated with fresh SDM-10 for 22 h for a total of 24 h of infection. The culture supernatant was removed and the cells were lysed by the addition of 200μl of sterile water for 10 minutes. The culture supernatant and lysate were combined and the number of bacterial CFUs in each well was determined by plating serial dilutions on TSA agar plates supplemented with 0.1 % cysteine, 10 μg ml⁻¹ tetracycline or 10 μg ml⁻¹ kanamycin for colony enumeration.

F. tularensis infection of D. melanogaster adult flies

Infections of D. melanogaster adult fly were performed as previously described (Santic et al., 2009) with minor changes. 7–14 days old flies were used in all our experiments. For D. melanogaster infections, adult female flies were anesthetized with CO₂, pricked in the dorsal thorax with a 30-gauge needle dipped into 1ml of bacteria culture diluted to 2×10⁶ ml⁻¹ in TSB-C broth, then transferred to their designated food vial. F. tularensis subsp novicida and its isogenic mutant iglC were used as positive and negative control, respectively. To screen for lethality effects of each mutant of interest upon infection of the adult flies, 20 flies were infected and housed in two different vials and their survival observed over a period of 15 days. The percent survival of flies was calculated as a ratio of flies surviving by day 15 to flies that were alive by day 1 post inoculation (day 15/day 1). To screen for bacterial growth, we performed viable cell count using 5 infected flies at day 0 and 10 infected flies at day 3 post-infection as previously described (Santic et al., 2009). Bacterial growth rate was calculated by dividing day 3 CFUs by day 0 CFUs. Groups of mutants were tested in independent experiments and wild type F tularensis subsp novicida was used as a control in all experiments. Thus to normalize bacterial growth rate of all mutants in order to compare them to each other, the results are expressed as the ratio of mutant bacterial load to wild type (control) bacterial load. The averages for bacterial cfu within the same test group were averaged using a trimmean, which excluded outliers (~20%) from each end of a data set.

Statistical Analyses

To analyze for statistically significant differences between two sets of data, student’s t-test was used and the p value was obtained. GraphPad Prism 5 was used for statistical analysis.

Acknowledgments

YAK is supported by Public Health Service Awards R01AI43965 and R01AI069321 from NIAID and by the commonwealth of Kentucky Research Challenge Trust Fund.

References

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[R1] Tularemia--United States, 1990–2000. MMWR Morb Mortal Wkly Rep. 2002;51:181–184. [PubMed] [Google Scholar]

[R2] Abd H, Johansson T, Golovliov I, Sandstrom G, Forsman M. Survival and growth of Francisella tularensis in Acanthamoeba castellanii. Appl Environ Microbiol. 2003;69:600–606. doi: 10.1128/AEM.69.1.600-606.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol. 2000;12:13–19. doi: 10.1016/s0952-7915(99)00045-x. [DOI] [PubMed] [Google Scholar]

[R4] Apidianakis Y, Rahme LG. Drosophila melanogaster as a model host for studying Pseudomonas aeruginosa infection. Nat Protoc. 2009;4:1285–1294. doi: 10.1038/nprot.2009.124. [DOI] [PubMed] [Google Scholar]

[R5] Baron GS, Nano FE. MglA and MglB are required for the intramacrophage growth of Francisella novicida. Mol Microbiol. 1998;29:247–259. doi: 10.1046/j.1365-2958.1998.00926.x. [DOI] [PubMed] [Google Scholar]

[R6] Bonquist L, Lindgren H, Golovliov I, Guina T, Sjostedt A. MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages. Infect Immun. 2008;76:3502–3510. doi: 10.1128/IAI.00226-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] Champion MD, Zeng Q, Nix EB, Nano FE, Keim P, Kodira CD, et al. Comparative genomic characterization of Francisella tularensis strains belonging to low and high virulence subspecies. PLoS Pathog. 2009;5:e1000459. doi: 10.1371/journal.ppat.1000459. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] Checroun C, Wehrly TD, Fischer ER, Hayes SF, Celli J. Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication. Proc Natl Acad Sci U S A. 2006;103:14578–14583. doi: 10.1073/pnas.0601838103. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] Cherry S. Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions? Curr Opin Microbiol. 2008;11:262–270. doi: 10.1016/j.mib.2008.05.007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] Chong A, Wehrly TD, Nair V, Fischer ER, Barker JR, Klose KE, Celli J. The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression. Infect Immun. 2008;76:5488–5499. doi: 10.1128/IAI.00682-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] Clemens DL, Lee BY, Horwitz MA. Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages. Infect Immun. 2004;72:3204–3217. doi: 10.1128/IAI.72.6.3204-3217.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] D’Argenio DA, Gallagher LA, Berg CA, Manoil C. Drosophila as a model host for Pseudomonas aeruginosa infection. J Bacteriol. 2001;183:1466–1471. doi: 10.1128/JB.183.4.1466-1471.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] Dennis DT, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Tularemia as a biological weapon: medical and public health management. JAMA. 2001;285:2763–2773. doi: 10.1001/jama.285.21.2763. [DOI] [PubMed] [Google Scholar]

[R14] Dionne MS, Ghori N, Schneider DS. Drosophila melanogaster is a genetically tractable model host for Mycobacterium marinum. Infect Immun. 2003;71:3540–3550. doi: 10.1128/IAI.71.6.3540-3550.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] Ellis J, Oyston PC, Green M, Titball RW. Tularemia. Clin Microbiol Rev. 2002;15:631–646. doi: 10.1128/CMR.15.4.631-646.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] Farlow J, Wagner DM, Dukerich M, Stanley M, Chu M, Kubota K, et al. Francisella tularensis in the United States. Emerg Infect Dis. 2005;11:1835–1841. doi: 10.3201/eid1112.050728. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] Gallagher LA, Ramage E, Jacobs MA, Kaul R, Brittnacher M, Manoil C. A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate. Proc Natl Acad Sci U S A. 2007;104:1009–1014. doi: 10.1073/pnas.0606713104. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] Goethert HK, Saviet B, Telford SR., 3rd Metapopulation structure for perpetuation of Francisella tularensis tularensis. BMC Microbiol. 2009;9:147. doi: 10.1186/1471-2180-9-147. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] Golovliov I, Baranov V, Krocova Z, Kovarova H, Sjostedt A. An attenuated strain of the facultative intracellular bacterium Francisella tularensis can escape the phagosome of monocytic cells. Infect Immun. 2003;71:5940–5950. doi: 10.1128/IAI.71.10.5940-5950.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] Hazlett KR, Cirillo KA. Environmental adaptation of Francisella tularensis. Microbes Infect. 2009 doi: 10.1016/j.micinf.2009.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA. Phylogenetic perspectives in innate immunity. Science. 1999;284:1313–1318. doi: 10.1126/science.284.5418.1313. [DOI] [PubMed] [Google Scholar]

[R22] Hopla CE. The ecology of tularemia. Adv Vet Sci Comp Med. 1974;18:25–53. [PubMed] [Google Scholar]

[R23] Keim P, Johansson A, Wagner DM. Molecular epidemiology, evolution, and ecology of Francisella. Ann N Y Acad Sci. 2007;1105:30–66. doi: 10.1196/annals.1409.011. [DOI] [PubMed] [Google Scholar]

[R24] Kim SH, Park SY, Heo YJ, Cho YH. Drosophila melanogaster-based screening for multihost virulence factors of Pseudomonas aeruginosa PA14 and identification of a virulence-attenuating factor, HudA. Infect Immun. 2008;76:4152–4162. doi: 10.1128/IAI.01637-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] Kraemer PS, Mitchell A, Pelletier MR, Gallagher LA, Wasnick M, Rohmer L, et al. Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice. Infect Immun. 2009;77:232–244. doi: 10.1128/IAI.00978-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] Kurz CL, Chauvet S, Andres E, Aurouze M, Vallet I, Michel GP, et al. Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening. EMBO J. 2003;22:1451–1460. doi: 10.1093/emboj/cdg159. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] Larsson P, Elfsmark D, Svensson K, Wikstrom P, Forsman M, Brettin T, et al. Molecular evolutionary consequences of niche restriction in Francisella tularensis, a facultative intracellular pathogen. PLoS Pathog. 2009;5:e1000472. doi: 10.1371/journal.ppat.1000472. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] Lauriano CM, Barker JR, Yoon SS, Nano FE, Arulanandam BP, Hassett DJ, Klose KE. MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc Natl Acad Sci U S A. 2004;101:4246–4249. doi: 10.1073/pnas.0307690101. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] Maier TM, Casey MS, Becker RH, Dorsey CW, Glass EM, Maltsev N, et al. Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages. Infect Immun. 2007;75:5376–5389. doi: 10.1128/IAI.00238-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] Mansfield BE, Dionne MS, Schneider DS, Freitag NE. Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster. Cell Microbiol. 2003;5:901–911. doi: 10.1046/j.1462-5822.2003.00329.x. [DOI] [PubMed] [Google Scholar]

[R31] Needham AJ, Kibart M, Crossley H, Ingham PW, Foster SJ. Drosophila melanogaster as a model host for Staphylococcus aureus infection. Microbiology. 2004;150:2347–2355. doi: 10.1099/mic.0.27116-0. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Molecular bases of proliferation of Francisella tularensis in Arthropod vectors

Rexford Asare

Christine Akimana

Snake Jones

Yousef Abu Kwaik

Summary

Introduction

Results and Discussion