The analysis of the intramacrophagic virulome of suis deciphers the environment encountered by the pathogen inside the host cell

Stephan Ko¨ hler*†‡, Vincent Foulongne†§, Safia Ouahrani-Bettache*, Gise` le Bourg§, Jacques Teyssier*, Michel Ramuz§, and Jean-Pierre Liautard*

*Institut National de la Sante´et de la Recherche Me´dicale U-431 (INSERM U-431), Universite´Montpellier II, 34095 Montpellier, France; and §INSERM U-431, Faculte´deMe´ decine, Avenue Kennedy, 30900 Nıˆmes,France

Edited by Roy Curtiss III, Washington University, St. Louis, MO, and approved September 30, 2002 (received for review July 31, 2002) The pathogen Brucella suis resides and multiplies within a phago- ability to infect animal cells (or protists for Legionella), because cytic vacuole of its host cell, the macrophage. The resulting com- they do not survive for protracted periods of time outside their plex relationship has been investigated by the analysis of the set hosts. These pathogens should therefore rather be considered of genes required for virulence, which we call intramacrophagic mainly as intracellular, facultatively extracellular organisms (4). virulome. Ten thousand two hundred and seventy-two miniTn5 The , to which they have adapted during evolution, is mutants of B. suis constitutively expressing gfp were screened by their natural niche. fluorescence microscopy for lack of intracellular multiplication in The identification of the whole set of genes involved in the human . One hundred thirty-one such mutants af- multiplication of Brucella spp. inside the macrophage is a fected in 59 different genes could be isolated, and a function was prerequisite to the understanding of the relation with the ascribed to 53 of them. We identified genes involved in (i) global macrophage. Signature-tagged mutagenesis and differential flu- adaptation to the intracellular environment, (ii) amino acid, and orescence induction approaches were recently used to identify (iii) nucleotide synthesis, (iv) sugar metabolism, (v) oxidoreduction, genes required for virulence in a murine model of infection and (vi) nitrogen metabolism, (vii) regulation, (viii) disulphide bond in human and murine macrophages (5–9). A large proportion of formation, and (ix) biosynthesis. Results led to these genes encode of various metabolic pathways. the conclusion that the replicative compartment of B. suis is poor However, the small number of mutants screened resulted in a in nutrients and characterized by low oxygen tension, and that limited output, making identification of the majority of the nitrate may be used for anaerobic respiration. Intramacrophagic virulence genes impossible. As a consequence, we decided to virulome analysis hence allowed the description of the nature of the replicative vacuole of the pathogen in the macrophage and design an alternative method. We screened 10,272 individual extended our understanding of the niche in which B. suis resides. Tn5 transposon mutants of B. suis for their inability to multiply We propose calling this specific compartment ‘‘brucellosome.’’ in a macrophage model of infection, and 131 attenuated mutants were detected. This analysis allowed us to define the environ- ment the encountered in the macrophage and to better nteractions between microorganisms and their hosts extend understand the nature of the phagosome. Ifrom acute infections to persistent infectious diseases or symbiosis. This type of interaction can result from a million years Materials and Methods of coevolution and coadaptation of the two organisms. Thus, the Bacterial Strains and Media. B. suis 1330 (American Type Culture biology of the interaction can be read, at least partially, in the Collection 23444) and all derived miniTn5 mutant strains were genome of the microorganism. In the specific case of pathogenic grown in tryptic soy broth at 37°C. The Escherichia coli donor bacteria, deciphering of the genes involved in the interaction and strain for conjugation SM10 ␭ pir containing a tagged miniTn5 analysis of their functions will shed light on the environment Km2 transposon in pUT (7, 10) was cultivated in LB broth. encountered by the parasite in the host and will contribute to the ␮ ͞ ␮ ͞ understanding of the complex relationship between two organ- Antibiotics were used at 50 g ml (kanamycin) and 25 g ml isms. As a name for the whole set of genes required for virulence, (nalidixic acid). i.e., involved in the invasion of the host by the bacteria and their adaptation to the environment provided by this host, we propose Acceptor Strain Construction and Mutagenesis. A constitutively virulome. In this study, we will perform a thorough analysis of the expressed copy of gfp was inserted by allelic exchange into bcsp31 intramacrophagic virulome of Brucella suis. on chromosome I (BMEI0796) of B. suis 1330. This gene is not Brucella spp. is an ␣ proteobacteriaceae that induces a per- involved in bacterial virulence (11). To this end, a previously sistent disease in some mammals, resulting in abortion. In described strong promoter isolated from B. suis (8) was cloned humans, initial septicemia may be followed by a subacute or a upstream of the gfp-cat reporter tandem into pBBR1-GFP (12), chronic infection (1). Brucella spp. is phyletically related as well reisolated in fusion with gfp-cat, and inserted into the unique to plant symbionts such as rhizobiaceae, as to rickettsiae, which EcoRV site of bcsp31 cloned in pUC18. The final construct, generate an acute infectious disease (2). In terms of virulence, which is a suicide vector in brucellae, was electroporated into B.

brucellae occupy an intermediate position where the adaptation suis 1330 (13), and clones that were fluorescent were further MICROBIOLOGY results in a mild disease that allows them to persist in mammal verified, confirming the insertion of gfp into chromosomal hosts. It is usually considered that, for a facultative intracellular bcsp31. A spontaneous NalR mutant of this strain was used as B. bacterium such as Brucella spp., which multiplies in trophoblasts suis acceptor strain for transposon mutagenesis performed as or macrophages (3), one of the challenges is to rapidly adapt to described (14). Five hundred B. suis clones were isolated per the intracellular settings but also to resist the harsh conditions generated by the immune system, including activation of the . However, the status of Brucella spp. and of some This paper was submitted directly (Track II) to the PNAS office. other bacteria such as Mycobacterium spp. or Legionella spp. is †S.K. and V.F. contributed equally to this work. more complex. Their persistence in nature depends only on their ‡To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.232454299 PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15711–15716 Downloaded by guest on September 29, 2021 conjugation, and a total of 22 conjugations were performed, databases. The identified virulence genes͞operons are distrib- yielding 10,272 mutants that were analyzed individually. uted evenly on the two chromosomes with no major clustering (Fig. 1). Screening of B. suis Tn5 Mutants for Attenuation in Human THP-1 Cells. The most intriguing result is the absence of virulence factors Macrophage-like THP-1 cells were infected by individual B. suis such as toxins or other exported proteins able to interfere with mutants and the acceptor strain as a control at a multiplicity of host cell functions, except the already known VirB secretion infection of 50 in 96-well plates (15). At 24 and 48 h postinfec- system, whose operon was targeted 33 times. None of the tion, the plates were examined by using fluorescence microscopy. mutants was affected in adherence or entry under the experi- Wells containing attenuated mutants showed no intracellular mental conditions used. The majority of the mutations resulting fluorescence, allowing their rapid identification. Attenuation of in intracellular growth defect affected genes acting in house- the selected mutants was validated in THP-1 infection experi- keeping functions, confirming the results previously obtained ments performed in duplicates in 24-well plates where the by signature-tagged mutagenesis or differential fluorescence number of intracellular bacteria was determined at 90 min and induction. 7, 24, and 48 h postinfection (15). Adaptation of the Bacteria to the Phagosome. Four mutations Analysis of Transposon Insertion Sites. Chromosomal DNA was affecting genes necessary for a global adaptation to an intracel- isolated from the attenuated mutants, and the sequence from the lular environment were detected, and an attenuated mutant of region flanking the transposon insertion site was obtained B. suis showed an insertion of Tn5 in a gene encoding a SpoT directly by using a PCR-based approach before automated homolog involved in the stringent response (Table 1). In E. coli, sequence analysis (Genaxis, Nıˆmes, France) by using primers P6 the two proteins RelA and SpoT regulate the concentration of and P7 (10). Sequence database searching was performed by (p)ppGpp, a global transcriptional activator of numerous met- using the BLASTN and BLASTX algorithms (16) and by comparison abolic pathways under starvation conditions (19). RelA is im- with the annotation for the genome of Brucella melitensis (17). portant for the virulence of Listeria monocytogenes (20), for the The presence of the identified genes in the genome of B. suis was activation of quorum sensing in Pseudomonas aeruginosa (21), verified by using the TIGR database (http:͞͞tigrblast.tigr.org͞ ͞ and for long-term survival of Mycobacterium tuberculosis (22). In ufmg ). This annotation was confirmed in several ways: search Brucella spp. and Sinorhizobium meliloti, only the gene coding for of functional motifs by alignment with other sequences, similar- a regulator closely related to SpoT exists and may be called rsh ity of secondary or tertiary structures of the homologous pro- for rel spo homolog. In S. meliloti, the rsh mutant does not induce teins, presence of the gene in an operon, and strong homologies nodulation and fails to grow in the host plant (23). In Legionella with phenotypically characterized genes in related bacteria such pneumophila, in contrast, RelA is not involved in intracellular as rhizobiaceae or Agrobacterium. replication (24). The attenuation of the B. suis mutant, unable to Results and Discussion induce these metabolic pathways, suggests that the phagosomal environment lacks the necessary precursors for these molecules, We reasoned that transposon mutagenesis was the best means to a hypothesis confirmed by the analysis of other mutants (see target genes, provided that enough individual clones are ana- below). lyzed. To perform this task, we labeled B. suis 1330 with Another attenuated mutant of B. suis was affected in the constitutively expressed gfp encoding GFP by direct insertion hfq into the genome, by using bcsp31 (11) as target. The resulting characterized virulence gene encoding Host Factor I. Mu- fluorescent B. suis was the acceptor strain for miniTn5 Km2 tation of hfq in Brucella abortus results in a lowered resistance to transposon (10), and 10,272 mutants were obtained. Statistical stress induced by peroxides and acidic conditions during sta- analysis showed that, based on the assumption that B. suis 1330 tionary phase (25). In Salmonella typhimurium, the transcription possesses Ϸ3,200 genes, the maximal probability to pick up a factor RpoS, which is required for the transcription of many gene at least once was 96.5%. The resulting mutants were genes expressed during the onset of stationary phase and for screened for their participation in virulence by infection of the virulence, is regulated by Host Factor I. Adaptation to nutrient macrophage cell line THP-1, as described (15, 18), and by limitation and starvation in E. coli depend on RpoS. However, individual analysis of intracellular multiplication by using fluo- there is no gene homologous to rpoS in B. suis. The attenuation of hfq mutants in Brucella can be explained either by the rescence microscopy, as described in Materials and Methods. The ␴ disrupted genes were identified by sequencing the chromosomal existence of a stationary-phase regulating factor different from DNA flanking Tn5 and homology search by using BLAST. RpoS or by its regulation of unknown virulence factors. One hundred thirty-one mutants unable to multiply in THP-1 We have isolated an attenuated Tn5 mutant affected in htrA. cells were selected. These 131 mutations affected 59 different The protease HtrA has been shown to be involved in the removal genes. Many of them were drawn several times, confirming that of proteins exposed to reactive oxygen intermediates. Contra- most of the genes were probably interrupted at least once by the dictory results on the role of htrA in virulence of B. abortus were transposon. However, a number of previously described genes reported: A htrA null mutant is not attenuated in a mouse model involved in virulence (5–7) were not picked up. Furthermore, the of infection (26). The same authors nevertheless confirm distribution was not isomorphic with the expected Poisson’s the attenuation of the mutant in isolated murine macrophages, distribution if we consider each gene 1,000 nucleotides in size. In obtaining results similar to ours. However, it has been shown that fact, the distribution is closer to one that discloses only Ϸ60% of brucellae penetrate the phagocytic cells without an oxidative the screened genes. We should interpret with caution the burst being detected (18). The biological role of HtrA in the hypothesis that most (Ϸ95%) of the genes were targeted by interaction with the host therefore remains obscure. miniTn5. The discrepancy between statistical analysis and the The stress-induced protease Lon was also found to be essential experimental results may be explained by a high threshold of for multiplication inside macrophages (Table 1). This protein has detection, a nonhomogeneous size of the genes, or a nonrandom been described as necessary for virulence in B. abortus (27) and insertion of Tn5. in S. typhimurium by controlling genes required for adherence We can ascribe a function to 53 of the 59 genes identified with and invasion (28). In S. meliloti, Lon, which shows 86% homology good confidence. The results are summarized in Table 1. Genes with the B. suis gene, regulates the synthesis of exopolysaccha- were classified according to the putative functions they encode. rides and is required for nodulation (29). Six genes did not yield homologous sequences in microbial All together, these results suggest that the stress response is

15712 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.232454299 Ko¨ hler et al. Downloaded by guest on September 29, 2021 Table 1. B. suis genes essential for intramacrophagic multiplication Attenuation at Mutated genes and 48 h postinfection SpoT Functional groups putative functions (log)* regulation† ORF number‡

Transporter cobW 2 II0308 virB (type IV secretion system) 5 II0025–0035 Amino acid synthesis§ thrA, thrC (T) 3 ϩ I0725, I1450 ilvC, ilvD, ilvI (I, L, V) 2.7; 5; 5 ϩ I0624, I1848, I0617 leuA, leuC (L) 2 I0451, I0157 hisD, hisF (H) 3; 1.7 ϩ I1668, I2041 glyA, serB (G, S) 2.4 I1192, I0615 lysA (K) 2.7 ϩ I0084 carAB (R) 2.8 I0526 gcvT (S) 2 II0559 aspB? (aminotransferase) 2 I0626 Sugar metabolism pyc (anaplerotic function) 3.9 I0266 eryB, eryC (erythritol) 2.3; 1.4 II0429, II0428 gnd (pentose phosphate pathway) 1.8 II1124 gtrB? (glycosyl ) 2.7 II1101 rbsK (ribokinase) 1.9 II0089 DNA͞RNA metabolism carAB (pyrimidines) 2.8 I0526 pyrB, pyrD (pyrimidines) 2.4; 3 Ϫ II0670, I1611 purD, purF (purines) 2.1; 2.5 Ϫ I1519, I1488 dut (dUTPase) 2.1 I0358 miaA (tRNA isopentenyl transferase) 2 I0616 Lipid metabolism aidB (acyl-CoA dehydrogenase) 1 II0671 Regulation SpotT/rsh (stringent response) 2.1 I1296 bvrR, bvrS (two component system) 2; 4.5 I2036, I2035 deoR (transcriptional regulator) 4.3 II1093 Stress proteins hfq (host factor I) 1.8 I0872 htrA (protease) 2.8 I1330 lon (protease) 1.8 ϩ I0876 Membrane structure uppS (peptidoglycan synthesis) 1.2 Ϫ I0827 Lipopolysaccharide wbdA (mannosyl transferase) 0.8 I0997 biosynthesis lpsA, lpsB (glycosyl ) 1.7; 5.9 I1326, I0509 manB (phosphomannomutase) 4.2 II0899 wbpW (phosphomannose ) 2 II0900 Oxidoreduction cydD (cytochrome oxydase) 2.5 II0762 narG (nitrate reductase) 2 II0950 caiB (carnitine ) 1.2 II1019 dsbA, dsbB (disulfide bond formation) 2.5; 5 I1440, I0384 Nitrogen metabolism glnA (glutamine synthetase) 3.3 ϩ I0979 glnD (PII uridylyl transferase) 3.2 I1804 glnL (NRII) 1.3 I1786 nifS (nitrogenase synthesis) 1.8 I1043 Unknown functions BMEI0085 1.5 I0085 BMEI0605 2 I0605 BMEI0671 2.5 I0671 BMEI1658 1.9 I1658 BMEI1859 2.3 I1859 BMEII1045 2.2 II1045

*As calculated from the comparison to the number of the intracellular B. suis control strain. †Genes reported to be under positive (ϩ) or negative (Ϫ) control during the stringent response in E. coli (19). ‡According to the nomenclature of the B. melitensis genome in GenBank (accession nos. NC003317 and NC003318). §In parentheses, amino acid single-letter code of the final pathway products.

crucial for the adaptation of brucellae to the intraphagosomal times in independent experiments. Synthesis of amino acids MICROBIOLOGY life style. starts from the five metabolites ␣ ketoglutarate, oxalacetate, pyruvate, 3-phosphoglyceric acid, and chorismate. The first four Genes Involved in Amino Acid Synthesis. The most striking feature pathways have been identified during mutagenesis but not the of the results obtained is the number of genes involved in the pathway for the synthesis of aromatic amino acids from choris- biosynthesis of amino acids (Table 1). Bacterial attenuation after mate. Furthermore, mutants affected in the synthesis of alanine disruption of these genes signifies that the corresponding me- from pyruvate, of methionine from oxalacetate, and of proline tabolites are not accessible to the bacteria inside the phagosome, from ␣ ketoglutarate were never isolated by this screening and that they are essential for the intramacrophagic growth. Of method. There are two possible explanations: (i) the screening the 15 mutated genes described, 11 were isolated two to five has not targeted all of the genes, and (ii) only some pathways are

Ko¨ hler et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15713 Downloaded by guest on September 29, 2021 uation of purE mutants from B. melitensis (31). This observation suggests that inside the phagosome, Brucella spp. cannot access the bases or the nucleotides necessary for its growth and has to synthesize them.

Genes Involved in Sugar Metabolism. Only a few genes involved in sugar metabolism have been identified as necessary for intram- acrophagic multiplication (Table 1; Fig. 2, which is published as supporting information on the PNAS web site). Among them is the operon encoding enzymes involved in erythritol degradation (32). We identified two genes, eryB and eryC, necessary for growth inside the macrophage. However, erythritol has not been described in human macrophages, making clear that the role of these enzymes in virulence is rather complex and awaits further analysis. We cannot rule out the possibility that EryB, homol- ogous to glycerol-3-phosphate dehydrogenases, and EryC, show- ing partial homology to hydrogenases (32), catabolize additional substrates present in human macrophages. The gnd gene encoding 6-phosphogluconate dehydrogenase is involved in the branching of sugar metabolism to the pentose phosphate pathway (Fig. 2, which is published as support- ing information on the PNAS web site). Brucella is known to have lost pfk and fda genes and thus is not able to use the Embden–Meyerhof pathway (glycolysis) to degrade glucose (17). The inactivation of gnd leaves the Entner–Doudoroff pathway as a shunt for sugar degradation (the homologues of the edd and eda genes are present on the Brucella chromosome II), explaining the slight attenuation in the macrophage model (1.8 log). Another remarkable gene involved in intracellular survival of B. suis is pyc encoding pyruvate carboxylase. This is involved in the synthesis of oxalacetate from pyruvate, restock- ing the tricarboxylic acid (TCA) cycle. The anaplerotic function of this enzyme confirms that metabolites of the TCA cycle are used for synthesis of amino acids and bases. However, the anaplerotic gene homologous to icl encoding isocitrate from the glyoxylate cycle is not involved in virulence of B. suis Fig. 1. Physical map of the two chromosomes of B. suis displaying the (S. K., unpublished work), in contrast to results published for M. distribution of the genes identified by Tn5 mutagenesis. tuberculosis (33). With Icl, acetyl-CoA is used, whereas in the reaction catalyzed by Pyc, CO2 and ATP are involved. In M. tuberculosis, Icl is necessary for persistence and is involved in the essential for intracellular brucellae. The second hypothesis is direct binding of acetyl-CoA obtained from the ␤ oxidation of unlikely, because (i) there is no evident rational means to draw fatty acids. According to our results, Brucella spp. seems to this conclusion; (ii) only a few mutations were found on genes behave differently and probably does not use mainly fatty acids encoding enzymes for a same specific pathway (histidine or as an energy source in the macrophage. threonine synthesis); and (iii) we and others have identified The carbon source of brucellae inside the macrophage has to genes involved in biosynthesis of amino acids not isolated in this be uncovered, but the necessity of a functional ribose kinase screening. AroC, for instance, necessary for the synthesis of (Table 1) suggests that brucellae import ribose and thus may use aromatic amino acids, has been shown to be required for pentoses as a source of carbon and energy. The pentose phos- intracellular multiplication (30). We conclude that not all path- phate pathway has already been recognized earlier as a major ways were targeted by the method used. Our results allow us to sugar degradation pathway in Brucella spp. (34). conclude that the synthesis of amino acids is absolutely necessary Oxidoreduction and Electron Transport. Functional analysis of the for the intracellular multiplication of Brucella spp., implying that genes necessary for virulence inside the macrophage reveals the amino acids are not available inside the phagosome. This finding presence of several genes involved in oxidoreduction, among confirms the need for a functional spoT gene for intracellular which is cydD, part of the operon cydDCAB. CydD participates multiplication, with (p)ppGpp inducing most of the amino acid in the assembly of the cytochrome bd oxidase encoded by cydAB, synthesis pathways identified as essential in our virulence screen- which is a terminal electron donor to oxygen under low oxygen ing (Table 1). A schematic presentation of these pathways with concentration conditions. The respiratory chain of B. suis con- the genes identified as essential for intracellular B. suis is shown tains two terminal oxidases, cytochrome bd and cytochrome bo in Fig. 2, which is published as supporting information on the oxidase (17). In E. coli, cytochrome bo oxidase is predominantly PNAS web site, www.pnas.org. expressed when oxygen is not limited, and cytochrome bd oxidase, which has a higher affinity for oxygen, is used under Genes Involved in Nucleotide Synthesis. Five genes engaged in the microaerophilic conditions (35). The importance of cydD is synthesis of purines (purD, purF) and pyrimidines (carAB, pyrB, corroborated by the seminal discovery of cydB as a virulence pyrD) are required for intracellular multiplication of B. suis gene in the mouse model of infection (36). The cydB mutant (Table 1; Fig. 2, which is published as supporting information on shows an increased sensitivity to oxygen radicals and to pH Յ3.6, the PNAS web site), confirming previous results on the atten- favoring the hypothesis that cytochrome bd oxidase participates

15714 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.232454299 Ko¨ hler et al. Downloaded by guest on September 29, 2021 in the resistance of Brucella spp. to the harsh conditions of the Role of Smooth Lipopolysaccharide in Intracellular Multiplication. phagosome (36). It has been shown for B. abortus that the switch Although the two species Brucella ovis and are from cytochrome bo oxidase to cytochrome bd oxidase can be naturally rough and respectively infect rams and dogs, it has been explained by lowered oxygen tension inside the macrophage shown that rough mutants from other species are avirulent (43, (37). A functional cytochrome bd oxidase is also required for 44). Among the attenuated mutants, five were affected in genes intracellular survival of Shigella flexneri (38). required at different stages of the lipopolysaccharide biosynthe- The genome of B. melitensis contains the genetic information sis (Table 1). Microscopic examination of infected macrophages necessary for the use of nitrate as electron acceptor, with confirms eradication of the rough bacteria, which is in agreement production of nitrite and nitrogen (17). Among the genes with results obtained in intracellular survival assays (not shown), involved in intramacrophagic multiplication, we have identified and which could be due to their sensitivity to defensin-like narG, coding for an essential component of the dissimilatory factors (43). nitrate reductase complex (Table 1). This complex is encoded by the narGHIJ locus, which is present in the B. suis genome Analytical Conclusion: The Nature of the B. suis Niche in the Macro- phage. together with the gene of the nitrite extrusion protein, narK. The This work, which was aimed at the definition and analysis of the intramacrophagic virulome of B. suis, reveals an extended narG mutant was unable to produce nitrite from nitrate (not picture of the niche in which B. suis resides in the macrophage. shown). The genes nir and nor necessary for nitrate respiration The pathogen remains enclosed in a vacuole throughout its are also present in the B. suis genome. The requirement of a intracellular life, but the nature of the phagosome is poorly functional narG gene for intracellular growth suggests that the defined, and its modifications during the activation of the vacuole containing B. suis is devoid of oxygen and can be are totally unknown. In macrophages, it has been interpreted as the ability of B. suis to use nitrate for respiration described that B. suis prevents the fusion of the phagosome with during intracellular growth. The impact of a mutation in caiB and that phagocytosis inhibits TNF␣ production and strengthens this hypothesis. In anaerobiosis, carnitine is de- of the cell (45–47) . graded to butyrylbetaine, which can be used as an electron Our results allow us to describe the environment encountered transporter. The three genes encoding the enzymes required for by the bacterium in the phagosome and, together with previously the carnitine metabolism, caiB, caiC, and caiD, are present as an published data, the strategy it uses to pervert the functions of the operon in the genome of B. suis. phagocyte. Complete genomic sequences of two Brucella species have confirmed the absence of ‘‘classical’’ virulence factors and Nitrogen Metabolism. B. suis has to synthesize all of the amino types I, II, and III protein secretion systems (17). Indeed, this acids and bases to multiply inside the phagosome. Thus, the bacterium enters the cell by rafts, using a lectin-like mechanism, nitrogen source cannot be imported as complex molecules, but and not by means of dedicated proteins such as internalin or B. melitensis contains genes encoding a putative assimilatory invasin (48). Previous work has demonstrated that an acid nitrate reductase and a copper-containing nitrite reductase (17). phagosomal pH of 3.5–4 is required for early steps of infection On the other hand, our results show that at least three enzymes within the cell, and neutralization of the phagosome after6hof ϩ involved in NH4 utilization (GlnA, GlnD, and NRII; Table 1) infection has no effect on intracellular growth of B. suis (49). are necessary for intracellular multiplication, the first being Synthesis of stress proteins such as DnaK, Lon, or Host Factor regulated by (p)ppGpp during the stringent response (see Table I are probably required at this stage (13, 25, 27). 1). glnA is also required for virulence of S. typhimurium (39), The 59 genes (virB operon ϭ 1) described here are necessary ϩ demonstrating that Brucella spp. uses NH4 , which may be for intracellular replication. Among the 131 attenuated mutants, obtained either by nitrate reduction or by direct uptake from the the virB operon, composed of 12 genes with a total size of 12 kb, phagosome, for amino acid or nucleotide synthesis. was targeted 33 times. The number of virB mutants was higher than expected, possibly due to an additional hot spot for Tn5 Genes Involved in Lipid Metabolism. The degradation of fatty acids insertion within the operon. The requirement of an acid pH for most likely does not play a crucial role for the growth of B. suis effective intracellular multiplication only during the first6hand inside the macrophage, as only one slightly attenuated mutant the demonstration that virB is induced under acidic conditions has been obtained in a gene encoding a putative acyl-CoA and in the phagosome (50) suggest that VirB might be important dehydrogenase. In total, eight enzymes with this potential func- only during the early phase of cellular infection. VirB may allow the bacterium to reach a particular cellular compartment by a tion are encoded in the genome of B. melitensis. If we associate yet-undiscovered mechanism and perhaps after several steps of this observation to the result that pyruvate carboxylase instead maturation, such as transit by multilamellar autophagosome-like of isocitrate lyase is crucial in anaplerotic reactions, it can be structures, as claimed earlier (51). inferred that fatty acids from the host cell are not available for Our observations are of importance in defining the environ- degradation by the pathogen. ment of the replicative niche containing Brucella spp., i.e., at 48 h postinfection. Before our work, the nature of the bacteria- Regulation. The sequence of 27 two-component regulation sys- containing phagosome was studied essentially by using cellular tems can be detected in the genome of Brucella. The only biology approaches (52). The characterization of the vacuoles is attenuated mutant selected by our technique and affected in mainly performed by colocalization with specific cellular mark- such a function is the bvrS-bvrR mutant. BvrR and BvrS are ers. Results obtained with this approach showed that in HeLa known to be required for intracellular multiplication (40). cells (nonprofessional phagocytes), B. abortus resides in a struc- MICROBIOLOGY ture that exhibits markers of the endoplasmic reticulum (ER) DsbAB Proteins, Assembly, and Exportation of Virulence Proteins. (51, 53, 54). A functional ER should contain proteins and DsbAB proteins are involved in disulphide bond formation in the peptides synthesized by the host cell, which would not be periplasm of Gram-negative bacteria, hence stabilizing the in- consistent with the shortage in amino acid availability revealed teractions of many proteins (41). DsbA has been lately described by our work. Furthermore, synthesis of proteins with a rapid as essential for the secretion of pertussis toxin (42), and it is turnover is not altered in cells infected by B. abortus (54), conceivable that attenuated B. suis dbsAB mutants may be indicating that the brucellae are probably not localized in the affected by the unfolding or lack of assembly of yet-to-be- same functional compartment. It could be put forward that the identified virulence factors. compartment containing Brucella spp. is different in HeLa cells

Ko¨ hler et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15715 Downloaded by guest on September 29, 2021 and macrophages. Indeed, results obtained on intracellular occupying the whole cytoplasm. During infection, the main trafficking of B. abortus in J774 macrophages report that only a functions of the cell seem to be unaffected, as suggested by few bacteria are localized in autophagosomes and ER-like inhibition of apoptosis (47). structures (55). However, partial analysis of the virulome of B. Altogether, these results demonstrate that brucellae actively melitensis has been performed in HeLa cells, and some amino build a pathogen-specific niche for multiplication within the acid biosynthesis pathways have been shown to be necessary (6, cells, different from all naturally existing cellular organelles, 56). In addition, recent observations that ER contributes to including those playing a vital physiological role to the unin- phagosome formation (57) and that ER markers colocalize with fected host. We therefore propose calling the vacuole in which containing latex beads (58) make it clear that a B. suis multiplies ‘‘brucellosome.’’ B. suis seems to behave vacuole cannot be defined only by the presence of specific furtively, disturbing the cell functions as little as possible. It proteins. The description of the nature of the replicative com- multiplies within the macrophage because of its resistance to the partment of B. suis in the late phase of infection can be different stress conditions encountered in the phagosome and complemented by our analysis of the intramacrophagic viru- because of its ability to synthesize all of the metabolites neces- lome: it is poor in nutrients, as shown by the requirement of sary for its survival and to use anaerobic respiration in the functional genes of various biosynthesis pathways; it most likely replicative compartment it creates. has a neutral pH; it is also characterized by low oxygen tension and, alternatively, nitrate ions are available for anaerobic res- This work was partly supported by Grant QLK2-CT-1999-00014 from the piration. In this compartment, B. suis multiplies actively, until European Union.

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