The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage 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 phagosome, 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 macrophages. 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) lipopolysaccharide biosynthesis. Results led to these genes encode enzymes 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 bacteria 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 phagocytes. 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).