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International audience; Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

International audience; Carbonic anhydrases have started to emerge as new potential antibacterial targets for several pathogens. Two β-carbonic anhydrases, denominated bsCA I and bsCA II, have been isolated and characterized from the bacterial pathogen Brucella suis, the causative agent of brucellosis or Malta fever. These enzymes have been investigated in detail and a wide range of classical aromatic and heteroaromatic sulfonamides as well as carbohydrate-based compounds have been found to inhibit selectively and efficiently Brucella suis carbonic anhydrases. Inhibition of these metalloenzymes constitutes a novel approach for the potential development of new anti-Brucella agents. This review aims at discussing the recent literature on this topic.

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

International audience; L-Histidinol dehydrogenase from Brucella suis (BsHDH) is an enzyme involved in the histidine biosynthesis pathway which is absent in mammals, thus representing a very interesting target for the development of anti-Brucella agents. In this paper we report the crystallographic structure of a mutated form of BsHDH both in its unbound form and in complex with a nanomolar inhibitor. These studies provide the first structural background for the rational design of potent HDH inhibitors, thus offering new hints for clinical applications.

Brucella suis histidinol dehydrogenase (HDH) can be efficiently targeted by substrate analogues. The growth of this pathogen in minimal medium was inhibited and the multiplication in human macrophages was totally abolished in the presence of the drugs. These effects have been shown to be correlated with the previously described inhibition of Brucella HDH activity.

A mutant of Brucella suis bearing a Tn 5 insertion in norD , the last gene of the operon norEFCBQD , encoding nitric oxide reductase, was unable to survive under anaerobic denitrifying conditions. The norD strain exhibited attenuated multiplication within nitric oxide-producing murine macrophages and rapid elimination in mice, hence demonstrating that norD is essential for Brucella virulence.

Persistence infection is the keystone of the ruminant and human diseases called brucellosis and Malta fever, respectively, and is linked to the intracellular tropism of Brucella spp. While described as non-motile, Brucella spp. have all the genes except the chemotactic system, necessary to assemble a functional flagellum. We undertook to determine whether these genes are expressed and are playing a role in some step of the disease process. We demonstrated that in the early log phase of a growth curve in 2YT nutrient broth, Brucella melitensis expresses genes corresponding to the basal (MS ring) and the distal (hook and filament) parts of the flagellar apparatus. Under these conditions, a polar and sheathed flagellar structure is visible by transmission electron microscopy (TEM). We evaluated the effect of mutations in flagellar genes of B. melitensis encoding various parts of the structure, MS ring, P ring, motor protein, secretion apparatus, hook and filament. None of these mutants gave a discernible phenotype as compared with the wild-type strain in cellular models of infection. In contrast, all these mutants were unable to establish a chronic infection in mice infected via the intraperitoneal route, raising the question of the biological role(s) of this flagellar appendage.

Brucella spp. are facultatively intracellular bacteria that persist and multiply in the macrophages of their mammalian hosts. The so-called phagosome to which they have adapted is their natural living niche. Characterization of this niche would facilitate an understanding of the true relationship between the host cell and the intracellular bacteria. This Opinion analyses and discusses the characteristic properties and genesis of this vacuole during phagocytosis as deduced from the virulence factors necessary for intracellular multiplication of the pathogen. We conclude that the replicative niche of Brucella spp.--the 'brucellosome'--differs from all other cellular organelles, and that it isolates the pathogen from certain cytoplasmic nutrients. Adaptation to the stress conditions encountered and the use of anaerobic respiration enable brucellae to replicate in the compartment they create.

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti , the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella , 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum , were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti , Brucella pinnipedialis , and newly described species ( B. microti , Brucella inopinata BO1, B. inopinata -like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.

BMC microbiology 13, 199 (2013). doi:10.1186/1471-2180-13-199, Published by BioMed Central [u.a.], London [u.a.]

BACKGROUND: Genome analysis indicated that the new species Brucella microti possesses a potentially functional glutamate decarboxylase (GAD) system involved in extreme acid resistance in several foodborne bacteria. The contribution of this system in adaptation of B. microti to an acidic environment, including the intracellular vacuole and stomach, was investigated. RESULTS: B. microti was GAD positive and able to export its product, γ-aminobutyrate, to the extracellular medium. The resistance of B. microti to acid stress (pH 2.5) was glutamate dependent. Mutants affected in the GAD system lost this resistance, demonstrating its direct involvement in survival under these conditions. The reciprocal heterologous complementation of mutants with the GAD systems of Escherichia coli or B. microti confirmed conserved functions in both bacterial species. A gad mutant was not attenuated during infection of macrophages, where Brucella resides in an acidified vacuole at a pH of 4-4.5 during the early phase of macrophage infection, but GAD contributed to the survival of B. microti in a murine model following oral infection. CONCLUSIONS: This work provides first evidence that the GAD system might play an essential role in the resistance of an environment-borne, pathogenic Brucella species to extreme acid shock and during passage through the host stomach following oral infection.

International audience; Brucella, a facultative intracellular pathogen, is one of the most common zoonotic diseases worldwide. Considering the alarming health problem caused by the emergence of resistance and multi-resistance of intracellular pathogen, the challenge is currently to identify and to validate novel pharmaceutical targets in this bacteria species. Brucella's genome encodes metalloproteins involved in various biosynthetic processes, some of them being essential during intracellular growth phase and virulence. The potential of prokaryotic zinc metalloproteins such as carbonic anhydrase (CA) and histidinol dehydrogenase (HDH) as anti-Brucella targets has only recently been taken into consideration in the search of novel anti-infective agents that lack of cross-resistance to existing drugs. These enzymes have a growing significance in modern medicine as they are required for growth and/or virulence in several intracellular pathogen species. This review illustrates and describes the progress which has been made in the design and the discovery of selective inhibitors of these bacterial enzymes as new potential anti-Brucella agents.

International audience; In murine infections, Brucella microti exhibits an atypical and highly pathogenic behavior resulting in a mortality of 82%. In this study, the possible involvement of the virB type IV secretion system, a key virulence factor of Brucella sp., in this lethal phenotype was investigated. As previously described for B. suis, expression of the virB operon of B. microti was induced in acid minimal medium, partially mimicking intracellular environment. Early neutralization of cellular compartments abolished intracellular replication of B. microti, showing that acidity of the Brucella-containing vacuole is an essential trigger. A ΔvirB mutant of B. microti exhibited strong attenuation in murine and human macrophages in vitro. Interestingly, infection with this mutant was not lethal in Balb/c mice and lacked the typical intrasplenic peak at 3 days post-infection, hence demonstrating that lethality of B. microti in murine infection absolutely requires a functional virB operon.

International audience; The facultative intracellular pathogen and zoonotic agent Brucella sp. possesses two carbonic anhydrases (CAs, EC 4.2.1.1), termed bsCA I and bsCA II (in Brucella suis), belonging to the β-class of these metalloproteins. These zinc enzymes, present in many other pathogenic bacteria, have been considered recently as potential antibacterial targets. The catalytic activity of bsCA II is higher than that of bsCA I (for the conversion of CO(2) to bicarbonate). Both enzymes were inhibited by the well-studied inhibitor acetazolamide, a sulfonamide drug. A library of 41 sulfonamides and one sulfamate, among which 12 clinically tested drugs, was used for inhibition studies with bsCA I and II. These compounds were generally much more potent inhibitors of bsCA I (K(Is) of 17-75 nM) than of bsCAII (K(I)s of 84-923nM). However, certain glycosidic sulfonamide derivatives exhibited the same strong inhibitory activity on both bsCA I and bsCA II (K(Is) of 8.9-20 nM). Furthermore, at least one of these glycosylsulfonamides showed a significant inhibition of B. suis growth after 8-11 days of culture in minimal medium. In conclusion, as β-CAs of Brucella are susceptible to inhibition by a wide range of aromatic and heteroaromatic sulfonamides, they may represent novel targets for the development of clinically useful antibacterial agents.

International audience; Low oxygen tension was proposed to be one of the environmental parameters characteristic of the patho-physiological conditions of natural infections by Brucella suis. We previously showed that various respiratory pathways may be used by B. suis in response to microaerobiosis and anaerobiosis. Here, we compare the whole proteome of B. suis exposed to such low-oxygenated conditions to that obtained from bacteria grown under ambient air using 2-D DIGE. Data showed that the reduction of basal metabolism was in line with low or absence of growth of B. suis. Under both microaerobiosis and anaerobiosis, glycolysis and denitrification were favored. In addition, fatty acid oxidation and possibly citrate fermentation could also contribute to energy production sufficient for survival under anaerobiosis. When oxygen availability changed and became limiting, basic metabolic processes were still functional and variability of respiratory pathways was observed to a degree unexpected for a strictly aerobic microorganism. This highly flexible respiration probably constitutes an advantage for the survival of Brucella under the restricted oxygenation conditions encountered within host tissue.

A 2-D DIGE approach allowed the characterization of the intramacrophagic proteome of the intracellular pathogen Brucella suis at the late stage of in vitro infection by efficient discrimination between bacterial and host cell proteins. Using a subtraction model, a total of 168 proteins showing altered concentrations in comparison with extracellularly grown, stationary-phase bacteria were identified. The majority of the 44 proteins significantly regulated at this stage of infection were involved in bacterial metabolism and 40% were present in lowered concentrations, supporting the hypothesis of an adaptive response by quantitative reduction of processes participating in energy, protein, and nucleic acid metabolism. In the future, the 2-D DIGE-based approach will permit to decipher specifically and quantitatively the intracellular proteomes of various pathogens during adaptation to their specific host cell environments.

Brucella spp. is the causative agent of brucellosis (Malta fever), which is the most widespread zoonosis worldwide. The pathogen is capable of establishing persistent infections in humans which are extremely difficult to eradicate even with antibiotic therapy. Moreover, Brucella is considered as a potential bioterrorism agent. Histidinol dehydrogenase (HDH, EC 1.1.1.23) has been shown to be essential for the intramacrophagic replication of this pathogen. It therefore constitutes an original and novel target for the development of anti-Brucella agents. In this work, we cloned and overexpressed the HDH-encoding gene from Brucella suis, purified the protein and evidenced its biological activity. We then investigated the inhibitory effects of a series of substituted benzylic ketones derived from histidine. Most of the compounds reported here inhibited B. suis HDH in the lower nanomolar range and constitute attractive candidates for the development of novel anti-Brucella agents.

Summary Physiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. One mechanism of adaptation, the stringent response, is induced by nutrient stress via its effector molecule (p)ppGpp, synthesized by the action of RelA/SpoT homologues. The intracellular pathogen Brucella spp., causative agent of brucellosis, pos- sesses a gene homologous to relA / spoT , named rsh , encoding a (p)ppGpp synthetase as confirmed by het- erologous complementation of a relA mutant of Sinorhizobium meliloti . The Rsh deletion mutants in Brucella suis and Brucella melitensis were character- ized by altered morphology, and by reduced survival under starvation conditions and in cellular and murine models of infection. Most interestingly, we evi- denced that expression of virB , encoding the type IV secretion system, a major virulence factor of Brucella , was Rsh-dependent. All mutant phenotypes, includ- ing lack of VirB proteins, were complemented with the rsh gene of Brucella . In addition, RelA of S. meliloti functionally replaced Brucella Rsh, describing the capacity of a gene from a plant symbiont to restore virulence in a mammalian pathogen. We therefore concluded that in the intramacrophagic environment encountered by Brucella , Rsh might participate in the adaptation of the pathogen to low-nutrient environ- ments, and indirectly in the VirB-mediated formation of the final replicative niche.

International audience; Brucellosis is not a sustainable disease in humans. The source of human infection always resides in domestic or wild animal reservoirs. The routes of infection are multiple: food-borne, occupational or recreational, linked to travel and even to bioterrorism. New Brucella strains or species may emerge and existing Brucella species adapt to changing social, cultural, travel and agricultural environment. Brucella melitensis is the most important zoonotic agent, followed by Brucella abortus and Brucella suis. This correlates with the fact that worldwide, the control of bovine brucellosis (due to B. abortus) has been achieved to a greater extent than the control of sheep and goat brucellosis (due to B. melitensis), these latter species being the most important domestic animals in many developing countries. The long duration and high cost of treatment of human brucellosis reduces the efficacy of the therapy. There is no human vaccine for brucellosis and the occurrence of brucellosis is directly linked to the status of animal brucellosis in a region. In this context, the Word Health Organization has defined the development of a human vaccine, besides the implementation of control and eradication programs in animals, as a high priority. The pathogenicity for humans of B. suis biovars 1, 3 and 4 is well established, whereas B. suis biovar 2 seems to be less pathogenic. Indeed, although hunters and pig farmers have repeatably experienced infectious contact with B. suis biovar 2 (found in wild boar and outdoor-rearing pigs in Europe), isolation of B. suis biovar 2 from human samples have only been seldom reported. Marine mammal brucellosis, due to two new proposed Brucella species i.e. B. cetaceae and B. pinnipediae, represents a new zoonotic threat but the pathogenicity for humans of the different Brucella species found in cetaceans and pinnipeds still has to be clearly established.

The heat shock protein DnaK is essential for intramacrophagic replication of Brucella suis . The replacement of the stress-inducible, native dnaK promoter of B. suis by the promoter of the constitutively expressed bla gene resulted in temperature-independent synthesis of DnaK. In contrast to a dnaK null mutant, this strain grew at 37°C, with a thermal cutoff at 39°C. However, the constitutive dnaK mutant, which showed high sensitivity to H 2 O 2 -mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction of dnaK is a crucial event in the intracellular replication of B. suis .

Brucellaspp. can establish themselves and cause disease in humans and animals. The mechanisms by whichBrucellaspp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-α) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) ofBrucella suis1330 (wild-type [WT]B. suis) on TNF-α production. For this purpose,omp25andomp31null mutants ofB. suis(Δomp25 B. suisand Δomp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-α. We showed that, in contrast to WTB. suisor Δomp31 B. suis, Δomp25 B. suisinduced TNF-α production when phagocytosed by human macrophages. The complementation of Δomp25 B. suiswith WTomp25(Δomp25-omp25 B. suismutant) significantly reversed this effect: Δomp25-omp25 B. suis-infected macrophages secreted significantly less TNF-α than did macrophages infected with the Δomp25 B. suismutant. Furthermore, pretreatment of WTB. suiswith an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-α production during macrophage infection. These observations demonstrated that Omp25 ofB. suisis involved in the negative regulation of TNF-α production upon infection of human macrophages.

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in Brucella suis , a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis , and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of B. suis was induced, and an isogenic B. suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress. In B. suis expressing gfp , ClpA but not ClpB participated in degradation of the green fluorescent protein at 42°C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.

The introduction into Brucella suis 1330 of a plasmid allowing the heterologous expression of a hybrid cytolysin containing listeriolysin from Listeria monocytogenes , and its export via the Escherichia coli hemolysin secretion pathway, resulted in secretion of active listeriolysin monitored by erythrocyte lysis. In contrast to observations with the nonhemolytic control strain, the phagosomes of infected human monocytes containing the hemolytic B. suis were partially disrupted, and this strain failed to multiply in human macrophage-like cells. These results added strong evidence supporting the proposal that the phagosome of the macrophage was the predominant niche of brucellae in their mammalian hosts.

The gram-negative bacteria Brucella spp. are the etiologic agents of brucellosis, a disease that is encountered worldwide and is endemic in many underdeveloped countries. Six distinct species have been identified in various mammalian hosts, including humans. In cattle, sheep, and goats, the disease still causes important economic losses. Among these species, Brucella melitensis, B. suis, and B. abortus are most frequently associated with pathogenicity in humans, characterized by undulant fever and other, less well-defined clinical symptoms. The infection can be asymptomatic, which may cause diagnostic difficulties and sometimes lead to chronic infections in bones, joints, and the central nervous system. Brucellae belong to the α-2 subdivision of the proteobacteria and are therefore phylogenetically related to the plant cell-associated species of the genera Rhizobium and Agrobacterium. Brucella spp. are facultative intracellular parasites that can survive within professional phagocytes. They are able to invade macrophages and to multiply inside acidified phagosomes (36). Among the mechanisms used by brucellae for intracellular survival are the inhibition of phagolysosomal fusion (35) and of tumor necrosis factor alpha production by macrophages (4), the activation of which under normal conditions is critical for the elimination of pathogens. Despite intensive work, the mechanisms allowing Brucella to behave as an intracellular parasite have not been elucidated. To date, little is known about the bacterial factors contributing to the persistence and multiplication of this pathogen within human phagocytes. Upon infection of the macrophage by Brucella spp., certain stress proteins also induced at low pH and at high temperature were found to be expressed. Among those are the proteins HtrA, GroEL, and DnaK, the latter having been shown to be essential for replication of B. suis in human macrophage-like cell lines (23). Genes encoding these stress proteins (12, 17, 26) were frequently identified by the use of heterologous probes designed from previously characterized genes of other species. More recently, a two-component regulatory system (41) and a locus homologous to the VirB type IV secretion system (34) have been demonstrated to play important roles in intracellular survival. In an attempt to identify the bacterial genes expressed during multiplication of brucellae in host cells, a genetic tool was developed using the green fluorescent protein (GFP) gene as a reporter gene fused to randomly cloned B. suis promoters (22). Characterization of these genes, especially inactivation, should give an insight into the mechanisms allowing Brucella spp. to adapt to host macrophages. Several clones were selected on the basis of the inducible expression of GFP fluorescence after infection of a macrophage cell line. Here, we describe the identification of a gfp-fused B. suis gene highly homologous to nikA, the first gene of the Escherichia coli ATP-binding cassette (ABC) transport system specific for nickel (33). Uptake of nickel by the periplasmic binding-protein-dependent transport system encoded by the nikABCDE operon is required for the synthesis and activities of the E. coli hydrogenase isoenzymes under anaerobic conditions (46). Transcription of this operon is activated by the general anaerobic transcriptional factor Fnr and repressed by the nickel-responsive regulator NikR when intracellular nickel concentrations are high (9, 46, 47). Many microorganisms incorporate this metal ion into enzymes participating in important metabolic reactions of hydrogen metabolism, ureolysis, methane biogenesis, and acetogenesis (20). Hydrolysis of urea is catalyzed by the nickel-dependent urease, an enzyme produced by all Brucella species (7). Our data show that B. suis possesses genes encoding counterparts highly similar to the five protein components of the E. coli NikABCDE permease and to the NikR regulator with a distinct genomic organization. We demonstrate that in vitro expression of the nikA promoter-gfp fusion is activated by low oxygen or nickel concentrations. We also show that inactivation of nikA severely alters the activity of the nickel metalloenzyme urease in this bacterium. In addition, successful reciprocal complementation of nik mutants of E. coli and B. suis with the nik operons supports the notion that the gene cluster of B. suis functions as a nickel transport system. Our data also suggest that, like E. coli and other bacteria, B. suis can use alternative ion transport systems for this metal when its concentration is high enough.

A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis , allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA to gfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B. suis . Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis -containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A 1 , a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis , in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.

In the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat-shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37 degrees C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937-derived phagocytes resulted in long-lasting DnaK induction in the wild-type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30 degrees C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild-type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37 degrees C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature-independent contribution of DnaK to intracellular multiplication of the pathogen B. suis.

Tumor necrosis factor alpha (TNF-alpha) plays a central role in activation of first-line defenses of a host against foreign organisms. To determine whether Brucella infection modulated TNF-alpha production, we measured the biological activity of this cytokine in supernatants of U937 cell-derived macrophages and of fresh human monocytes infected with Brucella spp. Neither the smooth nor rough Brucella strains used induced any measurable TNF-alpha excretion upon infection. On the contrary, as reported before for other gram-negative bacteria, phagocytosis of nonpathogenic Escherichia coli was followed by a rapid and transient induction of TNF-alpha release, suggesting an involvement of this cytokine in some autocrine process. As expected, the Brucella strains tested survived and/or multiplied within U937-derived macrophages, whereas E. coli was rapidly eliminated after phagocytosis. Immunoglobulin G opsonization of E. coli strains enhanced their intracellular killing and strongly potentiated TNF-alpha secretion. Immunoglobulin G opsonization of Brucella strains, in contrast, did not lead to TNF-alpha production, although their rate of intracellular multiplication was reduced. Killed brucellae, however, promoted a significant excretion of TNF-alpha from U937-derived macrophages into cell culture supernatants. We finally demonstrated that pretreatment of U937-derived macrophages with exogenous TNF-alpha significantly inhibited intracellular multiplication of Brucella spp. These results and experiments performed on fresh human monocytes or with isolated lipopolysaccharide (LPS) showed that (i) differences in TNF-alpha production observed during macrophage infection by Brucella spp. and E. coli were not due to differences in LPS structure but resulted from active inhibition of TNF-alpha production by a specific process linked to Brucella spp. and (ii) the capacity of Brucella spp. to use pathways avoiding TNF-alpha production during infection may be considered a major attribute of virulence.

In the study of interactions between facultative intracellular pathogens and macrophages, monocytic cell lines have the advantages of showing defined states of activation and lacking genetic variation among donors, thus yielding reproducible results. Nonpathogenic Escherichia coli K12 were killed at similar rates in the U937 cell line differentiated into macrophage-like cells by phorbol myristate acetate (PMA) or by the combination of retinoic acid (RA) and vitamin D3 (VD). Complete elimination was reached only when cells were activated by lipopolysaccharide for 30 min prior to infection, and it was further enhanced when bacteria were opsonized by specific immunoglobulin G. Both types of differentiation led to intracellular multiplication of virulent Listeria monocytogenes and to elimination of the animal pathogen Listeria ivanovii. For both strains, conditions for intracellular survival were more favorable in PMA-differentiated U937. During infection, RA/VD-differentiated U937 could discriminate between the human pathogen Brucella suis S1, which strongly multiplied, and the animal pathogen Brucella canis, which survived without multiplication. U937 cells differentiated by RA and VD therefore represent a basic model in bacteria-human macrophage interactions. J. Leukoc. Biol. 56: 174–181; 1994.

Facultatively intracellular bacteria express heat shock proteins after phagocytosis by macrophages. Using non-pathogenic Escherichia coli strains and the human monocytic cell line U937, we showed that deletion of the dnaK gene significantly increased the rate of initial intracellular killing of bacteria. trans-complementation of the deletion mutant with the dnaK / dnaJ operon of Brucella ovis restored the pattern of intracellular elimination of the control strain expressing dnaK. These differences were not observed using antibody-opsonized bacteria and activated cells. In vitro, strains expressing dnaK resisted hydrogen peroxide better than the deletion mutant; in contrast, the mutant complemented by dnaK / dnaJ of B. ovis tolerated low pH and low H2O2 better than the wild-type strain and the deletion mutant. Our results suggested the particvipation of DnaK on protection of intracellular bacteria against antimicrobial macrophage factors.

The genus Brucella currently comprises twelve species of facultative intracellular bacteria with variable zoonotic potential. Six of them have been considered as classical, causing brucellosis in terrestrial mammalian hosts, with two species originated from marine mammals. In the past fifteen years, field research as well as improved pathogen detection and typing have allowed the identification of four new species, namely Brucella microti, Brucella inopinata, Brucella papionis, Brucella vulpis, and of numerous strains, isolated from a wide range of hosts, including for the first time cold-blooded animals. While their genome sequences are still highly similar to those of classical strains, some of them are characterized by atypical phenotypes such as higher growth rate, increased resistance to acid stress, motility, and lethality in the murine infection model. In our review, we provide an overview of state-of-the-art knowledge about these novel Brucella sp., with emphasis on their phylogenetic positions in the genus, their metabolic characteristics, acid stress resistance mechanisms, and their behavior in well-established in cellulo and in vivo infection models. Comparison of phylogenetic classification and phenotypical properties between classical and novel Brucella species and strains finally lead us to propose a more adapted terminology, distinguishing between core and non-core, and typical versus atypical brucellae, respectively.