A novel multiplex and glycoprotein-based immunochromatographic serologic IgM test for the rapid diagnosis of Escherichia coli O157 and O145 causing bloody diarrhea and hemolytic uremic syndrome.

Shiga toxin-producing Escherichia coli (STEC) are the main etiological agents of hemolytic uremic syndrome (HUS). Good clinical management of STEC infections and HUS depends on early, rapid, and accurate diagnosis. Here, we have developed and evaluated the first multiplex and glycoprotein-based immunochromatographic test for the detection of IgM antibodies against the O-polysaccharide of the lipopolysaccharide of E. coli O157 and O145 in human serum samples. A retrospective study was carried out resulting in a diagnostic sensitivity of the E. coli O157/O145 LFIA (lateral flow immunoassay) of 97.1% and 98.9% for O157 and O145, respectively, and 97.9% for both serogroups. The diagnostic specificity was 98.7% for O157 and O145, and the overall specificity 97.4%. In samples obtained before 3 days after the onset of diarrhea, the detection percentage was 83%, increasing to 100% from 3 days onward. Finally, the association of bloody diarrhea (BD) or HUS cases to an STEC infection increased from 22.8% to 77.2% when stool culture and stx/Stx detection were combined with serology by LFIA. Our results demonstrate that the E. coli O157/O145 LFIA is a highly accurate and serospecific test for the early and rapid diagnosis of E. coli O157 and O145 infections in BD or HUS cases. This test allows the detection of specific IgM antibodies very early in the course of the infection, making it an ideal diagnostic tool to be implemented in pediatric emergencies and, thus, avoid delays in the application of the correct supportive or specific treatment and prevent complications associated with HUS.

The distinct cell physiology of Bradyrhizobium at the population and cellular level.

The α-Proteobacteria belonging to Bradyrhizobium genus are microorganisms of extreme slow growth. Despite their extended use as inoculants in soybean production, their physiology remains poorly characterized. In this work, we produced quantitative data on four different isolates: B. diazoefficens USDA110, B. diazoefficiens USDA122, B. japonicum E109 and B. japonicum USDA6 which are representative of specific genomic profiles. Notably, we found conserved physiological traits conserved in all the studied isolates: (i) the lag and initial exponential growth phases display cell aggregation; (ii) the increase in specific nutrient concentration such as yeast extract and gluconate hinders growth; (iii) cell size does not correlate with culture age; and (iv) cell cycle presents polar growth. Meanwhile, fitness, cell size and in vitro growth widely vary across isolates correlating to ribosomal RNA operon number. In summary, this study provides novel empirical data that enriches the comprehension of the Bradyrhizobium (slow) growth dynamics and cell cycle.

Development of a scalable recombinant system for cyclic beta-1,2-glucans production.

BACKGROUND: Cyclic ß-1,2-glucans (CßG) are bacterial cyclic homopolysaccharides with interesting biotechnological applications. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that CßG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, suggesting these molecules have great potential for their industrial and commercial exploitation. Nowadays, there is no method to produce CßG by chemical synthesis and bacteria that synthesize them are slow-growing or even pathogenic, which makes the scaling up of the process difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand and expand the repertoire of applications of CßG. RESULTS: We present the production of CßG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CßG synthesis, transport and succinilation. These strains produce different types of CßG: unsubstituted CßG, anionic CßG and CßG of high size. Unsubstituted CßG with a degree of polymerization of 17 to 24 glucoses were produced and secreted to the culture medium by one of the strains. Through high cell density culture (HCDC) of that strain we were able to produce 4,5 g of pure unsubstituted CßG /L in culture medium within 48 h culture. CONCLUSIONS: We have developed a new recombinant bacterial system for the synthesis of cyclic ß-1,2-glucans, expanding the use of bacteria as a platform for the production of new polysaccharides with biotechnological applications. This new approach allowed us to produce CßG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CßG availability for further research and the widespread use of these promising molecules across many application fields.

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

Development of a novel glycoprotein-based immunochromatographic test for the rapid serodiagnosis of bovine brucellosis.

AIMS: Bovine brucellosis is a worldwide zoonotic disease that causes important economic losses and public health concerns. Because control of the disease depends on vaccination, serodiagnosis and isolation of the infected animals, affordable, rapid and accurate point of care (POC) tests are needed. METHODS AND RESULTS: We developed and evaluated a novel glycoprotein-based immunochromatographic test for the detection of IgG antibodies against the O-polysaccharide of Brucella in bovine serum samples. Brucella GlycoStrip combines the power of immunochromatographic and bacterial glycoengineering technologies for the diagnosis of bovine brucellosis. The analysis of positive and negative reference samples indicated that the test has a diagnostic sensitivity and specificity of 96.9% (95% CI: 92.7%-100.0%) and 100%, respectively. CONCLUSIONS: Due to the recombinant glycoprotein-based antigen OAg-AcrA, which consists of the O-side chain of Brucella smooth lipopolysaccharide (sLPS) covalently linked to the carrier protein AcrA, the test is highly accurate, allows the differentiation of infected animals from those vaccinated with a rough strain or with a single dose of a smooth strain and fulfil the minimum diagnostic requirements established by the national and international regulations. SIGNIFICANCE AND IMPACT OF STUDY: This strip test could provide a rapid (10 min) and accurate diagnosis of bovine brucellosis in the field contributing to the control of the disease.

Macromolecular crowding links ribosomal protein gene dosage to growth rate in Vibrio cholerae.

BACKGROUND: In fast-growing bacteria, the genomic location of ribosomal protein (RP) genes is biased towards the replication origin (oriC). This trait allows optimizing their expression during exponential phase since oriC neighboring regions are in higher dose due to multifork replication. Relocation of s10-spc-α locus (S10), which codes for most of the RP, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction on its dosage, its expression, and bacterial growth rate. However, a mechanism linking S10 dosage to cell physiology has still not been determined. RESULTS: We hypothesized that S10 dosage perturbations impact protein synthesis capacity. Strikingly, we observed that in Vibrio cholerae, protein production capacity was independent of S10 position. Deep sequencing revealed that S10 relocation altered chromosomal replication dynamics and genome-wide transcription. Such changes increased as a function of oriC-S10 distance. Since RP constitutes a large proportion of cell mass, lower S10 dosage could lead to changes in macromolecular crowding, impacting cell physiology. Accordingly, cytoplasm fluidity was higher in mutants where S10 is most distant from oriC. In hyperosmotic conditions, when crowding differences are minimized, the growth rate and replication dynamics were highly alleviated in these strains. CONCLUSIONS: The genomic location of RP genes ensures its optimal dosage. However, besides of its essential function in translation, their genomic position sustains an optimal macromolecular crowding essential for maximizing growth. Hence, this could be another mechanism coordinating DNA replication to bacterial growth.

Brucella abortus Depends on l-Serine Biosynthesis for Intracellular Proliferation.

l-Serine is a nonessential amino acid and a key intermediate in several relevant metabolic pathways. In bacteria, the major source of l-serine is the phosphorylated pathway, which comprises three enzymes: d-3-phosphoglycerate dehydrogenase (PGDH; SerA), phosphoserine amino transferase (PSAT; SerC), and l-phosphoserine phosphatase (PSP; SerB). The Brucella abortus genome encodes two PGDHs (SerA-1 and SerA-2), involved in the first step in l-serine biosynthesis, and one PSAT and one PSP, responsible for the second and third steps, respectively. In this study, we demonstrate that the serA1 serA2 double mutant and the serC and serB single mutants are auxotrophic for l-serine. These auxotrophic mutants can be internalized but are unable to replicate in HeLa cells and in J774A.1 macrophage-like cells. Replication defects of auxotrophic mutants can be reverted by cell medium supplementation with l-serine at early times postinfection. In addition, the serB mutant is attenuated in the murine intraperitoneal infection model and has an altered lipid composition, since the lack of l-serine abrogates phosphatidylethanolamine synthesis in this strain. Taken together, these results reveal that limited availability of l-serine within the host cell impairs proliferation of the auxotrophic strains, highlighting the relevance of this biosynthetic pathway in Brucella pathogenicity.

Development and Evaluation of a Novel VHH-Based Immunocapture Assay for High-Sensitivity Detection of Shiga Toxin Type 2 (Stx2) in Stool Samples.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype. Therefore, Stx detection assays are considered the most suitable methods for the early detection of STEC infections. Single-domain antibodies from camelids (VHHs) exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis. In this work, we have exploited VHH technology for the development of an immunocapture assay for Stx2 detection. Thirteen anti-Stx2 VHHs previously obtained from a variable-domain repertoire library were selected and evaluated in 130 capture-detection pair combinations for Stx detection. Based on this analysis, two VHHs were selected and a double VHH-based biotin-streptavidin capture enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection was developed and optimized for Stx2 detection. This assay showed an excellent analytical and clinical sensitivity in both STEC culture supernatants and stool samples even higher than the sensitivity of a commercial ELISA. Furthermore, based on the analysis of stool samples, the VHH-based ELISA showed high correlation with stx2 detection by PCR and a commercial rapid membrane-based immunoassay. The intrinsic properties of VHHs (high target affinity and specificity, stability, and ease of expression at high yields in recombinant bacteria) and their optimal performance for Stx detection make them attractive tools for the diagnosis of HUS related to STEC (STEC-HUS).

Cyclic ß-glucans at the bacteria-host cells interphase: One sugar ring to rule them all.

Cyclic ß-1,2-D-glucans (CßG) are natural bionanopolymers present in the periplasmic space of many Proteobacteria. These molecules are sugar rings made of 17 to 25 D-glucose units linked exclusively by ß-1,2-glycosidic bonds. CßG are important for environmental sensing and osmoadaptation in bacteria, but most importantly, they play key roles in complex host-cell interactions such as symbiosis, pathogenesis, and immunomodulation. In the last years, the identification and characterisation of the enzymes involved in the synthesis of CßG allowed to know in detail the steps necessary for the formation of these sugar rings. Due to its peculiar structure, CßG can complex large hydrophobic molecules, a feature possibly related to its function in the interaction with the host. The capabilities of the CßG to function as molecular boxes and to solubilise hydrophobic compounds are attractive for application in the development of drugs, in food industry, nanotechnology, and chemistry. More importantly, its excellent immunomodulatory properties led to the proposal of CßG as a new class of adjuvants for vaccine development.

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity.

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (µ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At µmax , AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.

Glyco-iELISA: a highly sensitive and unambiguous serological method to diagnose STEC-HUS caused by serotype O157.

BACKGROUND: Providing proof of presence of Shiga toxin-producing E. coli (STEC) infection forms the basis for differentiating STEC-hemolytic uremic syndrome (HUS) and atypical HUS. As the gold standard to diagnose STEC-HUS has limitations, using ELISA to detect serum antibodies against STEC lipopolysaccharides (LPS) has proven additional value. Yet, conventional LPS-ELISA has drawbacks, most importantly presence of cross-reactivity due to the conserved lipid A part of LPS. The newly described glyco-iELISA tackles this issue by using modified LPS that eliminates the lipid A part. Here, the incremental value of glyco-iELISA compared to LPS-ELISA is assessed. METHODS: A retrospective study was performed including all pediatric patients (n = 51) presenting with a clinical pattern of STEC-HUS between 1990 and 2014 in our hospital. Subsequently, the diagnostic value of glyco-iELISA was evaluated in a retrospective nationwide study (n = 264) of patients with thrombotic microangiopathy (TMA). LPS- and glyco-iELISA were performed to detect IgM against STEC serotype O157. Both serological tests were compared with each other and with fecal diagnostics. RESULTS: Glyco-iELISA is highly sensitive and has no cross-reactivity. In the single-center cohort, fecal diagnostics, LPS-ELISA, and glyco-iELISA identified STEC O157 infection in 43%, 65%, and 78% of patients, respectively. Combining glyco-iELISA with fecal diagnostics, STEC infection due to O157 was detected in 89% of patients. In the nationwide cohort, 19 additional patients (8%) were diagnosed with STEC-HUS by glyco-iELISA. CONCLUSION: This study shows that using glyco-iELISA to detect IgM against STEC serotype O157 has clear benefit compared to conventional LPS-ELISA, contributing to optimal diagnostics in STEC-HUS.

A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis.

Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans.

Interaction network and localization of Brucella abortus membrane proteins involved in the synthesis, transport, and succinylation of cyclic ß-1,2-glucans.

UNLABELLED: Cyclic ß-1,2-glucans (CßG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction of Brucella with the host. Once synthesized in the cytoplasm by the CßG synthase (Cgs), CßG are transported to the periplasm by the CßG transporter (Cgt) and succinylated by the CßG modifier enzyme (Cgm). Here, we used a bacterial two-hybrid system and coimmunoprecipitation techniques to study the interaction network between these three integral inner membrane proteins. Our results indicate that Cgs, Cgt, and Cgm can form both homotypic and heterotypic interactions. Analyses carried out with Cgs mutants revealed that the N-terminal region of the protein (Cgs region 1 to 418) is required to sustain the interactions with Cgt and Cgm as well as with itself. We demonstrated by single-cell fluorescence analysis that in Brucella, Cgs and Cgt are focally distributed in the membrane, particularly at the cell poles, whereas Cgm is mostly distributed throughout the membrane with a slight accumulation at the poles colocalizing with the other partners. In summary, our results demonstrate that Cgs, Cgt, and Cgm form a membrane-associated biosynthetic complex. We propose that the formation of a membrane complex could serve as a mechanism to ensure the fidelity of CßG biosynthesis by coordinating their synthesis with the transport and modification. IMPORTANCE: In this study, we analyzed the interaction and localization of the proteins involved in the synthesis, transport, and modification of Brucella abortus cyclic ß-1,2-glucans (CßG), which play an important role in the virulence and interaction of Brucella with the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the existence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation in Brucella. We propose that the formation of this membrane complex could serve as a mechanism to ensure the fidelity of CßG biosynthesis by coordinating synthesis with the transport and modification.

Serogroup-specific bacterial engineered glycoproteins as novel antigenic targets for diagnosis of shiga toxin-producing-escherichia coli-associated hemolytic-uremic syndrome.

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coli O157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. coli lipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis.

A Brucella virulence factor targets macrophages to trigger B-cell proliferation.

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.

Brucella abortus induces collagen deposition and MMP-9 down-modulation in hepatic stellate cells via TGF-ß1 production.

In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-ß1 induction. In contrast, supernatants from B. abortus-infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus-infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-ß1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus-infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis.

Brucella alters the immune response in a prpA-dependent manner.

Brucellosis, a disease caused by the gram-negative bacterium Brucella spp., is a widespread zoonosis that inflicts important animal and human health problems, especially in developing countries. One of the hallmarks of Brucella infection is its capacity to establish a chronic infection, characteristic that depends on a wide repertoire of virulence factors among which are immunomodulatory proteins such as PrpA (encoding the proline racemase protein A or hydroxyproline-2-epimerase), involved in the establishment of the chronic phase of the infectious process that we have previously identified and characterized. We report here that, in vivo, Brucella abortus prpA is responsible for an increment in the B-cell number and in the specific antibody response and that these antibodies promote cell infection. We additionally found that Brucella alters the cytokine levels of IFN-γ, IL-10, TGFß1 and TNFα during the acute phase of the infectious process in a prpA dependent manner.

The 'ins and outs' of Brucella intracellular journey.

Members of the genus Brucella are the causative agents of brucellosis, a worldwide zoonosis affecting wild and domestic animals and humans. These facultative intracellular pathogens cause long-lasting chronic infections by evolving sophisticated strategies to counteract, evade, or subvert host bactericidal mechanisms in order to establish a secure replicative niche necessary for their survival. In this review, we present recent findings on selected Brucella effectors to illustrate how this pathogen modulates host cell signaling pathways to gain control of the vacuole, promote the formation of a safe intracellular replication niche, alter host cell metabolism to its advantage, and exploit various cellular pathways to ensure egress from the infected cell.

Identification and characterization of a high-affinity choline uptake system of Brucella abortus.

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.

A MarR-Type regulator directly activates transcription from the Brucella abortus virB promoter by sharing a redundant role with HutC.

Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the T4SS-encoding virB operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the virB genes that were isolated from total protein extracts of Brucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the virB promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA in vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated virB expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the virB promoter and is probably involved in coordinating gene expression in response to specific environmental signals.