Harmful algal bloom (HAB) formation leads to the eutrophication of water ecosystems and may render recreational lakes unsuitable for human use. We evaluated the applicability and comparison of metabarcoding, metagenomics, qPCR, and ELISA-based methods for cyanobacteria/cyanotoxin detection in bloom and non-bloom sites for the Great Lakes region. DNA sequencing-based methods robustly identified differences between bloom and non-bloom samples (e.g., the relative prominence of Anabaena and Planktothrix). Shotgun sequencing strategies also identified the enrichment of metabolic genes typical of cyanobacteria in bloom samples, though toxin genes were not detected, suggesting deeper sequencing or PCR methods may be needed to detect low-abundance toxin genes. PCR and ELISA indicated microcystin levels and microcystin gene copies were significantly more abundant in bloom sites. However, not all bloom samples were positive for microcystin, possibly due to bloom development by non-toxin-producing species. Additionally, microcystin levels were significantly correlated (positively) with microcystin gene copy number but not with total cyanobacterial 16S gene copies. In summary, next-generation sequencing-based methods can identify specific taxonomic and functional targets, which can be used for absolute quantification methods (qPCR and ELISA) to augment conventional water monitoring strategies.

Cyanobacteria (blue-green algae) are a diverse group of prokaryotic microorganisms that impact global biogeochemical cycles. Under eutrophic conditions, cyanobacterial species can produce cyanotoxins, resulting in harmful algal blooms (cHABs) that degrade water quality and result in economic and recreational losses. The Laurentian Great Lakes, a key global freshwater source, are increasingly affected by these blooms. To understand the underlying mechanisms in cHAB formation, we investigated microcystin levels, cyanotoxin genes/transcripts, and taxonomic/microcystin metabarcoding across three sampling locations in the Canadian Great Lakes region, including Hamilton Harbour, Bay of Quinte, and Three Mile Lake (Muskoka), to observe the regional and longitudinal cyanobacterial dynamics. The results revealed a positive correlation between microcystin levels, the occurrence of cyanobacterial taxonomic/cyanotoxin molecular markers, and the relative widespread abundance of specific dominant cyanobacterial taxa, including Planktothrix, Microcystis, and Dolichospermum. The Cyanobium genus was not observed in Hamilton Harbor samples during late summer (August to September), while it was consistently observed in the Three Mile Lake and Bay of Quinte samples. Notably, Dolichospermum and saxitoxin genes were predominantly higher in Three Mile Lake (an inland lake), suggesting site-specific characteristics influencing saxitoxin production. Additionally, among the potential microcystin producers, in addition to Microcystis, Hamilton Harbour and Bay of Quinte samples showed consistent presence of less dominant microcystin-producing taxa, including Phormidium and Dolichospermum. This study highlights the complexity of cHAB formation and the variability in cyanotoxin production in specific environments. The findings highlight regional and site-specific factors that can influence cyanobacterial taxonomic and molecular profiles, necessitating the integration of advanced molecular technologies for effective monitoring and targeted management strategies.

Cyanobacteria (blue-green algae) can accumulate to form harmful algal blooms (HABs) on the surface of freshwater ecosystems under eutrophic conditions. Extensive HAB events can threaten local wildlife, public health, and the utilization of recreational waters. For the detection/quantification of cyanobacteria and cyanotoxins, both the United States Environmental Protection Agency (USEPA) and Health Canada increasingly indicate that molecular methods can be useful. However, each molecular detection method has specific advantages and limitations for monitoring HABs in recreational water ecosystems. Rapidly developing modern technologies, including satellite imaging, biosensors, and machine learning/artificial intelligence, can be integrated with standard/conventional methods to overcome the limitations associated with traditional cyanobacterial detection methodology. We examine advances in cyanobacterial cell lysis methodology and conventional/modern molecular detection methods, including imaging techniques, polymerase chain reaction (PCR)/DNA sequencing, enzyme-linked immunosorbent assays (ELISA), mass spectrometry, remote sensing, and machine learning/AI-based prediction models. This review focuses specifically on methodologies likely to be employed for recreational water ecosystems, especially in the Great Lakes region of North America.

The performance of on-site wastewater treatment systems (OWTSs) can be improved by altering digester design and by manipulating environmental variables that affect microbial community composition. Community composition can potentially be assessed using high-throughput DNA sequencing, but the two most common methods of community DNA sequencing (16S and shotgun sequencing) generally yield different taxonomic identification profiles and can perform differently according to the sampled environment. To evaluate the use of these two approaches in monitoring OWTS operation, we conducted a comparative parallel analysis using both 16S rDNA and shotgun sequencing in a controlled field study. Results indicate that when examining microorganisms above 0.1% relative abundance, 16S and shotgun sequencing produced similar results in terms of individual sample species richness and between-sample community similarity. However, shotgun sequencing provided comparatively higher taxonomic richness for the bacterial communities with lower abundance in the OWTSs. In addition, 16S sequencing resolved only 48 out of 188 bacterial communities identified by shotgun sequencing (using a 0.1% abundance cutoff). Three key bacterial genera (Desulfomicrobium, Simplicispira, and Phenylobacterium) in anaerobic digestion processes were differentially abundant for both sequencing methods. These data indicate that both sequencing methods provide similar overall profiles for bacterial communities in anaerobic digestor systems. However, shotgun sequencing provides significantly (p-value < 0.01) higher taxonomic richness overall. Thus, shotgun sequencing provides a more robust taxonomic and functional profile that can be used for the optimization of anaerobic digestor systems.

Nonaqueous phase liquids (NAPL, e.g., hydrocarbons and chlorinated compounds) are common groundwater pollutants. Electrokinetic remediation of NAPLs uses electric fields to draw them toward electrodes and remove them from groundwater. The treatment requires NAPL mobility. Emulsification increases mobility, but at a risk for downstream receptors. We propose using alkaline aqueous solutions of zein and graphene nanoparticles (GNP) to form conductive materials, which could also act as barriers to control NAPL migration. Alkaline zein-GNP solutions can be injected in the polluted soil and solidified by neutralizing the pH (e.g., with glacial acetic acid, GAA). Shear rheology experiments showed that zein-GNP composites were cohesive, and voltammetry showed that GNP increased electrical conductivity of zein-based materials by 3.5 times. Gas chromatography-mass spectroscopy (GC-MS) demonstrated that the electrokinetic treatment of model sandy aquifers yielded >60% and ∼47% removal of emulsified toluene in freshwater and in salt solutions, respectively (with 30 min treatment using a 10 V differential voltage between a zein-GNP and an aluminum electrode. NaCl was used as model salt contaminant. The conductivity of surfactant solutions was lower in saline water than in freshwater, explaining differences in toluene removal. Toluene-water emulsions were stabilized using the natural surfactants lecithin and saponin. These surfactants acted synergistically in stabilizing emulsions in either freshwater or salt solutions. Lecithin and saponin likely interacted at toluene-water interfaces, as indicated by the morphology, interfacial tension and compressional rigidity of toluene-water interfaces with both components (relative to interfaces of either lecithin or saponin alone). The compressional behavior of interfacial films was well-described by the Marczak model. Electrokinetic treatment of saturated model sandy aquifers also decreased the turbidity of emulsions of water and either tricholoroethylene (TCE, by ∼41%) or diesel (by ∼75%), in the presence of a bacterial biosurfactant. This decrease was used as semiquantitative indicator of NAPL removal from water.

This study proposes injectable polymeric filters to prevent diesel migration during its bioremediation. Pseudomonas aeruginosa (Gram negative bacterium) was isolated from forest soil and used to biodegrade diesel. P. aeruginosa produced biosurfactants that emulsified diesel in water while degrading it, by decreasing the interfacial tension and forming skins around diesel droplets. In lab experiments, emulsified diesel droplets could migrate through model soil columns (sand and limestone) saturated with water (to mimic aquifers). These results indicate that clean aquifers downstream of diesel spills are at risk of becoming contaminated during diesel bioremediation by P. aeruginosa. Injectable filters were developed to mitigate this risk. Injectable filters were obtained by injecting into model soils aqueous solutions of quaternized hydroxyethylcellulose (HEC+, with a positive electrostatic charge). HEC + adsorbed onto model soils, yielding positively charged surfaces. Diesel droplets stabilized by P. aeruginosa had a negative electrostatic charge (as demonstrated through zeta potential measurements) and adhered onto HEC + coated soil, likely due to attractive electrostatic forces. As a result, diesel droplet mobility through soil columns decreased. Our proof of concept indicates that injectable HEC + filters can protect clean regions of aquifers during the bioremediation of diesel spills by P. aeruginosa.

Pseudomonas sp. bacteria were incorporated into a double emulsion system (water in oil in water, W/O/W), which can be injected in the subsurface to trap and biotreat naphthalene. The outermost phase of the W/O/W emulsion was an aqueous solution of chitosan (1.65 wt.%). Naphthalene could be dispersed in the aqueous chitosan phase of W/O/W emulsions. Two separate primary water in oil (W/O) emulsions were emulsified in chitosan, using Tween 20 as emulsifier. W/O emulsions were prepared with canola oil (containing ethylcellulose and calcium stearate as emulsifiers), and aqueous solutions of either Pseudomonas sp. in minimal broth or sodium citrate (crosslinker). The formation of W/O/W double emulsions was verified with optical and confocal microscopy. Without addition of triggers able to induce gelation on demand, W/O/W emulsions were stable and mainly viscous under shear. The shear elastic modulus (G' = 1.90 +/- 0.39 Pa) and the shear viscous modulus (G '' = 5.76 +/- 0.06 Pa) were low, to facilitate pumping in the subsurface. With addition of the surfactant cocamidopropyl betaine, W/O/W emulsions were destabilized on demand, thereby releasing citrate (crosslinker) and Pseudomonas sp. in the chitosan phase, where naphthalene was dispersed. Mixing chitosan with citrate led to its gelation, trapping naphthalene. Mixing naphthalene with Pseudomonas sp. led to its bioremediation. Gelation was demonstrated by the increase in the viscoelastic moduli, with G' = 111.02 +/- 1.67 Pa > G '' = 8.66 +/- 0.10 Pa. With naphthalene, Pseudomonas sp. survival in gelled chitosan was confirmed by plating bacteria sampled from the gel. Without naphthalene, Pseudomonas sp. did not survive in chitosan.

Residents in rural communities across Canada collect potable water from aquifers. Fecal contaminants from sewage and agricultural runoffs can penetrate aquifers, posing a public health risk. Standard methods for detecting fecal contamination test for fecal indicator bacteria (FIB), but the presence of these do not identify sources of contamination. In contrast, DNA-based diagnostic tools can achieve this important objective. We employed quantitative polymerase chain reaction (qPCR) and high-throughput DNA sequencing to trace fecal contamination sources in Wainfleet, a rural Ontario township that has been under the longest active boil water advisory in Canada due to FIB contamination in groundwater wells. Using traditional methods, we identified FIBs indicating persistent fecal pollution in well waters. We used 16S rRNA sequencing to profile groundwater microbial communities and identified Campylobacteraceae as a fecal contamination DNA marker in septic tank effluents (STEs). We also identified Turicibacter and Gallicola as a potential cow and chicken fecal contamination marker, respectively. Using human specific Bacteroidales markers, we identified leaking septic tanks as the likely primary fecal contamination source in some of Wainfleet's groundwater. Overall, the results support the use of sequencing-based methods to augment traditional water quality testing methods and help end-users assess fecal contamination levels and identify point and non-point pollution sources.

Environmental water monitoring is an important responsibility of municipal governments. In this study, we partnered with several municipalities in an extensive sampling program to investigate the effects of spatiotemporal and environmental factors on bacterial diversity in a complex watershed ecosystem containing specific environments including creeks, a river, canals, stormwater outfalls and freshwater lakes of the Niagara Peninsula. Samples were collected using standard municipal protocols and bacterial DNA extracted from these samples was sequenced using high-throughput DNA sequencing targeting the V3-V4 regions of the 16S rRNA gene. Average taxonomic richness and alpha diversity differed significantly between samples collected from lakes and creeks (P< 0.05), and between lakes and stormwater outfalls (P < 0.05). Beta diversity also differed significantly (P < 0.0001) between habitats suggesting that each of these habitats harbours distinct bacterial groups. Among the environmental factors examined, dissolved oxygen (DO) level was strongly associated (P < 0.001) with bacterial diversity. Using a Bayesian source tracking method, the proportional contribution of creeks, river, canals and stormwater outfall habitats in shaping lake bacterial community structure was quantified. Sequences associated with genera known to contain pathogens as well as fecal indicator bacteria were found in every habitat. This study demonstrates that DNA sequence analysis can augment traditional methods of watershed monitoring and management by providing additional information on bacteria of interest to water quality policy makers. Future work may integrate taxonomic and functional analyses to obtain a greater understanding of pathogen survival, nutrient cycling and microbial interactions in freshwater ecosystems. (C) 2018 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Halocarbons R-X are reduced to hydrocarbons R-H by folate model compounds under biomimetic conditions. The reactions correspond to a halide-hydride exchange with the methylenetetrahydrofolate (MTHF) models acting as hydride donors. The MTHF models are also functional equivalents of dehalohydrogenases but, unlike these enzymes, do not require a metal cofactor. The reactions suggest that halocarbons have the potential to act as endocrinological disruptors of biochemical pathways involving MTHF. As a case in point, we observe the rapid reaction of the MTHF models with the inhalation anaesthetic halothane. The ready synthetic accessibility of the MTHF models as well as their dehalogenation activity in the presence of air and moisture allow for the remediation of toxic, halogenated hydrocarbons.

Energy-sensing pathways, normally coordinated by 5′ AMP-activated protein kinase (AMPK), are dysregulated in renal cell carcinoma (RCC). Obesity can accentuate the pre-existing pro-tumorigenic metabolic machinery in RCC cells through its associated obesogenic hormonal milieu, characterized by lower circulating levels of adiponectin. In RCC patients, low adiponectin levels associate clinically with more aggressive disease. We investigated the adiponectin signaling pathway in RCC, focusing on adiponectin receptor 1 (AdipoR1) and associated activation of AMPK. AdipoR1 protein in RCC and normal surrounding renal tissues was determined by Western blot analysis and immunohistochemistry. Anti-tumorigenic effects of adiponectin in RCC cells in vitro were investigated via VEGF and MMP ELISA and invasion assays. Using in vivo models of RCC, the effect of AdipoR1-knockdown (shRNA) on tumor latency, growth and dissemination were determined. AdipoR1 protein was significantly reduced in clear cell RCC specimens. Adiponectin treatment inhibited VEGF, MMP-2 and MMP-9 secretion and activity and invasive and migratory capacities of RCC cells. AMPKα1-knockdown (shRNA) attenuated adiponectin’s effects. In cells stably expressing AdipoR1-specific shRNA, AMPK activation by adiponectin was significantly reduced compared to cells expressing control shRNA. In vivo, AdipoR1 knockdown increased the growth, dissemination and angiogenesis of RCC. These findings suggest that deficiencies in the entire adiponectin hormonal axis (the hormone and its receptor) result in underactivation of AMPK leading to increased angiogenic and invasive capacities of RCC. The established link between obesity and RCC can therefore be further explained by the adiponectin deficiency in obese individuals together with reduced AdipoR1 protein in RCC.

An established inverse clinical correlation between serum adiponectin levels and renal cell carcinoma (RCC) aggressiveness exists. We have recently demonstrated that adiponectin suppresses clear cell RCC (ccRCC) progression through interaction with its receptor, adiponectin receptor 1 (AdipoR1). ERp46 has been shown to inhibit adiponectin signaling via interaction with AdipoR1 in HeLa cells. However, the expression of ERp46 in RCC has not been described thus far. The objectives of this study were to investigate ERp46 in RCC, its expression, its effects on RCC growth in a mouse model and whether it interacts with AdipoR1. We demonstrated a higher ERp46/AdipoR1 expression ratio in metastatic compared to non-metastatic ccRCC, as determined by immunohistochemistry of tissue microarrays and subsequent image analysis. When ERp46 was stably knocked down using shRNA or overexpressed in murine RCC RAG cells, RCC growth after subcutaneous injection in BALB/c nude mice was inhibited and accelerated, respectively. In vitro analysis to determine the molecular interaction between AdipoR1 and ERp46 included co-immunoprecipitation using human ccRCC 786-O cells and a bacterial adenylate cyclase-based two hybrid system and demonstrated no sustained AdipoR1-ERp46 interaction. This is the first report to suggest a role for ERp46 as a potential therapeutic target in RCC given its expression profile in human RCC samples and its effect on in vivo RCC growth. Since a stable interaction with AdipoR1 could not be established, we suggest that the tumorigenic properties of ERp46 in RCC cells are not related to an inhibitory modulation of AdipoR1.

Enterohemorrhagic Escherichia coli O157:H7 is responsible for many outbreaks of gastrointestinal illness and hemolytic uremic syndrome worldwide. Monitoring this pathogen in food and water supplies is an important public health issue. Highly conserved genetic markers, which are characteristic for specific strains, can provide direct identification of target pathogens. In this study, we examined a new detection strategy for pathogenic strains of E. coli O157:H7 serotype based on a conserved signature insertion/deletion (CSI) located in the ybiX gene using TaqMan-probe-based quantitative PCR (qPCR). The qPCR assay was linear from 1.0 × 10(2) to 1.0 × 10(7) genome copies and was specific to O157:H7 when tested against a panel of 15 non-O157:H7 E. coli. The assay also maintained detection sensitivity in the presence of competing E. coli K-12, heterologous nontarget DNA spiked in at a 1000-fold and 800-fold excess of target DNA, respectively, demonstrating the assay's ability to detect E. coli O157:H7 in the presence of high levels of background DNA. This study thus validates the use of strain-specific CSIs as a new class of diagnostic marker for pathogen detection.

You have accessJournal of UrologyProstate Cancer: Basic Research (II)1 Apr 2013315 ERP46 MEDIATES PROSTATE CANCER TUMORIGENESIS IN VITRO BY INHIBITING ADIPONECTIN-INDUCED TUMOR-SUPPRESSIVE EFFECTS- LINKING OBESITY TO PROSTATE CANCER Jehonathan Pinthus, Sarah Hopmans, Athanasios Paschos, Richard Austin, and Wilhelmina Duivenvoorden Jehonathan PinthusJehonathan Pinthus Hamilton, Canada More articles by this author , Sarah HopmansSarah Hopmans Hamilton, Canada More articles by this author , Athanasios PaschosAthanasios Paschos Hamilton, Canada More articles by this author , Richard AustinRichard Austin Hamilton, Canada More articles by this author , and Wilhelmina DuivenvoordenWilhelmina Duivenvoorden Hamilton, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1700AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Several recent studies have documented that lower levels of adiponectin, a hormone secreted from adipocytes only, are associated with an increased risk of prostate cancer. Hypo-adiponectinemia is an endocrine hallmark of obesity. It has been demonstrated that adiponectin-induced tumor suppression is mediated downstream via AMPK. AMPK itself is a key regulator of mTOR. The endoplasmic reticulum (ER) protein ERp46, a member of the thioredoxin family of ER proteins, has been suggested as an inhibitor of this pathway. We examined this hypothesis in vitro using prostate cancer cells. METHODS Human prostate adenocarcinoma 22Rv1 cells are androgen-responsive, produce PSA and express AdipoR1, but little AdipoR2. For co-immunoprecipitation, an antibody against AdipoR1 covalently bound to magnetic Dynabeads was used, while an antibody against ERp46 served for detection. Gain- and loss-of-function experiments were done following stable ERp46 shRNA knockdown and ERp46 overexpression, respectively. RESULTS ERp46 been shown to interact with AdipoR1, leading to a decrease in the phosphorylation of AMPK. By co-immunoprecipitation, we show that ERp46 interacted with AdipoR1 in human 22Rv1 cells. We determined that treatment with adiponectin, at 1 and 20 μg/ml, significantly decreased PSA and VEGF secretion, and increased TIMP-1 secretion (ANOVA) in human prostate cancer 22Rv1 cells. Adiponectin, at 1 and 20 μg/ml for 1 hour also activated AMPK (Thr172 phosphorylation) and led to mTOR inhibition as determined by Western blot analysis. Stable knockdown of ERp46 (9-fold) increased phosphorylation of AMPKá in 22Rv1 cells compared to scrambled control, as determined by Western blot analysis, whereas stable overexpression of ERp46 (4-fold) led to a decrease in phosphorylation of AMPKá. When ERp46-manipulated cells were treated with 2.5 μg/mL human recombinant full-length adiponectin and the activation of AMPK was determined by immunoblotting, results confirmed an inhibitory role of ERp46 on AdipoR1 signaling as demonstrated by a decrease in phosphorylated AMPK. CONCLUSIONS ERp46 is a negative modulator of AdipoR1 activity in prostate cancer. Upon binding to AdipoR1, ERp46 inhibits the activation of AMPK and thus promotes tumorigenesis. Higher levels of adiponectin (inherently lower in obese individuals) may be needed to overcome this inhibition. Further characterization of this novel pathway may lead to better understanding of prostate cancer biology and to the development of novel targeted therapies. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e128 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.Metrics Author Information Jehonathan Pinthus Hamilton, Canada More articles by this author Sarah Hopmans Hamilton, Canada More articles by this author Athanasios Paschos Hamilton, Canada More articles by this author Richard Austin Hamilton, Canada More articles by this author Wilhelmina Duivenvoorden Hamilton, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Secretion systems translocate virulence factors of many bacterial pathogens, enabling their survival inside the host organism. Consequently, inhibition strongly attenuates pathogenicity and can be considered a target for novel antimicrobial drugs. The type IV secretion system (T4SS) of the intracellular pathogen Brucella is a prerequisite for its virulence, and in this work we targeted the interactions of the essential assembly factor protein, VirB8, using small-molecule inhibitors. High-throughput screening identified several potent and specific inhibitors, and the target-binding site of these inhibitors was identified by X-ray crystallography, in silico docking, and analysis of the derivates of the inhibitor B8I-2. VirB8 interaction inhibitors bind to a surface groove opposite to the dimerization interface, and by varying the binding-site residues, we were able to determine which residues are required for inhibitor activity. E115 and K182 were found to be especially important, and changes at R114, Y229, and L151 also reduced inhibitor efficiency.

Type IV secretion systems are macromolecular assemblies in the cell envelopes of bacteria that function in macromolecular translocation. Structural biology approaches have provided insights into the interaction of core complex components, but information about proteins that undergo transient interactions with membrane components has not been forthcoming. We have pursued an unbiased approach using peptide arrays and phage display to identify interaction partners and interaction domains of type IV secretion system assembly factor VirB8. These approaches identified the globular domain from the VirB5 protein to interact with VirB8. This interaction was confirmed in cross-linking, pull-down, and fluorescence resonance energy transfer (FRET)-based interaction assays. In addition, using phage display analysis, we identified different regions of VirB6 as potential interaction partners of VirB8. Using a FRET-based interaction assay, we provide the first direct experimental evidence of the interaction of a VirB6 periplasmic domain with VirB8. These results will allow us to conduct directed structural biological work and structure-function analyses aimed at defining the molecular details and biological significance of these interactions with VirB8 in the future.

ABSTRACT As bacterial pathogens develop resistance against most currently used antibiotics, novel alternatives for treatment of microbial infectious diseases are urgently needed. Targeting bacterial virulence functions in order to disarm pathogens represents a promising alternative to classical antibiotic therapy. Type IV secretion systems, which are multiprotein complexes in the cell envelope that translocate effectors into host cells, are critical bacterial virulence factors in many pathogens and excellent targets for such “antivirulence” drugs. The VirB8 protein from the mammalian pathogen Brucella was chosen as a specific target, since it is an essential type IV secretion system component, it participates in multiple protein-protein interactions, and it is essential for the assembly of this translocation machinery. The bacterial two-hybrid system was adapted to assay VirB8 interactions, and a high-throughput screen identified specific small-molecule inhibitors. VirB8 interaction inhibitors also reduced the levels of VirB8 and of other VirB proteins, and many of them inhibited virB gene transcription in Brucella abortus 2308, suggesting that targeting of the secretion system has complex regulatory effects in vivo. One compound strongly inhibited the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model. The results presented here show that in vivo screens with the bacterial two-hybrid assay are suited to the identification of inhibitors of Brucella type IV secretion system function.

VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein-protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure-function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein-protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein-protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens. Changes at Y105, T201, R230, and at several other residues impaired the in vivo function of VirB8, suggesting that we have identified interaction sites of relevance in the natural biological context.