Burkholderia Type VI Secretion Systems Have Distinct Roles in Eukaryotic and Bacterial Cell Interactions

Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans—leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections

Safety and immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in children in Sierra Leone: a randomised, double-blind, controlled trial

Background—Children account for a substantial proportion of cases and deaths from Ebola virus disease. We aimed to assess the safety and immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vectorbased vaccine, encoding glycoproteins from the Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in a paediatric population in Sierra Leone. Methods—This randomised, double-blind, controlled trial was done at three clinics in Kambia district, Sierra Leone. Healthy children and adolescents aged 1–17 years were enrolled in three age cohorts (12–17 years, 4–11 years, and 1–3 years) and randomly assigned (3:1), via computer-generated block randomisation (block size of eight), to receive an intramuscular injection of either Ad26.ZEBOV (5 × 1010 viral particles; first dose) followed by MVA-BN-Filo (1 × 108 infectious units; second dose) on day 57 (Ebola vaccine group), or a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo (second dose) on day 57 (control group). Study team personnel (except for those with primary responsibility for study vaccine preparation), participants, and their parents or guardians were masked to study vaccine allocation. The primary outcome was safety, measured as the occurrence of solicited local and systemic adverse symptoms during 7 days after each vaccination, unsolicited systemic adverse events during 28 days after each vaccination, abnormal laboratory results during the study period, and serious adverse events or immediate reportable events throughout the study period. The secondary outcome was immunogenicity (humoral immune response), measured as the concentration of Ebola virus glycoprotein-specific binding antibodies at 21 days after the second dose. The primary outcome was assessed in all participants who had received at least one dose of study vaccine and had available reactogenicity data, and immunogenicity was assessed in all participants who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response. This study is registered at ClinicalTrials.gov, NCT02509494. Findings—From April 4, 2017, to July 5, 2018, 576 eligible children or adolescents (192 in each of the three age cohorts) were enrolled and randomly assigned. The most common solicited local adverse event during the 7 days after the first and second dose was injection-site pain in all age groups, with frequencies ranging from 0% (none of 48) of children aged 1–3 years after placebo injection to 21% (30 of 144) of children aged 4–11 years after Ad26.ZEBOV vaccination. The most frequently observed solicited systemic adverse event during the 7 days was headache in the 12–17 years and 4–11 years age cohorts after the first and second dose, and pyrexia in the 1–3 years age cohort after the first and second dose. The most frequent unsolicited adverse event after the first and second dose vaccinations was malaria in all age cohorts, irrespective of the vaccine types. Following vaccination with MenACWY, severe thrombocytopaenia was observed in one participant aged 3 years. No other clinically significant laboratory abnormalities were observed in other study participants, and no serious adverse events related to the Ebola vaccine regimen were reported. There were no treatment-related deaths. Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second dose of the Ebola virus vaccine regimen were observed in 131 (98%) of 134 children aged 12–17 years (9929 ELISA units [EU]/mL [95% CI 8172–12 064]), in 119 (99%) of 120 aged 4–11 years (10 212 EU/mL [8419–12 388]), and in 118 (98%) of 121 aged 1–3 years (22 568 EU/mL [18 426–27 642]). Interpretation—The Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen was well tolerated with no safety concerns in children aged 1–17 years, and induced robust humoral immune responses, suggesting suitability of this regimen for Ebola virus disease prophylaxis in children

Safety and long-term immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Sierra Leone: a combined open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2 trial

Background The Ebola epidemics in west Africa and the Democratic Republic of the Congo highlight an urgent need for safe and effective vaccines to prevent Ebola virus disease. We aimed to assess the safety and long-term immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vector-based vaccine, encoding glycoproteins from Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in Sierra Leone, a country previously affected by Ebola. Methods The trial comprised two stages: an open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2. The study was done at three clinics in Kambia district, Sierra Leone. In stage 1, healthy adults (aged ≥18 years) residing in or near Kambia district, received an intramuscular injection of Ad26.ZEBOV (5×1010 viral particles) on day 1 (first dose) followed by an intramuscular injection of MVA-BN-Filo (1×108 infectious units) on day 57 (second dose). An Ad26.ZEBOV booster vaccination was offered at 2 years after the first dose to stage 1 participants. The eligibility criteria for adult participants in stage 2 were consistent with stage 1 eligibility criteria. Stage 2 participants were randomly assigned (3:1), by computer-generated block randomisation (block size of eight) via an interactive web-response system, to receive either the Ebola vaccine regimen (Ad26.ZEBOV followed by MVA-BN-Filo) or an intramuscular injection of a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo on day 57 (second dose; control group). Study team personnel, except those with primary responsibility for study vaccine preparation, and participants were masked to study vaccine allocation. The primary outcome was the safety of the Ad26.ZEBOV and MVA-BN-Filo vaccine regimen, which was assessed in all participants who had received at least one dose of study vaccine. Safety was assessed as solicited local and systemic adverse events occurring in the first 7 days after each vaccination, unsolicited adverse events occurring in the first 28 days after each vaccination, and serious adverse events or immediate reportable events occurring up to each participant’s last study visit. Secondary outcomes were to assess Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second vaccine in a per-protocol set of participants (ie, those who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response) and to assess the safety and tolerability of the Ad26.ZEBOV booster vaccination in stage 1 participants who had received the booster dose. This study is registered at ClinicalTrials.gov, NCT02509494. Findings Between Sept 30, 2015, and Oct 19, 2016, 443 participants (43 in stage 1 and 400 in stage 2) were enrolled; 341 participants assigned to receive the Ad26.ZEBOV and MVA-BN-Filo regimen and 102 participants assigned to receive the MenACWY and placebo regimen received at least one dose of study vaccine. Both regimens were well tolerated with no safety concerns. In stage 1, solicited local adverse events (mostly mild or moderate injection-site pain) were reported in 12 (28%) of 43 participants after Ad26.ZEBOV vaccination and in six (14%) participants after MVA-BN-Filo vaccination. In stage 2, solicited local adverse events were reported in 51 (17%) of 298 participants after Ad26.ZEBOV vaccination, in 58 (24%) of 246 after MVA-BN-Filo vaccination, in 17 (17%) of 102 after MenACWY vaccination, and in eight (9%) of 86 after placebo injection. In stage 1, solicited systemic adverse events were reported in 18 (42%) of 43 participants after Ad26.ZEBOV vaccination and in 17 (40%) after MVA-BN-Filo vaccination. In stage 2, solicited systemic adverse events were reported in 161 (54%) of 298 participants after Ad26.ZEBOV vaccination, in 107 (43%) of 246 after MVA-BN-Filo vaccination, in 51 (50%) of 102 after MenACWY vaccination, and in 39 (45%) of 86 after placebo injection. Solicited systemic adverse events in both stage 1 and 2 participants included mostly mild or moderate headache, myalgia, fatigue, and arthralgia. The most frequent unsolicited adverse event after the first dose was headache in stage 1 and malaria in stage 2. Malaria was the most frequent unsolicited adverse event after the second dose in both stage 1 and 2. No serious adverse event was considered related to the study vaccine, and no immediate reportable events were observed. In stage 1, the safety profile after the booster vaccination was not notably different to that observed after the first dose. Vaccine-induced humoral immune responses were observed in 41 (98%) of 42 stage 1 participants (geometric mean binding antibody concentration 4784 ELISA units [EU]/mL [95% CI 3736–6125]) and in 176 (98%) of 179 stage 2 participants (3810 EU/mL [3312–4383]) at 21 days after the second vaccination. Interpretation The Ad26.ZEBOV and MVA-BN-Filo vaccine regimen was well tolerated and immunogenic, with persistent humoral immune responses. These data support the use of this vaccine regimen for Ebola virus disease prophylaxis in adults

Forced resurgence and targeting of intracellular uropathogenic Escherichia coli reservoirs.

Intracellular quiescent reservoirs of uropathogenic Escherichia coli (UPEC), which can seed the bladder mucosa during the acute phase of a urinary tract infection (UTI), are protected from antibiotic treatments and are extremely difficult to eliminate. These reservoirs are a potential source for recurrent UTIs that affect millions annually. Here, using murine infection models and the bladder cell exfoliant chitosan, we demonstrate that intracellular UPEC populations shift within the stratified layers of the urothelium during the course of a UTI. Following invasion of the terminally differentiated superficial layer of epithelial cells that line the bladder lumen, UPEC can multiply and disseminate, eventually establishing reservoirs within underlying immature host cells. If given access, UPEC can invade the superficial and immature bladder cells equally well. As infected immature host cells differentiate and migrate towards the apical surface of the bladder, UPEC can reinitiate growth and discharge into the bladder lumen. By inducing the exfoliation of the superficial layers of the urothelium, chitosan stimulates rapid regenerative processes and the reactivation and efflux of quiescent intracellular UPEC reservoirs. When combined with antibiotics, chitosan treatment significantly reduces bacterial loads within the bladder and may therefore be of therapeutic value to individuals with chronic, recurrent UTIs

Chitosan-induced exfoliation of superficial BECs and analysis of UPEC reservoir populations.

<p>(<b>A</b> and <b>B</b>) Fluorescent images show the lumenal surfaces of mouse bladders fixed and stained with Hoechst dye following 20-min treatments with (<b>A</b>) phosphate buffer (pH 4.5) alone or with (<b>B</b>) 0.01% chitosan. Representative images of three independent experiments performed in duplicate. (<b>C</b>) The bladders of adult female CBA/J mice were inoculated with UTI89 via transurethral catheterization 4 h after a 20-min treatment of the mouse bladders with chitosan or phosphate buffer alone. Internalized bacteria were enumerated 1 h after initiation of the infection using <i>ex vivo</i> gentamicin protection assays. (<b>D</b> and <b>E</b>) Graphs show bacterial titers present in the bladders of mice treated with phosphate buffer or chitosan just before collection at (<b>D</b>) 3 d or (<b>E</b>) 14 d post-inoculation with UTI89. Bars indicate median values for each group; n = 11 mice. <i>P</i> values indicated were determined by the Mann-Whitney U-test.</p

UPEC persistence and recurrence within the bladder.

<p>(<b>1</b>) UPEC that gain entry into the bladder lumen can multiply within the urine and in association with the bladder surface. (<b>2</b>) Some bacteria enter host superficial cells and are trafficked into late endosome-like compartments. (<b>3</b>) Shearing forces from the flow of urine, secreted antimicrobial factors, and infiltrating neutrophils can eliminate many bacteria, while internalized UPEC are sheltered. Some intracellular bacteria may enter the host cytosol where they can rapidly multiply, forming IBCs. (<b>4</b>) Infection can induce the exfoliation of the superficial cells, exposing underlying immature BECs (<b>5</b>) that can rapidly differentiate to reestablish barrier function. Chitosan treatment can also stimulate exfoliation of superficial bladder cells and subsequent regenerative processes. Within both the superficial and immature cells of the bladder, UPEC can persist as quiescent reservoirs within membrane-bound compartments enmeshed in F-actin. Antibiotics that can effectively sterilize the urine are ineffective against the intracellular UPEC reservoirs. (<b>6</b>) Redistribution of actin and perhaps other signals associated with terminal differentiation of the BECs may elicit the resurgent growth of UPEC, resulting in bacterial release back into the bladder lumen. By promoting turnover of the urothelium, the intravesical delivery of chitosan can stimulate the resurgence of UPEC from intracellular reservoirs, making the bacteria more accessible to antibiotics.</p

Chitosan treatment enhances the efficacy of antibiotics in reducing bacterial titers within the bladder.

<p>Mice were infected with UTI89 via transurethral catheterization 3% chitosan. After a 20-min incubation and subsequent washes with PBS, mice were treated for 3 or 7 d with gentamicin (GENT), sparfloxacin (SPX), or ciprofloxacin (CIP), as indicated. Mice were sacrificed 3 d after cessation of antibiotics or mock treatments. An outline of the experimental setup is shown on top. Bars in the graph indicate median values for each sample group; n = 11 mice total from two independent assays. *<i>P</i><0.02, **<i>P</i><0.005, ***<i>P</i>≤0.0002; as determined by Mann-Whitney U-tests.</p

Chitosan treatment promotes the resurgence of UPEC reservoir populations into the bladder lumen.

<p>Adult female CBA/J mice were inoculated with UTI89 via transurethral catheterization or left uninfected. After 3 d, phosphate buffer alone (control) or 0.01% chitosan was instilled into the bladders for 20 min prior to rinses with PBS. Bladders were bisected, splayed and imaged 1 or 7 d later, as indicated, using (<b>A</b>) confocal fluorescence microscopy or (<b>B</b>) SEM. Images in (<b>B</b>) show F-actin (purple), host nuclei (blue), and bacteria (yellow). Scale bars, (<b>A</b>) 50 μm and (<b>B</b>) 10 μm. Images are representative of three independent experiments.</p

Chitosan affects UPEC growth, biofilm formation, and interactions with BECs.

<p>(<b>A</b>) Growth of UTI89 in modified M9 medium containing increasing concentrations of chitosan (0.00002, 0.0002, 0.002%) or phosphate buffer (pH 4.5). Data shown are representative of three independent experiments carried out in triplicate. (<b>B</b>) Graph shows chitosan effects on biofilm production by UTI89 relative to untreated controls, as measured by microtiter plate assays. Data represent mean results ± SEM from three separate experiments performed in quadruplicate. Inset shows an example of filamentous bacteria in 0.002% chitosan; scale bar, 10 μm. Chitosan (0.0002 or 0.002%) effects on (<b>C</b>) UTI89 growth in RPMI medium during a 2-h incubation with 5637 BECs, (<b>D</b>) bacterial attachment to BECs, (<b>E</b>) bacterial invasion of BECs, and (<b>F</b>) bacterial survival within BECs over a 14-h period were quantified relative to buffer-treated controls. The data in <b>F</b> were normalized by dividing the numbers of surviving intracellular bacteria recovered from BECs after the 14-h incubation with gentamicin by the numbers of invading bacteria present after the 2-h incubation with gentamicin. Graphs show mean results ± SEM from three independent experiments performed in triplicate. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus buffer-treated controls, as determined by Student’s <i>t</i> test with Welch’s correction when appropriate.</p