Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella.

Nontyphoidal Salmonellae cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM197 glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Salmonella Typhimurium and Enteritidis vaccines. Salmonella strains were chosen and tolR deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM197 Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce Salmonella infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with SalmonellaS Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. S. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal Salmonella vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.

Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence.

Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3'-5' exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.

Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis.

Invasive nontyphoidal Salmonella (iNTS) disease is a neglected disease with high mortality in children and HIV-positive individuals in sub-Saharan Africa, caused primarily by Africa-specific strains of Salmonella enterica serovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) fromS.Typhimurium andS.Enteritidis containing lipid A modifications to reduce potential in vivo reactogenicity is under development. GMMA with penta-acylated lipid A showed the greatest reduction in the level of cytokine release from human peripheral blood monocytes from that for GMMA with wild-type lipid A. Deletion of the lipid A modification genes msbB and pagP was required to achieve pure penta-acylation. Interestingly, ΔmsbBΔ pagP GMMA from S. Enteritidis had a slightly higher stimulatory potential than those from S. Typhimurium, a finding consistent with the higher lipopolysaccharide (LPS) content and Toll-like receptor 2 (TLR2) stimulatory potential of the former. Also, TLR5 ligand flagellin was found in Salmonella GMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from S. Typhimurium was added at a concentration as high as 10% of total protein, suggesting that flagellin impurities are not a major factor for GMMA-mediated immune stimulation. Overall, the stimulatory potential of S. Typhimurium and S. Enteritidis ΔmsbB ΔpagP GMMA was close to that of Shigella sonnei GMMA, which are currently in phase I clinical trials.

Small-molecular virulence inhibitors show divergent and immunomodulatory effects in infection models of Salmonella enterica serovar Typhimurium.

The virulence-associated Salmonella pathogenicity island 2 (SPI2) type III secretion system supports intracellular replication of Salmonella enterica serovar Typhimurium in macrophage-like RAW264.7 cells. In contrast, the salicylidene acylhydrazide INP0010 and the benzimidazole omeprazole prevent virulence factor-mediated replication of S. Typhimurium in these cells. Here we show that INP0010 enhances expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, the oxidative burst and tumour necrosis factor-alpha (TNFα) release in infected RAW264.7 cells. INP0010 also inhibited SPI2 activity in RAW264.7 cells. The ability of INP0010 to suppress bacterial intracellular replication correlated with NO production. The iNOS inhibitor N-monomethyl-l-arginine restored SPI2 activity and antagonised the bacteriostatic effect of INP0010. Omeprazole, which inhibited iNOS expression in RAW264.7 cells, likewise antagonised INP0010. In infected epithelioid MDCK cells that did not express NO upon infection, INP0010 enhanced bacterial intracellular replication. In Caenorhabditis elegans, INP0010 significantly attenuated the virulence of S. Typhimurium. In this infection model, the attenuating effect of INP0010 was further enhanced by omeprazole. These results demonstrate that chemically unrelated virulence inhibitors may act in an antagonistic or additive manner, that their effect depends on the infection model applied, and that the attenuating effects of INP0010 in part relate to its ability to promote the SPI2 antagonist NO.

Thioredoxin 1 participates in the activity of the Salmonella enterica serovar Typhimurium pathogenicity island 2 type III secretion system.

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.

Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium.

Colistin is a cyclic cationic peptide that kills gram-negative bacteria by interacting with and disrupting the outer membrane. We isolated 44 independent mutants in Salmonella enterica serovar Typhimurium with reduced susceptibility to colistin and identified 27 different missense mutations located in the pmrA and pmrB genes (encoding the regulator and sensor of a two-component regulatory system) that conferred increased resistance. By comparison of the two homologous sensor kinases, PmrB and EnvZ, the 22 missense mutations identified in pmrB were shown to be located in four different structural domains of the protein. All five pmrA mutations were located in the phosphate receiver domain of the regulator protein. The mutants appeared at a mutation rate of 0.6 x 10(-6) per cell per generation. The MICs of colistin for the mutants increased 2- to 35-fold, and the extent of killing was reduced several orders of magnitude compared to the susceptible strain. The growth rates of the mutants were slightly reduced in both rich medium and M9-glycerol minimal medium, whereas growth in mice appeared unaffected by the pmrA and pmrB mutations. The low fitness costs and the high mutation rate suggest that mutants with reduced susceptibility to colistin could emerge in clinical settings.

Omeprazole antagonizes virulence and inflammation in Salmonella enterica-infected RAW264.7 cells.

The proton pump inhibitor omeprazole reduced the intracellular replication of Salmonella enterica serovar Typhimurium in RAW264.7 cells without affecting bacterial growth in vitro or the viability of the host cells. The mechanism was bacteriostatic and interfered with replication mediated by the virulence-associated SPI2 type III secretion system. The proton pump inhibitor bafilomycin A(1), in contrast, mediated killing of intracellular bacteria and imposed a marked cytotoxicity on RAW264.7 cells. The two compounds also differentially affected the proinflammatory responses of the infected cells. Bafilomycin A(1) enhanced nitric oxide production, whereas omeprazole delayed IkappaB degradation and blocked nitric oxide production and the secretion of proinflammatory cytokines. These results imply that omeprazole can be used to block the virulence factor-mediated intracellular replication of S. Typhimurium, and that its mechanism of growth inhibition is different from that mediated by bafilomycin A(1).

Mechanism and fitness costs of PR-39 resistance in Salmonella enterica serovar Typhimurium LT2.

PR-39 is a porcine antimicrobial peptide that kills bacteria with a mechanism that does not involve cell lysis. Here, we demonstrate that Salmonella enterica serovar Typhimurium can rapidly acquire mutations that reduce susceptibility to PR-39. Resistant mutants appeared at a rate of 0.4 x 10(-6) per cell per generation. These mutants were about four times more resistant than the wild type and showed a greatly reduced rate of killing. Genetic analysis revealed mutations in the putative transport protein SbmA as being responsible for the observed resistance. These sbmA mutants were as fit as the wild-type parental strain as measured by growth rates in culture medium and mice and by long-term survival in stationary phase. These results suggest that resistance to certain antimicrobial peptides can rapidly develop without an obvious fitness cost for the bacteria and that resistance development could become a threat to the efficacy of antimicrobial peptides if used in a clinical setting.

Salicylidene acylhydrazides that affect type III protein secretion in Salmonella enterica serovar typhimurium.

A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector beta-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence.