Suitable ratio of dietary L-carnitine and α-ketoglutarate improves growth and health performance in Nile tilapia, Oreochromis niloticus.

L-carnitine (LC) and α-Ketoglutarate (AKG) are important growth promoters used in aquafeed. The study aimed to evaluate the incorporation of LC and AKG at different ratios in the diet of tilapia (initial weight 1.38 ± 0.03 g) in order to facilitate lipid utilization and protein synthesis. Fish were fed six isonitrogenous (~ 30 g/100 g CP) and isolipidic (~ 6 g/100 g CL) diets containing graded LC/AKG ratios of 0 (Control), 0.11, 0.42, 1.00, 2.33 and 9.00 in six treatments for 60 days. Fish fed with LC/AKG ratios 2.33 and 9.00 showed significantly (P < 0.05) higher percentage weight gain, specific growth rate and protein efficiency ratio. Feed conversion ratio in fish-fed diets with LC/AKG ratio 9.00 improved significantly (p < 0.05) than other treatments. The whole-body protein content of tilapia and digestive enzyme activity were significantly higher with higher weight gain. The body lipid content was significantly lower in the LC/AKG ratio 9.00. The liver antioxidant parameters and activity of the immune components were significantly higher in the LC/AKG ratio 9 group. The lower serum triglyceride and cholesterol level was also recorded in LC/AKG ratio 9 group. The histology of the intestine and liver showed increased villi area and decreased lipid droplets, respectively, in tilapia fed with higher LC/AKG ratios. It was concluded from the above results that the higher LC and lower AKG (LC/AKG ratio 9.00) combination attributed maximum lipid utilization and higher protein efficiency and thus better growth performance in tilapia. This was also reflected in activity of digestive enzymes, antioxidant enzymes and immune status in tilapia.

Highlights of the 12th International Bordetella Symposium.

To commemorate the 100th anniversary of the Nobel prize being awarded to Jules Bordet, the discoverer of Bordetella pertussis, the 12th International Bordetella Symposium was held from 9 to 12 April 2019 at the Université Libre de Bruxelles, where Jules Bordet studied and was Professor of Microbiology. The symposium attracted more than 300 Bordetella experts from 34 countries. They discussed the latest epidemiologic data and clinical aspects of pertussis, Bordetella biology and pathogenesis, immunology and vaccine development, and genomics and evolution. Advanced technological and methodological tools provided novel insights into the genomic diversity of Bordetella and a better understanding of pertussis disease and vaccine performance. New molecular approaches revealed previously unrecognized complexity of virulence gene regulation. Innovative insights into the immune responses to infection by Bordetella resulted in the development of new vaccine candidates. Such discoveries will aid in the design of more effective approaches to control pertussis and other Bordetella-related diseases.

Bordetella pertussis Lipid A Recognition by Toll-like Receptor 4 and MD-2 Is Dependent on Distinct Charged and Uncharged Interfaces.

Lipid A in LPS activates innate immunity through the Toll-like receptor 4 (TLR4)-MD-2 complex on host cells. Variation in lipid A has significant consequences for TLR4 activation and thus may be a means by which Gram-negative bacteria modulate host immunity. However, although even minor changes in lipid A structure have been shown to affect downstream immune responses, the mechanism by which the TLR4-MD-2 receptor complex recognizes these changes is not well understood. We previously showed that strain BP338 of the human pathogen Bordetella pertussis, the causative agent of whooping cough, modifies its lipid A by the addition of glucosamine moieties that promote TLR4 activation in human, but not mouse, macrophages. Using site-directed mutagenesis and an NFκB reporter assay screen, we have identified several charged amino acid residues in TLR4 and MD-2 that are important for these species-specific responses; some of these are novel for responses to penta-acyl B. pertussis LPS, and their mutation does not affect the response to hexa-acylated Escherichia coli LPS or tetra-acylated lipid IVA. We additionally show evidence that suggests that recognition of penta-acylated B. pertussis lipid A is dependent on uncharged amino acids in TLR4 and MD-2 and that this is true for both human and mouse TLR4-MD-2 receptors. Taken together, we have demonstrated that the TLR4-MD-2 receptor complex recognizes variation in lipid A molecules using multiple sites for receptor-ligand interaction and propose that host-specific immunity to a particular Gram-negative bacterium is, at least in part, mediated by very subtle tuning of one of the earliest interactions at the host-pathogen interface.

Minor modifications to the phosphate groups and the C3' acyl chain length of lipid A in two Bordetella pertussis strains, BP338 and 18-323, independently affect Toll-like receptor 4 protein activation.

Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4'-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3' acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399-BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3' acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.

Bordetella pertussis lipid A glucosamine modification confers resistance to cationic antimicrobial peptides and increases resistance to outer membrane perturbation.

Bordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

Anti-inflammatory effects of cannabidiol against lipopolysaccharides in cardiac sodium channels.

BACKGROUND: Sepsis, caused by a dysregulated response to infections, can lead to cardiac arrhythmias. However, the mechanisms underlying sepsis-induced inflammation, and how inflammation provokes cardiac arrhythmias, are not well understood. We hypothesized that cannabidiol (CBD) may ameliorate lipopolysaccharide (LPS)-induced cardiotoxicity, via Toll-like receptors (TLR4) and cardiac sodium channels (NaV 1.5). METHODS AND RESULTS: We incubated human immune cells (THP-1 macrophages) with LPS for 24 h, then extracted the THP-1 incubation media. ELISA assays showed that LPS (1 or 5 µg·ml-1 ), in a concentration-dependent manner, or MPLA (TLR4 agonist, 5 µg·ml-1 ) stimulated the THP-1 cells to release inflammatory cytokines (TNF-α and IL-6). Prior incubation (4 h) with CBD (5 µM) or C34 (TLR4 antagonist: 5 µg·ml-1 ) inhibited LPS and MPLA-induced release of both IL-6 and TNF-α. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were subsequently incubated for 24 h in the media extracted from THP-1 cells incubated with LPS, MPLA alone, or in combination with CBD or C34. Voltage-clamp experiments showed a right shift in the voltage dependence of NaV 1.5 activation, steady state fast inactivation (SSFI), increased persistent current and prolonged in silico action potential duration in hiSPC-CMs incubated in the LPS or MPLA-THP-1 media. Co-incubation with CBD or C34 rescued the biophysical dysfunction caused by LPS and MPLA. CONCLUSION: Our results suggest that CBD may protect against sepsis-induced inflammation and subsequent arrhythmias through (i) inhibition of the release of inflammatory cytokines, antioxidant and anti-apoptotic effects and/or (ii) a direct effect on NaV 1.5.

Variability in the lipooligosaccharide structure and endotoxicity among Bordetella pertussis strains.

Bordetella endotoxins show remarkable structural variability both among each other and in comparison to other gram-negative bacteria. Here we demonstrate that, in contrast to the common Bordetella pertussis laboratory strain and Tohama I derivative BP338, lipooligosaccharide from mouse challenge strain 18-323 is a poor inducer of inflammatory cytokines in human and murine macrophages, is greatly impaired in Toll-like receptor 4-mediated activation of nuclear factor-κB in transfected HEK-293 cells, and functions as a Toll-like receptor 4 antagonist. Comparison of lipid A and lipooligosaccharide structures of B. pertussis strains BP338 and 18-323 revealed that 18-323 (1) lacks the ability to modify its lipid A phosphate groups with glucosamine, (2) is distinct in its acylation at the C3' position of the lipid A diglucosamine backbone, and (3) expresses molecular lipooligosaccharide species that lack a terminal heptose. Our findings have important implications for interpreting previous studies of host defenses to B. pertussis infection in mice and in vitro.

RNase III and RNase E Influence Posttranscriptional Regulatory Networks Involved in Virulence Factor Production, Metabolism, and Regulatory RNA Processing in Bordetella pertussis.

Bordetella pertussis has been shown to encode regulatory RNAs, yet the posttranscriptional regulatory circuits on which they act remain to be fully elucidated. We generated mutants lacking the endonucleases RNase III and RNase E and assessed their individual impact on the B. pertussis transcriptome. Transcriptome sequencing (RNA-Seq) analysis showed differential expression of ∼25% of the B. pertussis transcriptome in each mutant, with only 28% overlap between data sets. Both endonucleases exhibited substantial impact on genes involved in amino acid uptake (e.g., ABC transporters) and in virulence (e.g., the type III secretion system and the autotransporters vag8, tcfA, and brkA). Interestingly, mutations in RNase III and RNase E drove the stability of many transcripts, including those involved in virulence, in opposite directions, a result that was validated by qPCR and immunoblotting for tcfA and brkA. Of note, whereas similar mutations to RNase E in Escherichia coli have subtle effects on transcript stability, a striking >20-fold reduction in four gene transcripts, including tcfA and vag8, was observed in B. pertussis. We further compared our data set to the regulon controlled by the RNA chaperone Hfq to identify B. pertussis loci influenced by regulatory RNAs. This analysis identified ∼120 genes and 19 operons potentially regulated at the posttranscriptional level. Thus, our findings revealed how changes in RNase III- and RNase E-mediated RNA turnover influence pathways associated with virulence and cellular homeostasis. Moreover, we highlighted loci potentially influenced by regulatory RNAs, providing insights into the posttranscriptional regulatory networks involved in fine-tuning B. pertussis gene expression. IMPORTANCE Noncoding, regulatory RNAs in bacterial pathogens are critical components required for rapid changes in gene expression profiles. However, little is known about the role of regulatory RNAs in the growth and pathogenesis of Bordetella pertussis. To address this, mutants separately lacking ribonucleases central to regulatory RNA processing, RNase III and RNase E, were analyzed by RNA-Seq. Here, we detail the first transcriptomic analysis of the impact of altered RNA degradation in B. pertussis. Each mutant showed approximately 1,000 differentially expressed genes, with significant changes in the expression of pathways associated with metabolism, bacterial secretion, and virulence factor production. Our analysis suggests an important role for these ribonucleases during host colonization and provides insights into the breadth of posttranscriptional regulation in B. pertussis, further informing our understanding of B. pertussis pathogenesis.

Substitution of the Bordetella pertussis lipid A phosphate groups with glucosamine is required for robust NF-kappaB activation and release of proinflammatory cytokines in cells expressing human but not murine Toll-like receptor 4-MD-2-CD14.

Bordetella pertussis endotoxin is a key modulator of the host immune response, mainly due to the role of its lipid A moiety in Toll-like receptor 4 (TLR4)-mediated signaling. We have previously demonstrated that the lipid A phosphate groups of B. pertussis BP338 can be substituted with glucosamine in a BvgAS-regulated manner. Here we examined the effect of this lipid A modification on the biological activity of B. pertussis endotoxin. We compared purified endotoxin and heat-killed B. pertussis BP338 whole cells that have modified lipid A phosphate groups to an isogenic mutant lacking this modification with respect to their capacities to induce the release of inflammatory cytokines by human and murine macrophages and to participate in the TLR4-mediated activation of NF-kappaB in transfected HEK-293 cells. We found inactivated B. pertussis cells to be stronger inducers of proinflammatory cytokines in THP-1-derived macrophages when lipid A was modified. Most notably, lack of lipid A modification abolished the ability of purified B. pertussis endotoxin to induce the release of inflammatory cytokines by human THP-1-derived macrophages but led to only slightly reduced inflammatory cytokine levels when stimulating murine (RAW 264.7) macrophages. Accordingly, upon stimulation of HEK-293 cells with inactivated bacteria and purified endotoxin, lack of lipid A modification led to impaired NF-kappaB activation only when human, and not when murine, TLR4-MD-2-CD14 was expressed. We speculate that in B. pertussis, lipid A modification has evolved to benefit the bacteria during human infection by modulating immune defenses rather than to evade innate immune recognition.

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins.

Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chloroflexus aurantiacus. Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides, nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA, and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the QA pocket of this heterologously expressed reaction center.

Crystallographic characterization of the passenger domain of the Bordetella autotransporter BrkA.

Autotransporters (ATs) are proteins that deliver effectors (the passenger domain) to the surface of Gram-negative bacteria by the type V secretion pathway. The passenger domain of BrkA, a Bordetella pertussis autotransporter mediating serum resistance and adherence, was cloned in a pET expression system and overexpressed in Escherichia coli. The gene product was correctly refolded, purified to homogeneity and crystallized. The crystals diffracted to 2.8 A resolution. The space group was assumed to be P4(1)2(1)2, with unit-cell parameters a = b = 108.19, c = 115.35 A.

Bacteriophage-mediated identification of bacteria using photoacoustic flow cytometry.

Infection with resistant bacteria has become an ever increasing problem in modern medical practice. Currently, broad spectrum antibiotics are prescribed until bacteria can be identified through blood cultures, a process that can take two to three days and is unable to provide quantitative information. To detect and quantify bacteria rapidly in blood samples, we designed a method using labeled bacteriophage in conjunction with photoacoustic flow cytometry (PAFC). PAFC is the generation of ultrasonic waves created by the absorption of laser light in particles under flow. Bacteriophage is a virus that infects bacteria and possesses the ability to discriminate bacterial surface antigens, allowing the bacteriophage to bind only to their target bacteria. Bacteria can be tagged with dyed phage and processed through a photoacoustic flow cytometer where they are detected by the acoustic response. We demonstrate that E. coli; can be detected and discriminated from Salmonella; using this method. Our goal is to develop a method to determine bacterial content in blood samples. We hope to develop this technology into future clinical use and decrease the time required to identify bacterial species from 3 to 4 days to less than 1 hour.

Type V protein secretion pathway: the autotransporter story.

Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function.

Glucosamine found as a substituent of both phosphate groups in Bordetella lipid A backbones: role of a BvgAS-activated ArnT ortholog.

Endotoxins are amphipathic lipopolysaccharides (LPSs), major constituents of the outer membrane of gram-negative bacteria. They consist of a lipid region, covalently linked to a core oligosaccharide, to which may be linked a repetitive glycosidic chain carrying antigenic determinants. Most of the biological activities of endotoxins have been associated with the lipid moiety of the molecule: unique to gram-negative bacteria, LPS is a ligand of the mammalian TLR4-MD2-CD14 pathogen recognition receptor complex. Lipid A preparations are often heterogeneous with respect to both the numbers and the lengths of fatty acids and the natures of substituents on the phosphate groups when present. The variants can significantly affect host immune responses. Nine species in the Bordetella genus have been described, and the fine LPS structures of seven of them have been published. In this report, lipids A from Bordetella pertussis Tohama I and B. bronchiseptica strain 4650 were further characterized and revealed to have a glucosamine substituting both lipid A phosphate groups of the diglucosamine backbone. These substitutions have not been previously described for bordetellae. Moreover, a B. pertussis transposon mutation that maps within a gene encoding a Bordetella ArnT (formerly PmrK) glycosyl transferase ortholog does not carry this substitution, thus providing a genetic basis for the modification. Reverse transcriptase PCR of this locus showed that it is Bvg regulated, suggesting that the ability of Bordetella to modify lipid A via this glucosamine modification is a potential virulence trait.

Protein secretion through autotransporter and two-partner pathways.

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.

Two-dimensional display and whole genome comparison of bacterial pathogen genomes of high G+C DNA content.

High-resolution comparison of bacterial genomes facilitates the identification of the genetic changes responsible for clinically relevant phenotypes. For this purpose we have established a method for the display and comparison of high G+C bacterial genomes in two dimensions. Here we describe the application of two-dimensional bacterial genomic display to resolve the genomes of Bordetella pertussis, Mycobacterium avium and Mycobacterium tuberculosis, and its utility in strain comparison and detection of insertion and substitution mutations.

Two-dimensional DNA displays for comparisons of bacterial genomes.

We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.