Phosphorylation of the lipid A region of meningococcal lipopolysaccharide: identification of a family of transferases that add phosphoethanolamine to lipopolysaccharide

A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions, N. meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A."     

Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon

A modified mariner transposon, miniHimar RB1, was generated to mutagenize cells of the metal-reducing bacterium Shewanella oneidensis. The use of this transposon led to the isolation of stable mutants and allowed rapid identification of disrupted genes. Fifty-eight mutants, including BG104 and BG148 with transposon insertions in the cytochrome c maturation genes ccmC and ccmF1, respectively, were analyzed. Both mutants were deficient in anaerobic respiration and cytochrome c production.     

How do bacteria resist human antimicrobial peptides?

Cationic antimicrobial peptides (CAMPs), such as defensins, cathelicidins and thrombocidins, are an important human defense mechanism, protecting skin and epithelia against invading microorganisms and assisting neutrophils and platelets. Staphylococcus aureus, Salmonella enterica and other bacterial pathogens have evolved countermeasures to limit the effectiveness of CAMPs, including the repulsion of CAMPs by reducing the net negative charge of the bacterial cell envelope through covalent modification of anionic molecules (e.g. teichoic acids, phospholipids and lipid A); expelling CAMPs through energy-dependent pumps; altering membrane fluidity; and cleaving CAMPs with proteases. Mutants susceptible to CAMPs are more efficiently inactivated by phagocytes and are virulence-attenuated, indicating that CAMP resistance plays a key role in bacterial infections.     

Helicobacter pylori mutagenesis by mariner in vitro transposition

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.     

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.     

O-Acetylation of the terminal N-acetylglucosamine of the lipooligosaccharide inner core in Neisseria meningitidis. Influence on inner core structure and assembly

O-Acetylation is a common decoration on endotoxins derived from many Gram-negative bacterial species, and it has been shown to be instrumental (e.g. in Salmonella typhimurium) in determining the final tertiary structure of the endotoxin and the immunogenicity of the molecule. Structural heterogeneity of endotoxins produced by mucosal pathogens such as Neisseria meningitidis is determined by decorations on the heptose inner core, including O-acetylation of the terminal N-acetylglucosamine (GlcNAc) attached to HepII. In this report, we show that O-acetylation of the meningococcal lipooligosaccharide (LOS) inner core has an important role in determining inner core assembly and immunotype expression. The gene encoding the LOS O-acetyltransferase, lot3, was identified by homology to NodX from Rhizobium leguminosarum. Inactivation of lot3 in strain NMB resulted in the loss of the O-acetyl group located at the C-3 position of the terminal GlcNAc of the LOS inner core. Inactivation of either lot3 or lgtG, which encodes the HepII glucosyltransferase, did not result in the appearance of the O-3-linked phosphoethanolamine (PEA) groups on the LOS inner core. Construction of a double mutant in which both lot3 and lgtG were inactivated resulted in the appearance of O-3-linked PEA groups on the LOS inner core. In conclusion, O-acetylation status of the terminal GlcNAc of the gamma-chain of the meningococcal LOS inner core is an important determinant for the appearance or exclusion of the O-3-linked PEA group on the LOS inner core and contributes to LOS structural diversity. O-Acetylation also likely influences resistance to complement-mediated lysis and may be important in LOS conjugate vaccine design.     

Shotgun cloning of transposon insertions in the genome of Caenorhabditis elegans

We present a strategy to identify and map large numbers of transposon insertions in the genome of Caenorhabditis elegans. Our approach makes use of the mutator strain mut-7, which has germline-transposition activity of the Tc1/mariner family of transposons, a display protocol to detect new transposon insertions, and the availability of the genomic sequence of C. elegans. From a pilot insertional mutagenesis screen, we have obtained 351 new Tc1 transposons inserted in or near 219 predicted C. elegans genes. The strategy presented provides an approach to isolate insertions of natural transposable elements in many C. elegans genes and to create a large-scale collection of C. elegans mutants.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis

The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.     

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.     

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.     

Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis

The difficulty in obtaining mutants in pathogenic Neisseria has limited the ability to genetically define determinants responsible for virulence as well as the ability to generate a genetic map. We show that the 16.5kb conjugative transposon Tn916 can be introduced into Neisseria meningitidis on the suicide vectors pAM120 and pAM170. After introduction, Tn916 transposed to different sites in the chromosome of recipient meningococci, apparently at random, and was stably incorporated. Following its integration into the meningococcal chromosome, Tn916 did not appear to readily express its conjugative and transpositional functions. However, chromosomal DNA from Tn916-carrying meningococci could be used to transform other meningococcal strains to tetracycline resistance. These studies indicate that Tn916 may be an important tool for genetic analysis of N. meningitidis.     

Insertional inactivation of the lpxA gene involved in the biosynthesis of lipid A in Neisseria meningitidis resulted in lpxA/lpxA::aph-3' heterodiploids

The lpxA gene is known to be involved in the biosynthesis of lipid A in Gram-negative bacteria and thought to be an essential gene. However, viable meningococcal lpxA mutants devoid of detectable endotoxin (lipooligosaccharide) have been reported. We characterised such mutants in strains of Neisseria meningitidis belonging to serogroups B and C using molecular and biochemical analysis. While lpxA mutants with no detectable or a low level of lipooligosaccharide could be obtained in N. meningitidis, the simple insertional inactivation of lpxA was not possible. In all mutants, we obtained lpxA/lpxA::aph-3' heterodiploids harbouring one copy of the wild-type lpxA gene and one copy of the inactivated lpxA gene by insertion of the kanamycin resistance cassette, aph-3'. The absence of lipooligosaccharide in these mutants may result from a negative transdominance effect of a truncated LpxA protein on the wild-type LpxA protein.     

Gradual pediocin PA-1 resistance in <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> confers cross-protection to diverse pore-forming cationic antimicrobial peptides displaying changes in cell wall and mannose PTS expression

Due to innate and acquired resistance in Enterococcus faecalis against most antibiotics, identification of new alternatives has increased interest in diverse populations of potent cationic antimicrobial peptides (CAMPs) for treatment and natural food biopreservation. The CAMPs, after crossing the cell wall to the periplasmic space, kill their target strain by forming pores in the cell membrane. However, reports of resistance against these CAMPs necessitated the understanding of step(s) interfered with while acquiring this resistance, for designing effective CAMP analogs. In this direction, we selected stable and gradual dose-dependent pediocin PA-1 single exposure resistant (Ped^r) mutants of E. faecalis, which conferred cross-protection to diverse CAMPs, viz., HNP-1, nisin and alamethicin but not to polymyxin B, lysozyme and vancomycin. With these Ped^r mutants of E. faecalis there was: a gradual neutralization in cell wall surface charge involving D-alanylation of wall teichoic acids (WTA) and lipoteichoic acids (LTA), increase in cell-surface hydrophobicity, increased cell aggregation and biofilm formation and ultra-structural changes in the cell wall, and a reduction of periplasmic space. In addition, a gradual decrease in expression of mannose PTS two (mpt) operon was also observed with distinct changes in growth rate achieving the same biomass production during the stationary phase. These results show that resistance to these CAMPs is not due to mpt directly acting as a docking molecule but due to changes in the cell wall, which increased the permeability barrier to CAMPs diffusion to reach the periplasmic space.