Extreme antimicrobial Peptide and polymyxin B resistance in the genus burkholderia

Cationic antimicrobial peptides and polymyxins are a group of naturally occurring antibiotics that can also possess immunomodulatory activities. They are considered a new source of antibiotics for treating infections by bacteria that are resistant to conventional antibiotics. Members of the genus Burkholderia, which includes various human pathogens, are inherently resistant to antimicrobial peptides. The resistance is several orders of magnitude higher than that of other Gram-negative bacteria such as Escherichia coli, Salmonella enterica, or Pseudomonas aeruginosa. This review summarizes our current understanding of antimicrobial peptide and polymyxin B resistance in the genus Burkholderia. These bacteria possess major and minor resistance mechanisms that will be described in detail. Recent studies have revealed that many other emerging Gram-negative opportunistic pathogens may also be inherently resistant to antimicrobial peptides and polymyxins and we propose that Burkholderia sp. are a model system to investigate the molecular basis of the resistance in extremely resistant bacteria. Understanding resistance in these types of bacteria will be important if antimicrobial peptides come to be used regularly for the treatment of infections by susceptible bacteria because this may lead to increased resistance in the species that are currently susceptible and may also open up new niches for opportunistic pathogens with high inherent resistance.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.

A system for the construction of targeted unmarked gene deletions in the genus Burkholderia

Burkholderia species are extremely multidrug resistant, environmental bacteria with extraordinary bioremediation and biocontrol properties. At the same time, these bacteria cause serious opportunistic infections in vulnerable patient populations while some species can potentially be used as bioweapons. The complete DNA sequence of more than 10 Burkholderia genomes provides an opportunity to apply functional genomics to a collection of widely adaptable environmental bacteria thriving in diverse niches and establishing both symbiotic and pathogenic associations with many different organisms. However, extreme multidrug resistance hampers genetic manipulations in Burkholderia. We have developed and evaluated a mutagenesis system based on the homing endonuclease I-SceI to construct targeted, non-polar unmarked gene deletions in Burkholderia. Using the cystic fibrosis pathogen Burkholderia cenocepacia K56-2 as a model strain, we demonstrate this system allows for clean deletions of one or more genes within an operon and also the introduction of multiple deletions in the same strain. We anticipate this tool will have widespread environmental and biomedical applications, facilitating functional genomic studies and construction of safe strains for bioremediation and biocontrol, as well as clinical applications such as live vaccines for Burkholderia and other Gram-negative bacterial species.

A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.

Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.

Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate

Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (hppD) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4-hydroxyphenylpyruvic acid dioxygenase (HppD) activity. Pigmentation could be restored by complementation providing hppD in trans. The hppD mutant was resistant to paraquat challenge but sensitive to H2O2 and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized in a dextran-positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Colocalization of the nonpigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by B. cenocepacia C5424 is a pyomelanin synthesized from an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

Characterization of SodC, a periplasmic superoxide dismutase from Burkholderia cenocepacia

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.

Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia

Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.

Micromethods for the characterization of lipid A-core and O-antigen lipopolysaccharide

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.

An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis

The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.

Identification of a UDP-Gal:GlcNAc-R galactosyltransferase activity in Escherichia coli VW187

A novel acceptor substrate for galactosyltransferase was synthesized containing GlcNAcalpha-pyrophosphate, covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-Phenyl). The new substrate was used to develop an assay for a galactosyltransferase activity from Escherichia coli strain VW187 that is involved in lipopolysaccharide synthesis and has not been studied by others. We showed that Gal was transferred from UDP-Gal to the novel acceptor substrate. This was a significant improvement over our previous preliminary assays of the enzyme using endogenous substrate, and showed that these synthetic substrates are useful for assaying enzymes that utilize lipid-bound substrates in O-chain synthesis in Gram-negative bacteria.

Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells

Bdellovibrio bacteriovorus is a Gram-negative bacterium that preys on other Gram-negative bacteria. The lifecycle of B. bacteriovorus alternates between an extracellular flagellated and highly motile non-replicative attack-phase cell and a periplasmic non-flagellated growth-phase cell. The prey bacterium containing periplasmic bdellovibrios becomes spherical but osmotically stable, forming a structure known as the bdelloplast. After completing the growth phase, newly formed bdellovibrios regain their flagellum and escape the bdelloplast into the environment, where they encounter more prey bacteria. The obligate predatory nature of B. bacteriovorus imposes a major difficulty to introducing mutations in genes directly involved in predation, since these mutants could be non-viable. This work reports the cloning of the B. bacteriovorus 109J motAB operon, encoding proteins from the flagellar motor complex, and a genetic approach based on the expression of a motA antisense RNA fragment to downregulate motility. Periplasmic bdellovibrios carrying the plasmid expressing antisense RNA displayed a marked delay in escaping from bdelloplasts, while the released attack-phase cells showed altered motility. These observations suggest that a functionally intact flagellar motor is required for the predatory lifecycle of B. bacteriovorus. Also, the use of antisense RNA expression may be a useful genetic tool to study the Bdellovibrio developmental cycle.

An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid

Nucleotide-binding oligomerization domain protein 1 (NOD1) belongs to a family that includes multiple members with NOD and leucine-rich repeats in vertebrates and plants. NOD1 has been suggested to have a role in innate immune responses, but the mechanism involved remains unknown. Here we report that NOD1 mediates the recognition of peptidoglycan derived primarily from Gram-negative bacteria. Biochemical and functional analyses using highly purified and synthetic compounds indicate that the core structure recognized by NOD1 is a dipeptide, gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Murine macrophages deficient in NOD1 did not secrete cytokines in response to synthetic iE-DAP and did not prime the lipopolysaccharide response. Thus, NOD1 mediates selective recognition of bacteria through detection of iE-DAP-containing peptidoglycan.

Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli

Lipopolysaccharide (LPS), a glycolipid molecule found on the outer leaflet of outer membranes of gram-negative bacteria, consists of three moieties: lipid A, core oligosaccharide, and the O-specific polysaccharide chain. The O-specific side chain, which extends to the extracellular milieu, plays an important role in pathogenicity, especially during the initial stages of infection, because of its ability to interact with serum complement. In recent years, several laboratories have used recombinant DNA tools to determine, at the molecular level, the organization, expression, and regulation of genes involved in LPS biosynthesis in Salmonella and Escherichia coli. An increased understanding of the molecular aspects of the O-specific side-chain genes will shed light on the intimate details related with the formation of the O-specific side chain, its assembly onto the lipid A--core, and the translocation and insertion of the complete LPS molecule into the outer membrane. It will also contribute to the understanding of the evolution of these genes and the correlation of chemical diversity of O-specific side chains with the genetic diversity of O-specific side-chain genes. In addition, since the O-specific side chains are involved in the pathogenicity of medically important gram-negative bacteria, a basic understanding of the regulation and expression of O-specific side chain LPS genes will contribute to the field of molecular pathogenesis. This article provides an overview of the role of O-specific side chains in septicemic infections and also discusses the current status of molecular genetic studies on O-specific side-chain genes from E. coli.