Shahar Molshanski-Mor

Reversing Bacterial Resistance to Antibiotics by Phage-Mediated Delivery of Dominant Sensitive Genes

ABSTRACT Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the genes sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phages ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

Assembly of the phagocyte NADPH oxidase complex : chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships

Phagocytes generate superoxide (O2.−) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH‐driven reduction of oxygen to O2.−, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67phox and Rac1. The prototype chimera p67phox (1–212)‐Rac1 (1–192) is a potent activator in a cell‐free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox‐Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X‐ray scattering, and gel filtration. Based on this, a “propagated wave” model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.

Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, p40phox, and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2)·RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP)·RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP)·RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2)·RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP)·RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP)·RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67phox, without the need for an anionic amphiphile, as activator. Both Rac1(GDP)·RhoGDI and Rac1(GMPPNP)·RhoGDI complexes elicited amphiphile-independent, p67phox-dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.

Cell-Free Assays

The superoxide (O2-)-generating enzyme complex of phagocytes, known as the NADPH oxidase, can be assayed in a number of in vitro cell-free (or broken cell) systems. These consist of a mixture of the individual components of the NADPH oxidase, derived from resting phagocytes or in the form of purified recombinant proteins, exposed to an activating agent (or situation), in the presence of NADPH and oxygen. O2- produced by the mixture is measured by being trapped immediately after its generation with an appropriate acceptor in a kinetic assay, which permits the calculation of the linear rate of O2- production over time. Cell-free assays are distinguished from whole-cell assays or assays performed on membranes derived from stimulated cells by the fact that all components in the reaction are derived from resting, nonstimulated cells and, thus, the steps of NADPH oxidase activation (precatalytic [assembly] and catalytic) occur in vitro. Cell-free assays played a paramount role in the identification of the components of the NADPH oxidase complex, the diagnosis of various forms of chronic granulomatous disease (CGD), and, more recently, the analysis of the domains present on the components of the NADPH oxidase participating in protein-protein interactions leading to the assembly of the active complex.

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division

Significance We have identified a phage-encoded inhibitor of the major cytoskeletal protein in bacterial division. The inhibition is shown to confer T7 bacteriophage with a significant growth advantage in dividing hosts. Our studies thus indicate a strategy in bacteriophages to maximize their progeny number by inhibiting escape of one of the daughter cells of an infected bacterium. Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage’s competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts’ cell resources.

Tripartite Chimeras Comprising Functional Domains Derived from the Cytosolic NADPH Oxidase Components p47phox, p67phox, and Rac1 Elicit Activator-independent Superoxide Production by Phagocyte Membranes AN ESSENTIAL ROLE FOR ANIONIC MEMBRANE PHOSPHOLIPIDS

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b559 with three cytosolic components: p47phox, p67phox, and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b559 within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47phox, the tetratricopeptide repeat and activation domains of p67phox, and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC50) for and formed a more stable complex (longer half-life) with cytochrome b559 compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22phox via Trp193 in the N-terminal SH3 domain of the p47phox segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67phox and Rac1 segments complementary to their physical fusion. The PX domain of the p47phox segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.

Extending the Host Range of Bacteriophage Particles for DNA Transduction

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Different approaches for using bacteriophages against antibiotic-resistant bacteria.

Bacterial resistance to antibiotics is an emerging threat requiring urgent solutions. Ever since their discovery, lytic bacteriophages have been suggested as therapeutic agents, but their application faces various obstacles: sequestration of the phage by the spleen and liver, antibodies against the phage, narrow host range, poor accessibility to the infected tissue, and bacterial resistance. Variations on bacteriophage use have been suggested, such as temperate phages as gene-delivery vehicles into pathogens. This approach, which is proposed to sensitize pathogens residing on hospital surfaces and medical personnels skin, and its prospects are described in this addendum. Furthermore, phage-encoded products have been proposed as weapons against antibiotic resistance in bacteria. We describe a new phage protein which was identified during basic research into T7 bacteriophages. This protein may serendipitously prove useful for treating antibiotic-resistant pathogens. We believe that further basic research will lead to novel strategies in the fight against antibiotic-resistant bacteria.

Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A “peptide walking” study

In this study, the “peptide walking” approach was applied to the DH region of Nox2 (residues 288–570) with the purpose of identifying domains of functional importance in the assembly and/or catalytic function of the NADPH oxidase complex of phagocytes. Ninety‐one overlapping 15‐mer peptides were synthesized to cover the full length of the Nox2 DH region, and these were tested for the ability to interfere with the activation of the oxidase in vitro in two semirecombinant cell‐free systems. The first consisted of phagocyte membranes p47phox, p67phox, and Rac1 and an amphiphile; the second was p47phox‐ and amphiphile‐free and contained prenylated Rac1. We identified 10 clusters of inhibitory peptides with IC50 values of 10 μM, all of which were inhibitory, also in the absence of p47phox. Based on the identification of residues shared by peptides in a particular cluster, we defined 10 functional domains in the Nox2 DH region. One domain corresponded to one FAD‐binding subdomain, and four domains overlapped parts of three NADPH‐binding subdomains. As expected, most inhibitory peptides acted only when added prior to the completion of oxidase assembly, but peptides associated with two NADPH‐binding subdomains were also active after assembly. Kinetic analysis demonstrated that inhibition by peptides was not explained by competition for substrates (FAD, NADPH) but was of a more complex nature: noncompetitive with respect to FAD and uncompetitive with respect to NADPH. We conclude that oxidase‐inhibitory peptides, in five out of 10 clusters identified, act by interfering with FAD‐ and NADPH‐related redox reactions.

Revealing bacterial targets of growth inhibitors encoded by bacteriophage T7

Significance Antibiotic resistance of pathogens is a growing threat to human health, requiring immediate action. Identifying new gene products of bacterial viruses and their bacterial targets may provide potent tools for fighting antibiotic-resistant strains. We show that a significant number of phage proteins are inhibitory to their bacterial host. DNA sequencing was used to map the targets of these proteins. One particular target was a key cytoskeleton protein whose function is impaired following the phage protein’s expression, resulting in bacterial death. Strikingly, in over 70 y of extensive research into the tested bacteriophage, this inhibition had never been characterized. We believe that the presented approach may be broadened to identify novel, clinically relevant bacteriophage growth inhibitors and to characterize their targets. Todays arsenal of antibiotics is ineffective against some emerging strains of antibiotic-resistant pathogens. Novel inhibitors of bacterial growth therefore need to be found. The target of such bacterial-growth inhibitors must be identified, and one way to achieve this is by locating mutations that suppress their inhibitory effect. Here, we identified five growth inhibitors encoded by T7 bacteriophage. High-throughput sequencing of genomic DNA of resistant bacterial mutants evolving against three of these inhibitors revealed unique mutations in three specific genes. We found that a nonessential host gene, ppiB, is required for growth inhibition by one bacteriophage inhibitor and another nonessential gene, pcnB, is required for growth inhibition by a different inhibitor. Notably, we found a previously unidentified growth inhibitor, gene product (Gp) 0.6, that interacts with the essential cytoskeleton protein MreB and inhibits its function. We further identified mutations in two distinct regions in the mreB gene that overcome this inhibition. Bacterial two-hybrid assay and accumulation of Gp0.6 only in MreB-expressing bacteria confirmed interaction of MreB and Gp0.6. Expression of Gp0.6 resulted in lemon-shaped bacteria followed by cell lysis, as previously reported for MreB inhibitors. The described approach may be extended for the identification of new growth inhibitors and their targets across bacterial species and in higher organisms.