Jarrod J. Mousa

MATE transport of the E. coli -derived genotoxin colibactin

Various forms of cancer have been linked to the carcinogenic activities of microorganisms1–3. The virulent gene island polyketide synthase (pks) produces the secondary metabolite colibactin, a genotoxic molecule(s) causing double-stranded DNA breaks4 and enhanced colorectal cancer development5,6. Colibactin biosynthesis involves a prodrug resistance strategy where an N-terminal prodrug scaffold (precolibactin) is assembled, transported into the periplasm and cleaved to release the mature product7–10. Here, we show that ClbM, a multidrug and toxic compound extrusion (MATE) transporter, is a key component involved in colibactin activity and transport. Disruption of clbM attenuated pks+ E. coli-induced DNA damage in vitro and significantly decreased the DNA damage response in gnotobiotic Il10−/− mice. Colonization experiments performed in mice or zebrafish animal models indicate that clbM is not implicated in E. coli niche establishment. The X-ray structure of ClbM shows a structural motif common to the recently described MATE family. The 12-transmembrane ClbM is characterized as a cation-coupled antiporter, and residues important to the cation-binding site are identified. Our data identify ClbM as a precolibactin transporter and provide the first structure of a MATE transporter with a defined and specific biological function.

Bbmsn2 acts as a pH‐dependent negative regulator of secondary metabolite production in the entomopathogenic fungus Beauveria bassiana

Fungal secondary metabolites are chemical compounds important for development, environmental adaptation and for potential biotechnological and pharmaceutical applications. Oosporein, a red-pigmented benzoquinone, produced by many fungal insect pathogenic Beauveria spp., shows remarkable functional diversity, displaying antimicrobial, antiviral and even anti-proliferative activities. A homologue of the msn2/seb1 transcription factor was identified in a Beauveria bassiana random T-DNA insertion library. Targeted gene-knockout of Bbmsn2 resulted in reduced growth and increased sensitivity to Calcofluor White, H2 O2 and Congo Red. However, when normalized to growth at 26°C, the ΔBbmsn2 mutant was more tolerant to high temperature (32°C) than the wild type parent. The ΔBbmsn2 mutant also displayed a pH-dependent growth phenotype, with little growth seen at pH < 5.0 but, better growth at alkaline conditions (pH > 8.0). Unexpectedly, a pH-dependent deregulation of a red pigment, identified as oosporein, was seen in the ΔBbmsn2 mutant. The ΔBbmsn2 strain was impaired in virulence in both topical and intrahaemocoel injection bioassays against Galleria mellonella. ΔBbmsn2 proliferation in the host haemolymph and conidiation on the host cadaver was reduced. These data indicate that Bbmsn2 acts as a negative regulator of oosporein production and contributes to virulence and growth in response to external pH in B. bassiana.

A novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein

Respiratory syncytial virus (RSV) remains a major human pathogen, infecting the majority of infants before age two and causing re-infection throughout life. Despite decades of RSV research, there is no licensed RSV vaccine. Most candidate vaccines studied to date have incorporated the RSV fusion (F) surface glycoprotein, because the sequence of F is highly conserved among strains of RSV. To better define the human B cell response to RSV F, we isolated from a single donor 13 new neutralizing human monoclonal antibodies (mAbs) that recognize the RSV F protein in the pre-fusion conformation. Epitope binning studies showed that the majority of neutralizing mAbs targeted a new antigenic site on the globular head domain of F, designated here antigenic site VIII, which occupies an intermediate position between the previously defined major antigenic sites II and site Ø. Antibodies to site VIII competed for binding with antibodies to both of those adjacent neutralizing sites. The new mAbs exhibited unusual breadth for pre-fusion F-specific antibodies, cross-reacting with F proteins from both RSV subgroups A and B viruses. We solved the X-ray crystal structure of one site VIII mAb, hRSV90, in complex with pre-fusion RSV F protein. The structure revealed a large footprint of interaction for hRSV90 on RSV F, in which the heavy chain and light chain both have specific interactions mediating binding to site VIII, the heavy chain overlaps with site Ø, and the light chain interacts partially with site II.

Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two closely related viruses that cause bronchiolitis and pneumonia in infants and the elderly1, with a significant health burden2–6. There are no licensed vaccines or small-molecule antiviral treatments specific to these two viruses at present. A humanized murine monoclonal antibody (palivizumab) is approved to treat high-risk infants for RSV infection7,8, but other treatments, as well as vaccines, for both viruses are still in development. Recent epidemiological modelling suggests that cross-immunity between RSV, HMPV and human parainfluenzaviruses may contribute to their periodic outbreaks9, suggesting that a deeper understanding of host immunity to these viruses may lead to enhanced strategies for their control. Cross-reactive neutralizing antibodies to the RSV and HMPV fusion (F) proteins have been identified10,11. Here, we examine the structural basis for cross-reactive antibody binding to RSV and HMPV F protein by two related, independently isolated antibodies, MPE8 and 25P13. We solved the structure of the MPE8 antibody bound to RSV F protein and identified the 25P13 antibody from an independent blood donor. Our results indicate that both antibodies use germline residues to interact with a conserved surface on F protein that could guide the emergence of cross-reactivity. The induction of similar cross-reactive neutralizing antibodies using structural vaccinology approaches could enhance intrinsic cross-immunity to these paramyxoviruses and approaches to controlling recurring outbreaks.

The PacC transcription factor regulates secondary metabolite production and stress response, but has only minor effects on virulence in the insect pathogenic fungus Beauveria bassiana

The PacC transcription factor is an important component of the fungal ambient pH-responsive regulatory system. Loss of pacC in the insect pathogenic fungus Beauveria bassiana resulted in an alkaline pH-dependent decrease in growth and pH-dependent increased susceptibility to osmotic (salt, sorbitol) stress and SDS. Extreme susceptibility to Congo Red was noted irrespective of pH, and ΔBbpacC conidia showed subtle increases in UV susceptibility. The ΔBbPacC mutant showed a reduced ability to acidify media during growth due to failure to produce oxalic acid. The ΔBbPacC mutant also did not produce the insecticidal compound dipicolinic acid, however, production of a yellow-colored compound was noted. The compound, named bassianolone B, was purified and its structure determined. Despite defects in growth, stress resistance, and oxalate/insecticidal compound production, only a small decrease in virulence was seen for the ΔBbpacC strain in topical insect bioassays using larvae from the greater waxmoth, Galleria mellonella or adults of the beetle, Tenebrio molitor. However, slightly more pronounced decreases were seen in virulence via intrahemcoel injection assays (G. mellonella) and in assays using T. molitor larvae. These data suggest important roles for BbpacC in mediating growth at alkaline pH, regulating secondary metabolite production, and in targeting specific insect stages.

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Significance Respiratory syncytial virus is a highly contagious human pathogen, infecting the majority of infants before age 2 y, and is the leading cause of viral bronchiolitis and viral pneumonia in infants and children. An approved prophylactic humanized mouse monoclonal antibody, palivizumab, is currently the standard-of-care treatment for immunocompromised individuals, and competition with palivizumab has been proposed as the basis for measuring effective immune responses for vaccine trials. Using a combination of X-ray crystallography, hydrogen-deuterium exchange, and saturation alanine mutagenesis scanning, we show the structural basis for neutralization by a human antibody at the palivizumab antigenic site. Furthermore, we describe nonneutralizing antibodies that directly compete with palivizumab and another human antibody 14N4. Taken together, the data presented provide unique concepts in structure-based vaccine design. Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix–loop–helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.

Structural and mechanistic diversity of multidrug transporters

Covering: 2009 to mid 2016Multidrug transporters are common and prevalent in all orders of life, having diverse functions from the removal of toxins, resistance to cytotoxins, and the transport of specific eluents. In addition, multidrug transporters pose a significant threat to modern medicine. Able to transport structurally diverse small molecule drugs, these transporters are implicated in antibiotic resistant strains of bacteria, as well as chemotherapeutic-resistance cancer cells. Although important in such resistance, a relatively small number of multidrug transporters have been structurally characterized, primarily due to the difficulty in purifying and crystallizing active membrane proteins and protein complexes. This review will cover recent structural breakthroughs in the past six years that have led to increased knowledge of the mechanisms of multidrug transporter chemistry, and the role of these transporters in exporting secondary metabolites.

ClbM is a versatile, cation-promiscuous MATE transporter found in the colibactin biosynthetic gene cluster

Multidrug transporters play key roles in cellular drug resistance to toxic molecules, yet these transporters are also involved in natural product transport as part of biosynthetic clusters in bacteria and fungi. The genotoxic molecule colibactin is produced by strains of virulent and pathobiont Escherichia coli and Klebsiella pneumoniae. In the biosynthetic cluster is a multidrug and toxic compound extrusion protein (MATE) proposed to transport the prodrug molecule precolibactin across the cytoplasmic membrane, for subsequent cleavage by the peptidase ClbP and cellular export. We recently determined the X-ray structure of ClbM, and showed preliminary data suggesting its specific role in precolibactin transport. Here, we define a functional role of ClbM by examining transport capabilities under various biochemical conditions. Our data indicate ClbM responds to sodium, potassium, and rubidium ion gradients, while also having substantial transport activity in the absence of alkali cations.

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.