Antibacterial phage ORFans of Pseudomonas aeruginosa phage LUZ24 reveal a novel MvaT inhibiting protein.

The functional elucidation of small unknown phage proteins ('ORFans') presents itself as one of the major challenges of bacteriophage molecular biology. In this work, we mined the Pseudomonas aeruginosa-infecting phage LUZ24 proteome for antibacterial and antibiofilm proteins against its host. Subsequently, their putative host target was identified. In one example, we observed an interaction between LUZ24 gp4 and the host transcriptional regulator MvaT. The polymerization of MvaT across AT-rich DNA strands permits gene silencing of foreign DNA, thereby limiting any potentially adverse effects of such DNA. Gel shift assays proved the inhibitory effect of LUZ24 gp4 on MvaT DNA binding activity. Therefore, we termed this gene product as Mip, the MvaT inhibiting protein. We hypothesize Mip prevents the AT-rich LUZ24 DNA from being physically blocked by MvaT oligomers right after its injection in the host cell, thereby allowing phage transcription and thus completion of the phage infection cycle.

Conserved Omp85 lid-lock structure and substrate recognition in FhaC.

Omp85 proteins mediate translocation of polypeptide substrates across and into cellular membranes. They share a common architecture comprising substrate-interacting POTRA domains, a C-terminal 16-stranded ß-barrel pore and two signature motifs located on the inner barrel wall and at the tip of the extended L6 loop. The observation of two distinct conformations of the L6 loop in the available Omp85 structures previously suggested a functional role of conformational changes in L6 in the Omp85 mechanism. Here we present a 2.5 Å resolution structure of a variant of the Omp85 secretion protein FhaC, in which the two signature motifs interact tightly and form the conserved 'lid lock'. Reanalysis of previous structural data shows that L6 adopts the same, conserved resting state position in all available Omp85 structures. The FhaC variant structure further reveals a competitive mechanism for the regulation of substrate binding mediated by the linker to the N-terminal plug helix H1.

Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of Pseudomonas aeruginosa.

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).

Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa.

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets.

Learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications.

The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application.

Substrate recognition by the POTRA domains of TpsB transporter FhaC.

Widespread in Gram-negative bacteria, the two-partner secretion (TPS) pathway mediates the secretion of large, ß-helical 'TpsA' proteins with various functions. TpsA proteins harbour a conserved, N-proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a ß barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a ß sheet edge and the flanking α helix, well-suited to accommodate extended, amphipathic strands of the substrate and consistent with ß augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat-rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion.

Functional importance of a conserved sequence motif in FhaC, a prototypic member of the TpsB/Omp85 superfamily.

In Gram-negative bacteria, the two-partner secretion pathway mediates the secretion of TpsA proteins with various functions. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their transport through a hydrophilic channel. The filamentous haemagglutinin adhesin (FHA)/FhaC pair represents a model two-partner secretion system, with the structure of the TpsB transporter FhaC providing the bases to decipher the mechanism of action of these proteins. FhaC is composed of a ß-barrel preceded by two periplasmic polypeptide-transport-associated (POTRA) domains in tandem. The barrel is occluded by an N-terminal helix and an extracellular loop, L6, folded back into the FhaC channel. In this article, we describe a functionally important motif of FhaC. The VRGY tetrad is highly conserved in the TpsB family and, in FhaC, it is located at the tip of L6 reaching the periplasm. Replacement by Ala of the invariant Arg dramatically affects the secretion efficiency, although the structure of FhaC and its channel properties remain unaffected. This substitution affects the secretion mechanism at a step beyond the initial TpsA-TpsB interaction. Replacement of the conserved Tyr affects the channel properties, but not the secretion activity, suggesting that this residue stabilizes the loop in the resting conformation of FhaC. Thus, the conserved motif at the tip of L6 represents an important piece of two-partner secretion machinery. This motif is conserved in a predicted loop between two ß-barrel strands in more distant relatives of FhaC involved in protein transport across or assembly into the outer membranes of bacteria and organelles, suggesting a conserved function in the molecular mechanism of transport.

First structural insights into the TpsB/Omp85 superfamily.

Proteins of the TpsB/Omp85 superfamily are involved in protein transport across, or assembly into, the outer membrane of Gram-negative bacteria, and their distant eukaryotic relatives exert similar functions in chloroplasts and mitochondria. The X-ray structure of one TpsB transporter, FhaC, provides the bases to decipher the mechanisms of action of these proteins. With two POTRA domains in the periplasm, a transmembrane beta barrel and a large loop harboring a functionally important motif, FhaC epitomizes the conserved features of the super-family.

Structure of the membrane protein FhaC: a member of the Omp85-TpsB transporter superfamily.

In Gram-negative bacteria and eukaryotic organelles, beta-barrel proteins of the outer membrane protein 85-two-partner secretion B (Omp85-TpsB) superfamily are essential components of protein transport machineries. The TpsB transporter FhaC mediates the secretion of Bordetella pertussis filamentous hemagglutinin (FHA). We report the 3.15 A crystal structure of FhaC. The transporter comprises a 16-stranded beta barrel that is occluded by an N-terminal alpha helix and an extracellular loop and a periplasmic module composed of two aligned polypeptide-transport-associated (POTRA) domains. Functional data reveal that FHA binds to the POTRA 1 domain via its N-terminal domain and likely translocates the adhesin-repeated motifs in an extended hairpin conformation, with folding occurring at the cell surface. General features of the mechanism obtained here are likely to apply throughout the superfamily.