Design and optimization of enzymatic activity in a de novo β-barrel scaffold

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds

Escherichia coli D-malate dehydrogenase, a generalist enzyme active in the leucine biosynthesis pathway.

The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of D-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on D-malate as a carbon source, the D-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB(-) strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (L(+)-tartrate, D-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution

Design and optimization of enzymatic activity in a de novo β-barrel scaffold

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded beta-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds.ISSN:1469-896XISSN:0961-836

Treponema pallidum subsp. pallidum with an Artificially impaired TprK antigenic variation system is attenuated in the Rabbit model of syphilis.

BackgroundThe TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum's ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen's surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted β-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis.Principal findingsA suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected.ConclusionThese data further support the critical role of TprK in T. pallidum virulence and persistence during infection

Heatmap of sequence diversity in TprK V3.

Deep sequencing of TprK V3 region showing persistence of inoculum variants and generation of non-inoculum variants in samples collected overtime from rabbits infected with the WT SS14 strain (“WT”-labeled samples, left side of the map) and form rabbits infected with the SS14-DCKO strain (“KO”-labeled samples, right side of the map), separated by a bold vertical dashed line. In each map, inoculum sequences for the WT strain are labeled as “WT Inoc B00 Day 00”, where WT Inoc indicates WT inoculum treponemes, B00 indicates that the sample was not obtained from a biopsy, and Day 00 indicates the experiment’s time 0. The same nomenclature was adopted for the SS14-DCKO inoculum, with the exception that KO replaced WT. Samples collected post-inoculation report rabbit number (R#), biopsy number (B#), and day post-inoculation the sample was obtained (Day#). Light gray dashed lines separate individual animals. Missing samples did not yield data. The same heatmap in interactive format is available at https://github.com/greninger-lab/Impaired-TprK-Antigenic-Variation. Peptide sequences and prevalence are also reported in S2 Table. (PDF)</p

TprK (sequence AIP85979.1) is predicted by AlphaFold2 to fold into a 20-strands beta-barrel structures with 10 surface-exposed loops.

Variable loops (V1-V7) are highlighted in colour. Conserved loops (C1-C3) are in white, and β-scaffolding are in light blue. OM: outer membrane; PS: periplasmic space.</p

Heatmap of sequence diversity in TprK V4.

Deep sequencing of TprK V4 region showing persistence of inoculum variants and generation of non-inoculum variants in samples collected overtime from rabbits infected with the WT SS14 strain (“WT”-labeled samples, left side of the map) and form rabbits infected with the SS14-DCKO strain (“KO”-labeled samples, right side of the map), separated by a bold vertical dashed line. In each map, inoculum sequences for the WT strain are labeled as “WT Inoc B00 Day 00”, where WT Inoc indicates WT inoculum treponemes, B00 indicates that the sample was not obtained from a biopsy, and Day 00 indicates the experiment’s time 0. The same nomenclature was adopted for the SS14-DCKO inoculum, with the exception that KO replaced WT. Samples collected post-inoculation report rabbit number (R#), biopsy number (B#), and day post-inoculation the sample was obtained (Day#). Light gray dashed lines separate individual animals. Missing samples did not yield data. The same heatmap in interactive format is available at https://github.com/greninger-lab/Impaired-TprK-Antigenic-Variation. Peptide sequences and prevalence are also reported in S2 Table. (PDF)</p

Primers used in this study.

BackgroundThe TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum’s ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen’s surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted β-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis.Principal findingsA suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected.ConclusionThese data further support the critical role of TprK in T. pallidum virulence and persistence during infection.</div

Barcoded primers used in this study for <i>tprk</i> deep sequencing.

Barcoded primers used in this study for tprk deep sequencing.</p

Sequence of the insert (yellow/green/gray bases) contained in the p<i>DC</i>arms-47p-<i>kan</i>^R suicide vector built using a pUC57 plasmid backbone, flanking regions (aqua), and primers used to evaluate the replacement of the DCs in the transformed <i>T</i>. <i>pallidum</i> strain.

Sequence of the insert (yellow/green/gray bases) contained in the pDCarms-47p-kanR suicide vector built using a pUC57 plasmid backbone, flanking regions (aqua), and primers used to evaluate the replacement of the DCs in the transformed T. pallidum strain.</p