Targeting Bacterial Virulence: The Coming Out of Type VII Secretion Inhibitors

Type VII (ESX) secretion systems of pathogenic mycobacteria, such as Mycobacterium tuberculosis, are crucial for intracellular survival, host cell lysis, and the subsequent cell-to-cell spread. In this issue of Cell Host & Microbe, Rybniker et al. (2014) have used these characteristics to identify two classes of type VII secretion inhibitors

Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella

Overproducing the BAM complex improves secretion of difficult-to-secrete recombinant autotransporter chimeras

Monomeric autotransporters have been used extensively to transport recombinant proteins or protein domains to the cell surface of Gram-negative bacteria amongst others for antigen display. Genetic fusion of such antigens into autotransporters has yielded chimeras that can be used for vaccination purposes. However, not every fusion construct is transported efficiently across the cell envelope. Problems occur in particular when the fused antigen attains a relatively complex structure in the periplasm, prior to its translocation across the outer membrane. The latter step requires the interaction with periplasmic chaperones and the BAM (β-barrel assembly machinery) complex in the outer membrane. This complex catalyzes insertion and folding of β-barrel outer membrane proteins, including the β-barrel domain of autotransporters. Here, we investigated whether the availability of periplasmic chaperones or the BAM complex is a limiting factor for the surface localization of difficult-to-secrete chimeric autotransporter constructs. Indeed, we found that overproduction of in particular the BAM complex, increases surface display of difficult-to-secrete chimeras. Importantly, this beneficial effect appeared to be generic not only for a number of monomeric autotransporter fusions but also for fusions to trimeric autotransporters. Therefore, overproduction of BAM might be an attractive strategy to improve the production of recombinant autotransporter constructs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01668-2

Combining Protein Ligation Systems to Expand the Functionality of Semi-Synthetic Outer Membrane Vesicle Nanoparticles

Bacterial outer membrane vesicles (OMVs) attract increasing interest as immunostimulatory nanoparticles for the development of vaccines and therapeutic agents. We previously engineered the autotransporter protein Hemoglobin protease (Hbp) into a surface display carrier that can be expressed to high density on the surface of Salmonella OMVs. Moreover, we implemented Tag-Catcher protein ligation technology, to obtain dense display of single heterologous antigens and nanobodies on the OMVs through coupling to the distal end of the Hbp passenger domain. Here, we aimed to further expand the versatility of the Hbp platform by enabling the coupling of heterologous proteins to internal sites of the Hbp passenger. Inserted SpyTags were shown to be accessible at the Salmonella OMV surface and to efficiently couple SpyCatcher-equipped fusion proteins. Next, we combined distally placed SnoopCatcher or SnoopTag sequences with internal SpyTags in a single Hbp molecule. This allowed the coupling of two heterologous proteins to a single Hbp carrier molecule without obvious steric hindrance effects. Since coupling occurs to Hbp that is already exposed on the OMVs, there are no limitations to the size and complexity of the partner proteins. In conclusion, we constructed a versatile modular platform for the development of bivalent recombinant OMV-based vaccines and therapeutics

Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier 11 Medical and Health Sciences 1107 Immunology

Background: Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results: We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion: We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development

Surface Labeling with Adhesion Protein FimH Improves Binding of Immunotherapeutic Agent Salmonella Ty21a to the Bladder Epithelium

BACKGROUND: Bladder cancer is the ninth most common cancer in men. 70% of these tumors are classified as non-muscle invasive bladder cancer and those patients receive 6 intravesical instillations with Mycobacterium bovis BCG after transurethral resection. However, 30% of patients show recurrences after treatment and experience severe side effects that often lead to therapy discontinuation. Recently, another vaccine strain, Salmonella enterica typhi Ty21a, demonstrated promising antitumor activity in vivo. Here we focus on increasing bacterial retention in the bladder in order to reduce the number of instillations required and improve antitumor activity. OBJECTIVE: To increase the binding of Ty21a to the bladder wall by surface labeling of the bacteria with adhesion protein FimH and to study its effect in a bladder cancer mouse model. METHODS: Binding of Ty21a with surface-labeled FimH to the bladder wall was analyzed in vitro and in vivo. The antitumor effect of a single instillation of Ty21a+FimH in treatment was determined in a survival experiment. RESULTS: FimH-labeled Ty21a showed significant (p < 0.0001) improved binding to mouse and human cell lines in vitro. Furthermore, FimH labeled bacteria showed ∼5x more binding to the bladder than controls in vivo. Enhanced binding to the bladder via FimH labeling induced a modest improvement in median but not in overall mice survival. CONCLUSIONS: FimH labeling of Ty21a significantly improved binding to bladder tumor cells in vitro and the bladder wall in vivo. The improved binding leads to a modest increase in median survival in a single bladder cancer mouse study

An anti-tuberculosis compound screen using a zebrafish infection model identifies an aspartyl-tRNA synthetase inhibitor

Finding new anti-tuberculosis compounds with convincing in vivo activity is an ongoing global challenge to fight the emergence of multidrug-resistant Mycobacterium tuberculosis isolates. In this study, we exploited the medium-throughput capabilities of the zebrafish embryo infection model with Mycobacterium marinum as a surrogate for M. tuberculosis. Using a representative set of clinically established drugs, we demonstrate that this model could be predictive and selective for antibiotics that can be administered orally. We further used the zebrafish infection model to screen 240 compounds from an anti-tuberculosis hit library for their in vivo activity and identified 14 highly active compounds. One of the most active compounds was the tetracyclic compound TBA161, which was studied in more detail. Analysis of resistant mutants revealed point mutations in aspS (rv2572c), encoding an aspartyl-tRNA synthetase. The target was genetically confirmed, and molecular docking studies propose the possible binding of TBA161 in a pocket adjacent to the catalytic site. This study shows that the zebrafish infection model is suitable for rapidly identifying promising scaffolds with in vivo activity