Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination.

Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.

Ligand-Receptor Interaction-Induced Intracellular Phase Separation: A Global Disruption Strategy for Resistance-Free Lethality of Pathogenic Bacteria.

Addressing the global challenge of bacterial resistance demands innovative approaches, among which multitargeting is a widely used strategy. Current strategies of multitargeting, typically achieved through drug combinations or single agents inherently aiming at multiple targets, face challenges such as stringent pharmacokinetic and pharmacodynamic requirements and cytotoxicity concerns. In this report, we propose a bacterial-specific global disruption approach as a vastly expanded multitargeting strategy that effectively disrupts bacterial subcellular organization. This effect is achieved through a pioneering chemical design of ligand-receptor interaction-induced aggregation of small molecules, i.e., DNA-induced aggregation of a diarginine peptidomimetic within bacterial cells. These intracellular aggregates display affinity toward various proteins and thus substantially interfere with essential bacterial functions and rupture bacterial cell membranes in an "inside-out" manner, leading to robust antibacterial activities and suppression of drug resistance. Additionally, biochemical analysis of macromolecule binding affinity, cytoplasmic localization patterns, and bacterial stress responses suggests that this bacterial-specific intracellular aggregation mechanism is fundamentally different from nonselective classic DNA or membrane binding mechanisms. These mechanistic distinctions, along with the peptidomimetic's selective permeation of bacterial membranes, contribute to its favorable biocompatibility and pharmacokinetic properties, enabling its in vivo antimicrobial efficacy in several animal models, including mice-based superficial wound models, subcutaneous abscess models, and septicemia infection models. These results highlight the great promise of ligand-receptor interaction-induced intracellular aggregation in achieving a globally disruptive multitargeting effect, thereby offering potential applications in the treatment of malignant cells, including pathogens, tumor cells, and infected tissues.

Multi-targeting oligopyridiniums: Rational design for biofilm dispersion and bacterial persister eradication.

The development of effective antibacterial drugs to combat bacterial infections, particularly the biofilm-related infections, remains a challenge. There are two important features of bacterial biofilms, which are well-known critical factors causing biofilms hard-to-treat in clinical, including the dense and impermeable extracellular polymeric substances (EPS) and the metabolically repressed dormant and persistent bacterial population embedded. These characteristics largely increase the difficulty for regular antibiotic treatment due to insufficient penetration into EPS. In addition, the dormant bacteria are insensitive to the growth-inhibiting mechanism of traditional antibiotics. Herein, we explore the potential of a series of new oligopyridinium-based oligomers bearing a multi-biomacromolecule targeting function as the potent bacterial biofilm eradication agent. These oligomers were rationally designed to be "charge-on-backbone" that can offer a special alternating amphiphilicity. This novel and unique feature endows high affinity to bacterial membrane lipids, DNAs as well as proteins. Such a broad multi-targeting nature of molecules not only enables its penetration into EPS, but also plays vital roles in the bactericidal mechanism of action that is highly effective against dormant and persistent bacteria. Our in vitro, ex vivo, and in vivo studies demonstrated that OPc3, one of the most effective derivatives, was able to offer excellent antibacterial potency against a variety of bacteria and effectively eliminate biofilms in zebrafish models and mouse wound biofilm infection models.

Chemical Biology Approach to Reveal the Importance of Precise Subcellular Targeting for Intracellular Staphylococcus aureus Eradication.

Intracellular bacterial pathogens, such as Staphylococcus aureus, that may hide in intracellular vacuoles represent the most significant manifestation of bacterial persistence. They are critically associated with chronic infections and antibiotic resistance, as conventional antibiotics are ineffective against such intracellular persisters due to permeability issues and mechanistic reasons. Direct subcellular targeting of S. aureus vacuoles suggests an explicit opportunity for the eradication of these persisters, but a comprehensive understanding of the chemical biology nature and significance of precise S. aureus vacuole targeting remains limited. Here, we report an oligoguanidine-based peptidomimetic that effectively targets and eradicates intracellular S. aureus persisters in the phagolysosome lumen, and this oligomer was utilized to reveal the mechanistic insights linking precise targeting to intracellular antimicrobial efficacy. The oligomer has high cellular uptake via a receptor-mediated endocytosis pathway and colocalizes with S. aureus persisters in phagolysosomes as a result of endosome-lysosome interconversion and lysosome-phagosome fusion. Moreover, the observation of a bacterium's altered susceptibility to the oligomer following a modification in its intracellular localization offers direct evidence of the critical importance of precise intracellular targeting. In addition, eradication of intracellular S. aureus persisters was achieved by the oligomer's membrane/DNA dual-targeting mechanism of action; therefore, its effectiveness is not hampered by the hibernation state of the persisters. Such precise subcellular targeting of S. aureus vacuoles also increases the agent's biocompatibility by minimizing its interaction with other organelles, endowing excellent in vivo bacterial targeting and therapeutic efficacy in animal models.

Structural insights into the roles of the IcmS-IcmW complex in the type IVb secretion system of Legionella pneumophila.

The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers ∼300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS-IcmW recognizes effectors, and what the roles of IcmS-IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/ß fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS-IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL-IcmS-IcmW complex reveals extensive and highly stable interactions between DotL and IcmS-IcmW. Moreover, IcmS-IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS-IcmW also functions as an inseparable integral component of the DotL-T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS-IcmW complex in T4BSSs.

Subversion of Macrophage Functions by Bacterial Protein Toxins and Effectors.

Macrophages represent one of the first lines of host immune defenses against the invasion of pathogenic bacteria. Many receptors, immune signaling pathways and cellular processes in macrophages, including Toll-like receptors, Nod-like receptors, phagocytosis, autophagy and programmed cell death, are involved in combating the infection of bacterial pathogens. For efficient colonization in the host, bacterial pathogens have evolved diverse mechanisms to interfere with macrophage functions to evade host defenses. The major weapons utilized by bacterial pathogens are protein toxins and effectors secreted via specific bacterial secretion systems, including type I-VII secretion apparatuses. In recent years, great advances have been achieved in understanding how bacterial toxins and effectors subvert immune signaling and cellular processes of macrophages. In this review, we focus on the toxins and effectors that modulate the phagocytosis, intracellular immune signaling pathways, autophagy and programmed cell death processes of macrophages from the bacterium Legionella pneumophila, Shigella flexneri, Listeria monocytogenes, Salmonella spp., Yersinia spp., enteropathogenic E. coli and Mycobacterium tuberculosis.

Sequential Immunizations with Influenza Neuraminidase Protein Followed by Peptide Nanoclusters Induce Heterologous Protection.

Enhancing cross-protections against diverse influenza viruses is desired for influenza vaccinations. Neuraminidase (NA)-specific antibody responses have been found to independently correlate with a broader influenza protection spectrum. Here, we report a sequential immunization regimen that includes priming with NA protein followed by boosting with peptide nanoclusters, with which targeted enhancement of antibody responses in BALB/c mice to certain cross-protective B-cell epitopes of NA was achieved. The nanoclusters were fabricated via desolvation with absolute ethanol and were only composed of composite peptides. Unlike KLH conjugates, peptide nanoclusters would not induce influenza-unrelated immunity. We found that the incorporation of a hemagglutinin peptide of H2-d class II restriction into the composite peptides could be beneficial in enhancing the NA peptide-specific antibody response. Of note, boosters with N2 peptide nanoclusters induced stronger serum cross-reactivities to heterologous N2 and even heterosubtypic N7 and N9 than triple immunizations with the prototype recombinant tetrameric (rt) N2. The mouse challenge experiments with HK68 H3N2 also demonstrated the strong effectiveness of the peptide nanocluster boosters in conferring heterologous protection.

Disulfide-stabilized trimeric hemagglutinin ectodomains provide enhanced heterologous influenza protection.

Influenza virus infection poses a continual menace to public health. Here, we developed soluble trimeric HA ectodomain vaccines by establishing interprotomer disulfide bonds in the stem region, which effectively preserve the native antigenicity of stem epitopes. The stable trimeric H1 ectodomain proteins exhibited higher thermal stabilities in comparison with unmodified HAs and showed strong binding activities towards a panel of anti-stem cross-reactive antibodies that recognize either interprotomer or intraprotomer epitopes. Negative stain transmission electron microscopy (TEM) analysis revealed the stable trimer architecture of the interprotomer disulfide-stapled WA11#5, NC99#2, and FLD#1 proteins as well as the irregular aggregation of unmodified HA molecules. Immunizations of mice with those trimeric HA ectodomain vaccines formulated with incomplete Freund's adjuvant elicited significantly more potent cross-neutralizing antibody responses and offered broader immuno-protection against lethal infections with heterologous influenza strains compared to unmodified HA proteins. Additionally, the findings of our study indicate that elevated levels of HA stem-specific antibody responses correlate with strengthened cross-protections. Our design strategy has proven effective in trimerizing HA ectodomains derived from both influenza A and B viruses, thereby providing a valuable reference for designing future influenza HA immunogens.

Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

Neuraminidase is suggested as an important component for developing a universal influenza vaccine. Targeted induction of neuraminidase-specific broadly protective antibodies by vaccinations is challenging. To overcome this, we rationally select the highly conserved peptides from the consensus amino acid sequence of the globular head domains of neuraminidase. Inspired by the B cell receptor evolution process, a reliable sequential immunization regimen is designed to result in immuno-focusing by steering bulk immune responses to a selected region where broadly protective B lymphocyte epitopes reside. After priming neuraminidase protein-specific antibody responses in C57BL/6 or BALB/c inbred mice strains by immunization or pre-infection, boost immunizations with certain neuraminidase-derived peptide-keyhole limpet hemocyanin conjugates significantly strengthened serum neuraminidase inhibition activities and cross-protections. Overall, this study provides proof of concept for a peptide-based sequential immunization strategy for achieving targeted induction of cross-protective antibody response, which provides references for designing universal vaccines against other highly variable pathogens.

Amphiphilic Nano-Swords for Direct Penetration and Eradication of Pathogenic Bacterial Biofilms.

Bacterial biofilms are major causes of persistent and recurrent infections and implant failures. Biofilms are formable by most clinically important pathogens worldwide, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, causing recalcitrance to standard antibiotic therapy or anti-biofilm strategies due to amphiphilic impermeable extracellular polymeric substances (EPS) and the presence of resistant and persistent bacteria within the biofilm matrix. Herein, we report our design of an oligoamidine-based amphiphilic "nano-sword" with high structural compacity and rigidity. Its rigid, amphiphilic structure ensures effective penetration into EPS, and the membrane-DNA dual-targeting mechanism exerts strong bactericidal effect on the dormant bacterial persisters within biofilms. The potency of this oligoamidine is shown in two distinct modes of application: it may be used as a coating agent for polycaprolactone to fully inhibit surface biofilm growth in an implant-site mimicking micro-environment; meanwhile, it cures model mice of biofilm infections in various ex vivo and in vivo studies.

Chimeric Virus-like Particles Co-Displaying Hemagglutinin Stem and the C-Terminal Fragment of DnaK Confer Heterologous Influenza Protection in Mice.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.

Cooperative Membrane Damage as a Mechanism for Pentamidine-Antibiotic Mutual Sensitization.

Most Gram-positive-selective antibiotics have low activity against Gram-negative bacteria due to the presence of an outer membrane barrier. There is, therefore, interest in developing combination therapies that can penetrate the outer membrane (OM) with known antibiotics coupled with membrane-active sensitizing adjuvants. However, two unanswered questions hinder the development of such combination therapies: the sensitization spectrum of the sensitizer and the mechanism of antibiotic-sensitizer mutual potentiation. Here, with pentamidine as an example, we screened a library of 170 FDA-approved antibiotics in combination with pentamidine, a compound known to disturb the OM of Gram-negative bacteria. We found that four antibiotics, minocycline, linezolid, valnemulin, and nadifloxacin, displaced enhanced activity in combination with pentamidine against several multidrug-resistant Gram-negative bacteria. Through a descriptor-based structural-activity analysis and multiple cell-based biochemical assays, we found that hydrophobicity, partial charge, rigidity, and surface rugosity were key factors that affected sensitization via a cooperative membrane damage mechanism in which lipopolysaccharides and phospholipids were identified as sites of synergy. Finally, in vitro experiments showed that the linezolid-pentamidine combination slowed the generation of drug resistance, and there was also potent activity in in vivo experiments. Overall, our results highlight the importance of the physicochemical properties of antibiotics and cooperative membrane damage for synergistic pentamidine-antibiotic drug combinations.

A bacterial effector deubiquitinase specifically hydrolyses linear ubiquitin chains to inhibit host inflammatory signalling.

Linear ubiquitin (Ub) chains regulate many cellular processes, including NF-κB immune signalling. Pathogenic bacteria have evolved to secrete effector proteins that harbour deubiquitinase activity into host cells to disrupt host ubiquitination signalling. All previously identified effector deubiquitinases hydrolyse isopeptide-linked polyubiquitin (polyUb). It has been a long-standing question whether bacterial pathogens have evolved an effector deubiquitinase to directly cleave linear Ub chains. In this study, we performed extensive screening of bacterial pathogens and found that Legionella pneumophila-the causative agent of human Legionnaire's disease-encodes an effector protein, RavD, which harbours deubiquitinase activity exquisitely specific for linear Ub chains. RavD hydrolyses linear Ub chains but not any type of isopeptide-linked polyUb. The crystal structure of RavD with linear diubiquitin reveals that RavD adopts a papain-like fold with a Cys-His-Ser catalytic triad. The Ub-binding surface and specific interacting residues in RavD determine its specificity for Met1 linkages. RavD prevents the accumulation of linear Ub chains on Legionella-containing vacuoles established by the pathogen in host cells to inhibit the NF-κB pathway during infection. This study identified a unique linear Ub chain-specific effector deubiquitinase and indicates its potential application as a tool to dissect linear polyUb-mediated signalling in mammalian cells.

Nε-Fatty acylation of Rho GTPases by a MARTX toxin effector.

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are a family of large toxins that are extensively distributed in bacterial pathogens. MARTX toxins are autocatalytically cleaved to multiple effector domains, which are released into host cells to modulate the host signaling pathways. The Rho guanosine triphosphatase (GTPase) inactivation domain (RID), a conserved effector domain of MARTX toxins, is implicated in cell rounding by disrupting the host actin cytoskeleton. We found that the RID is an Nε-fatty acyltransferase that covalently modifies the lysine residues in the C-terminal polybasic region of Rho GTPases. The resulting fatty acylation inhibited Rho GTPases and disrupted Rho GTPase-mediated signaling in the host. Thus, RID can mediate the lysine Nε-fatty acylation of mammalian proteins and represents a family of toxins that harbor N-fatty acyltransferase activities in bacterial pathogens.