Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Dendritic cell targeting peptide plus Salmonella FliCd flagellin fused outer membrane protein H (OmpH) demonstrated increased efficacy against infections caused by different Pasteurella multocida serogroups in mouse models.

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliCd flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation.

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Mucosal TLR5 activation controls healthspan and longevity.

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

Structural basis of the human NAIP/NLRC4 inflammasome assembly and pathogen sensing.

The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.

Agonistic effect of peptides derived from a truncated HMGB1 acidic tail sequence in TLR5 from Salmo salar.

Based on the structural knowledge of TLR5 surface and using blind docking platforms, peptides derived from a truncated HMGB1 acidic tail from Salmo salar was designed as TLR5 agonistic. Additionally, a template peptide with the native N-terminal of the acidic tail sequence as a reference was included (SsOri). Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. The best peptides, termed 6WK and 5LWK, were selected for chemical synthesis and experimental functional assay. The agonist activity by immunoblotting and immunocytochemistry was determined following the NF-κBp65 phosphorylation (p-NF-κBp65) and the nuclear translocation of the NF-κBp65 subunit from the cytosol, respectively. HeLa cells stably expressing a S. salar TLR5 chimeric form (TLR5c7) showed increased p-NF-κBp65 levels regarding extracts from flagellin-treated cells. No statistically significant differences (p > 0.05) were found in the detected p-NF-κBp65 levels between cellular extracts treated with peptides or flagellin by one-way ANOVA. The image analysis of NF-κBp65 immunolabeled cells obtained by confocal microscopy showed increased nuclear NF-κBp65 co-localization in cells both 5LWK and flagellin stimulated, while 6WK and SsOri showed less effect on p65 nuclear translocation (p < 0.05). Also, an increased transcript expression profile of proinflammatory cytokines such as TNFα, IL-1ß, and IL-8 in HKL cells isolated from Salmo salar was evidenced in 5LWK - stimulated by RT-PCR analysis. Overall, the result indicates the usefulness of novel peptides as a potential immunostimulant in S. salar.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice.

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

Enhanced immunogenicity elicited by a novel DNA vaccine encoding the SARS-CoV-2 S1 protein fused to the optimized flagellin of Salmonella typhimurium in mice.

IMPORTANCE: The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.

The NtDEGP5 gene improves drought tolerance in tobacco (Nicotiana tabacum L.) by dampening plastid extracellular Ca2+ and flagellin signaling and thereby reducing ROS production.

Tobacco is an essential cash crop, but drought has become a major factor in the decline of global tobacco production as a result of changes in the global climate. The HtrA protease is an oligomeric serine endopeptidase that responds to stress in plants. DEGP5 is a member of the gene family that encodes HtrA protease, which promotes plant adaptation to adversity. The aim of this study was to investigate the role and mechanism employed by the DEGP5 gene in response to drought stress in tobacco. NtDEGP5-overexpression lines were obtained by genetic transformation and the phenotypes and transcriptomes of NtDEGP5-overexpression lines and wild-type (K326) tobacco seedlings were compared under drought stress. The results demonstrated that plants overexpressing NtDEGP5 exhibited greater drought tolerance. The differentially expressed genes involved in the regulation of drought tolerance by DEGP5 were enriched in metabolic pathways, such as plant-pathogen interaction and glutathione metabolism, with the plant-pathogen interaction pathway having the most differentially expressed genes. An analysis of the plant-pathogen interaction pathway revealed that these genes contributed to the suppression of plastid extracellular Ca2+ signaling and flagellin signaling to inhibit reactive oxygen species production, and that lower levels of reactive oxygen species act as a signal to regulate the activation of the antioxidant system, further balancing the production and removal of reactive oxygen species in tobacco seedlings under drought stress. These findings suggest that the NtDEGP5 gene can enhance the drought tolerance of tobacco by regulating the homeostasis of reactive oxygen species by inhibiting extracellular plastids.

Phages on filaments: A genetic screen elucidates the complex interactions between Salmonella enterica flagellin and bacteriophage Chi.

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such as Salmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to the S. enterica flagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certain Salmonella serotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of the Salmonella flagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility in S. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host genera Salmonella, Escherichia, or Serratia.

Targeting Desulfovibrio vulgaris flagellin-induced NAIP/NLRC4 inflammasome activation in macrophages attenuates ulcerative colitis.

INTRODUCTION: The perturbations of gut microbiota could interact with excessively activated immune responses and play key roles in the etiopathogenesis of ulcerative colitis (UC). Desulfovibrio, the most predominant sulfate reducing bacteria (SRB) resided in the human gut, was observed to overgrow in patients with UC. The interactions between specific gut microbiota and drugs and their impacts on UC treatment have not been demonstrated well. OBJECTIVES: This study aimed to elucidate whether Desulfovibrio vulgaris (D. vulgaris, DSV) and its flagellin could activate nucleotide-binding oligomerization domain-like receptors (NLR) family of apoptosis inhibitory proteins (NAIP) / NLR family caspase activation and recruitment domain-containing protein 4 (NLRC4) inflammasome and promote colitis, and further evaluate the efficacy of eugeniin targeting the interaction interface of D. vulgaris flagellin (DVF) and NAIP to attenuate UC. METHODS: The abundance of DSV and the occurrence of macrophage pyroptosis in human UC tissues were investigated. Colitis in mice was established by dextran sulfate sodium (DSS) and gavaged with DSV or its purified flagellin. NAIP/NLRC4 inflammasome activation and macrophage pyroptosis were evaluated in vivo and in vitro. The effects of eugeniin on blocking the interaction of DVF and NAIP/NLRC4 and relieving colitis were also assessed. RESULTS: The abundance of DSV increased in the feces of patients with UC and was found to be associated with disease activity. DSV and its flagellin facilitated DSS-induced colitis in mice. Mechanistically, RNA sequencing showed that gene expression associated with inflammasome complex and pyroptosis was upregulated after DVF treatment in macrophages. DVF was further demonstrated to induce significant macrophage pyroptosis in vitro, depending on NAIP/NLRC4 inflammasome activation. Furthermore, eugeniin was screened as an inhibitor of the interface between DVF and NAIP and successfully alleviated the proinflammatory effect of DVF in colitis. CONCLUSION: Targeting DVF-induced NAIP/NLRC4 inflammasome activation and macrophage pyroptosis ameliorates UC. This finding is of great significance for exploring the gut microbiota-host interactions in UC development and providing new insights for precise treatment.

The evolution of archaeal flagellar filaments.

Flagellar motility has independently arisen three times during evolution: in bacteria, archaea, and eukaryotes. In prokaryotes, the supercoiled flagellar filaments are composed largely of a single protein, bacterial or archaeal flagellin, although these two proteins are not homologous, while in eukaryotes, the flagellum contains hundreds of proteins. Archaeal flagellin and archaeal type IV pilin are homologous, but how archaeal flagellar filaments (AFFs) and archaeal type IV pili (AT4Ps) diverged is not understood, in part, due to the paucity of structures for AFFs and AT4Ps. Despite having similar structures, AFFs supercoil, while AT4Ps do not, and supercoiling is essential for the function of AFFs. We used cryo-electron microscopy to determine the atomic structure of two additional AT4Ps and reanalyzed previous structures. We find that all AFFs have a prominent 10-strand packing, while AT4Ps show a striking structural diversity in their subunit packing. A clear distinction between all AFF and all AT4P structures involves the extension of the N-terminal α-helix with polar residues in the AFFs. Additionally, we characterize a flagellar-like AT4P from Pyrobaculum calidifontis with filament and subunit structure similar to that of AFFs which can be viewed as an evolutionary link, showing how the structural diversity of AT4Ps likely allowed for an AT4P to evolve into a supercoiling AFF.

Extracellular transfer of a conserved polymerization factor for multi-flagellin filament assembly in Caulobacter.

Unidirectional growth of filamentous protein assemblies including the bacterial flagellum relies on dedicated polymerization factors (PFs). The molecular determinants and structural transitions imposed by PFs on multi-subunit assembly are poorly understood. Here, we unveil FlaY from the polarized α-proteobacterium Caulobacter crescentus as a defining member of an alternative class of specialized flagellin PFs. Unlike the paradigmatic FliD capping protein, FlaY relies on a funnel-like ß-propeller fold for flagellin polymerization. FlaY binds flagellin and is secreted by the flagellar secretion apparatus, yet it can also promote flagellin polymerization exogenously when donated from flagellin-deficient cells, serving as a transferable, extracellular public good. While the surge in FlaY abundance precedes bulk flagellin synthesis, FlaY-independent filament assembly is enhanced by mutation of a conserved region in multiple flagellin paralogs. We suggest that FlaYs are (multi-)flagellin PFs that evolved convergently to FliDs yet appropriated the versatile ß-propeller fold implicated in human diseases for chaperone-assisted filament assembly.

Biophysical and proteomic analyses of Pseudomonas syringae pv. tomato DC3000 extracellular vesicles suggest adaptive functions during plant infection.

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

An unbroken network of interactions connecting flagellin domains is required for motility in viscous environments.

In its simplest form, bacterial flagellar filaments are composed of flagellin proteins with just two helical inner domains, which together comprise the filament core. Although this minimal filament is sufficient to provide motility in many flagellated bacteria, most bacteria produce flagella composed of flagellin proteins with one or more outer domains arranged in a variety of supramolecular architectures radiating from the inner core. Flagellin outer domains are known to be involved in adhesion, proteolysis and immune evasion but have not been thought to be required for motility. Here we show that in the Pseudomonas aeruginosa PAO1 strain, a bacterium that forms a ridged filament with a dimerization of its flagellin outer domains, motility is categorically dependent on these flagellin outer domains. Moreover, a comprehensive network of intermolecular interactions connecting the inner domains to the outer domains, the outer domains to one another, and the outer domains back to the inner domain filament core, is required for motility. This inter-domain connectivity confers PAO1 flagella with increased stability, essential for its motility in viscous environments. Additionally, we find that such ridged flagellar filaments are not unique to Pseudomonas but are, instead, present throughout diverse bacterial phyla.