Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence.

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein IpaD. Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

Catheter-associated urinary tract infection by Pseudomonas aeruginosa progresses through acute and chronic phases of infection.

Healthcare-associated infections are major causes of complications that lead to extended hospital stays and significant medical costs. The use of medical devices, including catheters, increases the risk of bacterial colonization and infection through the presence of a foreign surface. Two outcomes are observed for catheterized patients: catheter-associated asymptomatic bacteriuria and catheter-associated urinary tract infection (CAUTI). However, the relationship between these two events remains unclear. To understand this relationship, we studied a murine model of Pseudomonas aeruginosa CAUTI. In this model, we also observe two outcomes in infected animals: acute symptoms that is associated with CAUTI and chronic colonization that is associated with asymptomatic bacteriuria. The timing of the acute outcome takes place in the first week of infection, whereas chronic colonization occurs in the second week of infection. We further showed that mutants lacking genes encoding type III secretion system (T3SS), T3SS effector proteins, T3SS injection pore, or T3SS transcriptional activation all fail to cause acute symptoms of CAUTI. Nonetheless, all mutants defective for T3SS colonized the catheter and bladders at levels similar to the parental strain. In contrast, through induction of the T3SS master regulator ExsA, all infected animals showed acute phenotypes with bacteremia. Our results demonstrated that the acute symptoms, which are analogous to CAUTI, and chronic colonization, which is analogous to asymptomatic bacteriuria, are independent events that require distinct bacterial virulence factors. Experimental delineation of asymptomatic bacteriuria and CAUTI informs different strategies for the treatment and intervention of device-associated infections.

Mapping the functional B-cell epitopes of Shigella invasion plasmid antigen D (IpaD).

Shigella bacteria utilize the type III secretion system (T3SS) to invade host cells and establish local infection. Invasion plasmid antigen D (IpaD), a component of Shigella T3SS, has garnered extensive interest as a vaccine target, primarily due to its pivotal role in the Shigella invasion, immunogenic property, and a high degree of conservation across Shigella species and serotypes. Currently, we are developing an epitope- and structure-based multivalent vaccine against shigellosis and require functional epitope antigens of key Shigella virulence determinants including IpaD. However, individual IpaD B-cell epitopes, their contributions to the overall immunogenicity, and functional activities attributing to bacteria invasion have not been fully characterized. In this study, we predicted continuous B-cell epitopes in silico and fused each epitope to a carrier protein. Then, we immunized mice intramuscularly with each epitope fusion protein, examined the IpaD-specific antibody responses, and measured antibodies from each epitope fusion for the activity against Shigella invasion in vitro. Data showed that all epitope fusion proteins induced similar levels of anti-IpaD IgG antibodies in mice, and differences were noted for antibody inhibition activity against Shigella invasion. IpaD epitope 1 (SPGGNDGNSV), IpaD epitope 2 (LGGNGEVVLDNA), and IpaD epitope 5 (SPNNTNGSSTET) induced antibodies significantly better in inhibiting invasion from Shigella flexneri 2a, and epitopes 1 and 5 elicited antibodies more effectively at preventing invasion of Shigella sonnei. These results suggest that IpaD epitopes 1 and 5 can be the IpaD representative antigens for epitope-based polyvalent protein construction and protein-based cross-protective Shigella vaccine development.IMPORTANCEShigella is a leading cause of diarrhea in children younger than 5 years in developing countries (children's diarrhea) and continues to be a major threat to public health. No licensed vaccines are currently available against the heterogeneous Shigella species and serotype strains. Aiming to develop a cross-protective multivalent vaccine against shigellosis and dysentery, we applied novel multiepitope fusion antigen (MEFA) technology to construct a broadly immunogenic polyvalent protein antigen, by presenting functional epitopes of multiple Shigella virulence determinants on a backbone protein. The functional IpaD epitopes identified from this study will essentially allow us to construct an optimal polyvalent Shigella immunogen, leading to the development of a cross-protective vaccine against shigellosis (and dysentery) and the improvement of global health. In addition, identifying functional epitopes from heterogeneous virulence determinants and using them as antigenic representatives for the development of cross-protective multivalent vaccines can be applied generally in vaccine development.

Construction of a constitutively active type III secretion system for heterologous protein secretion.

Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells and inclusion bodies, purified, and resolubilized, which adds significant time and cost to the protein-manufacturing process. The Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) has been engineered to address these problems by secreting soluble, active proteins directly into the culture media, reducing the number of purification steps. However, the current best practices method of T3SS pathway activation is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer (IPTG) to the culture media. IPTG adds significant cost to production and plasmid-based expression is subject to instability in large-scale fermentation. Here, we modulate the upstream transcriptional regulator, hilD, to activate the T3SS via three distinct methods. In doing so, we develop a toolbox of T3SS activation methods and construct constitutively active T3SS strains capable of secreting a range of heterologous proteins at titers comparable to plasmid-based hilA overexpression. We also explore how each activation method in our toolbox impacts the SPI-1 regulatory cascade and discover an epistatic relationship between T3SS regulators, hilE and the hilD 3' untranslated region (hilD 3'UTR). Together, these findings further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. KEY POINTS: • Characterized 3 new type III secretion system (T3SS) activation methods for heterologous protein secretion, including 2 constitutive activation methods. • Eliminated the need for a second plasmid and a small molecule inducer to activate the system, making it more suitable for industrial production. • Discovered new regulatory insights into the SPI-1 T3SS, including an epistatic relationship between regulators hilE and the hilD 3' untranslated region.

A novel strategy to control Pseudomonas syringae through inhibition of type III secretion system.

Pseudomonas syringae (P. syringae) is a highly prevalent Gram-negative pathogen with over 60 pathogenic variants that cause yield losses of up to 80% in various crops. Traditional control methods mainly involve the application of antibiotics to inactivate pathogenic bacteria, but large-scale application of antibiotics has led to the development of bacterial resistance. Gram-negative pathogens including P. syringae commonly use the type III secretion system (T3SS) as a transport channel to deliver effector proteins into host cells, disrupting host defences and facilitating virulence, providing a novel target for antibacterial drug development. In this study, we constructed a high-throughput screening reporter system based on our previous work to screen for imidazole, oxazole and thiazole compounds. The screening indicated that the three compounds (II-14, II-15 and II-24) significantly inhibited hrpW and hrpL gene promoter activity without influencing the growth of P. syringae, and the inhibitory activity was better than that of the positive control sulforaphane (4-methylsulfinylbutyl isothiocyanate, SFN) at 50 µM. Three compounds suppressed the transcript levels of representative T3SS genes to different degrees, suggesting that the compounds may suppress the expression of T3SS by modulating the HrpR/S-HrpL regulatory pathway. Inoculation experiments indicated that all three compounds suppressed the pathogenicity of Pseudomonas syringae pv. tomato DC3000 in tomato and Pseudomonas syringae pv. phaseolicola 1448A in bean to varying degrees. One representative compound, II-15, significantly inhibited the secretion of the Pst DC3000 AvrPto effector protein. These findings provide a theoretical basis for the development of novel P. syringae T3SS inhibitors for application in disease prevention and control.

Host manipulation by bacterial type III and type IV secretion system effector proteases.

Proteases are powerful enzymes, which cleave peptide bonds, leading most of the time to irreversible fragmentation or degradation of their substrates. Therefore they control many critical cell fate decisions in eukaryotes. Bacterial pathogens exploit this power and deliver protease effectors through specialised secretion systems into host cells. Research over the past years revealed that the functions of protease effectors during infection are diverse, reflecting the lifestyles and adaptations to specific hosts; however, only a small number of peptidase families seem to have given rise to most of these protease virulence factors by the evolution of different substrate-binding specificities, intracellular activation and subcellular targeting mechanisms. Here, we review our current knowledge about the enzymology and function of protease effectors, which Gram-negative bacterial pathogens translocate via type III and IV secretion systems to irreversibly manipulate host processes. We highlight emerging concepts such as signalling by protease cleavage products and effector-triggered immunity, which host cells employ to detect and defend themselves against a protease attack. TAKE AWAY: Proteases irreversibly cleave proteins to control critical cell fate decisions. Gram-negative bacteria use type III and IV secretion systems to inject effectors. Protease effectors are integral weapons for the manipulation of host processes. Effectors evolved from few peptidase families to target diverse substrates. Effector-triggered immunity upon proteolytic attack emerges as host defence.

Insights into the structure-activity relationship of a type III secretion system inhibitor, aurodox.

Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.

Coordinate regulation of carbohydrate metabolism and virulence by PtsH in pathogen Edwardsiella piscicida.

Carbohydrate metabolism of bacterial pathogens conducts crucial roles in regulating pathogenesis but the molecular mechanisms by which metabolisms and virulence are been modulated and coordinated remain to be illuminated. Here, we investigated in this regard Edwardsiella piscicida, a notorious zoonotic pathogen previously named E. tarda that could ferment very few PTS sugars including glucose, fructose, mannose, N-acetylglucosamine, and N-acetylgalactosamine. We systematically characterized the roles of each of the predicted 23 components of phosphotransferase system (PTS) with the respective in-frame deletion mutants and defined medium containing specific PTS sugar. In addition, PtsH was identified as the crucial PTS component potentiating the utilization of all the tested PTS sugars. Intriguingly, we also found that PtsH while not Fpr was involved in T3SS gene expression and was essential for the pathogenesis of E. piscicida. To corroborate this, His15 and Ser46, the two established PtsH residues involved in phosphorylation cascade, showed redundant roles in regulating T3SS yields. Moreover, PtsH was shown to facilitate mannose uptake and transform it into mannose-6-phosphate, an allosteric substrate established to activate EvrA to augment bacterial virulence. Collectively, our observations provide new insights into the roles of PTS reciprocally regulating carbohydrate metabolism and virulence gene expression. KEY POINTS: • PTS components' roles for sugar uptake are systematically determined in Edwardsiella piscicida. • PtsH is involved in saccharides uptake and in the regulation of E. piscicida's T3SS expression. • PtsH phosphorylation at His15 and Ser46 is essential for the T3SS expression and virulence.

Type III effector provides a novel symbiotic pathway in legume-rhizobia symbiosis.

Rhizobia form nodules on the roots of legumes and fix atmospheric nitrogen into ammonia, thus supplying it to host legumes. In return, plants supply photosynthetic products to maintain rhizobial activities. In most cases, rhizobial Nod factors (NFs) and their leguminous receptors (NFRs) are essential for the establishment of symbiosis. However, recent studies have discovered a novel symbiotic pathway in which rhizobia utilize the type III effectors (T3Es) similar to the pathogenic bacteria to induce nodulation. The T3Es of rhizobia are thought to be evolved from the pathogen, but they have a unique structure distinct from the pathogen, suggesting that it might be customized for symbiotic purposes. This review will focus on the recent findings from the study of rhizobial T3Es, discussing their features on a symbiont and pathogen, and the future perspectives on the role of rhizobial T3Es in symbiosis control technology.

Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in elongation factor, fusA1.

Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression.

The bacterial metalloprotease NleD selectively cleaves mitogen-activated protein kinases that have high flexibility in their activation loop.

Microbial pathogens often target the host mitogen-activated protein kinase (MAPK) network to suppress host immune responses. We previously identified a bacterial type III secretion system effector, termed NleD, a metalloprotease that inactivates MAPKs by specifically cleaving their activation loop. Here, we show that NleDs form a growing family of virulence factors harbored by human and plant pathogens as well as insect symbionts. These NleDs disable specifically Jun N-terminal kinases (JNKs) and p38s that are required for host immune response, whereas extracellular signal-regulated kinase (ERK), which is essential for host cell viability, remains intact. We investigated the mechanism that makes ERK resistant to NleD cleavage. Biochemical and structural analyses revealed that NleD exclusively targets activation loops with high conformational flexibility. Accordingly, NleD cleaved the flexible loops of JNK and p38 but not the rigid loop of ERK. Our findings elucidate a compelling mechanism of native substrate proteolysis that is promoted by entropy-driven specificity. We propose that such entropy-based selectivity is a general attribute of proteolytic enzymes.

Genomic analysis of the nomenclatural type strain of the nematode-associated entomopathogenic bacterium Providencia vermicola.

BACKGROUND: Enterobacteria of the genus Providencia are mainly known as opportunistic human pathogens but have been isolated from highly diverse natural environments. The species Providencia vermicola comprises insect pathogenic bacteria carried by entomoparasitic nematodes and is investigated as a possible insect biocontrol agent. The recent publication of several genome sequences from bacteria assigned to this species has given rise to inconsistent preliminary results. RESULTS: The genome of the nematode-derived P. vermicola type strain DSM_17385 has been assembled into a 4.2 Mb sequence comprising 5 scaffolds and 13 contigs. A total of 3969 protein-encoding genes were identified. Multilocus sequence typing with different marker sets revealed that none of the previously published presumed P. vermicola genomes represents this taxonomic species. Comparative genomic analysis has confirmed a close phylogenetic relationship of P. vermicola to the P. rettgeri species complex. P. vermicola DSM_17385 carries a type III secretion system (T3SS-1) with probable function in host cell invasion or intracellular survival. Potentially antibiotic resistance-associated genes comprising numerous efflux pumps and point-mutated house-keeping genes, have been identified across the P. vermicola genome. A single small (3.7 kb) plasmid identified, pPVER1, structurally belongs to the qnrD-type family of fluoroquinolone resistance conferring plasmids that is prominent in Providencia and Proteus bacteria, but lacks the qnrD resistance gene. CONCLUSIONS: The sequence reported represents the first well-supported published genome for the taxonomic species P. vermicola to be used as reference in further comparative genomics studies on Providencia bacteria. Due to a striking difference in the type of injectisome encoded by the respective genomes, P. vermicola might operate a fundamentally different mechanism of entomopathogenicity when compared to insect-pathogenic Providencia sneebia or Providencia burhodogranariea. The complete absence of antibiotic resistance gene carrying plasmids or mobile genetic elements as those causing multi drug resistance phenomena in clinical Providencia strains, is consistent with the invertebrate pathogen P. vermicola being in its natural environment efficiently excluded from the propagation routes of multidrug resistance (MDR) carrying genetic elements operating between human pathogens. Susceptibility to MDR plasmid acquisition will likely become a major criterion in the evaluation of P. vermicola for potential applications in biological pest control.

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles.

Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell's metabolism for the benefit of the bacterium to bypassing the host's defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.

Type III Secretion: Building and Operating a Remarkable Nanomachine.

The Type III secretion system (T3SS) is a protein export pathway that is widespread in Gram-negative bacteria and delivers effector proteins directly into eukaryotic cells. At its core lie the injectisome (a sophisticated transmembrane secretion apparatus) and a complex network of specialized chaperones that target secretory proteins to the antechamber of the injectisome. The assembly of the system, and the subsequent secretion of proteins through it, undergo fine-tuned, hierarchical regulation. Here, we present the current understanding of the injectisome assembly process, secretion hierarchy, and the role of chaperones. We discuss these events in light of available structural and biochemical dissection and propose future directions essential to revealing mechanistic insight into this fascinating nanomachine.

Chaperone-mediated secretion switching from early to middle substrates in the type III secretion system encoded by Salmonella pathogenicity island 2.

The bacterial type III secretion system (T3SS) delivers virulence proteins, called effectors, into eukaryotic cells. T3SS comprises a transmembrane secretion apparatus and a complex network of specialized chaperones that target protein substrates to this secretion apparatus. However, the regulation of secretion switching from early (needle and inner rod) to middle (tip/filament and translocators) substrates is incompletely understood. Here, we investigated chaperone-mediated secretion switching from early to middle substrates in the T3SS encoded by Salmonella pathogenicity island 2 (SPI2), essential for systemic infection. Our findings revealed that the protein encoded by ssaH regulates the secretion of an inner rod and early substrate, SsaI. Structural modeling revealed that SsaH is structurally similar to class III chaperones, known to associate with proteins in various pathogenic bacteria. The SPI2 protein SsaE was identified as a class V chaperone homolog and partner of SsaH. A pulldown analysis disclosed that SsaH and SsaE form a heterodimer, which interacted with another early substrate, the needle protein SsaG. Moreover, SsaE also helped stabilize SsaH and a middle substrate, SseB. We also found that SsaE regulates cellular SsaH levels to translocate the early substrates SsaG and SsaI and then promotes the translocation of SseB by stabilizing it. In summary, our results indicate that the class III chaperone SsaH facilitates SsaI secretion, and a heterodimer of SsaH and the type V chaperone SsaE then switches secretion to SsaG. This is the first report of a chaperone system that regulates both early and middle substrates during substrate switching for T3SS assembly.

The cytoplasmic domain of MxiG interacts with MxiK and directs assembly of the sorting platform in the Shigella type III secretion system.

Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion. Studies using bacterial "minicells" have revealed an unprecedented level of structural detail of the sorting platform; however, many of the structure-function relationships within this complex remain enigmatic. Here, we report on improved cryo-electron tomographic approaches to enhance the resolution of the Shigella T3SA sorting platform (at ≤2 nm resolution) done in concert with biochemical and genetic methods to define the sorting platform interactome and interactions with the T3SA inner membrane ring (IR). We observed that the sorting platform consists of "pods" with 6-fold symmetry that interact with the Spa47 ATPase via radial extensions comprising MxiN. Most importantly, MxiK maintained an interaction with the IR via specific interactions with the cytoplasmic domain of the IR protein MxiG (MxiGC), which is a noncanonical forkhead-associated domain, and MxiK has an elongated structure that interacts with the IR via MxiGC T4 lysozyme-mediated insertional mutagenesis of MxiK revealed its orientation within the sorting platform and enabled disruption of interactions with its binding partners, which abolished sorting platform assembly. Finally, a comparison with the homologous interactions in the Salmonella T3SS sorting platform revealed clear differences in their IR-sorting platform interfaces that have possible mechanistic implications.

The Sinorhizobium fredii HH103 type III secretion system effector NopC blocks nodulation with Lotus japonicus Gifu.

The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis.

Biochemical properties and in planta effects of NopM, a rhizobial E3 ubiquitin ligase.

Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

My winding trail while fulfilling my love for science and family.

My winding path toward a career in science was awkward, like an adolescent finding an identity. It did not follow a classic course; it had many interruptions, complications, and challenges. It also involved a bit of luck and extremely supportive colleagues, mentors, and family, including my husband, children, and in-laws. I was inspired to tell my story here because I met a young woman interviewing in 2018 for graduate school who is growing up with the same complicated family expectations, social challenges, love for science, and desire to be a scientist as I had four decades ago. Her future is uncertain, because her chosen academic path is not encouraged by those around her. We, as a society, must find ways to encourage, promote, enable, and give strength to those who want to follow their dreams, despite facing many challenges in their lives. Here are some things I learned on my career path that I hope might be helpful for others.

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin.

Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 3'5'-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.