Biology subjects

Wu, C.-F.

Publications and source records attributed to Wu, C.-F..

18 recordsLinked to original sources

bioRxiv2026

Abiotic cues modulate exopolysaccharides to influence type VI secretion system-mediated antagonism

The type VI secretion system (T6SS) enables Gram-negative bacteria to inject toxic effectors into neighboring cells, mediating contact-dependent antagonism and interbacterial competition. How the T6SS-mediated attack responds to environmental cues varies and remains unclear among different bacteria. Here, using Agrobacterium fabrum C58, a soil-borne phytopathogenic bacterium, we investigated the impact of osmolarity, moisture, surface stiffness, and glucose on T6SS-mediated antagonism. We show that these abiotic factors influenced the production of two polysaccharides, cyclic-{beta}-(1,2)-glucan (C{beta}G) and succinoglycan (SG), and modulated the T6SS-killing outcome. Mechanistically, high osmolarity inhibits C{beta}G production, thereby enhancing the expression and secretion of the T6SS. In contrast, SG biosynthesis, in response to moisture, surface stiffness, and glucose, does not impact T6SS expression and function but decreases T6SS-mediated killing efficacy. Electron microscopy revealed that SG creates a physical barrier between bacterial cells. Such physical distancing not only hinders the T6SS attack from Agrobacterium, but also confers protection against other competitors at both intra- and inter-species levels. Our results unravel the complexity of how specific environmental factors modulate the contact-dependent antagonism and highlight a balance between offensive and defensive behaviors. Significance StatementBacteria live in polymicrobial communities where they often need to fight off competitors to survive. One well-characterized weapon is the type VI secretion system (T6SS), a nanomachine mediating contact-dependent antagonism. In this study, we aim to study how the T6SS attack is influenced by environmental cues. We discovered that carbon sources, osmolarity, and surface stiffness modulate the T6SS efficacy through the secretion of sugar chains outside the cell. This sugar secretion increases the physical distance between cells, protecting against T6SS-mediated attack. However, such physical distancing also hinders the efficacy of the T6SS attack originating from Agrobacterium itself. Our results reveal a previously understudied offense-defense tradeoff between EPS production and T6SS-mediated attack. This finding underscores the intricate balance between bacterial offense and defense strategies.

microbiology↗

bioRxiv2026

Effects of oxidative stress and aging on nerve, muscle, and synapse in a male-specific abdominal neuromuscular junction in Drosophila

Defects in Drosophila Cu2+/Zn2+ superoxide dismutase (encoded by the gene Sod1) lead to elevated oxidative stress and a drastically shortened lifespan. To contrast the effects of aging and oxidative stress on nerve conduction, synaptic transmission, and muscle excitability, we developed an easily accessible adult abdominal neuromuscular preparation, utilizing the male-specific Muscle of Lawrence (MOL) in Drosophila. The large size of MOL facilitated analyses of presynaptic nerve signals and postsynaptic responses that could result in sizable excitatory junctional potentials (EJPs) evoking full-blown muscle action potentials (APs) which were terminated rapidly by a characteristic afterhyperpolarization (AHP). Aged wild-type (WT) individuals (80 days or older) exhibited diminished neuromuscular transmission, mainly reflecting declines in motor axon conduction, with synaptic transmission remaining largely intact (since robust EJPs could still be evoked when nerve terminals were directly stimulated electrotonically). Additionally, muscle APs showed enhanced depolarizing peaks and weakened AHPs during current injection, suggesting weakening in repolarizing K+ currents. Chronologically younger Sod1 mutants (up to 30 days) displayed similar trends of neuromuscular changes, confirming a major role of oxidative stress in aging. However, certain distinctions exist in muscle membrane properties and transmitter release machinery. A clear increase in muscle membrane resistance was seen in Sod1 but not in aged WT. Additionally, unlike normal spontaneous release of synaptic vesicles leading to miniature EJPs (mEJPs), extremely enlarged spontaneous transmitter discharges occurred in aged WT but was never seen in Sod1, indicating a distinct, aging-specific alteration in transmitter release regulation. Notably, our work revealed considerable variation among individuals, ranging from transmission failure to largely intact neuromuscular functions, demonstrating the stochastic nature of functional declines due to aging and oxidative stress. Moreover, this study uncovered a well-defined common vulnerability, i.e. weakening of the Ca2+-activated BK current that caused drastic reduction in AHP in both aged WT and Sod1 mutants, as confirmed by their diminishing sensitivity to the BK channel blocker paxilline, which caused striking alterations in the AHP in WT control.

neuroscience↗

bioRxiv2025

Genetic vitamin B6 deficiency and alcohol interaction in behavior and metabolism

Alcohol abuse is a leading cause of preventable deaths, affecting brain function and metabolism, including GABA transmission and vitamin B6 (VB6) levels. However, the interaction between genetic VB6 deficiency and alcohol consumption remains unexplored. Here, we utilized Drosophila models with mutations in pyridox(am)ine-5-phosphate oxidase (PNPO), a key enzyme in VB6 metabolism, to examine this interaction at behavioral and biochemical levels. Our findings demonstrate that PNPO deficiency reduces alcohol aversion, increases consumption, and alters locomotor behavior. Biochemically, PNPO deficiency and alcohol exposure converge on amino acid metabolism, elevating inhibitory neurotransmitters GABA and glycine. Moreover, both PNPO deficiency and alcohol exposure lead to lethality with significant interaction, which can be rescued by VB6 supplementation. These results highlight a functional interaction between genetic VB6 deficiency and alcohol, suggesting potential therapeutic strategies for alcohol-related behaviors.

animal behavior and cognition↗

bioRxiv2024

Identification of a novel mycovirus belonging to the ''flexivirus''-related family with icosahedral virion

The order Tymovirales currently comprises five viral families with positive-sense RNA ((+)RNA) genomes that infect plants, fungi, and insects. Virion morphologies within the order Tymovirales differ between families, with icosahedral virions in the Tymoviridae and filamentous virions in the other "flexi"viridae families. Despite their different morphologies, these viruses are placed in the same order based on phylogenetic analyses of replicase-associated polyproteins. However, one of the families in the Tymovirales, Deltaflexiviridae, is considered to be capsidless because there have been no published reports of virion isolation. Here we report that a new "flexivirus"-related (+)RNA virus, prospectively named Fusarium oxysporum icosahedral virus 1 (FoIV1), is icosahedral, and that most deltaflexiviruses may have icosahedral virions. Phylogenetic analyses based on replicase-associated polyproteins indicated that FoIV1 forms a distinct group in the Tymovirales with some viruses originally assigned to the Deltaflexiviridae. Electron microscopy, protein analysis, and protein structure predictions indicate that FoIV1 ORF4 encodes a single jelly-roll (SJR)-like coat protein (CP) that constitutes the icosahedral virions. Results of clustering analyses based on amino acid sequences and predicted CP structures suggested that most of the deltaflexiviruses have icosahedral virions composed of SJR-like CPs as in FoIV1, rather than having filamentous virions or capsidless. These results challenge the conventional understanding of viruses in the order Tymovirales, with important implications for revising its taxonomic framework and providing insights into the evolutionary relationships within this diverse and broad host range group of (+)RNA viruses.

microbiology↗

bioRxiv2024

Simultaneous GCaMP imaging and focal recording of tonic and phasic synapses: Probing short-term plasticity within a defined microenvironment

Key point summaryO_LIWe developed a protocol for stable simultaneous focal recording and GCaMP imaging on identified motor synaptic boutons in the Drosophila neuromuscular preparation with minimized muscular movements in extended concentration ranges of extracellular Ca2+ and Sr2+. C_LIO_LIThis approach directly demonstrated temporal correlation between the dynamics of cytosolic residual Ca2+ and activity-dependent synaptic plasticity in tonic and phasic synapses. C_LIO_LIOur data demonstrated presynaptic GCaMP signals markedly lagged behind and poorly correlated with the concurrent transmitter release, but reliably indicated the immediate states of short-term plasticity. GCaMPs physical-chemical properties allow information extraction on cytosolic Ca2+ levels during facilitation and depression phases. C_LIO_LIThe decay phase of GCaMP signal often coincided with lingering vesicular releases after stimulation, more pronounced in phasic than tonic synapses and exaggerated in Sr2+-containing saline. Lingering releases were coupled with the tendency of asynchronous transmission. C_LI GCaMP fluorescence has been widely used to monitor intracellular Ca2+. However, the physiological significance of the GCaMP signal in presynaptic terminals remains to be further elucidated. We investigated how the dynamics of GCaMP signals correlates with the activity dependence of short-term plasticity in synaptic transmission. We devised a local manipulation protocol that minimizes interference from muscular contraction during simultaneous Ca2+ imaging and focal recording at the Drosophila larval neuromuscular junction (NMJ), where the tonic and phasic excitatory synapses can be compared side-by-side. By confining the local ionic microenvironment, this protocol enabled stable measurements across extended concentration ranges of Ca2+ or Sr2+ in saline. Compared to tonic synapses, phasic synapses displayed stronger GCaMP signals, along with faster facilitation and more severe depression. Upon repetitive stimulation (40 Hz), facilitation of transmission occurred during or immediately prior to the early rising phase (0.25 s) of the GCaMP signal, which could subsequently convert into a depression phase of transmission decline, most evident during a steeper and longer rise of GCaMP signals in higher Ca2+ saline. Typically, deepest depression occurred when GCaMP signals rose to a plateau. Phasic synapses with stronger GCaMP signal and deeper depression, more often exhibited lingering post-stimulation releases. In both tonic and phasic synapses, replacing Ca2+ with Sr2+ induced extreme asynchronous transmission coupled with post-stimulation lingering releases during the decay of GCaMP signals. Further applications of this focal recording-local manipulation protocol may help to probe additional mechanisms underlying synaptic transmission and plasticity.

neuroscience↗

bioRxiv2024

Cell-type-specific fluorescent tagging of endogenous target proteins reveals synaptic enrichment and dynamic regulations of dopamine receptors

Dopamine can play opposing physiological roles depending on the receptor subtype. In the fruit fly Drosophila melanogaster, Dop1R1 and Dop2R encode the D1- and D2-like receptors, respectively, and are reported to oppositely regulate intracellular cAMP levels. Here, we profiled the expression and subcellular localization of endogenous Dop1R1 and Dop2R in specific cell types in the mushroom body circuit. For cell-type-specific visualization of endogenous proteins, we employed reconstitution of split-GFP tagged to the receptor proteins. We detected dopamine receptors at both presynaptic and postsynaptic sites in multiple cell types. Quantitative analysis revealed enrichment of both receptors at the presynaptic sites, with Dop2R showing a greater degree of localization than Dop1R1. The presynaptic localization of Dop1R1 and Dop2R in dopamine neurons suggests dual feedback regulation as autoreceptors. Furthermore, we discovered a starvation-dependent, bidirectional modulation of the presynaptic receptor expression in the PAM and PPL1 clusters, two distinct subsets of dopamine neurons, suggesting regulation of appetitive behaviors. Our results highlight the significance of the co-expression of the two opposing dopamine receptors in the spatial and conditional regulation of dopamine responses in neurons.

neuroscience↗

bioRxiv2022

Intense light unleashes male courtship behaviorin wild-type Drosophila

Drosophila courtship studies have elucidated several principles of the neurogenetic organization of complex behavior. Through an integration across sensory modalities, males perform stereotypic patterns of chasing, courtship song production, and copulation attempts. Here we report a serendipitous finding that intense light not only enhances courtship toward female targets but also triggers unexpected courtship behaviors among male flies. Strikingly, in wild-type male-only chambers, we observed extreme behavioral manifestations, such as "chaining" and "wheeling", resembling previously reported male-male courtship behaviors in fruitless mutants and in transformants with ectopic mini-white+ overexpression. This male-male courtship was greatly diminished in a variety of visual system mutants, including disrupted phototransduction (norpA), eliminated eye-color screening pigments (white), or deletion of the R7 photoreceptor cells (sevenless). However, light-induced courtship was unhampered in wing-cut flies, despite their inability to produce courtship song, a major acoustic signal during courtship. Unexpectedly the olfactory mutants orco and sbl displayed unrestrained male-male courtship. Particularly, orco males attained maximum courtship scores under either dim or intense light conditions. Together, our observations support the notion that the innate male courtship behavior is restrained by olfactory cues under normal conditions but can be unleashed by strong visual stimulation in Drosophila.

neuroscience↗

bioRxiv2022

Abnormal larval neuromuscular junction morphology and physiology in Drosophila Prickle isoform mutants with defective axonal transport and adult seizure behavior

Previous studies have demonstrated that mutations of the Drosophila planar cell polarity gene prickle (pk) result in altered microtubule-mediated vesicular transport in larval motor axons, as well as adult neuronal circuit hyperexcitability and epileptic behavior. It is also known that mutant alleles of the prickle-prickle (pkpk) and prickle-spiny-legs (pksple) isoforms differ in phenotype but display isoform counterbalancing effects in heteroallelic pkpk/pksple flies to ameliorate adult motor circuit and behavioral hyperexcitability. We have further investigated the larval neuromuscular junction (NMJ) and uncovered robust phenotypes in both pkpk and pksple alleles (heretofore referred to as pk and sple alleles, respectively), including synaptic terminal overgrowth, as well as irregular motor axon terminal excitability, poor vesicle release synchronicity, and altered efficacy of synaptic transmission. We observed significant increase in whole-cell excitatory junctional potential (EJP) in pk homozygotes, which was restored to near WT level in pk/sple heterozygotes. We further examined motor terminal excitability sustained by presynaptic Ca2+ channels, under the condition of pharmacological blockade of Na+ and K+ channel function. Such manipulation revealed extreme Ca2+ channel-dependent nerve terminal excitability in both pk and sple mutants. However, when combined in pk/sple heterozygotes, such terminal hyper-excitability was restored to nearly normal. Focal recording from individual synaptic boutons revealed asynchronous vesicle release in both pk and sple homozygotes, which nevertheless persisted in pk/sple heterozygotes without indications of isoform counter-balancing effects. Similarly, the overgrowth at NMJs was not compensated in pk/sple heterozygotes, exhibiting an extremity comparable to that in pk and sple homozygotes. Our observations uncovered differential roles of the pk and sple isoforms and their distinct interactions in the various structural and functional aspects of the larval NMJ and adult neural circuits.

neuroscience↗

bioRxiv2021

Diet modifies allele-specific phenotypes in Drosophila carrying epilepsy-associated PNPO variants

Pyridox(am)ine 5-phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters GABA and monoamines. Pathogenic variants in PNPO have been repeatedly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities, including developmental impairment and intellectual disability. It is unclear how seizure types and associated comorbidities are linked to specific PNPO alleles and to what degree diet can modify their expression. Furthermore, the molecular characteristics of PNPO variants have not been examined in model systems. Using CRISPR/Cas9, we generated four knock-in Drosophila alleles, hWT, hR116Q, hD33V, and hR95H, in which the endogenous Drosophila PNPO (sugarlethal) was replaced by wild-type human PNPO cDNA and epilepsy-associated variants corresponding to R116Q, D33V, and R95H, respectively. We examined these knock-in flies at the molecular, circuitry, and behavioral levels. Collectively, we found a wide range of phenotypes in an allele- and diet-dependent manner. Specifically, the D33V mutation reduces the mRNA level, R95H reduces the protein stability, and R116Q alters the protein localization of PNPO in the brain. D33V and R95H mutations lead to partial and complete lethality during development, respectively and R116Q and D33V mutations shorten lifespan. At the behavioral level, hD33V/hR95H trans-heterozygous flies are hypoactive on all tested diets whereas hR116Q flies show diet-dependent locomotor activities. At the circuitry level, hD33V homozygotes show rhythmic burst firing and hD33V/hR95H trans-heterozygotes exhibit spontaneous seizure discharges. In hR95H homozygotes rescued with PLP supplementation, we uncovered that PLP deficiency abolishes development and causes extreme seizures in adults. Lastly, genetic and electrophysiological analyses demonstrate that hWT/hR95H heterozygous flies are prone to seizures due to a dominant-negative effect of hR95H on hWT, highlighting the possibility that human R95H carriers may also be susceptible to epilepsy. Together, this study demonstrates that human PNPO variants interact with diet to contribute to phenotypic variations; and that the knock-in Drosophila model offers a powerful approach to systematically examine clinical manifestations and the underlying mechanisms of human PNPO deficiency.

neuroscience↗

bioRxiv2021

Species boundaries in the Agrobacterium tumefaciens complex and multi-level modular evolution of their antibacterial type VI secretion system and tumor-inducing plasmids

BackgroundMany bacterial taxa are species complexes and uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized Agrobacterium tumefaciens, a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. ResultsWe utilized 35 genome assemblies to achieve a comprehensive and balanced sampling of A. tumefaciens. Our confident inference of gene content and core-genome phylogeny supported a quantitative guideline for delineating 12 species and allowed for robust investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. ConclusionsWe demonstrated that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. Our strategies for multi-level investigations at scales that range from whole-genomes to intragenic domains and phylogenetic depths of between- and within-species are applicable to other bacteria. Finally, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

genomics↗

bioRxiv2021

Fly seizure EEG: field potential activity in the Drosophila brain

Hypersynchronous neural activity is a characteristic feature of seizures. Although many Drosophila mutants of epilepsy-related genes display clear behavioral spasms and motor unit hyperexcitability, field potential measurements of aberrant hypersynchronous activity across brain regions during seizures have yet to be described. Here, we report a straightforward method to observe local field potentials (LFPs) from the Drosophila brain to monitor ensemble neural activity during seizures in behaving tethered flies. High frequency stimulation across the brain reliably triggers a stereotypic sequence of electroconvulsive seizure (ECS) spike discharges readily detectable in the dorsal longitudinal muscle (DLM) and coupled with behavioral spasms. During seizure episodes, the LFP signal displayed characteristic large-amplitude oscillations with a stereotypic temporal correlation to DLM flight muscle spiking. ECS-related LFP events were clearly distinct from rest- and flight-associated LFP patterns. We further characterized the LFP activity during different types of seizures originating from genetic and pharmacological manipulations. In the bang-sensitive sodium channel mutant bangsenseless (bss), the LFP pattern was prolonged, and the temporal correlation between LFP oscillations and DLM discharges was altered. Following administration of the pro-convulsant GABAA blocker picrotoxin, we uncovered a qualitatively different LFP activity pattern, which consisted of a slow (1-Hz), repetitive, waveform, closely coupled with DLM bursting and behavioral spasms. Our approach to record brain LFPs presents an initial framework for electrophysiological analysis of the complex brain-wide activity patterns in the large collection of Drosophila excitability mutants.

neuroscience↗

bioRxiv2021

Differential effects on neuromuscular physiology between Sod loss-of-function mutation and paraquat-induced oxidative stress in Drosophila

Oxidative stress is thought to be a major contributor to aging processes. Here, we report differential effects on neurotransmission caused by loss-of-function mutations of Superoxide dismutase (Sod) and by paraquat (PQ) feeding in Drosophila. We demonstrated alterations in Sod mutants; the larval neuromuscular junction displayed supernumerary discharges and the adult giant-fiber escape pathway showed increased latency and poor response to repetitive high-frequency stimulation. Even though the concentrations used led to motor coordination defects and lethality, PQ feeding failed to reproduce such performance deficits in these larval and adult preparations, indicating mechanistic distinctions between genetic and pharmacological manipulation of oxidative stress.

neuroscience↗

bioRxiv2020

Investigations into modifications of neuromuscular physiology by axonal transport disruptions in Drosophila SOD1 mutants

The reactive oxygen species (ROS)-scavenging enzyme Cu/Zn superoxide dismutase (SOD1) is an evolutionarily conserved mechanism for the maintenance of oxidative homeostasis, and missense mutations in the human SOD1 gene are associated with the motor neuron degenerative disease amyotrophic lateral sclerosis (ALS). Mutations in the Drosophila melanogaster SOD1 gene (Sod) shorten fly lifespan, attenuate motor function, and induce developmental mortality. We have previously found morphological and physiological defects at the neuromuscular junctions (NMJs) of hypomorphic Sodn108 and ALS model SodG85R mutant larvae. Here, we report genetic interactions causing striking modifications of Sod mutant phenotypes by mutations in the gene Prickle (Pk), which are linked to planar cell polarity, epilepsy, and axonal transport disruptions. Pk is expressed as two isoforms prickle-spiny-legs (sple) and prickle-prickle (pk), which are each suppressed by specific hypomorphic mutations (sple1 and pk1). Interestingly, Sod phenotypes are distinctly modified depending on both the Pk isoform suppressed, and whether said suppression is heterozygous or homozygous. Heterozygous sple1 and pk1 improved the developmental survival of Sod mutants, whereas homozygous sple1 and pk1 drastically increased mortality. Further, only heterozygous sple1 and pk1 clearly ameliorated morphological defects at the neuromuscular junctions of Sod mutant larvae. Pharmacological treatment of Sod mutants reveals allele-specific motor neuron terminal hyperexcitability, characterized by synaptic transmissions of extended duration and abnormal presynaptic Ca2+ transients. Heterozygous sple1 mutation suppresses this Sod mutant terminal hyperexcitability, but pk1 does not. We reversed this suppressive effect of sple1 by pharmacological blockade of Ca2+-activated K+ channel slowpoke. Altogether, this study builds on our prior knowledge of Sod mutant development and physiology to show that axonal transport-linked gene mutations strikingly modify Sod mutant phenotypes, providing strong evidence for a role of intracellular transport in alterations of neuromuscular morphology and physiology by SOD1 mutations.

neuroscience↗

bioRxiv2020

Aging-vulnerable and -resilient trajectories of specific motor circuit function in oxidation- and temperature-stressed Drosophila

We examined several sensory-motor processing circuits in Drosophila across the lifespan and uncovered distinctive age-resilient and age-vulnerable trajectories in their established functional properties. We observed relatively little deterioration toward the end of lifespan in the giant-fiber (GF) and downstream circuit elements responsible for the jump-and-flight escape reflex. In contrast, we found substantial age-dependent modifications in the performance of GF inputs and other circuits driving flight motoneuron activities. Importantly, in high temperature (HT)-reared flies (29 {degrees}C), the characteristic age-dependent progression of these properties was largely maintained, albeit over a compressed time scale, lending support for the common practice of expediting Drosophila aging studies by HT rearing. We discovered shortened lifespans in Cu2+/Zn2+ Superoxide Dismutase 1 (Sod) mutant flies were accompanied by alterations distinct from HT-reared flies, highlighting differential effects of oxidative vs temperature stressors. This work also establishes several age-vulnerable parameters that may serve as quantitative neurophysiological landmarks for aging in Drosophila.

neuroscience↗

bioRxiv2019

Redundancy and specificity of type VI secretion vgrG loci in antibacterial activity of Agrobacterium tumefaciens 1D1609 strain

Type VI secretion system (T6SS) is a contractile nanoweapon employed by many Proteobacteria to deliver effectors to kill or inhibit their competitors. One T6SS gene, vgrG, encodes a spike protein for effector translocation and is often present as multiple copies in bacterial genomes. Our phylogenomic analyses sampled 48 genomes across diverse Proteobacteria lineages and found [~]70% of them encode multiple VgrGs, yet only four genomes have nearly identical paralogs. Among these four, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Compared to A. tumefaciens model strain C58 which harbors two vgrG genes, 1D1609 encodes four vgrG genes (i.e. vgrGa-d) with each adjacent to different putative effector genes. Thus, 1D1609 was selected to investigate the functional redundancy and specificity of multiple vgrG genes and their associated effectors. Secretion assay of single and multiple vgrG deletion mutants demonstrated that these four vgrGs are functionally redundant in mediating T6SS secretion. By analyzing various vgrG mutants, we found that all except for the divergent vgrGb could contribute to 1D1609s antibacterial activity. Further characterizations of putative effector-immunity gene pairs revealed that vgrGa-associated gene 2 (v2a) encodes an AHH family nuclease and serves as the major antibacterial toxin. Interestingly, C58s VgrG2 shares 99% amino acid sequence identity with 1D1609s VgrGa, VgrGc and VgrGd. This high sequence similarity allows 1D1609 to use an exogenous VgrG delivered from C58 to kill another competing bacterium. Taken together, Agrobacterium can use highly similar VgrGs, either produced endogenously or injected from its close relatives, for T6SS-mediated interbacterial competition. Authors SummarySelective pressure drives bacteria to develop adaptive strategies, which include competitive and cooperative behaviors. Type VI secretion system (T6SS) is one powerful antibacterial and anti-host nanoweapon employed by many Gram-negative bacteria for growth advantages or pathogenesis. A T6SS-harboring bacterium can encode one to multiple VgrG proteins for delivery of cognate effector(s) but the prevalence and biological significance of having sequence redundant vgrGs have not been comprehensively explored. In this study, we investigated the extensiveness of having multicopy vgrG genes for effector delivery among diverse Proteobacteria with T6SS. Moreover, a plant pathogenic bacterium Agrobacterium tumefaciens strain 1D1609 with highest vgrG redundancy was selected for detailed characterization of the roles of multiple VgrGs in T6SS secretion and antibacterial activity. We revealed that the majority of Proteobacterial genomes harbor multiple copies of vgrG and the expansion of vgrG gene clusters contributed to effector diversity and functional redundancy. Furthermore, the near identical VgrG proteins between 1D1609 and its sibling strain C58 can be exchanged for effector delivery in killing another competing bacterium. Such strategy in using exchangeable effector carriers injected from its isogenic sibling or close relatives during T6SS attacks may be a beneficial strategy for agrobacteria to compete in their ecological niche.

microbiology↗

bioRxiv2019

Pyridox(am)ine 5’-phosphate oxidase deficiency induces seizures in Drosophila melanogaster

Pyridox(am)ine 5-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5-phosphate (PLP), the biologically active form of VB6, and involved in the synthesis of neuro-transmitters including GABA, dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously we identified a c.95 C > A missense mutation in sgll - the Drosophila homolog of human PNPO (hPNPO) and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here we report the establishment of both sgll95 and ubiquitous sgll knockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Both sgll95 and sgll KD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-type sgll or hPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specific sgll KD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, the PNPO gene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

neuroscience↗

bioRxiv2019

Effector loading onto VgrG spike proteins is critical for the assembly of the type VI secretion system in Agrobacterium tumefaciens

The type VI secretion system (T6SS) is used by many bacteria to engage in social behaviors with others and can directly or indirectly affect the health of plants and animals. Because activities associated with T6SS are often costly, the assembly and activation of the T6SS must be highly regulated. However, our knowledge regarding how T6SS assembly and contraction are regulated remains limited. Here we show that the loading of effectors onto their cognate carriers is critical for the assembly of a functional T6SS in Agrobacterium tumefaciens. A. tumefaciens strain C58 encodes one T6SS and two Tde DNase toxin effectors used as major weapons for interbacterial competition. We found that loading of Tde effectors onto their cognate carrier, the VgrG spike, is required for active T6SS secretion. Our data also suggest the assembly of the TssBC contractile sheath occurs only after Tde effectors are loaded onto the VgrG spike. The requirement of effector loading for efficient T6SS secretion was also validated in other A. tumefaciens strains. Such a mechanism may be used by bacteria as a strategy for efficacious T6SS firing. Given the prevalence of T6SS-encoding loci in host-associated bacteria, these findings inform on mechanisms that influence the composition of microbial communities and the services provided to hosts.

microbiology↗

bioRxiv2018

Distinctions among electroconvulsion- and proconvulsant-induced seizure discharges and native motor patterns during flight and grooming: Quantitative spike pattern analysis in Drosophila flight muscles

In Drosophila, high-frequency electrical stimulation across the brain triggers a highly stereotypic repertoire of spasms known as electroconvulsive seizures (ECS). The distinctive ECS spiking discharges manifest across the nervous system and can be stably assessed throughout the seizure repertoire in the large indirect flight muscles (DLMs). ECS discharges in DLMs have been extensively used to monitor seizure activities, notably in stress ( bang)-sensitive mutants. However, the relationships between ECS-spike patterns and native motor programs, including flight and grooming, are not known and their similarities and distinctions remain to be characterized. We employed quantitative spike pattern analyses including: 1) overall firing frequency, 2) spike timing between contralateral fibers, and 3) short-term variability in spike interval regularity (CV2) and instantaneous firing frequency (ISI-1) to reveal distinctions amongst ECS discharges, flight and grooming motor patterns. We then examined DLM firing phenotypes in well-established mutants in excitatory cholinergic (Cha), inhibitory GABAergic (Rdl) and electrical (ShakB) synaptic transmission. The results provide an initial glimpse on the vulnerability of individual motor patterns to perturbations of respective synaptic transmission systems. We found marked alterations of ECS discharge spike patterns in terms of either seizure threshold, spike frequency or spiking regularity. In contrast, no gross alterations during grooming and only a minor reduction of firing frequency during Rdl mutant flight were observed, suggesting a role for GABAergic modulation of flight motor programs. Picrotoxin (PTX), a known pro-convulsant that inhibits GABAA receptors, induced DLM seizure patterns that displayed some features, e.g. left-right coordination and ISI-1 range, that could be found in flight or grooming, but distinct from ECS discharges. Our results indicate that these quantitative techniques may be employed to reveal overlooked relationships among aberrant motor patterns and native DLM motor programs in genetic and pharmacological analyses of underlying cellular and neural circuit function.

neuroscience↗