EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation.

The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, possibly, ASDs.

The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular, labile iron.

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.

Evolution of Osteocrin as an activity-regulated factor in the primate brain.

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.

Functional comparison of pacifiers using finite element analysis.

BACKGROUND: Pacifiers have been shown to affect maxillary growth related to the anatomic structure of the palate and forces placed upon it during sucking. This study compares and evaluates the mechanical behavior of pacifiers of different design and size (i.e., fit), identified by brand and size, positioned in age-specific palatal models with respect to both contact area and force when subjected to peristaltic tongue function and intraoral pressure related to non-nutritive sucking. METHODS: Nonlinear finite element analyses were used to simulate dynamic mechanical interaction between the pacifiers and palates. Time-varying, external pressure loads were applied which represent intraoral pressure arising from non-nutritive sucking and peristaltic behavior of the tongue. The silicone rubber pacifier bulb was represented using a hyperelastic material model. RESULTS: Results from the finite element analyses include deformation, stress, strain, contact area, and contact force. Mechanical interaction was evaluated in terms of the spatial distribution of the contact area and force between the pacifier and the palate. The resulting palatal interaction profiles were quantitatively compared to assess how pacifier fit specifically affects the support provided to two areas of the palate, the palatal vault and the Tektal wall. CONCLUSIONS: Pacifiers interact with the palate differently based on their fit (i.e., design and size) regardless of whether they are labeled conventional or orthodontic. Finite element analysis is an effective tool for evaluating how a pacifier's design affects functional mechanics and for providing guidance on biometric sizing.

Small molecular modulators of JMJD1C preferentially inhibit growth of leukemia cells.

Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP+ and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.

The Oral Bacterium Fusobacterium nucleatum Binds Staphylococcus aureus and Alters Expression of the Staphylococcal Accessory Regulator sarA.

Staphylococcus aureus, an opportunistic pathogen member of the nasal and skin microbiota, can also be found in human oral samples and has been linked to infectious diseases of the oral cavity. As the nasal and oral cavities are anatomically connected, it is currently unclear whether S. aureus can colonize the oral cavity and become part of the oral microbiota, or if its presence in the oral cavity is simply transient. To start addressing this question, we assessed S. aureus ability to directly bind selected members of the oral microbiota as well as its ability to integrate into a human-derived complex oral microbial community in vitro. Our data show that S. aureus forms aggregates with Fusobacterium nucleatum and Porphyromonas gingivalis and that it can incorporate into the human-derived in vitro oral community. Further analysis of the F. nucleatum-S. aureus interaction revealed that the outer-membrane adhesin RadD is partially involved in aggregate formation and that the RadD-mediated interaction leads to an increase in expression of the staphylococcal global regulator gene sarA. Our findings lend support to the notion that S. aureus can become part of the complex microbiota of the human mouth, which could serve as a reservoir for S. aureus. Furthermore, direct interaction with key members of the oral microbiota could affect S. aureus pathogenicity contributing to the development of several S. aureus associated oral infections.

Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR.

Rett syndrome (RTT) is an X-linked human neurodevelopmental disorder with features of autism and severe neurological dysfunction in females. RTT is caused by mutations in methyl-CpG-binding protein 2 (MeCP2), a nuclear protein that, in neurons, regulates transcription, is expressed at high levels similar to that of histones, and binds to methylated cytosines broadly across the genome. By phosphotryptic mapping, we identify three sites (S86, S274 and T308) of activity-dependent MeCP2 phosphorylation. Phosphorylation of these sites is differentially induced by neuronal activity, brain-derived neurotrophic factor, or agents that elevate the intracellular level of 3',5'-cyclic AMP (cAMP), indicating that MeCP2 may function as an epigenetic regulator of gene expression that integrates diverse signals from the environment. Here we show that the phosphorylation of T308 blocks the interaction of the repressor domain of MeCP2 with the nuclear receptor co-repressor (NCoR) complex and suppresses the ability of MeCP2 to repress transcription. In knock-in mice bearing the common human RTT missense mutation R306C, neuronal activity fails to induce MeCP2 T308 phosphorylation, suggesting that the loss of T308 phosphorylation might contribute to RTT. Consistent with this possibility, the mutation of MeCP2 T308A in mice leads to a decrease in the induction of a subset of activity-regulated genes and to RTT-like symptoms. These findings indicate that the activity-dependent phosphorylation of MeCP2 at T308 regulates the interaction of MeCP2 with the NCoR complex, and that RTT in humans may be due, in part, to the loss of activity-dependent MeCP2 T308 phosphorylation and a disruption of the phosphorylation-regulated interaction of MeCP2 with the NCoR complex.

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform.

An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE: This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

Protein acetylation dynamics in response to carbon overflow in Escherichia coli.

In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. As glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism.

Wnt5a-Ror-Dishevelled signaling constitutes a core developmental pathway that controls tissue morphogenesis.

Wnts make up a large family of extracellular signaling molecules that play crucial roles in development and disease. A subset of noncanonical Wnts signal independently of the transcription factor ß-catenin by a mechanism that regulates key morphogenetic movements during embryogenesis. The best characterized noncanonical Wnt, Wnt5a, has been suggested to signal via a variety of different receptors, including the Ror family of receptor tyrosine kinases, the Ryk receptor tyrosine kinase, and the Frizzled seven-transmembrane receptors. Whether one or several of these receptors mediates the effects of Wnt5a in vivo is not known. Through loss-of-function experiments in mice, we provide conclusive evidence that Ror receptors mediate Wnt5a-dependent processes in vivo and identify Dishevelled phosphorylation as a physiological target of Wnt5a-Ror signaling. The absence of Ror signaling leads to defects that mirror phenotypes observed in Wnt5a null mutant mice, including decreased branching of sympathetic neuron axons and major defects in aspects of embryonic development that are dependent upon morphogenetic movements, such as severe truncation of the caudal axis, the limbs, and facial structures. These findings suggest that Wnt5a-Ror-Dishevelled signaling constitutes a core noncanonical Wnt pathway that is conserved through evolution and is crucial during embryonic development.

Activity-dependent regulation of inhibitory synapse development by Npas4.

Neuronal activity regulates the development and maturation of excitatory and inhibitory synapses in the mammalian brain. Several recent studies have identified signalling networks within neurons that control excitatory synapse development. However, less is known about the molecular mechanisms that regulate the activity-dependent development of GABA (gamma-aminobutyric acid)-releasing inhibitory synapses. Here we report the identification of a transcription factor, Npas4, that plays a role in the development of inhibitory synapses by regulating the expression of activity-dependent genes, which in turn control the number of GABA-releasing synapses that form on excitatory neurons. These findings demonstrate that the activity-dependent gene program regulates inhibitory synapse development, and suggest a new role for this program in controlling the homeostatic balance between synaptic excitation and inhibition.

Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus.

While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process.

Acetylation of the response regulator RcsB controls transcription from a small RNA promoter.

Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154.

Attrition from paediatric weight management impacts anthropometric outcomes at 2 years, but not health-related quality of life.

The study objective is to evaluate the influence of attrition from a paediatric weight management program (PWM) on health indicators over a 2-year period. In this observational study, children and youth with obesity were recruited at entry into a family-based behaviour modification PWM and had four research study visits, independent of clinic visits, over 2 years. Participants were divided into attrition groups based on length of clinic enrolment. Body composition, cardiometabolic health and health-related quality of life (HRQoL) were assessed. Among 269 children enrolled, 19% had no clinic treatment visit, 16% had treatment visits only up to 6 months, 23% up to 1 year and 42% had at least one clinic visit after 1 year (No Attrition). Greater declines in BMI z-score and body fat were seen at 2 years in children with No Attrition, while improvements in HRQoL were similar for all attrition groups. Children who attended at least one treatment visit reported improved HRQoL up to 2 years, regardless of duration in clinic. In contrast, declines in body fat and BMI z-score were greater at 2 years for those with at least one visit after 1 year. Continued efforts to reduce attrition are likely to improve anthropometric health outcomes during PWM.

The NALCN channel regulates metastasis and nonmalignant cell dissemination.

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.

Bacterial protein acetylation: the dawning of a new age.

Protein acetylation has historically been considered a predominantly eukaryotic phenomenon. Recent evidence, however, supports the hypothesis that acetylation broadly impacts bacterial physiology. To explore more rapidly the impact of protein acetylation in bacteria, microbiologists can benefit from the strong foundation established by investigators of protein acetylation in eukaryotes. To help advance this learning process, we will summarize the current understanding of protein acetylation in eukaryotes, discuss the emerging link between acetylation and metabolism and highlight the best-studied examples of protein acetylation in bacteria.

Retrospective analysis of complications related to endoscopic retrograde cholangio-pancreatography in patients with cirrhosis vs patients without cirrhosis.

BACKGROUND: There is minimal objective data regarding adverse events related to endoscopic retrograde cholangio-pancreatography (ERCP) in patients with cirrhosis compared to those without cirrhosis and even fewer data comparing complications among cirrhosis patients based on severity of cirrhosis. AIM: To determine if patients with cirrhosis are at increased risk of adverse events related to ERCP: mainly pancreatitis, bleeding, perforation, cholangitis, and mortality; And to see if higher Child-Pugh (CP) score and Model for End-Stage Liver Disease (MELD) score are associated with higher post-ERCP complications. METHODS: We performed a retrospective analysis of 692 patients who underwent ERCP and analyzed the impact of cirrhosis etiology, gender, type of sedation used during procedure, interventions performed, and co-morbidities on the rate of complications in cirrhosis patients as compared to non-cirrhosis patients. RESULTS: Overall complications were higher in those with cirrhosis as compared to those without cirrhosis (P = 0.015 at significance level of 0.05). CP class, especially CP class C, was shown to be associated with a significantly higher rate of ERCP complications as compared to CP class A and CP class B (P = 0.010 at significance level of 0.05). CONCLUSION: The results of our study reaffirm that liver cirrhosis has an impact on the occurrence of complications during ERCP. Our study shows that CP class seems to be more reliable as compared to MELD score in predicting complications of ERCP in cirrhosis patients.

Modulators of histone demethylase JMJD1C selectively target leukemic stem cells.

Leukemic stem cells (LSCs) comprise a very rare cell population that results in the development of acute myeloid leukemia. The selective targeting of drivers in LSCs with small molecule inhibitors holds promise for treatment of acute myeloid leukemia. Recently, we reported the identification of inhibitors of the histone lysine demethylase JMJD1C that preferentially kill MLL rearranged acute leukemia cells. Here, we report the identification of jumonji domain modulator #7 (JDM-7). Surface plasmon resonance analysis showed that JDM-7 binds to JMJD1C and its family homolog JMJD1B. JDM-7 did not significantly suppress cell proliferation in liquid cell culture at higher doses, although it led to a significant decrease in semi-solid colony formation experiments at lower concentrations. Moreover, low doses of JDM-7 did not suppress the proliferation of erythroid progenitor cells. We identified that JDM-7 downregulates the LSC self-renewal gene HOXA9 in leukemia cells. We further found that the structure of JDM-7 is similar to that of tadalafil, a drug approved by the US Food and Drug Administration. Molecular docking and surface plasmon resonance analysis showed that tadalafil binds to JMJD1C. Moreover, similar to JDM-7, tadalafil suppressed colony formation of leukemia cells in semi-solid cell culture at a concentration that did not affect primary umbilical cord blood cells. In summary, we have identified JDM-7 and tadalafil as potential JMJD1C modulators that selectively inhibit the growth of LSCs.

Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation.

Mutations or duplications in MECP2 cause Rett and Rett-like syndromes, neurodevelopmental disorders characterized by mental retardation, motor dysfunction, and autistic behaviors. MeCP2 is expressed in many mammalian tissues and functions as a global repressor of transcription; however, the molecular mechanisms by which MeCP2 dysfunction leads to the neural-specific phenotypes of RTT remain poorly understood. Here, we show that neuronal activity and subsequent calcium influx trigger the de novo phosphorylation of MeCP2 at serine 421 (S421) by a CaMKII-dependent mechanism. MeCP2 S421 phosphorylation is induced selectively in the brain in response to physiological stimuli. Significantly, we find that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription. These findings suggest that, by triggering MeCP2 phosphorylation, neuronal activity regulates a program of gene expression that mediates nervous system maturation and that disruption of this process in individuals with mutations in MeCP2 may underlie the neural-specific pathology of RTT.

Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC.

Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain.IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.