Synthesis and Biophysical and Biological Studies of N-Phenylbenzamide Derivatives Targeting Kinetoplastid Parasites.

The AT-rich mitochondrial DNA (kDNA) of trypanosomatid parasites is a target of DNA minor groove binders. We report the synthesis, antiprotozoal screening, and SAR studies of three series of analogues of the known antiprotozoal kDNA binder 2-((4-(4-((4,5-dihydro-1H-imidazol-3-ium-2-yl)amino)benzamido)phenyl)amino)-4,5-dihydro-1H-imidazol-3-ium (1a). Bis(2-aminoimidazolines) (1) and bis(2-aminobenzimidazoles) (2) showed micromolar range activity against Trypanosoma brucei, whereas bisarylimidamides (3) were submicromolar inhibitors of T. brucei, Trypanosoma cruzi, and Leishmania donovani. None of the compounds showed relevant activity against the urogenital, nonkinetoplastid parasite Trichomonas vaginalis. We show that series 1 and 3 bind strongly and selectively to the minor groove of AT DNA, whereas series 2 also binds by intercalation. The measured pKa indicated different ionization states at pH 7.4, which correlated with the DNA binding affinities (ΔTm) for series 2 and 3. Compound 3a, which was active and selective against the three parasites and displayed adequate metabolic stability, is a fine candidate for in vivo studies.

CRISPR-Cas-Guided Mutagenesis of Chromosome and Virulence Plasmid in Shigella flexneri by Cytosine Base Editing.

Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large-scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas-dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by single guide RNA (sgRNA) to introduce targeted C→T transitions, resulting in internal stop codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both Escherichia coli and Shigella flexneri with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion was in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Furthermore, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella flexneri genes and found the mutant phenotype to be similar to those of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well-plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days. IMPORTANCE Loss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches systems biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen-Shigella. This pathogen causes high morbidity and mortality in children, and antibiotic-resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of the utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method has the potential to be programmed to generate multiple mutants in a single, massively parallel reaction. By virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.

Methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate: A new reagent for the synthesis of N-phenylpyridinecarboxamidine ("arylimidamide") DNA-minor groove binders from poorly nucleophilic amines.

We report the synthesis and use of methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate as new reagent for the preparation of N-phenylpyridinecarboxamidines ("arylimidamides"), a class of DNA minor groove binding molecules with antiprotozoal activity. This versatile reagent allowed the access to electron-deficient halogen-containing bis(arylimidamides) that could not be obtained with the classical methods reported in the literature. With this two-step protocol, the N-Boc-protected arylimidamide intermediate, which is soluble in organic solvents, can be purified by centrifugal preparative thin layer chromatography on silica and/or by reverse-phase (C-18) chromatography. The target N-phenylpyridinecarboxamidines are obtained as salts by smooth hydrolysis of the Boc-protecting group with TFA. This methodology allows the synthesis of a pharmaceutically important class of antiparasitic compounds otherwise inaccessible.

Infection-induced membrane ruffling initiates danger and immune signaling via the mechanosensor PIEZO1.

Microorganisms are generally sensed by receptors recognizing microbial molecules, which evoke changes in cellular activities and gene expression. Bacterial pathogens induce secretion of the danger signal ATP as an early alert response of intestinal epithelial cells, initiating overt inflammation. However, what triggers ATP secretion during infection is unclear. Here we show that the inherently mechanosensitive plasma membrane channel PIEZO1 acts as a sensor for bacterial entry. PIEZO1 is mechanically activated by invasion-induced membrane ruffles upstream of Ca2+ influx and ATP secretion. Mimicking mechanical stimuli of pathogen uptake with sterile beads equally elicits ATP secretion. Chemical or genetic PIEZO1 inactivation inhibits mechanically induced ATP secretion. Moreover, chemical or mechanical PIEZO1 activation evokes gene expression in immune and barrier pathways. Thus, mechanosensation of invasion-induced plasma membrane distortion initiates immune signaling upon infection, independently of detection of microbial molecules. Hence, PIEZO1-dependent detection of infection is driven by physical signals instead of chemical ligands.

Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a.

BACKGROUND: Functionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci. RESULTS: Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator's sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase. CONCLUSIONS: We conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless ΔatpIBEFHAGDC mutant.

Imidazoline- and Benzamidine-Based Trypanosome Alternative Oxidase Inhibitors: Synthesis and Structure-Activity Relationship Studies.

The trypanosome alternative oxidase (TAO), a mitochondrial enzyme involved in the respiration of the bloodstream form trypomastigotes of Trypanosoma brucei, is a validated drug target against African trypanosomes. Earlier series of TAO inhibitors having a 2,4-dihydroxy-6-methylbenzoic acid scaffold ("head") and a triphenylphosphonium or quinolin-1-ium cation as a mitochondrion-targeting group ("tail") were shown to be nanomolar inhibitors in enzymatic and cellular assays. We investigated here the effect of different mitochondrion-targeting cations and other scaffold modifications on the in vitro activity of this class of inhibitors. Low micromolar range activities were obtained, and the structure-activity relationship studies showed that modulation of the tail region with polar substituents is generally detrimental to the enzymatic and cellular activity of TAO inhibitors.

Enhanced arthrocentesis of the effusive knee with pneumatic compression.

AIM: Complete arthrocentesis of the effusive knee ameliorates patient pain, reduces intra-articular and intraosseous pressure, removes inflammatory cytokines, and has been shown to substantially improve the therapeutic outcomes of intra-articular injections. However, conventional arthrocentesis incompletely decompresses the knee, leaving considerable residual synovial fluid in the intra-articular space. The present study determined whether external pneumatic circumferential compression of the effusive knee permitted more successful arthrocentesis and complete joint decompression. METHODS: Using a paired sample design, 50 consecutive effusive knees underwent conventional arthrocentesis and then arthrocentesis with pneumatic compression. Pneumatic compression was applied to the superior knee using a conventional thigh blood pressure cuff inflated to 100 mm Hg which compressed the suprapatellar bursa and patellofemoral joint, forcing fluid from the superior knee to the anterolateral portal where the fluid could be accessed. Arthrocentesis success and fluid yield in mL before and after pneumatic compression were determined. RESULTS: Successful diagnostic arthrocentesis (≥3 mL) of the effusive knee was 82% (41/50) with conventional arthrocentesis and increased to 100% (50/50) with pneumatic compression (P = .001). Synovial fluid yields increased by 144% (19.8 ± 17.1 mL) with pneumatic compression (conventional arthrocentesis; 13.7 ± 16.4 mL, pneumatic compression: 33.4 ± 26.5 mL; 95% CI: 10.9 < 19.7 < 28.9 mL, P < .0001). CONCLUSIONS: Conventional arthrocentesis routinely does not fully decompress the effusive knee. External circumferential pneumatic compression markedly improves arthrocentesis success and fluid yield, and permits complete decompression of the effusive knee. Pneumatic compression of the effusive knee with a thigh blood pressure cuff is an inexpensive and widely available technique to improve arthrocentesis outcomes.

Atypical Chronic Inflammatory ANCA-Positive Deforming Arthritis After Cocaine-Levamisole Exposure.

BACKGROUND/OBJECTIVE: Immunostimulatory drugs including immune checkpoint inhibitors and levamisole can induce inflammatory disease including vasculitis, rashes, tissue necrosis, and arthritis. METHODS: This prospective cohort study determined the 5-year outcomes of cocaine-levamisole-induced inflammatory disease as to outcomes and survival. Thirty-one consecutive cocaine-levamisole autoimmune patients and 45 primary vasculitis patients were characterized as to clinical differentiating features, antineutrophil cytoplasmic antibody (ANCA) status, treatment, the presence of acute and chronic arthritis, and 5-year outcome. RESULTS: Seventy-one percent (22/31) of cocaine-levamisole vasculopathy cases were ANCA positive (86% p-ANCA and 14% c-ANCA), whereas 53% (23/45) of the primary vasculitis were ANCA positive (p = 0.04). The ANCA-positive cocaine-levamisole cohort at onset were characterized by younger age (45 ± 12 vs 53 ± 14 years, p = 0.04), superficial skin necrosis (82% vs 54%, p = 0.036), depressed complement C3 (27% vs 4%, p = 0.33), antiphospholipid antibodies (50% vs 4%, p < 0.001), neutropenia (18% vs 0%, p = 0.044), and elevated antimyeloperoxidase (MPO) antibody levels (100% vs 67%, p < 0.001). Chronic cocaine-levamisole disease was characterized by severe cicatrical deformities of the face and extremities (45.5% vs 8.3%, p = 0.005). Arthralgias (71% vs 82%, p = 0.19) and acute arthritis (33% vs 32%, p = 0.25) were similar between the 2 groups. However, a substantial proportion cocaine-levamisole-induced autoimmune patients (18% vs 0%, p = 0.045) developed a chronic deforming inflammatory arthritis that was rheumatoid factor, anti-cyclic-citrillinated antibody antibody, and HLA-B27 negative, but p-ANCA-and MPO antibody positive. CONCLUSIONS: Patients exposed to cocaine-levamisole may develop serious chronic sequelae including cicatrical cutaneous and facial deformities and an atypical seronegative, p-ANCA and MPO antibody-positive, HLA-B27-negative chronic deforming inflammatory arthritis.

PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection.

Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

Rapid movement and transcriptional re-localization of human cohesin on DNA.

The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

Sororin actively maintains sister chromatid cohesion.

Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome-spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin-induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin's SMC3 subunit, and that DNA replication is also required for stable cohesin-chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.

Different tradeoffs result from alternate genetic adaptations to a common environment.

Fitness tradeoffs are often assumed by evolutionary theory, yet little is known about the frequency of fitness tradeoffs during stress adaptation. Even less is known about the genetic factors that confer these tradeoffs and whether alternative adaptive mutations yield contrasting tradeoff dynamics. We addressed these issues using 114 clones of Escherichia coli that were evolved independently for 2,000 generations under thermal stress (42.2 °C). For each clone, we measured their fitness relative to the ancestral clone at 37 °C and 20 °C. Tradeoffs were common at 37 °C but more prevalent at 20 °C, where 56% of clones were outperformed by the ancestor. We also characterized the upper and lower thermal boundaries of each clone. All clones shifted their upper boundary to at least 45 °C; roughly half increased their lower niche boundary concomitantly, representing a shift of thermal niche. The remaining clones expanded their thermal niche by increasing their upper limit without a commensurate increase of lower limit. We associated these niche dynamics with genotypes and confirmed associations by engineering single mutations in the rpoB gene, which encodes the beta subunit of RNA polymerase, and the rho gene, which encodes a termination factor. Single mutations in the rpoB gene exhibit antagonistic pleiotropy, with fitness tradeoffs at 18 °C and fitness benefits at 42.2 °C. In contrast, a mutation within the rho transcriptional terminator, which defines an alternative adaptive pathway from that of rpoB, had no demonstrable effect on fitness at 18 °C. This study suggests that two different genetic pathways toward high-temperature adaptation have contrasting effects with respect to thermal tradeoffs.

¹H, ¹5N and ¹³C resonance assignments of PpdD, a type IV pilin from enterohemorrhagic Escherichia coli.

Bacterial type 4 pili (T4P) are long flexible fibers involved in adhesion, DNA uptake, phage transduction, aggregation and a flagella-independent movement called "twitching motility". T4P comprise thousands of copies of the major pilin subunit, which is initially inserted in the plasma membrane, processed and assembled into dynamic helical filaments. T4P are crucial for host colonization and virulence of many Gram-negative bacteria. In enterohemorrhagic Escherichia coli the T4P, called hemorrhagic coli pili (HCP) promote cell adhesion, motility, biofilm formation and signaling. To understand the mechanism of HCP assembly and function, we analyzed the structure of the major subunit prepilin peptidase-dependent protein D (PpdD) (also called HcpA), a 15 kDa pilin with two potential disulfide bonds. Here we present the (1)H, (15)N and (13)C backbone and side chain resonance assignments of the C-terminal globular domain of PpdD as a first step to its structural determination.

Wapl is an essential regulator of chromatin structure and chromosome segregation.

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

The type II secretion system - a dynamic fiber assembly nanomachine.

Type II secretion systems (T2SSs) share common origins and structure with archaeal flagella (archaella) and pili, bacterial competence systems and type IV pili. All of these systems use a conserved ATP-powered machinery to assemble helical fibers that are anchored in the plasma membrane. The T2SSs assemble pseudopili, periplasmic filaments that promote extracellular secretion of folded periplasmic proteins. Comparative analysis of T2SSs and related fiber assembly nanomachines might provide important clues on their functional specificities and dynamics. This review focuses on recent developments in the study of pseudopilus structure and biogenesis, and discusses mechanistic models of pseudopilus function in protein secretion.

Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation.

In Gram-negative bacteria, type IV pilus assembly (T4PS) and type II secretion (T2SS) systems polymerize inner membrane proteins called major pilins or pseudopilins respectively, into thin filaments. Four minor pilins are required in both systems for efficient fibre assembly. Escherichia coli K-12 has a set of T4PS assembly genes that are silent under standard growth conditions. We studied the heterologous assembly of the E. coli type IV pilin PpdD by the Klebsiella oxytoca T2SS called the Pul system. PpdD pilus assembly in this context depended on the expression of the K. oxytoca minor pseudopilin genes pulHIJK or of the E. coli minor pilin genes ppdAB-ygdB-ppdC. The E. coli minor pilins restored assembly of the major pseudopilin PulG in a pulHIJK mutant, but not the secretion of the T2SS substrate pullulanase. Thus, minor pilins and minor pseudopilins are functionally interchangeable in initiating major pilin assembly, further extending the fundamental similarities between the two systems. The data suggest that, in both systems, minor pilins activate the assembly machinery through a common self-assembly mechanism. When produced together, PulG and PpdD assembled into distinct homopolymers, establishing major pilins as key determinants of pilus elongation and structure.

Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation.

In Gram-negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston-like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two-hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1-nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self-assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.

Detailed structural and assembly model of the type II secretion pilus from sparse data.

Many gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.