Type IV pili of Enterobacteriaceae species.

Type IV pili (T4Ps) are surface filaments widely distributed among bacteria and archaea. T4Ps are involved in many cellular functions and contribute to virulence in some species of bacteria. Due to the diversity of T4Ps, different properties have been observed for homologous proteins that make up T4Ps in various organisms. In this review, we highlight the essential components of T4Ps, their functions, and similarities to related systems. We emphasize the unique T4Ps of enteric pathogens within the Enterobacteriaceae family, which includes pathogenic strains of Escherichia coli and Salmonella. These include the bundle-forming pilus (BFP) of enteropathogenic E. coli (EPEC), longus (Lng) and colonization factor III (CFA/III) of enterotoxigenic E. coli (ETEC), T4P of Salmonella enterica serovar Typhi, Colonization Factor Citrobacter (CFC) of Citrobacter rodentium, T4P of Yersinia pseudotuberculosis, a ubiquitous T4P that was characterized in enterohemorrhagic E. coli (EHEC), and the R64 plasmid thin pilus. Finally, we highlight areas for further study.

High throughput and targeted screens for prepilin peptidase inhibitors do not identify common inhibitors of eukaryotic gamma-secretase.

INTRODUCTION: Prepilin peptidases (PPP) are essential enzymes for the biogenesis of important virulence factors, such as type IV pili (T4P), type II secretion systems, and other T4P-related systems of bacteria and archaea. PPP inhibitors could be valuable pharmaceuticals, but only a few have been reported. Interestingly, PPP share similarities with presenilin enzymes from the gamma-secretase protease complex, which are linked to Alzheimer's disease. Numerous gamma-secretase inhibitors have been reported, and some have entered clinical trials, but none has been tested against PPP. OBJECTIVE: The objective of this study is to develop a high-throughput screening (HTS) method to search for inhibitors of PPP from various chemical libraries and reported gamma-secretase inhibitors. METHOD: More than 15,000 diverse compounds, including 13 reported gamma-secretase inhibitors and other reported peptidase inhibitors, were screened to identify potential PPP inhibitors. RESULTS: The authors developed a novel screening method and screened 15,869 compounds. However, the screening did not identify a PPP inhibitor. Nevertheless, the study suggests that gamma-secretase is sufficiently different from PPP that specific inhibitors may exist in a larger chemical space. CONCLUSION: The authors believe that the HTS method that they describe has numerous advantages and encourage others to consider its application in the search for PPP inhibitors.

Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.

Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

Landmark Discoveries and Recent Advances in Type IV Pilus Research.

Type IV pili (T4P) are retractable multifunctional nanofibers present on the surface of numerous bacterial and archaeal species. Their importance to microbiology is difficult to overstate. The scientific journey leading to our current understanding of T4P structure and function has included many innovative research milestones. Although multiple T4P reviews over the years have emphasized recent advances, we find that current reports often omit many of the landmark discoveries in this field. Here, we attempt to highlight chronologically the most important work on T4P, from the discovery of pili to the application of sophisticated contemporary methods, which has brought us to our current state of knowledge. As there remains much to learn about the complex machine that assembles and retracts T4P, we hope that this review will increase the interest of current researchers and inspire innovative progress.

Draft Genome Sequence of Escherichia coli Strain UMD142.

Escherichia coli can be a harmless commensal organism or cause a range of diseases in humans, including diarrhea, urinary tract infections, meningitis, sepsis, and skin and soft tissue infections. Here, we describe the genome of an isolate that was associated with necrotizing fasciitis and the decompensation of previously undiagnosed cirrhosis.

Draft genome sequences of concurrent Escherichia coli blood and fecal isolates from a patient with bacteremia and diarrhea belie BioFire-based detection of fecal enteropathogenic E. coli.

The BioFire FilmArray® Gastrointestinal panel is a multiplex PCR assay widely used to determine the etiology of infectious gastroenteritis directly from stool specimens. Recently a positive BioFire result for fecal enteropathogenic Escherichia coli (EPEC) was reported by a clinical microbiology laboratory for an adult patient with diarrhea and bacteremia. Since EPEC infrequently infects adults and rarely causes bacteremia, we isolated fecal E. coli and characterized the patient's blood and fecal E. coli isolates. Draft genome sequencing using a combination of methods indicated that the blood and fecal strains are virtually identical, are from sequence type 963 (phylogroup D) and exhibit neither the virulence genes characteristic of EPEC and extraintestinal pathogenic E. coli (ExPEC) nor classic EPEC-associated phenotypes. These findings support a gut source for the patient's bacteremia but exclude EPEC as the causative organism, and suggest that results of multiplex PCR assays from complex samples can be misleading, and should be interpreted with caution when they are discordant with clinical information. BioProject accession numbers for strains MVAST5574 and MVAST5635 genomes are PRJNA611789 and PRJNA611804, respectively.

Attaching-and-Effacing Pathogens Exploit Junction Regulatory Activities of N-WASP and SNX9 to Disrupt the Intestinal Barrier.

BACKGROUND & AIMS: Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a key regulator of the actin cytoskeleton in epithelial tissues and is poised to mediate cytoskeletal-dependent aspects of apical junction complex (AJC) homeostasis. Attaching-and-effacing (AE) pathogens disrupt this homeostasis through translocation of the effector molecule early secreted antigenic target-6 (ESX)-1 secretion-associated protein F (EspF). Although the mechanisms underlying AJC disruption by EspF are unknown, EspF contains putative binding sites for N-WASP and the endocytic regulator sorting nexin 9 (SNX9). We hypothesized that N-WASP regulates AJC integrity and AE pathogens use EspF to induce junction disassembly through an N-WASP- and SNX9-dependent pathway. METHODS: We analyzed mice with intestine-specific N-WASP deletion and generated cell lines with N-WASP and SNX9 depletion for dynamic functional assays. We generated EPEC and Citrobacter rodentium strains complemented with EspF bearing point mutations abolishing N-WASP and SNX9 binding to investigate the requirement for these interactions. RESULTS: Mice lacking N-WASP in the intestinal epithelium showed spontaneously increased permeability, abnormal AJC morphology, and mislocalization of occludin. N-WASP depletion in epithelial cell lines led to impaired assembly and disassembly of tight junctions in response to changes in extracellular calcium. Cells lacking N-WASP or SNX9 supported actin pedestals and type III secretion, but were resistant to EPEC-induced AJC disassembly and loss of transepithelial resistance. We found that during in vivo infection with AE pathogens, EspF must bind both N-WASP and SNX9 to disrupt AJCs and induce intestinal barrier dysfunction. CONCLUSIONS: Overall, these studies show that N-WASP critically regulates AJC homeostasis, and the AE pathogen effector EspF specifically exploits both N-WASP and SNX9 to disrupt intestinal barrier integrity during infection.

Genomic diversity of EPEC associated with clinical presentations of differing severity.

Enteropathogenic Escherichia coli (EPEC) are diarrhoeagenic E. coli, and are a significant cause of gastrointestinal illness among young children in developing countries. Typical EPEC are identified by the presence of the bundle-forming pilus encoded by a virulence plasmid, which has been linked to an increased severity of illness, while atypical EPEC lack this feature. Comparative genomics of 70 total EPEC from lethal (LI), non-lethal symptomatic (NSI) or asymptomatic (AI) cases of diarrhoeal illness in children enrolled in the Global Enteric Multicenter Study was used to investigate the genomic differences in EPEC isolates obtained from individuals with various clinical outcomes. A comparison of the genomes of isolates from different clinical outcomes identified genes that were significantly more prevalent in EPEC isolates of symptomatic and lethal outcomes than in EPEC isolates of asymptomatic outcomes. These EPEC isolates exhibited previously unappreciated phylogenomic diversity and combinations of virulence factors. These comparative results highlight the diversity of the pathogen, as well as the complexity of the EPEC virulence factor repertoire.

Pilin Vaccination Stimulates Weak Antibody Responses and Provides No Protection in a C57Bl/6 Murine Model of Acute Clostridium difficile Infection.

Clostridium difficile is the leading cause of nosocomial infections in the United States, adding billions of dollars per year to health care costs. A vaccine targeted against the bacterium would be extremely beneficial in decreasing the morbidity and mortality caused by C. difficile-associated disease; a vaccine directed against a colonization factor would hinder the spread of the bacterium as well as prevent disease. Type IV pili (T4Ps) are extracellular appendages composed of protein monomers called pilins. They are involved in adhesion and colonization in a wide variety of bacteria and archaea, and are putative colonization factors in C. difficile. We hypothesized that vaccinating mice with pilins would lead to generation of anti-pilin antibodies, and would protect against C. difficile challenge. We found that immunizing C57Bl/6 mice with various pilins, whether combined or as individual proteins, led to low anti-pilin antibody titers and no protection upon C. difficile challenge. Passive transfer of anti-pilin antibodies led to high serum anti-pilin IgG titers, but to undetectable fecal anti-pilin IgG titers and did not protect against challenge. The low antibody titers observed in these experiments may be due to the particular strain of mice used. Further experiments, possibly with a different animal model of C. difficile infection, are needed to determine if an anti-T4P vaccine would be protective against C. difficile infection.

Type IV pili promote early biofilm formation by Clostridium difficile.

Increasing morbidity and mortality from Clostridium difficile infection (CDI) present an enormous challenge to healthcare systems. Clostridium difficile express type IV pili (T4P), but their function remains unclear. Many chronic and recurrent bacterial infections result from biofilms, surface-associated bacterial communities embedded in an extracellular matrix. CDI may be biofilm mediated; T4P are important for biofilm formation in a number of organisms. We evaluate the role of T4P in C. difficile biofilm formation using RNA sequencing, mutagenesis and complementation of the gene encoding the major pilin pilA1, and microscopy. RNA sequencing demonstrates that, in comparison to other growth phenotypes, C. difficile growing in a biofilm has a distinct RNA expression profile, with significant differences in T4P gene expression. Microscopy of T4P-expressing and T4P-deficient strains suggests that T4P play an important role in early biofilm formation. A non-piliated pilA1 mutant forms an initial biofilm of significantly reduced mass and thickness in comparison to the wild type. Complementation of the pilA1 mutant strain leads to formation of a biofilm which resembles the wild-type biofilm. These findings suggest that T4P play an important role in early biofilm formation. Novel strategies for confronting biofilm infections are emerging; our data suggest that similar strategies should be investigated in CDI.

BfpI, BfpJ, and BfpK Minor Pilins Are Important for the Function and Biogenesis of Bundle-Forming Pili Expressed by Enteropathogenic Escherichia coli.

UNLABELLED: Enteropathogenic Escherichia coli (EPEC) remains a significant cause of infant diarrheal illness and associated morbidity and mortality in developing countries. EPEC strains are characterized by their ability to colonize the small intestines of their hosts by a multistep program involving initial loose attachment to intestinal epithelial cells followed by an intimate adhesion phase. The initial loose interaction of typical EPEC with host intestinal cells is mediated by bundle-forming pili (BFP). BFP are type 4b pili (T4bP) based on structural and functional properties shared with T4bP expressed by other bacteria. The major structural subunit of BFP is called bundlin, a T4b pilin expressed from the bfpA gene in the BFP operon, which contains three additional genes that encode the pilin-like proteins BfpI, BfpJ, and BfpK. In this study, we show that, in the absence of the BFP retraction ATPase (BfpF), BfpI, BfpJ, and BfpK are dispensable for BFP biogenesis. We also demonstrate that these three minor pilins are incorporated along with bundlin into the BFP filament and contribute to its structural integrity and host cell adhesive properties. The results confirm that previous findings in T4aP systems can be extended to a model T4bP such as BFP. IMPORTANCE: Bundle-forming pili contribute to the host colonization strategy of enteropathogenic Escherichia coli. The studies described here investigate the role for three minor pilin subunits in the structure and function of BFP in EPEC. The studies also suggest that these subunits could be antigens for vaccine development.

Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes.

A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli.

Bacterial Factors Associated with Lethal Outcome of Enteropathogenic Escherichia coli Infection: Genomic Case-Control Studies.

BACKGROUND: Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome. METHODS: We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality. RESULTS: Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains. CONCLUSIONS: Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes.

Type IV pilus secretins have extracellular C termini.

UNLABELLED: Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer. IMPORTANCE: Secretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.

Signature-tagged mutagenesis and co-infection studies demonstrate the importance of P fimbriae in a murine model of urinary tract infection.

Escherichia coli is the leading cause of urinary tract infections (UTIs), one of the most common infections in humans. P fimbria was arguably the first proposed virulence factor for uropathogenic E. coli, based on the capacity of E. coli isolated from UTIs to adhere to exfoliated epithelial cells in higher numbers than fecal strains of E. coli. Overwhelming epidemiologic evidence has been presented for involvement of P fimbriae in colonization. It has been difficult, however, to demonstrate this requirement for uropathogenic strains in animal models of infections or in humans. In this study, a signature-tagged mutagenesis screen identified a P-fimbrial gene (papC) and 18 other genes as being among those required for full fitness of cystitis isolate E. coli F11. A P-fimbrial mutant was outcompeted by the wild-type strain in cochallenge in the murine model of ascending UTI, and this colonization defect could be complemented with the cloned pap operon. To our knowledge, this study is the first to fulfill molecular Koch's postulates in which a pathogenic strain was attenuated by mutation of pap genes and then complemented to restore fitness, confirming P fimbria as a virulence factor in a pathogenic clinical isolate.

Structural and evolutionary analyses show unique stabilization strategies in the type IV pili of Clostridium difficile.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion, and horizontal gene transfer. However, many Gram-positive species, including Clostridium difficile, also produce type IV pili. Here, we identify the major subunit of the type IV pili of C. difficile, PilA1, and describe multiple 3D structures of PilA1, demonstrating the diversity found in three strains of C. difficile. We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the type IVb pilins from Gram-negative bacteria that suggest that the type IV pili of C. difficile are involved in microcolony formation.

Enteropathogenic Escherichia coli inhibits type I interferon- and RNase L-mediated host defense to disrupt intestinal epithelial cell barrier function.

Enteropathogenic Escherichia coli (EPEC) primarily infects children in developing countries and causes diarrhea that can be deadly. EPEC pathogenesis occurs through type III secretion system (T3SS)-mediated injection of effectors into intestinal epithelial cells (IECs); these effectors alter actin dynamics, modulate the immune response, and disrupt tight junction (TJ) integrity. The resulting compromised barrier function and increased gastrointestinal (GI) permeability may be responsible for the clinical symptoms of infection. Type I interferon (IFN) mediates anti-inflammatory activities and serves essential functions in intestinal immunity and homeostasis; however, its role in the immune response to enteric pathogens, such as EPEC, and its impact on IEC barrier function have not been examined. Here, we report that IFN-ß is induced following EPEC infection and regulates IEC TJ proteins to maintain barrier function. The EPEC T3SS effector NleD counteracts this protective activity by inhibiting IFN-ß induction and enhancing tumor necrosis factor alpha to promote barrier disruption. The endoribonuclease RNase L is a key mediator of IFN induction and action that promotes TJ protein expression and IEC barrier integrity. EPEC infection inhibits RNase L in a T3SS-dependent manner, providing a mechanism by which EPEC evades IFN-induced antibacterial activities. This work identifies novel roles for IFN-ß and RNase L in IEC barrier functions that are targeted by EPEC effectors to escape host defense mechanisms and promote virulence. The IFN-RNase L axis thus represents a potential therapeutic target for enteric infections and GI diseases involving compromised barrier function.

Identification, immunogenicity, and cross-reactivity of type IV pilin and pilin-like proteins from Clostridium difficile.

The Gram-positive anaerobe Clostridium difficile is the major cause of nosocomial diarrhea; manifestations of infection include diarrhea, pseudomembranous colitis, and death. Genes for type IV pili, a bacterial nanofiber often involved in colonization and until relatively recently described only in Gram-negatives, are present in all members of the Clostridiales. We hypothesized that any pilins encoded in the C. difficile genome would be immunogenic, as has been shown with pilins from Gram-negative organisms. We describe nine pilin or pilin-like protein genes, for which we introduce a coherent nomenclature, in the C. difficile R20291 genome. The nine predicted pilin or pilin-like proteins have relatively conserved N-terminal hydrophobic regions, but diverge at their C-termini. Analysis of synonymous and nonsynonymous substitutions revealed evidence of diversifying selective pressure in two pilin genes. Six of the nine identified proteins were purified and used to immunize mice. Immunization of mice with each individual protein generated antibody responses that varied in titer and cross-reactivity, a notable result given the low amino acid sequence identity among the pilins. Further studies in other small mammals mirrored our results in mice. Our results illuminate components of the C. difficile type IV pilus and help identify targets for an anti-C. difficile vaccine.

Structure of Clostridium difficile PilJ exhibits unprecedented divergence from known type IV pilins.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface.