Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.

Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.

It takes two to attach - endo-1,3-ß-d-glucanase as a potential receptor of mannose-independent, FimH-dependent Salmonella Typhimurium binding to spinach leaves.

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-ß-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.

Understanding a Core Pilin of the Type IVa Pili of Acidithiobacillus thiooxidans, PilV.

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

Up-regulation of sortase-dependent pili in Bifidobacterium longum BBMN68 in response to bile stress enhances its adhesion to HT-29 cells.

Adhesion to gastrointestinal tract is crucial for bifidobacteria to exert their probiotic effects. Our previous work found that bile salts significantly enhance the adhesion ability of Bifidobacterium longum BBMN68 to HT-29 cells. In this study, trypsin-shaving and LC-MS/MS-based surface proteomics were employed to identify surface proteins involved in bile stress response. Among the 829 differentially expressed proteins, 56 up-regulated proteins with a fold change >1.5 were subjected to further analysis. Notably, the minor pilin subunit FimB was 4.98-fold up-regulated in response to bile stress. In silico analysis and RT-PCR confirmed that gene fimB, fimA and srtC were co-transcribed and contributed to the biosynthesis of sortase-dependent pili Pil1. Moreover, scanning electron microscopy and immunogold electron microscopy assays showed increased abundance and length of Pil1 on BBMN68 under bile stress. As the major pilin subunit FimA serves as adhesion component of Pil1, an inhibition assay using anti-FimA antibodies further confirmed the critical role of Pil1 in mediating the adhesion of BBMN68 to HT-29 cells under bile stress. Our findings suggest that the up-regulation of Pil1 in response to bile stress enhances the adhesion of BBMN68 to intestinal epithelial cells, highlighting a novel mechanism of gut persistence in B. longum strains.

Systematic functional analysis of the Com pilus in Streptococcus sanguinis: a minimalistic type 4 filament dedicated to DNA uptake in monoderm bacteria.

IMPORTANCE: Type 4 filaments (T4F) are nanomachines ubiquitous in prokaryotes, centered on filamentous polymers of type 4 pilins. T4F are exceptionally versatile and widespread virulence factors in bacterial pathogens. The mechanisms of filament assembly and the many functions they facilitate remain poorly understood because of the complexity of T4F machineries. This hinders the development of anti-T4F drugs. The significance of our research lies in characterizing the simplest known T4F-the Com pilus that mediates DNA uptake in competent monoderm bacteria-and showing that four protein components universally conserved in T4F are sufficient for filament assembly. The Com pilus becomes a model for elucidating the mechanisms of T4F assembly.

Stochastic chain termination in bacterial pilus assembly.

Adhesive type 1 pili from uropathogenic Escherichia coli strains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reaction in vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembled in vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction. In addition, we demonstrate that FimI contributes to anchoring the pilus to the outer membrane and report the crystal structures of (i) FimI in complex with the assembly chaperone FimC, (ii) the FimI-FimC complex bound to the N-terminal domain of FimD, and (iii) a ternary complex between FimI, FimA and FimC that provides structural insights on pilus assembly termination and pilus anchoring by FimI.

Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms.

Streptococcus pyogenes harboring an FCT type 3 genomic region display pili composed of three types of pilins. In this study, the structure of the base pilin FctB from a serotype M3 strain (FctB3) was determined at 2.8 Å resolution. In accordance with the previously reported structure of FctB from a serotype T9 strain (FctB9), FctB3 was found to consist of an immunoglobulin-like domain and proline-rich tail region. Data obtained from structure comparison revealed main differences in the omega (Ω) loop structure and the proline-rich tail direction. In the Ω loop structure, a differential hydrogen bond network was observed, while the lysine residue responsible for linkage to growing pili was located at the same position in both structures, which indicated that switching of the hydrogen bond network in the Ω loop without changing the lysine position is advantageous for linkage to the backbone pilin FctA. The difference in direction of the proline-rich tail is potentially caused by a single residue located at the root of the proline-rich tail. Also, the FctB3 structure was found to be stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. Comparisons of the FctB3 and FctA structures indicated that the FctA structure is more favorable for linkage to FctA. In addition, the heterodimer formation of FctB with Cpa or FctA was shown to be mediated by the putative chaperone SipA. Together, these findings provide an alternative FctB structure as well as insight into the interactions between pilin proteins.

Presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli isolates recovered from an apex medical institute in North India.

PURPOSE: To detect the presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli (UPEC) isolates recovered from patients coming to the out patient department (OPD) of our hospital. METHODS: E. coli isolates recovered from patients who had symptoms of urinary tract infection (UTI) were processed for the presence of fimH and iss genes. DNA was extracted using an in house method after which conventional PCR using forward and reverse primers targeting the four genes was carried out. The amplified products were electrophoresed and visualized in a gel documentation imager. Relevant demographic details of the patients were recorded on a pre-designed pro-forma and antimicrobial susceptibility testing of the isolates was done by disc diffusion method. RESULTS: fimH was present in 87.5% of UPEC isolates whereas iss type 1 was seen in 7.3%, type 2 in 4.2% and iss type 3 in 71.9% isolates. Age of the patients ranged from 3 months to 82 â€‹yrs (mean 43.5 SD â€‹± â€‹18.20). UTI was more common in females (60.2%) as compared to males patients (39.8%). Dysuria (66.7%) was the most common symptom in the studied subjects and diabetes mellitus (42.6%) the most common co-morbidity. A total of 56.5% patients gave a history of prior antibiotic intake. The UPEC isolates were resistant to most of the antibiotics tested. However all the isolates were sensitive to polymyxin B and colistin. Fosfomycin resistance was seen in 9.5% of the UPEC isolates harbouring fimH gene. CONCLUSION: This is the first study that highlights the presence of iss type 3 gene in UPEC isolates along with the fimH and iss type 1 and 2 genes. The results of this study can serve as a stepping stone for future in depth research into the significance of the iss genes in causing UTI.

Unraveling the immunopotentiation of P. aeruginosa PAPI-1 encoded pilin: From immunoinformatics survey to active immunization.

For protection against Pseudomonas aeruginosa strains, a number of vaccine candidates have been introduced thus far. However, despite significant attempts in recent years, there are currently no effective immunogenic Bacteria components against this pathogen on the market. P. aeruginosa encoding a number of different virulence characteristics, as well as the rapid growth in multiple drug-resistant forms, has raised numerous health issues throughout the world. This pathogen expresses three different subtypes of T4P, including IVa, IVb, and Tad which are involved in various cellular processes. Highly virulent strains of P. aeruginosa can encode well-conserved PAPI-1 associated PilS2 pilus. Designing an efficient pili-based immunotherapy approach targeting P. aeruginosa pilus has remained controversial due to the variability heterogeneousness and hidden well-preserved binding site of T4aP and no approved human study is commercially based on IVa pilin. In this investigation, for the first time, through analytical immunoinformatics, we designed an effective chimeric PilS2 immunogen against numerous clinically important P. aeruginosa strains. Through active immunization against the extremely conserved region of the chimeric PilS2 pilin, we showed that PilS2 chimeric pilin whether administered alone or formulated with alum as an adjuvant could substantially stimulate humoral immunological responses in BALB/c mice. Based on these findings, we conclude that PilS2 pilin is therapeutically effective against a variety of highly virulent strains of P. aeruginosa and can act as a new immunogen for more research towards the creation of efficient immunotherapy techniques against the P. aeruginosa as a dexterous pathogen.

Tad and toxin-coregulated pilus structures reveal unexpected diversity in bacterial type IV pili.

Type IV pili (T4P) are ubiquitous in both bacteria and archaea. They are polymers of the major pilin protein, which has an extended and protruding N-terminal helix, α1, and a globular C-terminal domain. Cryo-EM structures have revealed key differences between the bacterial and archaeal T4P in their C-terminal domain structure and in the packing and continuity of α1. This segment forms a continuous α-helix in archaeal T4P but is partially melted in all published bacterial T4P structures due to a conserved helix breaking proline at position 22. The tad (tight adhesion) T4P are found in both bacteria and archaea and are thought to have been acquired by bacteria through horizontal transfer from archaea. Tad pilins are unique among the T4 pilins, being only 40 to 60 residues in length and entirely lacking a C-terminal domain. They also lack the Pro22 found in all high-resolution bacterial T4P structures. We show using cryo-EM that the bacterial tad pilus from Caulobacter crescentus is composed of continuous helical subunits that, like the archaeal pilins, lack the melted portion seen in other bacterial T4P and share the packing arrangement of the archaeal T4P. We further show that a bacterial T4P, the Vibrio cholerae toxin coregulated pilus, which lacks Pro22 but is not in the tad family, has a continuous N-terminal α-helix, yet its α1 s are arranged similar to those in other bacterial T4P. Our results highlight the role of Pro22 in helix melting and support an evolutionary relationship between tad and archaeal T4P.

Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.

Screening of antigenic epitopes related to the adhesion of the avian Escherichia coli Type 1 Fimbrial Agglutinin Domain.

BACKGROUND: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli. RESULTS: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111-124 and 154-162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111-124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154-162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111-124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein. CONCLUSIONS: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111-124 and 154-162 could inhibit the adhesion of E.coli to the chicken trachea.

Pilus of Streptococcus pneumoniae: structure, function and vaccine potential.

The pilus is an extracellular structural part that can be detected in some Streptococcus pneumoniae (S. pneumoniae) isolates (type I pili are found in approximately 30% of strains, while type II pili are found in approximately 20%). It is anchored to the cell wall by LPXTG-like motifs on the peptidoglycan. Two kinds of pili have been discovered, namely, pilus-1 and pilus-2. The former is encoded by pilus islet 1 (PI-1) and is a polymer formed by the protein subunits RrgA, RrgB and RrgC. The latter is encoded by pilus islet 2 (PI-2) and is a polymer composed mainly of the structural protein PitB. Although pili are not necessary for the survival of S. pneumoniae, they serve as the structural basis and as virulence factors that mediate the adhesion of bacteria to host cells and play a direct role in promoting the adhesion, colonization and pathogenesis of S. pneumoniae. In addition, as candidate antigens for protein vaccines, pili have promising potential for use in vaccines with combined immunization strategies. Given the current understanding of the pili of S. pneumoniae regarding the genes, proteins, structure, biological function and epidemiological relationship with serotypes, combined with the immunoprotective efficacy of pilins as protein candidates for vaccines, we here systematically describe the research status and prospects of S. pneumoniae pili and provide new ideas for subsequent vaccine research and development.

The role of the minor colonization factor CS14 in adherence to intestinal cell models by geographically diverse ETEC isolates.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low- to middle-income countries. ETEC adheres to small intestinal epithelia via colonization factors (CFs) and secretes heat-stable toxin and/or heat-labile toxin, causing dysregulated ion transport and water secretion. There are over 30 CFs identified, including major CFs associated with moderate-to-severe diarrhea (MSD) and minor CFs for which a role in pathogenesis is less clear. The Global Enteric Multicenter Study identified CS14, a class 5a fimbriae, as the only minor CF significantly associated with MSD and was recommended for inclusion in ETEC vaccines. Despite detection of CS14 in ETEC isolates, the sequence conservation of the CS14 operon, its role in adherence, and functional cross-reactivity to other class 5a fimbriae like CFA/I and CS4 are not understood. Sequence analysis determined that the CS14 operon is >99.9% identical among seven geographically diverse isolates with expanded sequence analysis demonstrating SNPs exclusively in the gene encoding the tip adhesin CsuD. Western blots and electron microscopy demonstrated that CS14 expression required the growth of isolates on CFA agar with the iron chelator deferoxamine mesylate. CS14 expression resulted in significantly increased adherence to cultured intestinal cells and human enteroids. Anti-CS14 antibodies and anti-CS4 antibodies, but not anti-CFA/I antibodies, inhibited the adherence of a subset of ETEC isolates, demonstrating CS14-specific inhibition with partial cross-reactivity within the class 5a fimbrial family. These data provide support for CS14 as an important fimbrial CF and its consideration as a vaccine antigen in future strategies. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) infection causes profuse watery diarrhea in adults and children in low- to middle-income countries and is a leading cause of traveler's diarrhea. Despite increased use of rehydration therapies, young children especially can suffer long-term effects including gastrointestinal dysfunction as well as stunting and malnutrition. As there is no licensed vaccine for ETEC, there remains a need to identify and understand specific antigens for inclusion in vaccine strategies. This study investigated one adhesin named CS14. This adhesin is expressed on the bacterial surface of ETEC isolates and was recently recognized for its significant association with diarrheal disease. We demonstrated that CS14 plays a role in bacterial adhesion to human target cells, a critical first step in the disease process, and that adherence could be blocked by CS14-specific antibodies. This work will significantly impact the ETEC field by supporting inclusion of CS14 as an antigen for ETEC vaccines.

Uptake of environmental DNA in Bacillus subtilis occurs all over the cell surface through a dynamic pilus structure.

At the transition to stationary phase, a subpopulation of Bacillus subtilis cells can enter the developmental state of competence, where DNA is taken up through the cell envelope, and is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC molecules changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data support a model in which transport of environmental DNA occurs through the entire cell surface by a dynamic pilus, mediating efficient uptake through the cell wall into the periplasm, where DNA diffuses to a cell pole containing the localized transport machinery mediating passage into the cytosol.

Visualizing dynamic competence pili and DNA capture throughout the long axis of Bacillus subtilis.

The first step in the process of bacterial natural transformation is DNA capture. Although long hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA binding had not yet been visualized for Bacillus subtilis. Here, we visualize functional competence pili in Bacillus subtilis using fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy. In strains that produce pilin monomers within tenfold of wild-type levels, the median length of detectable pili is 300 nm. These pili are retractile and associate with DNA. The analysis of pilus distribution at the cell surface reveals that they are predominantly located along the long axis of the cell. The distribution is consistent with localization of proteins associated with subsequent transformation steps, DNA binding, and DNA translocation in the cytosol. These data suggest a distributed model for B. subtilis transformation machinery, in which initial steps of DNA capture occur throughout the long axis of the cell and subsequent steps may also occur away from the cell poles. IMPORTANCE This work provides novel visual evidence for DNA translocation across the cell wall during Bacillus subtilis natural competence, an essential step in the natural transformation process. Our data demonstrate the existence of natural competence-associated retractile pili that can bind exogenous DNA. Furthermore, we show that pilus biogenesis occurs throughout the cell long axis. These data strongly support DNA translocation occurring all along the lateral cell wall during natural competence, wherein pili are produced, bind to free DNA in the extracellular space, and finally retract to pull the bound DNA through the gap in the cell wall created during pilus biogenesis.

siRNA targeting Atp5a1 gene encoding ATPase α, the ligand of Peg fimbriae, reduced Salmonella Enteritidis adhesion.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a zoonotic pathogen that can infect both humans and animals. Among the 13 types of fimbrial operons in S. Enteritidis, the highly conserved Peg fimbriae play a crucial role in the adhesion and invasion of S. Enteritidis into host cells but are not well studied. In this study, we identified the ATP synthase subunit alpha (ATPase α) as a ligand of Peg fimbriae using ligand blotting and mass spectrometry techniques. We confirmed the in vitro binding of ATPase α to the purified adhesion protein (PegD). Furthermore, we used siRNA to suppress the expression of ATPase α gene Atp5a1 in Leghorn male hepatoma (LMH) cells, which resulted in a significant reduction in the adhesion rate of S. Enteritidis to the cells (P < 0.05). The findings in this study provide insight into the mechanism of S. Enteritidis infection through Peg fimbriae and highlight the importance of ATPase α in the adhesion process.RESEARCH HIGHLIGHTS Ligand blotting was performed to screen the ligand of S. Enteritidis Peg fimbriae.Binding assay confirmed that ATPase α is the ligand of the Peg fimbriae.siRNA targeting ATPase α gene (Atp5a1) significantly reduced S. Enteritidis adhesion.

High-Frequency Changes in Pilin Glycosylation Patterns during Neisseria meningitidis Serogroup a Meningitis Outbreaks in the African Meningitis Belt.

In the meningitis belt of sub-Saharan Africa, there are cyclic meningococcal epidemics that coincide with clonal waves of Neisseria meningitidis carriage and invasive disease. In the framework of longitudinal colonization and disease studies in Ghana and Burkina Faso, meningococcal isolates belonging to the closely related hypervirulent A:ST-5, A:ST-7, and A:ST-2859 clones have been collected from 1998 to 2011 during meningococcal outbreaks. A comparative whole-genome sequencing study with 100 of these isolates identified the pilin glycosylation (pgl) locus as one hot spot of recombination. Frequent exchange of pgl genes in N. meningitidis by lateral gene transfer results in differences in the glycosylation patterns of pilin and other cell surface glycoproteins. In this study, we looked at both recombination and phase variation of the pgl genes of these clinical isolates and analyzed the glycan structures resulting from different pgl alleles and their variable expression. Our results indicate that the basal O-linked sugar of the glycans expressed by these isolates is masked by various additional mono- or disaccharide structures whose expression is highly variable due to the phase-variable expression of pgl genes. We also observed a distinct glycoform in two isolates with pgl loci that were modified by recombination. These data suggest that variation in N. meningitidis protein glycosylation could be crucial for bacterial adaptation to evade herd immunity in semi-immune populations. Investigating pilin glycosylation in N. meningitidis can shed light on the mechanisms by which this pathogen evades the host immune response, and may help identify potential targets for novel therapies and vaccines.