Piliated Strains Research Articles

The Molecular Epidemiology of Pneumococcal Strains Isolated from the Nasopharynx of Preschool Children 3 Years after the Introduction of the PCV Vaccination Program in Poland.

The genetic mechanisms of resistance, clonal composition, and the occurrence of pili were analyzed in 39 pneumococcal strains isolated from healthy children in the southeastern region of Poland. Strains with resistance to combinations of erythromycin, clindamycin, and tetracycline were found in clonal groups (CGs) related to Tennessee 23F-4 and Taiwan 19F-14 clones. Capsular switching possibly occurred in the Spain 9V-3 clone and its variants to serotypes 35B and 6A, as well as DLVs of Tennessee 23F-4 to serotype 23A. The double-locus variants of Colombia 23F-26 presented serotype 23B. The major transposons carrying the erythromycin and tetracycline resistance genes were Tn6002 (66.6%), followed by Tn916 (22.2%) and Tn2009 (11.1%). The macrolide efflux genetic assembly (MEGA) element was found in 41.7% of all erythromycin-resistant isolates. The majority of the isolates carrying the PI-1 gene belonged to the CGs related to the Spain 9V-3 clone expressing serotypes 35B and 6A, and the presence of both PI-1 and PI-2 was identified in CG4 consisting of the isolates related to the Taiwan 19F-14 clone expressing serotypes 19F and 19A. Importantly, in the nearest future, the piliated strains of serogroups 23B, 23A, and 35B may be of concern, being a possible origin of the emerging clones of piliated non-vaccine pneumococcal serotypes in Poland. This study reveals that nasopharyngeal carriage in children is an important reservoir for the selection and spreading of new drug-resistant pneumococcal clones in the community after the elimination of vaccine serotypes.

MP34-15 THE ROLE OF FERROPTOSIS IN NEUTROPHILS-MEDIATED INNATE IMMUNE DEFENSE AGAINST URINARY TRACT INFECTION

You have accessJournal of UrologyCME1 Apr 2023MP34-15 THE ROLE OF FERROPTOSIS IN NEUTROPHILS-MEDIATED INNATE IMMUNE DEFENSE AGAINST URINARY TRACT INFECTION Yan Liu, Hongying Huang, Herbert Lepor, Xue-Ru Wu, and Ellen Shapiro Yan LiuYan Liu More articles by this author , Hongying HuangHongying Huang More articles by this author , Herbert LeporHerbert Lepor More articles by this author , Xue-Ru WuXue-Ru Wu More articles by this author , and Ellen ShapiroEllen Shapiro More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003268.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ferroptosis is a recently discovered programmed cell death involved in disorders including infectious disease, autoimmune disease, and cancer. It is characterized by iron accumulation and reactive oxygen species (ROS)-induced lipid peroxidation. Neutrophils are the first line of innate defense against urinary tract infection (UTI), and their interaction with uropathogenic E.coli (UPEC) triggers generation of ROS and leads to cell death. However, whether UPECs induce ferroptosis in neutrophils remains elusive. This study aims to elucidate the role of ferroptosis in neutrophils-mediated host defense against UTI. METHODS: Human myeloblastic cells HL-60 were induced to terminally differentiated neutrophils by 1.5% DMSO for 72 hours. The differentiated cells were challenged by type 1 piliated UPEC strains (UTI89, NU14), isogenic FimH adhesin deletion strain (NU14-1), or non-fimbriated laboratory strain (HB101) at a multiplicity of infection of 40. ROS level was examined by H2DCFDA cellular ROS assay. Iron level was measured by Phen Green FL diacetate assay. Cell membrane integrity was checked by DiO cell labeling. Lipid peroxidation was detected by cell-based lipid peroxidation assay. The cells were exposed to UPEC virulent factors lipopolysaccharide (LPS) and flagellin. HL-60 cells pretreated with or without the ferroptosis inhibitor ferrostatin-1 were infected, and cell suspensions were lysed, diluted, and plated to assess viable UPECs. RESULTS: Type 1 piliated UPEC strains UTI89 and NU14 induced a rapid elevation in ROS and iron accumulation in differentiated HL-60 cells in a dose- and time-dependent manner and accompanied by lipid peroxidation with multiple perforations on the membrane, all of which are hallmarks of ferroptosis. On the contrary, pretreatment of HL-60 cells with ferroptosis inhibitor ferrostatin-1 reduced 20% of UPEC-induced ROS production, alleviated lipid peroxidation, and led to a 36% higher number of viable UPECs compared to the controls. Furthermore, FimH deletion isogenic strain NU14-1 and non-fimbriated laboratory strain HB101 did not elicit activation of ferroptosis in HL-60 cells, indicating FimH-mediated adherence is required for inducing ferroptosis. Lastly, UPEC virulent factors LPS or flagellin alone did not evoke ferroptosis. CONCLUSIONS: Our data demonstrate for the first time that UPECs induce ferroptosis in neutrophils, and the induction depends on the adhesion of UPECs to the cell surface. The results also suggest that enhancing ferroptosis holds the therapeutic potential for improved clearance of UPECs. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e464 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yan Liu More articles by this author Hongying Huang More articles by this author Herbert Lepor More articles by this author Xue-Ru Wu More articles by this author Ellen Shapiro More articles by this author Expand All Advertisement PDF downloadLoading ...

Association of pili with widespread multidrug-resistant genetic lineages of non-invasive pediatric Streptococcus pneumoniae isolates.

The study aimed to evaluate the presence of pili in non-invasive pediatric pneumococcal isolates and to elucidate possible links with genetic lineages, serotypes, and antimicrobial resistance. We examined 147 Streptococcus pneumoniae isolates from children with respiratory tract infections and acute otitis media. Serotyping was performed by latex agglutination and capsule swelling reaction. Serogroup 6 was subjected to PCR-serotyping. Minimum inhibitory concentrations were determined according to EUCAST breakpoints. PCRs for rlrA and pitB genes were performed to detect a presence oftype 1 and type 2 pili. MLST was conducted to define the clonal structure of the piliated strains. Almost all children (96.5%) were vaccinated with the pneumococcal conjugate vaccine PCV10. We detected 76.8% non-PCV10 - serotypes (NVTs) and 14.3% PCV10 serotypes. The predominant serotypes were NVTs: 19A (14.3%), 6C (12.2%), 3 (9.5%), 15A (7.5%) and 6A (6.8%). PI-1 was detected among 10.9% non-PCV10 serotypes 6A, 6C, and 19A and 6.1% PCV10 serotypes 19F and 23F. Type 2 pili were not found in the studied population. High levels of antimicrobial nonsusceptibility to erythromycin (58.5%), oral penicillin (55.8%), clindamycin (46.9%), trimethoprim-sulfamethoxazole (45.6%), tetracycline (39.5%) and ceftriaxone (16.3%) were revealed. The multidrug-resistant strains (MDR) were 55.1%. MLST represented 18 STs and three CCs among the piliated pneumococci: CC386, CC320, and CC81. More than half of the piliated strains (56.0%) belonged to successfully circulating international clones. PI-1 was associated mainly with MDR 6A, 6C, 19A, 19F, and 23F isolates from the widespread CC386, CC320, and CC81.

Antimicrobial susceptibility, serotype distribution, virulence profile and molecular typing of piliated clinical isolates of pneumococci from east coast, Peninsular Malaysia

Pilus has been recently associated with pneumococcal pathogenesis in humans. The information regarding piliated isolates in Malaysia is scarce, especially in the less developed states on the east coast of Peninsular Malaysia. Therefore, we studied the characteristics of pneumococci, including the piliated isolates, in relation to antimicrobial susceptibility, serotypes, and genotypes at a major tertiary hospital on the east coast of Peninsular Malaysia. A total of 100 clinical isolates collected between September 2017 and December 2019 were subjected to serotyping, antimicrobial susceptibility test, and detection of pneumococcal virulence and pilus genes. Multilocus sequence typing (MLST) and phylogenetic analysis were performed only for piliated strains. The most frequent serotypes were 14 (17%), 6A/B (16%), 23F (12%), 19A (11%), and 19F (11%). The majority of isolates were resistant to erythromycin (42%), tetracycline (37%), and trimethoprim-sulfamethoxazole (24%). Piliated isolates occurred in a proportion of 19%; 47.3% of them were multidrug-resistant (MDR) and a majority had serotype 19F. This study showed ST236 was the most predominant sequence type (ST) among piliated isolates, which was related to PMEN clone Taiwan19F-14 (CC271). In the phylogenetic analysis, the piliated isolates were grouped into three major clades supported with 100% bootstrap values. Most piliated isolates belonged to internationally disseminated clones of S. pneumoniae, but pneumococcal conjugate vaccines (PCVs) have the potential to control them.

Pili and other surface proteins influence the structure and the nanomechanical properties of Lactococcus lactis biofilms

Lactic acid bacteria, in particular Lactococcus lactis, are widely used in the food industry, for the control and/or the protection of the manufacturing processes of fermented food. While L. lactis has been reported to form compact and uniform biofilms it was recently shown that certain strains able to display pili at their surface form more complex biofilms exhibiting heterogeneous and aerial structures. As the impact of those biofilm structures on the biomechanical properties of the biofilms is poorly understood, these were investigated using AFM force spectroscopy and imaging. Three types of strains were used i.e., a control strain devoid of pili and surface mucus-binding protein, a strain displaying pili but no mucus-binding proteins and a strain displaying both pili and a mucus-binding protein. To identify potential correlations between the nanomechanical measurements and the biofilm architecture, 24-h old biofilms were characterized by confocal laser scanning microscopy. Globally the strains devoid of pili displayed smoother and stiffer biofilms (Young Modulus of 4–100 kPa) than those of piliated strains (Young Modulus around 0.04–0.1 kPa). Additional display of a mucus-binding protein did not affect the biofilm stiffness but made the biofilm smoother and more compact. Finally, we demonstrated the role of pili in the biofilm cohesiveness by monitoring the homotypic adhesion of bacteria to the biofilm surface. These results will help to understand the role of pili and mucus-binding proteins withstanding external forces.

Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy.

Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing pili or not as model bacteria. Moreover the force-distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strains showed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7 × 10-18 J, respectively). On the contrary, the piliated strains showed high adhesion levels with adhesion forces and adhesion work over 200 pN and 50 × 10-18 J, respectively. The force-extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical worm-like chain model to get fundamental knowledge on the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucus-binding protein at its surface and two derivative mutants revealed the capacity of pili to interact with other surface proteins including mucus-binding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.

The impact of PCV7/13 on the distribution of carried pneumococcal serotypes and on pilus prevalence; 14 years of repeated cross-sectional surveillance

BackgroundS. pneumoniae carriage by children is a major source of pneumococcal transmission, and the initial step prior to infection. Pilus type 1, reported in ~30% of pneumococcal strains in the pre-vaccine era, contributes to pneumococcal colonization and virulence. In this study, we report the impact of the pneumococcal conjugate vaccine (PCV), PCV7/PCV13 sequential implementation on serotype distribution, and on the prevalence of piliated strains among carried pneumococci during the pre- and post-vaccine eras. MethodsDuring 2002-2016, 12 repeated cross-sectional surveillances of nasopharyngeal S. pneumoniae carriage were conducted among 8,473 children <5.5 years old visiting primary care physicians in Central Israel. Seven biannual surveillances in the pre-PCV period, 2 surveillances after PCV7 was licensed but before implementation in the National Immunization Plan, and 3 additional surveillances in the post-PCV period. S. pneumoniae serotype distribution and prevalence of piliated strains were assessed. ResultsCarriage of S. pneumoniae was relatively stable (45.4%). The prevalence of serotypes included in PCV13 was 65.7%, in the pre-vaccine period and the pilus was present in 26.4% of isolates. The distribution of serotypes and the pilus prevalence in the pre-PCV period was relatively stable except for a decrease in prevalence of piliated 19F, observed following the first study year. Following PCV7/PCV13 implementation, vaccine type 13 (VT13) strains were nearly eliminated to 3.3% by 2016. Piliated strains, which were primarily of VT13 serotypes, initially followed a similar trend and were nearly eliminated by 2014 (1.7%). Yet, two years later, pilus prevalence re-emerged among non-VT strains to 12.8% of all pneumococci. ConclusionsFollowing PCV implementation, a dramatic and rapid decrease in VT strains prevalence was observed with a concomitant increase in non-VT strains. Piliated strains were nearly eliminated, yet re-emerged 7 years following PCV7/PCV13 implementation in various non-VT strains. This suggests that the pilus confers an advantage in colonization.

Immune exclusion by naturally acquired secretory IgA against pneumococcal pilus-1.

Successful infection by mucosal pathogens requires overcoming the mucus barrier. To better understand this key step, we performed a survey of the interactions between human respiratory mucus and the human pathogen Streptococcus pneumoniae. Pneumococcal adherence to adult human nasal fluid was seen only by isolates expressing pilus-1. Robust binding was independent of pilus-1 adhesive properties but required Fab-dependent recognition of RrgB, the pilus shaft protein, by naturally acquired secretory IgA (sIgA). Pilus-1 binding by specific sIgA led to bacterial agglutination, but adherence required interaction of agglutinated pneumococci and entrapment in mucus particles. To test the effect of these interactions in vivo, pneumococci were preincubated with human sIgA before intranasal challenge in a mouse model of colonization. sIgA treatment resulted in rapid immune exclusion of pilus-expressing pneumococci. Our findings predict that immune exclusion would select for nonpiliated isolates in individuals who acquired RrgB-specific sIgA from prior episodes of colonization with piliated strains. Accordingly, genomic data comparing isolates carried by mothers and their children showed that mothers are less likely to be colonized with pilus-expressing strains. Our study provides a specific example of immune exclusion involving naturally acquired antibody in the human host, a major factor driving pneumococcal adaptation.

Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting.

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect 2.5 × 103 CFU/ml of pLR colonies spiked in 106 CFU/ml of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

Vaccine escape of piliated Streptococcus pneumoniae strains

IntroductionType1-pilus proteins were suggested as targets of future protein-based vaccines. Here we studied the effect of pneumococcal-conjugate vaccine (PCV7) implementation on the prevalence of piliated strains in a unique study setting which controls for typical confounders; the Palestinian-Israeli Collaborative Research (PICR). MethodsAnnual cross-sectional surveys of pneumococcal carriage were performed during 2009–2011 among two closely related population that live under different health policies (a) Palestinian-Authority (PA) (n=1773), where PCV7 was not yet introduced (b) East-Jerusalem (EJ) (n=983) where PCV7 was rapidly implemented. Clinical data were collected, pneumococci identified and characterized and the presence of Type1-pilus genes was determined by rrgC PCR. ResultsFollowing PCV7 implementation in EJ, overall carriage prevalence did not change (∼30%), but VT7 strains decreased from 61.5% to 33.8%. While prevalence of non-piliated-VT7 isolates decreased from 37% to 10%, p<0.001, the prevalence of piliated-VT7 strains persisted ∼25%. Additionally, piliated non-VT13 strains emerged (1–15%, p<0.001). These changes were not observed in PA. These dynamics were independent of the bacteria's resistance pattern. ConclusionsA differential effect of PCV7 was observed with a relative resistance of piliated strains to the vaccine. This suggests that Type1-pilus confers an intrinsic advantage for colonization and may be an attractive vaccine target.

Neisseria gonorrhoeae Metalloprotease NGO1686 Is Required for Full Piliation, and Piliation Is Required for Resistance to H 2 O 2 - and Neutrophil-Mediated Killing

ABSTRACTThe sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2 and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants of N. gonorrhoeae are less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates of N. gonorrhoeae.

Biofilm formation and binding specificities of CFA/I, CFA/II and CS2 adhesions of Enterotoxigenic Escherichia coli and CfaE-R181A mutant

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF'lacI (q)/∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF'lacI (q)/pEU5881) showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.

Biofilm formation and binding specificities of CFA/I, CFA/II and CS2 adhesions of enterotoxigenic Escherichia coli and Cfae-R181A mutant

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF’lacIq/∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF’lacIq/pEU5881) showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.

RrgB321, a Fusion Protein of the Three Variants of the Pneumococcal Pilus Backbone RrgB, Is ProtectiveIn Vivoand Elicits Opsonic Antibodies

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.

The genetic analysis of the flp locus of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae, one of the most important porcine respiratory pathogens, exhibits tight adherence to cell surfaces. The Flp pilus, which is assembled by the proteins encoded by the flp (fimbrial low-molecular-weight protein) operon, may play an important role in the bacterial adherence. In this study, the flp operons of twelve A. pleuropneumoniae serotype reference strains were sequenced and analyzed. The phenotypic diversity of fimbriae was observed using transmission electron microscopy, and the adherence ability was tested against a porcine lung epithelial cell line. The complete flp operon was identified in the reference strains of serotypes 1, 4, 5, 7, 12, and 13, consisting of 14 genes (flp1-flp2-tadV-rcpCAB-tadZABCDEFG). Fimbriae were observed protruding from the bacterial cell surfaces of these strains. In contrast, the flp promoter was absent in serotypes 2, 3, 6, 9, and 11, and the flp1 gene was truncated in serotypes 10 and 15. No pilus was observed on the surfaces of these strains. The piliated strains have higher efficiency of adhesion than the pilus-negative strains. Our data demonstrated that the Flp pili are involved in A. pleuropneumoniae adherence. The genetic diversity of the flp operons among different strains may contribute, at least in part, to the variation in virulence of Actinobacillus pleuropneumoniae.

Re-emergence of the type 1 pilus among Streptococcus pneumoniae isolates in Massachusetts, USA

Pneumococcal type 1 pilus proteins have been proposed as potential vaccine candidates. Following conjugate pneumococcal vaccination, the prevalence of the pneumococcal type 1 pilus declined dramatically, a decline associated with the elimination of vaccine-type (VT) strains. Here we show that between 2004 and 2007, there has been a significant increase in pilus prevalence, now exceeding rates from the pre-conjugate vaccine era. This increase is primarily due to non-VT strains. These emerging piliated non-VT strains are mostly novel clones, with some exceptions. The rise in pilus type 1 frequency across multiple distinct genetic backgrounds suggests that the pilus may confer an intrinsic advantage.

Increased expression of the type IV secretion system in piliated Neisseria gonorrhoeae variants.

Neisseria gonorrhoeae produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either traD expression or growth rate. A mutant with a deletion in traD was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that traD is transcriptionally and translationally coupled to traI, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing traI and traD. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of traD and traI.

Adherence of Streptococcus pyogenes to human epithelial cells is modulated by Haemophilus influenzae

Haemophilus influenzae and Streptococcus pyogenes colonize overlapping regions of the nasopharynx and oropharynx. The effect of 10 H. influenzae strains (5 non-piliated parent strains and 5 piliated daughter strains) on adherence of S. pyogenes to human respiratory epithelial cells was assessed in this study. A 39.6% and 49.0% increase in the number of adherent S. pyogenes was observed in Chang epithelial cells if the cells were already colonized with 2 out of 5 non-piliated strains of H. influenzae (p <0.05). Four out of 5 piliated strains of H. influenzae enhanced the adherence of S. pyogenes to Chang cells by 103.5%, 113.6%, 109.7% (p<0.01) and 53.4% (p<0.05). With A549 epithelial cells, non-piliated strains of H. influenzae did not affect the adherence of S. pyogenes (p>0.05), but a 36.9% (p<0.05), 62.9% (p<0.01) and 32.1% (p<0.05) increase in the number of adherent S. pyogenes was found with 3 out of 5 piliated strains of H. influenzae. These results indicate that increases of S. pyogenes adherence to epithelial cells may be modulated species-specifically by H. influenzae, especially by piliated H. influenzae.

Identification of a Putative Acetyltransferase Gene, MMP0350, Which Affects Proper Assembly of both Flagella and Pili in the ArchaeonMethanococcus maripaludis

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukaryotic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis, a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslational modifications.

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