Surface Pili Research Articles

EbsA is essential for both motility and biofilm formation in the filamentous cyanobacterium Nostoc punctiforme.

Many cyanobacteria, both unicellular and filamentous, exhibit surface motility driven by type IV pili (T4P). While the component parts of the T4P machinery described in other prokaryotes are largely conserved in cyanobacteria, there are also several T4P proteins that appear to be unique to this phylum. One recently discovered component is EbsA, which has been characterized in two unicellular cyanobacteria. EbsA was found to form a complex with other T4P proteins and is essential for motility. Additionally, deletion of ebsA in one of these strains promoted the formation of biofilms. To expand the understanding of ebsA in cyanobacteria, its role in motility and biofilm formation were investigated in the model filamentous cyanobacterium Nostoc punctiforme. Expression of ebsA was strictly limited to hormogonia, the motile filaments of N. punctiforme. Deletion of ebsA did not affect hormogonium development but resulted in the loss of motility and the failure to accumulate surface pili or produce hormogonium polysaccharide (HPS), consistent with pervious observations in unicellular cyanobacteria. Protein-protein interaction studies indicated that EbsA directly interacts with PilB, and the localization of EbsA-GFP resembled that previously shown for both PilB and Hfq. Collectively, these results support the hypothesis that EbsA forms a complex along with PilB and Hfq that is essential for T4P extension. In contrast, rather than enhancing biofilm formation, deletion of both ebsA and pilB abolish biofilm formation in N. punctiforme, implying that distinct modalities for the relationship between motility, T4P function and biofilm formation may exist in different cyanobacteria.

Bidirectional pilus processing in the Tad pilus system motor CpaF

The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.

Detection of Cell Wall-Anchoring Machinery by Immunogold-Labeling Thin-Section Electron Microscopy.

Cell wall anchoring of surface proteins and pili in Gram-positive bacteria is mediated by sortase - a highly conserved transpeptidase enzyme. Early studies have demonstrated the membrane-associated nature of this enzyme in close proximity with its cognate substrates, using immunogold-labeling thin-section electron microscopy. Here, we provide a detail protocol of this methodology, including specimen preparation, ultrathin sectioning, and immunogold-labeling electron microscopic procedures, with an experimental model of sortase enzymes from Actinomyces oris. In principle, this protocol can be employed for any bacterial ultrathin-section samples to detect subcellular localization of proteins and organelles by immuno-electron microscopy.

Determination of the Crystal Structure of the Cell Wall-Anchored Proteins and Pilins.

Surface proteins and pili (or pilus) anchored on the Gram-positive bacterial cell wall play a vital role in adhesion, colonization, biofilm formation, and immunomodulation. The pilus consists of building blocks called pilins or pilus subunits. The surface proteins and pilins share some common sequences and structural features. They contain an N-terminal signal sequence and the C-terminal cell wall sorting region, enabling their transportation across the membrane and covalent attachment to the bacterial cell wall, respectively. The transpeptidase enzymes called sortases facilitate the covalent links between the pilins during the pilus assembly and between surface proteins or basal subunits of pili and peptidoglycan-bridge during the cell wall anchoring. Thus, elucidating three-dimensional structures for the surface proteins and pilins at the atomic level is essential for understanding the mechanism of adhesion, pilus assembly, and host interaction. This chapter aims to provide a general protocol for crystal structure determination of surface proteins and pilins anchored on the Gram-positive bacterial cell wall and substrates for sortases. The protocol involves the production of recombinant protein, crystallization, and structure determination by X-ray crystallography technique.

Assembly mechanism of a Tad secretion system secretin-pilotin complex

The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.

Deciphering the substrate specificity of housekeeping sortase A and pilus-specific sortase C of probiotic bacterium Lactococcus lactis

Several strains and species of lactic acid bacteria (LAB) are widely used in fermented foods, including dairy products and also as probiotics, because of their contribution to various health benefits in humans. Sortase enzymes decorate the bacterial cell wall with different surface proteins and pili for facilitating the interactions with host and environment for the colonization and beneficial effects. While the sortases and sortase anchored proteins from pathogens have been the prime focus of the research in the past, sortases from many non-pathogenic bacteria, including LAB strains, have attracted attention for their potential applications in vaccine delivery and other clinical interventions. Here, we report the purification and functional characterization of two sortases (housekeeping SrtA and pilus-specific SrtC) from a probiotic Lactococcus lactis. The purified sortases were found to be active against the putative LPXTG motif-based peptide substrates, albeit with differences. The in-silico analysis provides insights into the residues involved in substrate binding and specificity. Overall, this study sheds new light on the aspects of structure, substrate specificity, and function of sortases from non-pathogenic bacteria, which may have physiological ramifications as well as their applications in sortase-mediated protein bioconjugation.

Group B streptococcal infections in infants in Iceland: clinical and microbiological factors.

IntroductionGroup B streptococcus (GBS) is a leading cause of invasive neonatal infections. These have been divided into early-onset disease (EOD; <7 days) and late-onset disease (LOD; 7–89 days), with different GBS clonal complexes (CCs) associated with different disease presentations.Hypothesis/Gap StatementDifferent GBS CCs are associated with timing of infection (EOD or LOD) and clinical presentation (sepsis, meningitis or pneumonia).AimTo study infant GBS infections in Iceland from 1975 to 2019. Are specific GBS CCs related to disease presentation? Is CC17 overrepresented in infant GBS infections in Iceland?MethodologyAll culture-confirmed invasive GBS infections in infants (<90 days) in Iceland from 1975 to 2019 were included. Clinical information was gathered from medical records.ResultsA total of 127 invasive GBS infections in infants were diagnosed, but 105 infants were included in the study. Of these, 56 had EOD and 49 had LOD. The incidence of GBS infections declined from 2000 onwards but increased again at the end of the study period. Furthermore, there was a significant increase in LOD over the study period (P=0.0001). The most common presenting symptoms were respiratory difficulties and fever and the most common presentation was sepsis alone. Approximately one-third of the cases were caused by GBS CC17 of serotype III with surface protein RIB and pili PI-1+PI-2b or PI-2b. CC17 was significantly associated with LOD (P<0.001).ConclusionCC17 is a major cause of GBS infection in infants in Iceland. This clone is associated with LOD, which has been increasing in incidence. Because intrapartum antibiotic prophylaxis only prevents EOD, it is important to continue the development of a GBS vaccine in order to prevent LOD infections.

Exploiting pilus-mediated bacteria-host interactions for health benefits

Surface pili (or fimbriae) are an important but conspicuous adaptation of several genera and species of Gram-negative and Gram-positive bacteria. These long and non-flagellar multi-subunit adhesins mediate the initial contact that a bacterium has with a host or environment, and thus have come to be regarded as a key colonization factor for virulence activity in pathogens or niche adaptation in commensals. Pili in pathogenic bacteria are well recognized for their roles in the adhesion to host cells, colonization of tissues, and establishment of infection. As an ‘anti-adhesive’ ploy, targeting pilus-mediated attachment for disruption has become a potentially effective alternative to using antibiotics. In this review, we give a description of the several structurally distinct bacterial pilus types thus far characterized, and as well offer details about the intricacy of their individual structure, assembly, and function. With a molecular understanding of pilus biogenesis and pilus-mediated host interactions also provided, we go on to describe some of the emerging new approaches and compounds that have been recently developed to prevent the adhesion, colonization, and infection of piliated bacterial pathogens.

Crystal structure of the usher chaperone YadV reveals a monomer with the proline lock in closed conformation suggestive of an intermediate state.

Cell surface pili assembled by the chaperone-usher (CU) pathway play a crucial role in the adhesion of uropathogenic Escherichiacoli. YadV is the chaperone component of the CU pathway of Yad pili. Here, we report the crystal structure of YadV from E.coli. In contrast to major usher chaperones, YadV is a monomer in solution as well as in the crystallographic symmetry, and the monomeric form is a preferred state for interacting with pilus subunits. Moreover, we observed a closed conformation for the proline lock, a crucial structural element for chaperone-pilus subunit interaction. MD simulation shows that the closed state of the proline lock is not energetically stable. Thus, the structure of monomeric YadV with its closed proline lock may serve as an intermediate state to provide suitable access to pilus subunits.

Kinetics and Optimization of the Lysine-Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae.

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.

Molecular characterization of Streptococcus agalactiae isolated from pregnant women and newborns at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia

BackgroundStreptococcus agalctiae (Group B Streptococcus, GBS) is a perinatal pathogen and a leading cause of neonatal infections worldwide. Serotype, sequence type, clonality, antibiotic resistance genes and surface protein profiles of GBS are scarce in Ethiopia, a reason that this study was planned to investigate. .MethodsSixteen colonizing GBS isolates obtained from recto-vaginal swabs of pregnant women and body surfaces of newborns were further analyzed. Minimum inhibitory concentration (MIC) test, and whole genome sequence (WGS) methods were done for antibiotic susceptibility test, and molecular characterization of the isolates.ResultsAll the GBS isolates analyzed were belonged to four capsular serotypes: II, 11/16(68.8%), V, 3/16(18.8%), Ia and VI each with 1/16(6.3%) and five sequence type (ST-2, ST-10, ST-14, ST-569 and ST-933). Sequence type-10 was the most predominant ST followed by ST-569. The five STs were grouped into the four clonal complexes (CC - 1, CC-10, CC-19, and CC-23). Different surface proteins and pili families such as ALP1, ALPHA, ALP23, PI-1 / PI-2A1, PI-1 / PI-2B, and Srr1 were detected from WGS data. All isolates were found to be susceptible to the tested antibiotics except for tetracycline in MIC and WGS test methods used. Tetracycline resistant determinant genes such as TETM and TETL / TETM combination were identified.ConclusionFurther studies on serotype and molecular epidemiology will provide a comprehensive data of the GBS capsular serotype and clones available in Ethiopia.

A comprehensive in silico analysis of sortase superfamily.

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.

Increasing macrolide resistance among Streptococcus agalactiae causing invasive disease in non-pregnant adults was driven by a single capsular-transformed lineage, Portugal, 2009 to 2015.

We characterised Lancefield group B streptococcal (GBS) isolates causing invasive disease among non-pregnant adults in Portugal between 2009 and 2015. All isolates (n = 555) were serotyped, assigned to clonal complexes (CCs) by multilocus sequence typing and characterised by surface protein and pilus island gene profiling. Antimicrobial susceptibility was tested by disk diffusion and resistance genotypes identified by PCR. Overall, serotype Ia was most frequent in the population (31%), followed by serotypes Ib (24%) and V (18%). Serotype Ib increased significantly throughout the study period (p < 0.001) to become the most frequent serotype after 2013. More than 40% of isolates clustered in the CC1/alp3/PI-1+PI-2a genetic lineage, including most isolates of serotypes Ib (n = 110) and V (n = 65). Erythromycin and clindamycin resistance rates were 35% and 34%, respectively, both increasing from 2009 to 2015 (p < 0.010) and associated with CC1 and serotype Ib (p < 0.001). The Ib/CC1 lineage probably resulted from acquisition of the type Ib capsular operon in a single recombination event by a representative of the V/CC1 macrolide-resistant lineage. Expansion of the new serotype Ib/CC1 lineage resulted in increased macrolide resistance in GBS, causing invasive disease among adults in Portugal. The presence of this clone elsewhere may predict more widespread increase in resistance.

The glycointeractome of serogroup B Neisseria meningitidis strain MC58

Neisseria meningitidis express numerous virulence factors that enable it to interact with diverse microenvironments within the host, during both asymptomatic nasopharyngeal colonization and invasive disease. Many of these interactions involve bacterial or host glycans. In order to characterise the meningococcal glycointeractome, glycan arrays representative of structures found on human cells, were used as a screening tool to investigate host glycans bound by N. meningitidis. Arrays probed with fluorescently labelled wild-type MC58 revealed binding to 223 glycans, including blood group antigens, mucins, gangliosides and glycosaminoglycans. Mutant strains lacking surface components, including capsule, lipooligosaccharide (LOS), Opc and pili, were investigated to identify the factors responsible for glycan binding. Surface plasmon resonance and isothermal calorimetry were used to confirm binding and determine affinities between surface components and host glycans. We observed that the L3 LOS immunotype (whole cells and purified LOS) bound 26 structures, while L8 only bound 5 structures. We further demonstrated a direct glycan-glycan interaction between purified L3 LOS and Thomsen–Friedenreich (TF) antigen, with a KD of 13 nM. This is the highest affinity glycan-glycan interaction reported to date. These findings highlight the diverse glycointeractions that may occur during different stages of meningococcal disease, which could be exploited for development of novel preventative and therapeutic strategies.

Human Gut-Commensalic Lactobacillus ruminis ATCC 25644 Displays Sortase-Assembled Surface Piliation: Phenotypic Characterization of Its Fimbrial Operon through In Silico Predictive Analysis and Recombinant Expression in Lactococcus lactis.

Sortase-dependent surface pili (or fimbriae) in Gram-positive bacteria are well documented as a key virulence factor for certain harmful opportunistic pathogens. However, it is only recently known that these multi-subunit protein appendages are also belonging to the “friendly” commensals and now, with this new perspective, they have come to be categorized as a niche-adaptation factor as well. In this regard, it was shown earlier that sortase-assembled piliation is a native fixture of two human intestinal commensalics (i.e., Lactobacillus rhamnosus and Bifidobacterium bifidum), and correspondingly where the pili involved have a significant role in cellular adhesion and immunomodulation processes. We now reveal that intestinal indigenous (or autochthonous) Lactobacillus ruminis is another surface-piliated commensal lactobacillar species. Heeding to in silico expectations, the predicted loci for the LrpCBA-called pili are organized tandemly in the L. ruminis genome as a canonical fimbrial operon, which then encodes for three pilin-proteins and a single C-type sortase enzyme. Through electron microscopic means, we showed that these pilus formations are a surface assemblage of tip, basal, and backbone pilin subunits (respectively named LrpC, LrpB, and LrpA) in L. ruminis, and also when expressed recombinantly in Lactococcus lactis. As well, by using the recombinant-piliated lactococci, we could define certain ecologically relevant phenotypic traits, such as the ability to adhere to extracellular matrix proteins and gut epithelial cells, but also to effectuate an induced dampening on Toll-like receptor 2 signaling and interleukin-8 responsiveness in immune-related cells. Within the context of the intestinal microcosm, by wielding such niche-advantageous cell-surface properties the LrpCBA pilus would undoubtedly have a requisite functional role in the colonization dynamics of L. ruminis indigeneity. Our study provides only the second description of a native-piliated Lactobacillus species, but at the same time also involves the structural and functional characterization of a third type of lactobacillar pilus.

INFLUENCE OF PROBIOTICS ON CYTOKINE PRODUCTION IN THE IN VITRO AND IN VIVO SYSTEMS

Modulatory effects of three probiotic bacterial strains (Lactobacillus rhamnosus K32 (L), Bifidobacterium longum GT15 (B, Enterococcus faecium L-3 (E) on expression level and contents of key cytokines were studied using PCR techniques with reverse transcription, and enzyme-linked immunosorbent assay. Both cell cultures and an experimental model of intestinal dysbiosis were used in this study. The genes encoding bacteriocins, surface membrane component, pili and exopolysaccharides involved in host immune system modulation were previously identified in the B and Ebacterial strains. Investigation of probiotic strains and effects of their supernatants expression of cytokines in cell cultures of promonocyte origin (HTP-1) showed increased expression of TNFα, due to E and L supernatants. Moreover, the Bl culture induced IL-8 and IL-10 expression. In a model of Wistar rats with ampicillinand metronidazole-induced intestinal dysbiosis corrected with probiotics we have shown that the dysbiosis was accompanied by sufficient alterations in microbiota composition (Klebsiella spp. overgrowth and low contents of Faecalobacterium prausnitzii) that were observed only in the animals untreated with probiotics (control), or after administration of L. In contrast to these results, the animals treated with E and B, the following changes were revealed: 1) low expression of proinflammatory cytokines IL-8, TNFα, MCP-1 inmesenteric lymph nodes and appropriate changes of their serum contents, 2) increased serum content of the anti-inflammatory TGFβ cytokine. Hence, the present study, having used two complementary models, has detected some individual features of immune modulation produced by the probiotictic strains of L. rhamnosus K32, B. longum GT15 и E. faecium L-3 which exert differential effects upon the intestinal microbiota.

The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus.

The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens.

Pilins in gram-positive bacteria: A structural perspective.

Pilins or fimbrilins are a class of proteins found in bacterial surface pilus, a hair-like surface appendage. Both the Gram-negative and -positive bacteria produce pilins to assemble pili on their cell-surface for different purposes including adherence, twitching motility, conjugation, immunomodulation, biofilm formation, and electron transfer. Immunogenic properties of the pilins make them attractive vaccine candidates. The polymerized pilins play a key role in the initiation of host adhesion, which is a critical step for bacterial colonization and infection. Because of their key role in adhesion and exposure on the cell surface, targeting the pilins-mediated adhesion (anti-adhesion therapy) is also seen as a promising alternative approach for preventing and treating bacterial infections, one that may overcome their ever-increasing repertoires of resistance mechanisms. Individual pilins interact with each other non-covalently to assemble the pilus fiber with the help of associated proteins like chaperones and Usher in Gram-negative bacteria. In contrast, the pilins in Gram-positive bacteria often connect with each other covalently, with the help of sortases. Certain unique structural features present on the pilins distinguish them from one another across different bacterial strains, and these dictate their cellular targets and functions. While the structure of pilins has been extensively studied in Gram-negative pathogenic bacteria, the pilins in Gram-positive pathogenic bacteria have been in only during the last decade. Recently, the discovery of pilins in non-pathogenic bacteria, such as Lactobacillus rhamnosus GG, has received great attention, though traditionally the attention was on pathogenic bacteria. This review summarizes and discusses the current structural knowledge of pilins in Gram-positive bacteria with emphasis on those pilins which are sortase substrates.

Structure and assembly of group B streptococcus pilus 2b backbone protein.

Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.

Neisseria gonorrhoeae MutS affects pilin antigenic variation through mismatch correction and not by pilE guanine quartet binding.

Many pathogens use homologous recombination to vary surface antigens to avoid immune surveillance. Neisseria gonorrhoeae achieves this in part by changing the properties of its surface pili in a process called pilin antigenic variation (AV). Pilin AV occurs by high-frequency gene conversion reactions that transfer silent pilS sequences into the expressed pilE locus and requires the formation of an upstream guanine quartet (G4) DNA structure to initiate this process. The MutS and MutL proteins of the mismatch correction (MMC) system act to correct mismatches after replication and prevent homeologous (i.e., partially homologous) recombination, but MutS orthologs can also bind to G4 structures. A previous study showed that mutation of MutS resulted in a 3-fold increase in pilin AV, which could be due to the loss of MutS antirecombination properties or loss of G4 binding. We tested two site-directed separation-of-function MutS mutants that are both predicted to bind to G4s but are not able to perform MMC. Pilus phase variation assays and DNA sequence analysis of pilE variants produced in these mutants showed that all three mutS mutants and a mutL mutant had similar increased frequencies of pilin AV. Moreover, the mutS mutants all showed similar increased levels of pilin AV-dependent synthetic lethality. These results show that antirecombination by MMC is the reason for the effect that MutS has on pilin AV and is not due to pilE G4 binding by MutS. Neisseria gonorrhoeae continually changes its outer surface proteins to avoid recognition by the immune system. N. gonorrhoeae alters the antigenicity of the pilus by directed recombination between partially homologous pilin copies in a process that requires a guanine quartet (G4) structure. The MutS protein of the mismatch correction (MMC) system prevents recombination between partially homologous sequences and can also bind to G4s. We confirmed that loss of MMC increases the frequency of pilin antigenic variation and that two MutS mutants that are predicted to separate the two different functions of MutS inhibit pilin variation similarly to a complete-loss-of-function mutant, suggesting that interaction of MutS with the G4 structure is not a major factor in this process.