Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

Meningococcal disease: A paradigm of type-IV pilus dependent pathogenesis.

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for two devastating forms of invasive diseases: purpura fulminans and meningitis. Interaction with both peripheral and cerebral microvascular endothelial cells is at the heart of meningococcal pathogenesis. During the last two decades, an essential role for meningococcal type IV pili in vascular colonisation and disease progression has been unravelled. This review summarises 20 years of research on meningococcal type IV pilus-dependent virulence mechanisms, up to the identification of promising anti-virulence compounds that target type IV pili.

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa.

Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.

Role of Cyclic Di-GMP and Exopolysaccharide in Type IV Pilus Dynamics.

For Pseudomonas aeruginosa, levels of cyclic di-GMP (c-di-GMP) govern the transition from the planktonic state to biofilm formation. Type IV pili (T4P) are crucial determinants of biofilm structure and dynamics, but it is unknown how levels of c-di-GMP affect pilus dynamics. Here, we scrutinized how c-di-GMP affects molecular motor properties and adhesive behavior of T4P. By means of retraction, T4P generated forces of ∼30 pN. Deletion mutants in the proteins with known roles in biofilm formation, swarming motility, and exopolysaccharide (EPS) production (specifically, the diguanylate cyclases sadC and roeA or the c-di-GMP phosphodiesterase bifA) showed only modest effects on velocity or force of T4P retraction. At high levels of c-di-GMP, the production of exopolysaccharides, particularly of Pel, is upregulated. We found that Pel production strongly enhances T4P-mediated surface adhesion of P. aeruginosa, suggesting that T4P-matrix interactions may be involved in biofilm formation by P. aeruginosa Finally, our data support the previously proposed model of slingshot-like "twitching" motility of P. aeruginosaIMPORTANCE Type IV pili (T4P) play various important roles in the transition of bacteria from the planktonic state to the biofilm state, including surface attachment and surface sensing. Here, we investigate adhesion, dynamics, and force generation of T4P after bacteria engage a surface. Our studies showed that two critical components of biofilm formation by Pseudomonas aeruginosa, T4P and exopolysaccharides, contribute to enhanced T4P-mediated force generation by attached bacteria. These data indicate a crucial role for the coordinated impact of multiple biofilm-promoting factors during the early stages of attachment to a surface. Our data are also consistent with a previous model explaining why pilus-mediated motility in P. aeruginosa results in characteristic "twitching" behavior.

PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1-12)). Structural analysis, multiangle light scattering coupled with size exclusion chromatography, and bacterial two-hybrid data revealed that PilM forms dimers mediated by the binding of a novel conserved motif in the N terminus of PilM, and binding PilN abrogates this binding interface, resulting in PilM monomerization. Structural comparison of PilM with PilM·PilN(1-12) revealed that upon PilN binding, there is a large domain closure in PilM that alters its ATP binding site. Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. Bacterial two-hybrid data suggested the connectivity of the cytoplasmic and inner membrane components of the type IVa pilus machinery in P. aeruginosa, with PilM binding to PilB, PilT, and PilC in addition to PilN. Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. We propose a working model in which dynamic binding of PilN facilitates functionally relevant structural changes in PilM.

Type 4 pili are dispensable for biofilm development in the cyanobacterium Synechococcus elongatus.

The hair-like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo-proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm-promoting function in type IV pili-producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non-piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well-studied type IV pili-producing heterotrophic bacteria.

Uropathogenic Escherichia coli Express Type 1 Fimbriae Only in Surface Adherent Populations Under Physiological Growth Conditions.

BACKGROUND: Most uropathogenic Escherichia coli (UPEC) strains harbor genes encoding adhesive type 1 fimbria (T1F). T1F is a key factor for successful establishment of urinary tract infection. However, UPEC strains typically do not express T1F in the bladder urine, and little is understood about its induction in vivo. METHODS: A flow chamber infection model was used to grow UPEC under conditions simulating distinct infection niches in the bladder. Type 1 fimbriation on isolated UPEC was subsequently determined by yeast cell agglutination and immunofluorescence microscopy, and the results were correlated with the ability to adhere to and invade cultured human bladder cells. RESULTS: Although inactive during planktonic growth in urine, T1F expression occurs when UPEC settles on and infects bladder epithelial cells or colonizes catheters. As a result, UPEC in these sessile populations enhances bladder cell adhesion and invasion potential. Only T1F-negative UPEC are subsequently released to the urine, thus limiting T1F expression to surface-associated UPEC alone. CONCLUSIONS: Our results demonstrate that T1F expression is strictly regulated under physiological growth conditions with increased expression during surface growth adaptation and infection of uroepithelial cells. This leads to separation of UPEC into low-expression planktonic populations and high-expression sessile populations.

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile.

UNLABELLED: The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called "hypervirulent" epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile. We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile. The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCE: How Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by C. difficile. TFP are also required for surface motility, which has not previously been demonstrated for C. difficile. Furthermore, an epidemic-associated C. difficile strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

Earthquake-like dynamics in Myxococcus xanthus social motility.

Myxococcus xanthus is a bacterium capable of complex social organization. Its characteristic social ("S")-motility mechanism is mediated by type IV pili (TFP), linear actuator appendages that propel the bacterium along a surface. TFP are known to bind to secreted exopolysaccharides (EPS), but it is unclear how M. xanthus manages to use the TFP-EPS technology common to many bacteria to achieve its unique coordinated multicellular movements. We examine M. xanthus S-motility, using high-resolution particle-tracking algorithms, and observe aperiodic stick-slip movements. We show that they are not due to chemotaxis, but are instead consistent with a constant TFP-generated force interacting with EPS, which functions both as a glue and as a lubricant. These movements are quantitatively homologous to the dynamics of earthquakes and other crackling noise systems. These systems exhibit critical behavior, which is characterized by a statistical hierarchy of discrete "avalanche" motions described by a power law distribution. The measured critical exponents from M. xanthus are consistent with mean field theoretical models and with other crackling noise systems, and the measured Lyapunov exponent suggests the existence of highly branched EPS. Such molecular architectures, which are common for efficient lubricants but rare in bacterial EPS, may be necessary for S-motility: We show that the TFP of leading "locomotive" cells initiate the collective motion of follower cells, indicating that lubricating EPS may alleviate the force generation requirements on the lead cell and thus make S-motility possible.

Calcium binding properties of the Kingella kingae PilC1 and PilC2 proteins have differential effects on type IV pilus-mediated adherence and twitching motility.

Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, which is dependent on type IV pili and is influenced by two PilC-like proteins called PilC1 and PilC2. Production of either PilC1 or PilC2 is necessary for K. kingae piliation and bacterial adherence. In this study, we set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional 9-amino-acid calcium-binding (Ca-binding) site with homology to the Pseudomonas aeruginosa PilY1 Ca-binding site and that PilC2 contains a functional 12-amino-acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we showed that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in K. kingae and provide insights into the biology of the PilC-like family of proteins.

Involvement of T6 pili in biofilm formation by serotype M6 Streptococcus pyogenes.

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.

Evolution of the chaperone/usher assembly pathway: fimbrial classification goes Greek.

Many Proteobacteria use the chaperone/usher pathway to assemble proteinaceous filaments on the bacterial surface. These filaments can curl into fimbrial or nonfimbrial surface structures (e.g., a capsule or spore coat). This article reviews the phylogeny of operons belonging to the chaperone/usher assembly class to explore the utility of establishing a scheme for subdividing them into clades of phylogenetically related gene clusters. Based on usher amino acid sequence comparisons, our analysis shows that the chaperone/usher assembly class is subdivided into six major phylogenetic clades, which we have termed alpha-, beta-, gamma-, kappa-, pi-, and sigma-fimbriae. Members of each clade share related operon structures and encode fimbrial subunits with similar protein domains. The proposed classification system offers a simple and convenient method for assigning newly discovered chaperone/usher systems to one of the six major phylogenetic groups.

Fast uncoiling kinetics of F1C pili expressed by uropathogenic Escherichia coli are revealed on a single pilus level using force-measuring optical tweezers.

Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force-measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-versus-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e., type 1, P, and S, with force-induced elongation in three regions, one of which represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed as 26.4 ±1.4 pN, which is similar to those of other pili (which range from 21 pN for S(I) to 30 pN for type 1). The corner velocity for dynamic response (1,400 nm/s) was found to be larger than those of the other pili (400-700 nm/s for S and P pili, and 6 nm/s for type 1). The kinetics were found to be faster, with a thermal opening rate of 17 Hz, a few times higher than S and P pili, and three orders of magnitude higher than type 1. These data suggest that F1C pili are, like P and S pili, evolutionarily selected to primarily withstand the conditions expressed in the upper urinary tract.

Pili allow dominant marine cyanobacteria to avoid sinking and evade predation.

How oligotrophic marine cyanobacteria position themselves in the water column is currently unknown. The current paradigm is that these organisms avoid sinking due to their reduced size and passive drift within currents. Here, we show that one in four picocyanobacteria encode a type IV pilus which allows these organisms to increase drag and remain suspended at optimal positions in the water column, as well as evade predation by grazers. The evolution of this sophisticated floatation mechanism in these purely planktonic streamlined microorganisms has important implications for our current understanding of microbial distribution in the oceans and predator-prey interactions which ultimately will need incorporating into future models of marine carbon flux dynamics.

The Superior Adherence Phenotype of E. coli O104:H4 is Directly Mediated by the Aggregative Adherence Fimbriae Type I.

Whereas the O104:H4 enterohemorrhagic Escherichia coli (EHEC) outbreak strain from 2011 expresses aggregative adherence fimbriae of subtype I (AAF/I), its close relative, the O104:H4 enteroaggregative Escherichia coli (EAEC) strain 55989, encodes AAF of subtype III. Tight adherence mediated by AAF/I in combination with Shiga toxin 2 production has been suggested to result in the outbreak strain's exceptional pathogenicity. Furthermore, the O104:H4 outbreak strain adheres significantly better to cultured epithelial cells than archetypal EAEC strains expressing different AAF subtypes. To test whether AAF/I expression is associated with the different virulence phenotypes of the outbreak strain, we heterologously expressed AAF subtypes I, III, IV, and V in an AAF-negative EAEC 55989 mutant and compared AAF-mediated phenotypes, incl. autoaggregation, biofilm formation, as well as bacterial adherence to HEp-2 cells. We observed that the expression of all four AAF subtypes promoted bacterial autoaggregation, though with different kinetics. Disturbance of AAF interaction on the bacterial surface via addition of α-AAF antibodies impeded autoaggregation. Biofilm formation was enhanced upon heterologous expression of AAF variants and inversely correlated with the autoaggregation phenotype. Co-cultivation of bacteria expressing different AAF subtypes resulted in mixed bacterial aggregates. Interestingly, bacteria expressing AAF/I formed the largest bacterial clusters on HEp-2 cells, indicating a stronger host cell adherence similar to the EHEC O104:H4 outbreak strain. Our findings show that, compared to the closely related O104:H4 EAEC strain 55989, not only the acquisition of the Shiga toxin phage, but also the acquisition of the AAF/I subtype might have contributed to the increased EHEC O104:H4 pathogenicity.

Type IV Pili of Streptococcus sanguinis Contribute to Pathogenesis in Experimental Infective Endocarditis.

Streptococcus sanguinis is a common cause of infective endocarditis (IE). Efforts by research groups are aimed at identifying and characterizing virulence factors that contribute to the ability of this organism to cause IE. This Gram-positive pathogen causes heart infection by gaining access to the bloodstream, adhering to host extracellular matrix protein and/or platelets, colonizing the aortic endothelium, and incorporating itself into the aortic vegetation. While many virulence factors have been reported to contribute to the ability of S. sanguinis to cause IE, it is noteworthy that type IV pili (T4P) have not been described to be a virulence factor in this organism, although S. sanguinis strains typically encode these pili. Type IV pili are molecular machines that are capable of mediating diverse virulence functions and surface motility. T4P have been shown to mediate twitching motility in some strains of S. sanguinis, although in most strains it has been difficult to detect twitching motility. While we found that T4P are dispensable for direct in vitro platelet binding and aggregation phenotypes, we show that they are critical to the development of platelet-dependent biofilms representative of the cardiac vegetation. We also observed that T4P are required for in vitro invasion of S. sanguinis into human aortic endothelial cells, which indicates that S. sanguinis may use T4P to take advantage of an intracellular niche during infection. Importantly, we show that T4P of S. sanguinis are critical to disease progression (vegetation development) in a native valve IE rabbit model. The results presented here expand our understanding of IE caused by S. sanguinis and identify T4P as an important virulence factor for this pathogen. IMPORTANCE This work provides evidence that type IV pili produced by Streptococcus sanguinis SK36 are critical to the ability of these bacteria to attach to and colonize the aortic heart valve (endocarditis). We found that an S. sanguinis type IV pili mutant strain was defective in causing platelet-dependent aggregation in a 24-h infection assay but not in a 1-h platelet aggregation assay, suggesting that the type IV pili act at later stages of vegetation development. In a rabbit model of disease, a T4P mutant strain does not develop mature vegetations that form on the heart, indicating that this virulence factor is critical to disease and could be a target for IE therapy.

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.

Antiadhesive properties of a quaternary structure-specific hybridoma antibody against type 1 fimbriae of Escherichia coli.

The relationship between the structure and biological function of type 1 fimbriae of Escherichia coli was investigated using a set of monoclonal antibodies directed against conformation-specific antigenic determinants. Of three monoclonal antibodies tested, only one (clone CD3) prevented adhesion of the vaccine strain to epithelial cells or guinea pig erythrocytes. The antibody produced by CD3, but not that produced by the other two hybridoma clones (AA8 and GG1), precipitated isolated fimbriae by double diffusion in agar gel and was shown to bind in a highly discrete, periodic manner along the length of each of the fimbriae by immunoelectron microscopy. Immunoelectroblots of type 1 fimbrial subunits and polymers electrophoresed in SDS-gels indicated that the antibodies in AA8 and GG1 reacted only with fimbrial monomers (mol wt 17,000), whereas the antibody in CD3 reacted only with polymers of mol wt 102,000 (hexamers) or higher. ELISA inhibition assays demonstrated that dissociated fimbrial subunits lost their reactivity with antibody CD3 but gained reactivity with antibodies AA8 and GG1. Conversely, when allowed to reassemble in vitro in the presence of 5 mM MgCl2, the reassembled fimbriae lost their reactivity with antibodies AA8 and GG1 but regained reactivity with antibody CD3. These results demonstrated that certain antigenic epitopes are dependent on quaternary structural determinants, whereas others are independent of quaternary fimbrial structure and also are inaccessible for antibody binding in fimbriae once they have been assembled. These monoclonal antibodies should prove useful in studies of the structural determinants of the biological function of type 1 fimbriae as well as in studies of fimbrial synthesis, transport, and assembly.

Roles of oral bacteria in cardiovascular diseases--from molecular mechanisms to clinical cases: Involvement of Porphyromonas gingivalis in the development of human aortic aneurysm.

Accumulating evidence suggests the involvement of Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen, in cardiovascular diseases. Clinical specimens of aneurysmal tissue and dental plaque collected from patients infected with or without P. gingivalis were analyzed. The number of aneurysms in the distal aorta in the P. gingivalis-infected group was significantly higher than that in the non-infected group. Cellular accumulation of adipocytes in aneurysms was less frequently identified in the infected group. The expression of embryonic myosin heavy chain isoform, a phenotypic marker for proliferative smooth muscle cells, was higher in the P. gingivalis-infected group than the non-infected group. Clinical and histopathological features of aortic aneurysms associated with P. gingivalis infection are different from those present in non-infected patients. The major characteristic of P. gingivalis infection associated with aneurysms is smooth muscle cell proliferation in the distal aorta.

Species-Specific Recognition of Sulfolobales Mediated by UV-Inducible Pili and S-Layer Glycosylation Patterns.

The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA to repair DNA double-strand breaks via homologous recombination. Substitution of the Sulfolobus acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N-glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N-glycan composition to ensure aggregation and, consequently, also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination.IMPORTANCE Type IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The UV-inducible pili system of Sulfolobales (Ups) pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, thereby assisting in genome stability. With this work, we show that different Sulfolobus species have specific regions in their Ups pili subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species have unique surface-layer N-glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition.