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In general, the periodontal pathogen Porphyromonas gingivalis expresses distinct FimA and Mfa1 fimbriae. Each of these consists of five FimA–E and five Mfa1–5 proteins encoded by the fim and mfa gene clusters, respectively. The main shaft portion comprises FimA and Mfa1, whereas FimB and Mfa2 are localized on the basal portion and function as anchors and elongation terminators. FimC–E and Mfa3–5 participate in the assembly of an accessory protein complex on the tips of each fimbria. Hence, they serve as ligands for the receptors on host cells and other oral bacterial species. The crystal structures of FimA and Mfa1 fimbrial proteins were recently elucidated and new insights into the localization, function, and biogenesis of these proteins have been reported. Several studies indicated a correlation between P. gingivalis pathogenicity and the fimA genotype but not the mfa1 genotype. We recently revealed polymorphisms of all genes in the fim and mfa gene clusters. Intriguingly, mfa5 occurred in numerous different forms and underwent duplication. Detailed structural and functional knowledge of the fimbrial proteins in the context of the entire filament could facilitate the development of innovative therapeutic strategies for structure-based drug design.

To evaluate the ability of inulin to enhance the immune response of a ptfA gene DNA vaccine for avian Pasteurella multocida, inulin was added as an adjuvant to the ptfA-DNA vaccine, obtaining an inulin-adjuvant DNA vaccine. The DNA vaccine was administered to chickens; a fimbria protein vaccine and an attenuated live vaccine were used as positive controls. The levels of the serum antibody and concentrations of interferon-γ (IFN-γ), interleukin-2 (IL-2), and interleukin-4 (IL-4) were determined, and a lymphocyte proliferation assay was performed. After being challenged with virulent P. multocida, the protective efficacy was evaluated. The results showed that the serum antibodies induced by the ptfA-DNA vaccine were not enhanced by inulin. The stimulation index values and the concentrations of IL-2 and IFN-γ in chickens vaccinated with inulin-adjuvant DNA vaccine were significantly higher than those in chickens vaccinated with the DNA vaccine, those with the fimbria protein vaccine, and the chickens gavaged with inulin. The concentrations of IL-4 in the inulin-adjuvant DNA vaccine group and the fimbria protein vaccine group were higher than those in the DNA vaccine group and the inulin-gavage group. The protective efficacy rates of the attenuated live vaccine group, the fimbria protein vaccine group, the DNA vaccine group, the inulin-adjuvant DNA vaccine group, and the inulin-gavage group were 90, 70, 55, 65, and 55%, respectively.

Outflow obstruction of a catheter for peritoneal dialysis (PD) is one of the most common complications in patients who are undergoing PD for renal failure. Although the cause of catheter obstruction is variable, a choked catheter caused by a fallopian tube fimbria is quite rare and only five pediatric cases were reported. We report a case of a five-month-old female who suffered from recurrent catheter obstruction by a fallopian tube fimbria. Laparoscopic surgery was performed to recanalize the catheter, but the catheter obstruction recurred three days after the surgery. We abandoned the use of the catheter that had been inserted into the Douglas fossa and a new catheter was inserted into the left para-colonic groove. After the second surgery, catheter obstruction did not occur and PD has been continued without any complications.

Bacterial virulence factors (VFs) influence the site and severity of urinary tract infections (UTI) and further leading to sepsis infection. Phenotypic characterisation of VFs specific to sepsis Escherichia coli strains has not been characterized in Indian population till date. In this data article, we have described important VFs of uropathogenic E. coli (UPEC) that is P fim, Type-1 fim, cell surface hydrophobicity, mannose resistant haemagglutination/mannose sensitive haemagglutination (MRHA/MSHA) expression and α-haemolysin production. The data includes a profile of the five VFs investigated in E. coli isolates from sepsis patients (N=78) and control group (N=50) from non-sepsis subjects. We found that P fim phenotype was expressed in 25.3% of E. coli isolates from sepsis patients, whereas Type-1 fimbriae was detected in 30.5%. Cell surface hydrophobicity phenotype was present in 30.5%, α-haemolysin in 26.3% and MRHA/MSHA in 22.1% of sepsis E. coli isolates. None of the control E. coli isolates showed presence of these phenotypes. The combined phenotypic profile of all the five VFs was significantly higher in sepsis patients as compared to the control group. Keywords: Escherichia coli, Phenotypes, Sepsis

Mucinous adenocarcinoma of the fallopian tube is exceptionally rare and the detailed clinicopathologic features of these tumors have not yet been reported in English literature. Here we report a moderately differentiated mucinous adenocarcinoma arising in the tubal fimbria in a 70-year-old woman. Patient had a history of cholecystectomy for gallstones and gastric banding who presented with gastrointestinal discomfort and was found to have a large adnexal mass on imaging studies. Serum CA-125 was moderately elevated. Recent mammography, upper endoscopy and colonoscopy were completely normal. She underwent surgical staging for the adnexal mass. Frozen section and final pathology diagnosis revealed moderately differentiated adenocarcinoma arising in the left fimbria. Carcinoma had spread to the ipsilateral ovary and pelvic soft tissue at the time of her presentation. Tumor was strongly immunoreactive to CK7 and CEA, and was negative for CK20, CDX-2, PAX-8, WT-1, p16, ER, and vimentin. TP53 showed wild-type phenotype by immunohistochemistry. Molecular studies showed no mutation in codon 12 and 13 of the k-ras gene, and no mutation was detected in the BRAF and EGFR genes. In addition, the non-tumorous fimbria epithelium showed a spectrum of mucinous alterations with variable nuclear atypia: cytologically bland areas that were reminiscent of mucinous metaplasia were positive for p53 and showed minimal proliferation as assessed by Ki-67, and cytologically atypical stratified mucinous epithelium that was positive for p53 and Ki-67. The patient received 3 cycles of Folfox and was regularly followed at a 3–6 month interval. Her carcinoma recurred in abdomen at 32 months post surgery. After excluding the possibility of an extra-gynecologic tract primary through extensive clinical investigations and post-surgical follow-up, we concluded that this tumor most likely represented a mucinous adenocarcinoma of tubal origin.

Animal, 8 (6), ISSN:1751-7311, ISSN:1751-732X

Type IV pili, a special class of bacterial surface filaments, are key behavioral mediators for many important human pathogens. However, we know very little about the role of these structures in the lifestyles of plant-associated bacteria. Over the past few years, several groups studying the extensive genus of Xanthomonas spp. have gained insights into the roles of played by type IV pili in bacteria-host interactions and pathogenesis, motility, biofilm formation, and interactions with bacteriophages. Protein-protein interaction studies have identified T4P regulators and these, along with structural studies, have begun to reveal some of the possible molecular mechanisms that may control the extension/retraction cycles of these dynamic filaments. Fil: Dunger, Ricardo German. Universidade de Sao Paulo; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina Fil: Llontop, Edgar. Universidade de Sao Paulo; Brasil Fil: Guzzo, Cristiane R.. Universidade de Sao Paulo; Brasil Fil: Farah, Chuck S.. Universidade de Sao Paulo; Brasil

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI.

The pathogenesis of disease caused by H. influenzae type b and non-typeable H. influenzae involves multiple steps. Although similar mechanisms are used to promote colonization of the nasopharynx, both organisms express unique virulence determinants that dictate the spectrum of disease they are able to cause. A major difference is the expression of a PRP capsule by type b strains. This alone is sufficient to facilitate intravascular survival and the onset of invasive disease. Although it is a major determinant of virulence, the capsule has played a useful role as the basis of the highly successful H. influenzae vaccine. In contrast, no such highly conserved virulence determinant is common to non-typeable strains of H. influenzae, which complicates the search for a candidate vaccine for this group of organisms. As a consequence, while disease due to type b stains is now rare, non-typeable strains continue to be a significant cause of morbidity and even mortality in children. New insights into non-typeable H. influenzae virulence factors may give opportunities for vaccine or other strategies to prevent disease.

Klebsiella pneumoniae is a Gram-negative bacterium that is frequently found in the flora of the mouth, skin and intestines as well as in natural environments. K. pneumoniae is an opportunistic pathogen that can cause severe hospital-acquired infections such as septicaemia, pneumonia, urinary tract infection and soft tissue infection in debilitated individuals. This species is well-known for its ability to accumulate and transfer drug-resistance determinants such as extended-spectrum β-lactamase (ESBL). Treatment of serious infection with ESBL-producing K. pneumoniae is extremely difficult because the organisms are increasingly resistant to multiple antibiotics, which may be caused by very complex mechanisms. The pathogenicity of K. pneumoniae is multifactorial including LPS, capsule, urease, adhesins, outer-membrane proteins and biofilms. Vaccines have been tested but no effective products are in use.

Research on the function and assembly of extracellular fibres in Gram-negative pathogenic bacteria has provided insights into some of the most basic principles of molecular biology: how a protein folds into domains that serve as assembly modules for building up large supramolecular structures. Studies of the chaperone–usher pathway (CUP) pili have elucidated a reaction called donor strand complementation, in which the chaperone mediates pilus subunit folding, and a reaction called donor strand exchange, in which subunits of a pilus polymerize into a fibre with the aid of the usher, an outer-membrane-gated channel. CUP pili are ubiquitous in Gram-negative bacteria, with many genomes encoding ten or more types, all containing dedicated adhesins that function in adherence, invasion of host tissues, and biofilm formation on medical devices and in various niches and body habitats. Many Gram-negative bacteria also use specific molecular machinery to direct production of amyloid fibres called curli, which can provide structural, adhesive and protective properties to a biofilm. Frequent and long-term prophylactic use of antimicrobial agents has contributed to a looming worldwide crisis of multi-drug resistance that has spawned the need for new ways of thinking about drug development, including the targeting of bacterial molecular machines that catalyse the biogenesis of virulence-associated extracellular fibres.

The binding of bacterial adhesins to host receptors is a dynamic process occurring in several steps, which involve complex bacteria–host cell interaction. Initial weak physical interactions lead to more specific adhesion mechanisms that may be shared by several organisms, but eventually to species-specific adhesins that may elicit both bacterial and host factors leading to host cell damage, induction of inflammation and disease. Species-specific fimbrial adhesins may be viewed as direct mediators of bidirectional signalling between bacteria and host cells. Understanding of this process has been highly informative for the design of novel strategies to modulate these signalling pathways and to curb bacterial infections and their harmful sequelae. Development of mixtures of inhibitors or a polyvalent inhibitor is under investigation, since many infectious agents express multiple specificities. Multiple molecular mechanisms of adhesion are required to initiate infection, and effective anti-adhesion strategies will need to address both bacterial and host site particularities.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of life-threatening diarrheal disease around the world. The major aspects of ETEC virulence are colonization of the small intestine and the secretion of enterotoxins which elicit diarrhea. Intestinal colonization is mediated, in part, by adhesins displayed on the bacterial cell surface. As colonization of the intestine is the critical first step in the establishment of an infection, it represents a potential point of intervention for the prevention of infections. Therefore, colonization factors (CFs) have been important subjects of research in the field of ETEC virulence. Research in this field has revealed that ETEC possesses a large array of serologically distinct CFs that differ in composition, structure, and function. Most ETEC CFs are pili (fimbriae) or related fibrous structures, while other adhesins are simple outer membrane proteins lacking any macromolecular structure. This chapter reviews the genetics, structure, function, and regulation of ETEC CFs and how such studies have contributed to our understanding of ETEC virulence and opened up potential opportunities for the development of preventive and therapeutic interventions.

Urinary tract infections (UTIs) belong to the most common infectious diseases worldwide. The most frequently isolated pathogen from uncomplicated UTIs is Escherichia coli. To establish infection in the urinary tract, E. coli has to overcome several defence strategies of the host, including the urine flow, exfoliation of urothelial cells, endogenous antimicrobial factors and invading neutrophils. Thus, uropathogenic E. coli (UPEC) harbour a number of virulence and fitness factors enabling the bacterium to resist and overcome these different defence mechanisms. There is no particular factor which allows the identification of UPEC among the commensal faecal flora apart from the ability to enter the urinary tract and cause an infection. Many of potential virulence or fitness factors occur moreover with high redundancy. Fimbriae are inevitable for adherence to and invasion into the host cells; the type 1 pilus is an established virulence factor in UPEC and indispensable for successful infection of the urinary tract. Flagella and toxins promote bacterial dissemination, while different iron-acquisition systems allow bacterial survival in the iron-limited environment of the urinary tract. The immune response to UPEC is primarily mediated by toll-like receptors recognising lipopolysaccharide, flagella and other structures on the bacterial surface. UPEC have the capacity to subvert this immune response of the host by means of actively impacting on pro-inflammatory signalling pathways, or by physical masking of immunogenic structures. The large repertoire of bacterial virulence and fitness factors in combination with host-related differences results in a complex interaction between host and pathogen in the urinary tract.

Uropathogenic E. coli (UPEC) are a genotypically diverse subset of extraintestinal pathogenic E. coli that cause urinary tract infections. UPEC have evolved from commensal E. coli by acquisition of virulence factors through horizontal gene transfer. Through reductive evolution, asymptomatic bacteriuria strains have evolved from UPEC to live a commensal-like lifestyle in the urinary tract. UPEC, unlike diarrheagenic E. coli, have no core set of virulence factors; instead, this pathotype uses flagella to ascend the urinary tract, and a variety of adhesins, iron acquisition systems, and toxins to colonize the bladder and kidneys. While distinct virulence factors have been associated with uropathogenicity, there is also functional redundancy. Some virulence factor genes such as those encoding type 1 and P fimbriae undergo phase variation, leading to a bacterial population with mixed expression. Strain variation resulting from both a heterogeneous set of virulence factors and phase variation leads to a phenotypic diversity that complicates vaccine development against UPEC and likely allows for recurrent infections.

Enteroaggregative Escherichia coli (EAEC) are among the most prevalent enteric pathogens worldwide. The pathotype has been implicated in endemic diarrhea in both developing and industrialized populations, epidemic diarrhea, and traveler’s diarrhea. A recent outbreak of Shiga toxin-producing EAEC adds a new dimension to the epidemiology of this pathotype. Although the pathogenesis of this pathotype is not fully understood, a number of putative virulence factors have been described. The aggregative adherence fimbriae (AAF) are mucosal adhesins that also elicit inflammatory responses from infected mucosal surfaces. Expression of AAF adhesins is induced by the transcriptional activator AggR, which activates expression of several other secreted, but as yet relatively cryptic, proteins. EAEC-induced histopathology includes exfoliation of enterocytes, which has been linked to the action of serine protease autotransporter toxins. Epidemiologic evidence supports a model of EAEC pathogenesis comprising the concerted action of multiple virulence factors.

The enterotoxigenic Escherichia coli are a pervasive cause of serious diarrheal illness in developing countries. Presently, there is no vaccine to prevent these infections, and many features of the basic pathogenesis of these organisms remain poorly understood. Until very recently most pathogenesis studies had focused almost exclusively on a small subset of known “classical” virulence genes, namely fimbrial colonization factors and the heat-labile (LT) and heat stable (ST) enterotoxins. However, recent investigations of pathogen-host interactions reveal a surprisingly complex and intricately orchestrated engagement involving the interplay of classical and “novel” virulence genes, as well as participation of genes highly conserved in the E. coli species. These studies may inform further rational approaches to vaccine development for these important pathogens.

Enteropathogenic Escherichia coli (EPEC) strains are a leading cause of diarrhea among infants in developing countries. These bacteria spread via fecal–oral transmission and colonize the small intestine. Typical EPEC differs from atypical strains in that they possess the bfp operon and generate a type IV-B class of fimbriae known as the bundle-forming pilus (BFP). EPEC uses BFP to attach to intestinal epithelium. Upon attachment, EPEC translocates effector proteins into the host cell via a type 3 secretion system, which is encoded on a pathogenicity island known as the locus of enterocyte effacement. Translocated effectors modulate the host response in a number of ways, including subversion of immune responses and hijacking of the cytoskeletal system. By inducing actin rearrangement, EPEC causes the formation of actin pedestals onto which it sits, and leads to the effacement of microvilli, giving this bacterium a characteristic attaching and effacing phenotype. EPEC infections are typically self-limiting, with rehydration therapy constituting primary treatment, but they can be protracted and lethal. Breastfeeding provides a great protective effect, validating the use of a vaccine to induce a humoral response in mother or child as a potential means of preventative action. This chapter provides a detailed discussion on EPEC pathogenesis and host cell responses and summarizes the current detection and treatment options available.

Bacteria and Archaea expose on their outer surfaces a variety of thread-like proteinaceous organelles with which they interact with their environments. These structures are repetitive assemblies of covalently or non-covalently linked protein subunits, organized into filamentous polymers known as pili ("hair"), flagella ("whips") or injectisomes ("needles"). They serve different roles in cell motility, adhesion and host invasion, protein and DNA secretion and uptake, conductance, or cellular encapsulation. Here we describe the functional, morphological and genetic diversity of these bacterial filamentous protein structures. The organized, multi-copy build-up and/or the natural function of pili and flagella have lead to their biotechnological application as display and secretion tools, as therapeutic targets or as molecular motors. We review the documented and potential technological exploitation of bacterial surface filaments in light of their structural and functional traits.

Colibacillosis appears to be of increasing importance in layer flocks. The aim of this study was to determine characteristics of avian pathogenic Escherichia coli associated with the occurrence of colibacillosis outbreaks at flock level. Forty E. coli strains originating from layers from healthy flocks (‘control isolates’), consisting of 25 caecal and 15 extra-intestinal isolates, were compared with 40 strains isolated from layers originating from colibacillosis-affected flocks (‘outbreak isolates’), consisting of 20 caecal and 20 extra-intestinal isolates. The examined characteristics were adhesins, invasivity in T84 cell culture, serum resistance, iron uptake, colicin production, and toxinogenicity. The following traits were significantly more often detected in the outbreak isolates than in the control isolates: tsh, iss, iucA, iutA, irp2, fyuA, iroC, cvaC, colicin and colicin V production. A comparison of the extra-intestinal outbreak isolates and the caecal control isolates yielded the same results as when the caecal isolates, extra-intestinal isolates and total number of isolates of the outbreak and the control group were compared. When comparing the caecal and extra-intestinal isolates within the control and within the outbreak group, no significant differences were detected. The O78 and O2 groups showed significant differences with other O-types and NT strains for prevalence of most of the same characteristics. The combination of type 1 fimbriae, tsh, serum resistance, iss, traT, iucA, fyuA, iroC and colicin or colicin V production was significantly more often present in extra-intestinal outbreak isolates than in extra-intestinal control isolates. Only the combination of serum resistance, fyuA and colicin production was present in all outbreak isolates, with a significantly lower prevalence in the control isolates. None of the characteristics or combinations examined were exclusive to the outbreak isolates.

Innate defense mechanisms help to define the minimal requirements for successful colonization by commensal and pathogenic organisms—those that cannot adapt to these conditions will be incapable of maintaining themselves within the host. Phagocytes represent a major component of innate defense at the cellular level, and all classes of phagocytes, including macrophages, neutrophils, eosinophils, and mast cells, occur within the mucosal tissues. Although the activities of phagocytes may be largely confined to the tissues themselves rather than taking place within the lumen—the microenvironment close to the mucosal surface may permit phagocytic activity. As most secretions are hypotonic, phagocytes probably do not survive with functional activity for long in the bulk fluid phase. Innate immune recognition is mediated by a series of germline–encoded receptors, known as pattern-recognition receptors (PRRs)—which detect virulent microorganisms through recognition of invariant pathogen-associated molecular patterns (PAMPs). Studies in wild-type and PRR-deficient mice as well as human macrophages activated by lipopolysaccharide (LPS), taxol, or bacterial fimbriae, suggest that—TLRs, CD14, and β 2 integrins form a functional multireceptor complex that coordinates induction of intracellular signals.

Publisher Summary This chapter discusses the strategies to prevent colonization of the gut by enterotoxigenic E. coli (ETEC). The chapter describes the attachment of E. coli, by virtue of fimbriae, to the intestinal wall, and the molecules involved both at the bacterial surface and on the intestinal epithelium. ETEC are pathogens that cause diarrhoea in humans and livestock. These bacteria colonize the small intestinal epithelium by of expressing adhesins (lectins) in the form of long proteinaceous appendages that protrude from their surface, and that are called fimbriae. Because of their size, architecture and extracellular expression, fimbriae can easily be obtained in a highly purified state and in appreciable quantities to be used as vaccine components. Purified fimbriae have proven to be strong immunogens, and humoral antibodies are readily generated in rodents, chickens and farm animals. Plant lectins are potential candidates to block intestinal mucosal E. coli lectin receptors, because most plant lectins are rather resistant to proteolytic degradation in the stomach and small intestine, and, can bind to glycoconjugates present in the intestinal mucosa. The beneficial effect of probiotics in protecting the host from intestinal disorders and/or colonization of the intestine is because of one or a combination of the following effects: (1) stimulation of the immune system; (2) competitive inhibition for bacterial adhesion sites on the intestinal surface; (3) degradation of intestinal toxin receptors; and (4) production of inhibitory substances such as bacteriocins, hydrogen peroxide or organic acids.

Publisher Summary Haemophilus influenzae (H. influenzae) is a small Gram-negative, nonspore-forming coccobacillus that belongs to the family Pasteurellaceae. H. influenzae is an obligate commensal of humans that resides predominantly in the upper respiratory tract and is best known for occasional associated diseases. A strategy has been adopted by H. influenzae to facilitate acclimation to their host. This is to increase the effective mutation rate of genes that encode antigens critical for interactions with the host. Capsular polysaccharide is known to be a critical determinant for invasive systemic disease caused by H. influenzae. Strains of H. influenzae may express one of six serologically distinct capsular types, each of which comprises polymers of distinct sugars in specific linkages. In H. influenzae, the capacity to generate diversity and antigenic variations of cell-surface expressed molecules is largely mediated through specialized DNA sequences and runs of repetitive DNA (microsatellites). An example of phase variation in H. influenzae that results from altered transcription is the expression of pili (or fimbriae). These long filamentous appendages can act as an adhesin that mediates attachment to respiratory epithelia or other host-cell types and are targets of the host immune response.

Type IV pili (Tfps) or fimbriae essentially refer to nonflagellar, filamentous surface appendages that are distributed widely among bacterial species. By mediating interactions to other bacterial cells of the same species, to eukaryotic cells, or to other surfaces, Tfps functions in diverse processes such as motility, virulence, biofilm formation, and horizontal gene transfer. Tfps are mainly homopolymeric structures consisting of a structural subunit that is synthesized as a preprotein with a short, positively charged leader peptide. This chapter focuses on the Tfps of pathogenic E.coli . The best-studied system is the bundle-forming pilus (BFP) of enteropathogenic E.coli (EPEC), which mediates reversible bacterial aggregation and the localized adherence phenotype. Two other Tfps are described for human enterotoxigenic E. coli (ETEC). It is observed that both ETEC and Tfps are encoded on large virulence plasmids and although closely related, they represent different pilus types because their expression is induced under different conditions. This chapter discusses the distribution, function, and biogenesis of Tfps, with emphasis on the bundle-forming pilus (BFP) of EPEC. This chapter also summarizes the current knowledge of these organelles with reference to other well-known Tfps and related systems.

Publisher Summary Pili or fimbriae are proteinaceous, filamentous, polymeric organdies expressed on the surface of bacteria. Pilus has become a generic term used to describe all types of nonflagellar filamentous appendages, and it is used interchangeably with the term “fimbriae.” Pili can act as receptors for bacteriophages, facilitate DNA uptake and transfer (conjugation), and at least one type of pilus, type 4, call function in cellular motility. The primary function of most pili, however, is to act as scaffolding for the presentation of specific adhesive moieties. Adhesive pilus subunits (adhesins) are often incorporated as minor components into the tips of pili, but major structural subunits can also function as adhesins. Adhesins can mediate the interaction of bacteria with each other, with inanimate surfaces, and with tissues and cells in susceptible host organisms. For many bacterial pathogens, adhesive pili play a key role in the colonization of host tissues. Type 1 pill-mediated attachment to bladder epithelial cells is critical in the establishment of urinal tract infections by uropathogenic E. coli. Similarly, binding of P pili to Galα(1–4)Gal sugar moieties in the kidney are necessary for development of pyelonephritis.

Publisher Summary The genetic and phenotypic analysis of muilticellular behavior in salmonella typhimurium. According to Costerton et al. , biofilms can be defined in a narrower sense as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface,” or in a broader sense as “matrix-enclosed bacterial populations adherent to each other and/or to surfaces or interfaces.” In Salmonella typhimurium, a morphotype in which cells are acting as a community is identified. This morphotype displayed various modes of multicellular behavior in stationary phase, such as cell clumping in aerated, liquid culture, pellicle formation in standing liquid culture, formation of a rigid cell network accompanied by surface translocation on solid medium, adhesion to biological macromolecules, and biofilm formation on various abiotic surfaces such as glass, polystyrene (PS), and stainless steel. On solid medium, the colony bound the dye Congo red (CR) included in the agar plates, thereby leading to the pronounced display of a peculiar morphotype that is an irregularly shaped, flat, wrinkled colony with undulate margin. Referring to the morphotype on plates, the complete multicellular behavior has been called rdar—red, dry, and rough—morphotype. Genetic and electron microscopy analyses revealed that the extracellular matrix consists of at least two components; thin aggregative fimbriae (agf); and an unknown substance.

The ability of bacterial pathogens to bind to the host mucosa is a critical step in the pathogenesis of many bacterial infections and, for Escherichia coli, a large number of different fimbrial adhesins have been implicated as virulence factors. In this chapter, our current understanding of the regulatory mechanisms that control the expression of two of the best characterized fimbrial adhesins, pyelonephritis-associated pilus (encoded by pap) and the type 1 fimbria (encoded by fim), will be described. The expression of both fimbrial adhesins is controlled by phase variation (the reversible and apparently random switching between expressing ('on') and non-expressing ('off') states), and is regulated in response to environmental conditions. The phase variation of pap (and of some other fimbriae in Escherichia coli) is determined by the formation of alternative nucleoprotein complexes that either activate (phase 'on') or suppress (phase 'off') transcription of the fimbria genes. Formation of each complex protects a single Dam methylation site (5' GATC) from modification (GATCdist in phase 'on' cells and GATCprox in phase 'off' cells). Furthermore, complex formation is inhibited by methylation of the two 5' GATC sites. Both the phase variation of pap and the transcription of the pap genes in phase 'on' cells, are regulated and expression is subject to both positive and negative feedback control. In contrast to pap, the phase variation of fim is determined by the site-specific inversion of a short element of DNA (the fim switch). In phase 'on' cells, a promoter within the invertible element directs the transcription of the fim structural genes, whereas in phase 'off' cells transcription of the fimbrial genes is silenced. Despite the very different molecular mechanisms controlling the expression of pap and fim, the two systems share many features in common and have probably evolved to fulfill the same function. In addition to details about the molecular mechanisms that control pap and fim, the possible physiological significance of the observed regulation will be discussed.

Publisher Summary Two of the most important factors that dictate survivability in biofilms are the ability to adhere there and the ability to utilize available nutrients in the matrix. This chapter describes methods that probe the biofilm matrix that determines whether its complex polysaccharides are being utilized and examine nutrient fluxes there. The biofilm matrix is composed of substantial quantities of capsular exopolymeric substances (EPS) that in turn normally contain substantial quantities of capsular polysaccharide (CPS). The survival value of capsular EPS has been reported. Survival functions fall under the headings of (a) barrier, including protection from predation and resistance to toxins, antibiotics, and poisons; (b) cell–cell interaction and recognition, including symbiosis; and (c) adhesion and biofilm formation. Along with fimbriae, EPS may tether the organism to the surface, thereby serving as a primary adhesin.

Publisher Summary This chapter discusses an overview of strategies for the study of interactions between bacteria and their hosts. They range from assays using isolated bacterial and host cell molecules to experimental infections in whole animals. For most bacteria to cause a productive infection, they must first colonize their host. To accomplish this bacteria utilize surface proteins termed “adhesins,” which is organized as complex macromolecular structures known as pili or fimbriae, or as single molecules called a fimbrial adhesins. The involvement of fimbriae in adhesion is extensively studied and in a number of cases the receptor that binds to the fimbrial adhesin is fully characterized. In most cases, fimbriae are lectins that recognize the carbohydrate moiety of glycolipids or glycoproteins. How bacteria affect host cells is also described in depth.

Publisher Summary This chapter discusses an overview of fimbriae; it also discusses its detection, purification, and characterization. Fimbriae are adhesive bacterial surface structures that enable bacteria to target and colonize particular host tissues, due to specific receptor recognition. Fimbrial-associated receptor recognition permits bacteria to adhere to diverse targets ranging from inorganic substances to highly complex biomolecules. This capacity is of paramount importance in bacterial colonization of a given surface, whether inanimate or a particular tissue in a mammalian host. The interplay between a fimbrial adhesin and its cognate receptor plays a significant role in determining host and tissue tropisms in pathogenic bacteria. Consequently, fimbriae are often recognized as virulence factors, and this aspect has spurred intensive research into the molecular biology, biochemistry, and genetics of these surface structures. In general, fimbriae are heteropolymers, consisting of a major structural protein and a small percentage of minor component proteins, one of which is the fimbrial adhesin.

Publisher Summary The ability to colonize the small intestine is an important virulence attribute of enterotoxigenic Escherichia coli (ETEC), bacteria that cause diarrhea in humans and animals. Bacterial adhesins, known as “fimbriae” or “pili,” facilitate intestinal colonization by promoting attachment of ETEC to specific receptors on the villous epithelium. With the development of immunoblot and overlay techniques for identifying fimbriae-specific receptors, there have been numerous descriptions of glycoproteins and glycolipids that bind ETEC fimbriae in vitro . It is important to determine if the putative receptors are relevant in intestinal colonization. This chapter summarizes the immunoblot techniques used to identify receptors for 987P fimbriae in porcine small intestines and to demonstrate a correlation between the distribution of intestinal receptors for 987P and age-related susceptibility to 987P-mediated ETEC diarrhea. With some modifications, the methods are applicable for studying other intestinal receptors for other ligands.

Publisher Summary This chapter focuses on the adhesion of oral bacteria to soft tissue and describes procedures utilized to study this adhesion. The focus is on Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis . Adhesion is a prerequisite for colonization of oral bacteria as the flow of saliva removes exposed bacteria that are not firmly bound. Bacteria adhere to oral surfaces in a highly specific manner. The physical conditions in the mouth and components derived from the bacterium, from the host, from the diet of the host, and from other bacteria are responsible for the specificity. Most bacteria that colonize the soft tissues of the mouth are slow growing and fastidious. Components on the surface of bacteria that are associated with adhesion include fimbriae, flagella, lipopolysaccharide, polysaccharide, microvesicles, and outer membranes. Adhesion of oral bacteria to soft tissue is likely mediated by specific and nonspecific interactions of one or more of the adhesion determinants. Components of saliva have molecules that mimic those of receptors found on oral epithelial cells. The interaction of bacterial adhesins with receptors in secretions diminishes or precludes interaction with receptors on oral epithelial cells. The ability of saliva and antibodies to inhibit adhesion may represent important antiadhesion mechanisms for oral bacteria.

Publisher Summary Understanding the physiology of bacteria grown in vivo in various hosts has been a difficult task. By using the adhesive properties of the microorganism, it is possible to isolate certain bacteria from their natural environment for further investigations. Pure cultures so obtained do not require growth on an artificial laboratory medium. The chapter describes the method for adhesin-dependent isolation and characterization of bacteria from their natural environment. It is performed by using a streptomycin-treated mouse as a host for a human Escherichia coli strain, producing type 1 fimbriae in vivo . Type 1 fimbriae bind specifically to D-mannose moieties. In this context, experiments were performed in which sepharose beads coupled with D-mannose were used for isolating bacterial cells from a fecal suspension, and/or from cecal mucus and cecal contents. Then, the isolated intact bacterial cells can be characterized by techniques such as electron microscopy, flow cytometry, and in situ hybridization. By electron microscopy, cell shape and surface components can be observed. A flow cytometer can be used for determining rapidly and with high precision the cell size of individual bacterial cells and the cell-size distribution in the isolated population. DNA content can be measured and the number of genomes per cell calculated. By in situ hybridization with labeled ribosomal RNA probes, the rRNA can be measured, which will reflect the metabolic activity of the bacterial cell in the host.

Publisher Summary This chapter discusses the strategies for employing molecular genetics to study tip adhesions. Molecular genetic approaches make it possible to analyze fimbrial determinants of different species and to determine genes responsible for tip adhesins. To determine the regions responsible for adhesion, specific mutations are introduced into the genes by site-directed mutagenesis. Various integration systems can be used to introduce adhesin-specific mutations into the chromosome of wild-type pathogens. Thus, the role of tip adhesins in vivo can be analyzed. The chapter focuses on three of the best studied tip-adhesin systems among pathogenic bacteria. All three adhesion factors are produced by extrainintestinal Escherichia coli strains. P fimbriae, which are preferentially produced by uropathogenic E. coli (UPEC), are able to bind to α-D-Gal-I,4-β-D-Gal–specific receptor structures. The Gal-Gal-specific adhesin, termed “PapG,” is located at the tip of the fimbrial rod, where it forms a tip-located fibrillum. S fimbriae are preferentially produced by UPEC and by strains causing meningitis. The S-fimbrial adhesion factors (Sfa proteins) are able to bind to sialic acid–containing glycoproteins. Type 1 fimbriae (Fim proteins) are produced by pathogenic and nonpathogenic bacteria. The adhesion protein FimH, which binds to mannose-containing receptors, has been located at the tip of the fimbriae as well as in the fimbrial rod.

Publisher Summary The brain-derived neurotrophic factor (BDNF) was first isolated from pig brain. In vivo , BDNF increases the survival of embryonic neurons in the dorsal root ganglia and nodose ganglia, as well as developing and axotomized motoneurons. It also prevents the degeneration of medial septal cholinergic neurons following fimbria transection. BDNF null-mutated mice develop several sensory deficits; however, no obvious defects were seen for motoneurons or central dopaminergic neurons. In both the rat embryo and the adult animal, NT-4 mRNA expression appears to be more general than expression of mRNAs for the other three neurotrophins. Low but detectable levels of NT-4 mRNA were seen at all the developmental stages in all the 12 non-neural tissues analyzed. In the pituitary and submandibular glands, the expression level of NT-4 mRNA was the highest in the one-week-old animal and the amount declined progressively during the onset of development.

Publisher Summary This chapter presents methods and approaches to study and quantify the adhesion of bacteria to human phagocytic cells, specifically polymorphonuclear neutrophils (PMNs), monocytes, and macrophages. Adherent bacteria are often, but not always, internalized by the phagocytes to which they adhere. The assays presented in the chapter consist of incubating bacteria and phagocytes, sometimes with serum or other body fluids, and then quantifying adhesion. Bacteria attach to phagocytes by two broad mechanisms: (1) directly, by using specific molecules expressed on outer cell surfaces such as fimbriae (pili) and nonfimbrial ligands, often called “adhesins,” such as the Opa proteins of Neisseria gonorrhoeae , and (2) indirectly, by binding host components, most often immunoglobulins and complement components, which subsequently bind to phagocyte receptors. Regardless of the mechanism, the methods used to quantify adhesion are similar. The ability of bacteria to bind to phagocytes can vary tremendously depending on the bacterial strain or variant—and growth conditions and growth phase—among other variables. Many bacterial surface ligands vary independently of growth conditions or phase; such variations are not rare events.

Publisher Summary This chapter presents the experimental procedures employed in studies on Escherichia coli lectinlike adhesins, focusing on models for testing adhesins and for determining glycoprotein receptors of the adhesins. Bacterial lectinlike adhesins are proteinaceous structures located on the surface of bacterial cells that mediate the specific adhesion of the bacteria to the host cells. These recognizing proteins bind to the sugar components of glycoproteins or glycolipids on the surface of target cells. The capacity of bacteria to bind specifically to host cells is considered an important virulence factor involved in the initial step of infection. The hemagglutination (HA) test is used for the detection and characterization of lectins. The large natural variability of glycoproteins and glycolipids on the surface of erythrocytes (RBCs) of various animal species provides a tool for evaluating the specificity of bacterial adherence. The HA activity of group I strains of E. coli , defined as carrying type I fimbriae, is best developed in a stationary phase of bacterial culture grown in liquid medium at 37°. The HA activity may be expressed, albeit less strongly, in a culture grown at 20 ° or on solid medium. The hemagglutinins (adhesins) of E. coli are usually associated with fimbrial structures protruding from bacterial surfaces.

Publisher Summary This chapter discusses the phage typing of Klebsiella . Strains of Klebsiella are responsible for a wide variety of diseases of man. The increasing number of Klebsiella infections occurring, particularly those of the respiratory, urinary, and alimentary tracts, besides specific diseases like rhinoscleroma and ozaena justify the attempts at typing Klebsiella by bacteriophages. Klebsiella are non-motile rods with well developed capsules, some with fimbriae. One of the characteristic features of Klebsiella is the production of well developed capsules. These are antigenic and constitute the basis for serotyping of Klebsiella according to K-antigens. Klebsiella can be subdivided on the basis of their morphological, physiological, and antigenic characteristics, their resistance to antibiotics, and sensitivity to bacteriocins (pneumocins) and bacteriophages. The practical value of these different methods of typing should be the subject of further clinical and epidemiological evaluations.

Publisher Summary This chapter focuses on Cyanobacterial Fimbriae. Fimbriae are stiff, tubular filaments of uniform thickness that rise from the bacterial cell surface. They are usually 1-2 μm in length and 5-7 nm in diameter and comprise hundreds of identical subunits, fimbrillins, helically arranged along the filament. Fimbrillins are type specific, and apparent mass range from 8000 to 20,000 has been determined. In one respect, however, all fimbrillins are similar: they are particularly rich in hydrophobic amino acids, which results in increased hydrophobicity of fimbriate cells. Fimbriae are best visualized by using negative staining. This technique involves drying a small quantity of the sample on a grid for electron microscopy and staining it with an electron-dense material such as phosphotungstic acid. Regions of the specimen that the stain cannot infiltrate remain electron-transparent and are seen with high resolution against the electron-dense background. Phosphotungstic acid is recommended for routine staining of cyanobacterial fimbriae.

Publisher Summary This chapter discusses structure–function analysis of group A streptococcal M proteins with hybridoma antibodies. The M protein on the surface of group A streptococci is the major virulence factor of the organism. M protein, which is the type-specific substance of group A streptococci, is a fibrous molecule that partially constitutes the fimbriae or fuzzy layer on the outermost surface of the bacteria. Organisms that are rich in M protein are resistant to phagocytosis in the nonimmune host. In the immune host, antibodies against only M protein opsonize and protect against subsequent infections by related serotypes of streptococci. All cases of acute rheumatic fever are preceded by group A streptococcal pharyngitis; however, only a very small percentage of individuals with streptococcal sore throat subsequently develop rheumatic fever. To maintain the functional characteristics of M protein, the seven-residue periodicity is retained from one serotype to another, while antigenicity is altered to enhance the survivability of the organism in the host.

Publisher Summary This chapter reviews the current knowledge of E. coli adhesions and their receptors. The main emphasis is given on the genetic and physiological aspects of adhesin production, the primary structure of adhesion subunits, their adhesive properties, and the characterization of adhesion receptors. Adhesins are macromolecular structures present on the bacterial cell and mediate the attachment of these cells to a surface. Pathogenic E. coli strains associated with intestinal or urinary tract infections are able to produce one or more different types of fimbrial adhesins that enable the bacteria to colonize the host epithelia. The majority of these fimbriae are composed of a single repeating protein subunit, but some fimbriae may contain other polypeptides as well. It is noted that all fimbriae appear to recognize and bind to relatively simple glycolipids or to glycoproteins present on the host epithelial cell surface or on certain types of erythrocytes. The chapter also discusses the classification of fimbriae according to their affinity for a particular oligosaccharide structure present on these glycoconjugates.

An abnormal distribution in IgG- or IgA-subclass antibody producing cells may contribute to the development of diseases involving chronic inflammation. The advanced stage of Adult Periodontitis (AP) is characterized by local accumulation of plasma cells, lymphocytes and monocytes (macrophages and granulocytes). In order to study both total and antigen-specific IgG and IgA subclass-producing cells, surgically removed gingival tissues were treated with the neutral protease enzyme Dispase®, for isolation of viable mononuclear cells (MC). When the frequency of IgM, IgG and IgA producing cells, including IgG and IgA subclasses were examined by ELISPOT assay, gingival MC of AP contained high numbers of IgG1-producing cells followed by IgG2, IgG3 and IgG4 cells. IgA1-producing cells exceeded IgA2, however, the frequency of the latter cells increased as the disease progressed. Few IgM-producing cells were detected. In the case of antigen-specific antibody-producing cells, approximately 5% of IgG- and IgA-secreting cells were Bacteroides gingivalis fimbriae (Bg-f)-specific. Further analysis of IgG subclass distribution of Bg-f specific antibody-producing cells showed a response of IgG4 > IgG1 > IgG3 > IgG2. Bg-f-specific IgA1-secreting cells predominated in advanced AP with a relative increase in IgA2 cells. In summary, these results suggest that increased numbers of IgG and IgA subclass-secreting cells including those to specific antigen may be involved in the development of AP

Publisher Summary This chapter reviews and discusses the structure and function of pili in light of recent advances employing biochemical, immunological, and genetic approaches. Bacterial “fimbriae” or “pili” are thin (2-1 2 nm diameter) non-flagellar protein filaments found on the surfaces of many types of bacteria. The adhesive properties of pili allow them to bind to other bacteria, bacteriophages, mammalian cells, and inert surfaces. The chapter discusses the classification scheme for pili based on morphology, function, and biochemical properties. It describes three major groups of pilus—namely, conjugative, adhesive, and N-methylphenylalanine (NMePhe) pili. The simplest classification of pili on the basis of function is the division into two broad groups: “conjugative” and “adhesive” pili. It is noted that the biochemical properties help to identify the subpopulations of these pili. The conjugative pili show variable preferences for promoting bacterial mating in liquid or on solid media. Flexible pili generally confer the “universal mating type” and promote mating in liquid media and on solid surfaces equally well, whereas rigid pili are usually associated with “surface preferred” or “surface obligatory” mating types.

Publisher Summary This chapter discusses the preparation of hybridoma cell lines, which secrete monoclonal antibodies cell surface antigens from two gram-positive bacteria, Streptococcus mutans and Actinomyces viscosus, associated with dental caries formation. A. viscosus monoclonal antibodies were prepared against fimbriae found on the surface of virulent strains of this bacterium. The antibodies were specific for the type 1 fimbriae that mediate binding of the cells to the tooth surface and type 2 fimbriae that promote the cell–cell binding of this bacterium to oral streptococci. Hybridoma cell lines which make monoclonal antibodies to species-specific and serospecific antigens of Actinobacillus actinomycetemcomitans, the primary etiological agent in localized juvenile periodontitis were also produced.