Recombinant Pili Research Articles

Type I pili, CsuA/B and FimA induce a protective immune response against Acinetobacter baumannii

BackgroundAcinetobacter baumannii, a nosocomial pathogen, is considered as a common cause of hospital and community-acquired infections. Emerging multidrug-resistance in this pathogen followed by subsequent problems in treatment has been increasing to alarming levels that warrant investigation of new therapeutic approaches. One strategy to reduce antibiotic resistance is to use of vaccines. Although there is no vaccine currently in development for this pathogen, different attempts have been made to develop one. MethodsIn this study, we used two different recombinant pilus proteins (CsuA/B and FimA) either singly or in combination to evaluate protective efficacy against A. baumannii in lethal and sub-lethal murine sepsis models. FindingsActive immunization with recombinant proteins in combination elicited high levels of IgG antibody after the first immunization and provided 62% (five of eight mice; p < 0·001) protection against a lethal dose (1·2 × 106 CFU) of A. baumannii along with efficient clearance of bacteria in internal organs viz. spleen, liver, and lungs at sub-lethal challenge. Immunization with CsuA/B alone conferred partial protection as demonstrated by low survival rate (three [37%] of eight mice; p < 0·05) after lethal challenge and reduction of bacteria in internal organs of the mice after 24 h post-sub-lethal infection. Immunization with FimA, in comparison to CsuA/B, showed better protection (four [50%] of eight mice; p < 0·01) and reduction in CFU after 14 h. InterpretationOur results showed that pilus proteins in combination as a single immunogen could potentially impart protection against A. baumannii. FundingShahed University.

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

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

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion.

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable fibronectin-binding, collagen-binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT-6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain-of-function mutants we show that, unlike other GAS pili, the FCT-6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT-6 pili.

Structural and functional specificity of Dichelobacter nodosus Pili: Synthesis of pilin protein D. nodosus in various recombinant bacterial strains

Sheep hoof rot is a serious infectious disease specific to this type of animal; its pathogenic agent is anaerobic gram-negative bacillus Dichelobacter nodosus. Very strict requirements for cultivation conditions and slow rate of accumulation of bacterial mass make the vaccine and diagnistcs production laborious and expensive. Fimbriae (pili) are one of the main factors of D. nodosus virulence, since they have both antigenic and immunogenic properties that allow them to be added to vaccines to intensify immunogenicity. After it was determined that the fimA gene, which encodes the fimbrial subunit, attempts were made to obtain recombinant pili. The production of genetically engineered fimbriae serves to simplify and lower the cost of the process of vaccine production, as well as to improve immune-protective qualities of the obtained preparations. The first system of bacterial expression was selected for E. coli, however, the assembly of D. nodosus pili on its surface failed due to significant differences in the biogenesis and fimbria structure of these microorganisms. The morphogenesis of D. nodosus pili succeeded in Pseudomonas aeruginosa, which is characterized by the most similar structure of pilin subunits. Sheep’s immunity after injection of recombinant fimbria, native fimbria, and whole cells of D. nodosus was almost analogous in intensity. The article presents a review of data from publications on the production of recombinant cultures of P. aeruginosa with D. nodosus pili and expression of pilin genes in various bacterial systems.

Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilus-negative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three-dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild-type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GAS-mediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.

Streptococcus pneumoniae Pilus Subunits Protect Mice against Lethal Challenge

Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.

Use ofLactococcus lactisExpressing Pili from Group BStreptococcusas a Broad‐Coverage Vaccine against Streptococcal Disease

Recent data indicate that the human pathogen group B Streptococcus (GBS) produces pilus-like structures encoded in genomic islands with similar organization to pathogenicity islands. On the basis of the amino acid sequence of their protein components, 3 different types of pili have been identified in GBS, at least 1 of which is present in all isolates. We recently demonstrated that recombinant pilus proteins protect mice from lethal challenge with GBS and are thus potential vaccine candidates. Here, we show that GBS pilin island 1, transferred into the nonpathogenic microorganism Lactococcus lactis, leads to pilus assembly. We also show that systemically or mucosally delivered Lactococcus expressing pilin island 1 protects mice from challenge with GBS isolates carrying pilus 1. Furthermore, lactococci engineered to express hybrid pili containing GBS pilus 1 and pilus 2 components confer protection against strains expressing either of the 2 pilus types. These data pave the way to the design of pilus-based, multivalent live vaccines against streptococcal pathogens.

Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens

Although pili have long been recognized in Gram-negative pathogens as important virulence factors involved in adhesion and invasion, very little is known about extended surface organelles in Gram-positive pathogens. Here we report that Group A Streptococcus (GAS), a Gram-positive human-specific pathogen that causes pharyngitis, impetigo, invasive disease, necrotizing fasciitis, and autoimmune sequelae has long, surface-exposed, pilus-like structures composed of members of a family of extracellular matrix-binding proteins. We describe four variant pili and show that each is recognized by a specific serum of the Lancefield T-typing system, which has been used for over five decades to characterize GAS isolates. Furthermore, we show that immunization of mice with a combination of recombinant pilus proteins confers protection against mucosal challenge with virulent GAS bacteria. The data indicate that induction of a protective immune response against these structures may be a useful strategy for development of a vaccine against disease caused by GAS infection.

Enterotoxigenic K99 +Escherichia coli attachment to host cell receptors inhibited by recombinant pili protein

Most enterotoxigenic Escherichia coli (ETEC) isolated from neonatal cattle with diarrhea (enteric colibacillosis) exhibit the colonization factor antigen, K99. The K99 pili are necessary for the bacteria to bind to a receptor, N-glycolylneuraminic acid-GM3 on the host cells in the small intestine where the bacteria multiply and secrete toxins that cause the diarrhea. When the attachment of the ETEC to host cell is inhibited, the bacteria do not accumulate sufficiently in the gut to cause disease. Since purified K99 pili block K99 + ETEC from binding to host epithelia, three recombinant K99 proteins of different sizes were developed and produced to demonstrate inhibition with in vitro competitive binding assays. The full-length recombinant protein, rK99-476 inhibited the binding of ETEC with an activity similar to that of the native purified K99, whereas the truncated recombinant K99 protein had no inhibitory activity. Thus this binding activity of rK99-476, which is specific and effective in blocking the receptors on the host cells, may be able to competitively inhibit K99 + ETEC infections in cattle.

Epitopes fused to F-pilin are incorporated into functional recombinant pili

In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin ( traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope ( myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.

Antigens for serological diagnosis of ovine footrot

An antigen extracted from Dichelobacter nodosus with potassium thiocyanate (KSCN) is currently used in enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of ovine footrot, but the test lacks specificity in mature sheep. Other antigens were therefore evaluated for use in this test. Structural components of the cell envelope of D. nodosus including outer membrane, cytoplasmic membrane, lipopolysaccharide and pilus and extracellular proteases were purified from cultured D. nodosus while recombinant membrane proteins, protease and pilus antigens were also evaluated. Many antigenic components of D. nodosus participated in reactions in ELISA that were not specific for infection with D. nodosus and apart from pilus, none of the antigens resulted in improved specificity of the ELISA. Using a positive-negative cut-off to yield sensitivity of 70%, ELISA using pili from cultured D. nodosus serogroup A had a specificity of 98.3% compared with 89.7% for the ELISA with KSCN-extract as antigen ( P < 0.001). Recombinant pili morphogenetically expressed in Pseudomonas aeruginosa were unsuitable for use in ELISA due to copurification of Pseudomonas antigens to which apparently healthy sheep directed antibodies. The application of ELISA with D. nodosus pilus as antigen in footrot control programs is discussed.

Antigenic competition in a multivalent foot rot vaccine

The antigenic competition that occurs when pilus antigens of different serogroups are combined in multivalent vaccines for foot rot has been investigated using recombinant pilus antigens. Our prototype vaccine contains pili from nine serogroups of Dichelobacter nodosus which are expressed in Pseudomonas aeruginosa. Sheep inoculated with this multivalent vaccine were not as well protected against foot rot as those given the monovalent vaccine. Levels of agglutinating and total antibody specific for any particular pili serogroup were found to be significantly reduced in sheep vaccinated with six or more closely related pili. This effect was more pronounced for agglutinating antibody, which is thought to mediate protection, but was also observed with total antibody levels measured by ELISA. The antigenic competition was not associated with the total antigen load as a tenfold higher dose of monovalent pili induced high titres of antibody. Furthermore, distributing the vaccine to four sites, each draining to a different lymph node, failed to overcome the competition. Experiments with mixtures of monospecific sera indicate that the phenomenon is unlikely to be due to blocking of serogroup-specific protective antibodies by an excess of cross-reactive non-protective antibody elicited by heterologous pili.

Cloning of DNA sequences encoding foreign peptides and their expression in the K88 pili

A genetic system that allows the cloning of a peptide-coding sequence in the Escherichia coli K88ac and K88ad pilin genes and their expression as recombinant pili has been constructed. Two insertion vectors were created by subcloning the pilin genes in a pBR322 plasmid and replacing the coding sequence of two nonconserved clusters by a linker. The K88ac helper genes were subcloned in the compatible pACYC184 plasmid, and expression of pili by bacteria carrying both plasmids occurred by complementation. Two peptide-coding sequences of the influenza hemagglutinin were cloned in both insertion vectors, and recombinant pilins were shown to be assembled in pili. One recombinant pilus was shown to elicit antibodies against the synthetic peptide in immunized rats. The somatostatin-coding sequence was cloned in both vectors and led in one case to detectable pilus production. The fused somatostatin was shown to be recognized by specific monoclonal and polyclonal antibodies.