Mannose-sensitive Pili Research Articles

Multiplex real-time PCR detection of Vibrio cholerae

Cholera is an important enteric disease, which is endemic to different regions of the world and has historically been the cause of severe pandemics. Vibrio cholerae is a natural inhabitant of the aquatic environment and the toxigenic strains are causative agents of potentially life-threatening diarrhoea. A multiplex, real-time detection assay was developed targeting four genes characteristic of potentially toxigenic strains of V. cholerae, encoding: repeat in toxin ( rtxA), extracellular secretory protein ( epsM), mannose-sensitive pili ( mshA) and the toxin coregulated pilus ( tcpA). The assay was developed on the Cepheid Smart Cycler using SYBR Green I for detection and the products were differentiated based on melting temperature ( T m) analysis. Validation of the assay was achieved by testing against a range of Vibrio and non- Vibrio species. The detection limit of the assay was determined to be 10 3 CFU using cells from pure culture. This assay was also successful at detecting V. cholerae directly from spiked environmental water samples in the order of 10 4 CFU, except from sea water which inhibited the assay. The incorporation of a simple DNA purification step prior to the addition to the PCR increased the sensitivity 10 fold to 10 3 CFU. This multiplex real-time PCR assay allows for a more reliable, rapid detection and identification of V. cholerae which is considerably faster than current conventional detection assays.

SUPPRESSION OF RENAL INFLAMMATION WITH VITAMINS A AND E IN ASCENDING PYELONEPHRITIS IN RATS

We evaluated the effects of vitamin A and E supplementation alone or in combination with non-steroidal anti-inflammatory drugs (NSAIDs) on the development of inflammation in an animal model of ascending pyelonephritis. Ascending pyelonephritis was induced in adult rats by surgical bladder inoculation with P-pili-forming Escherichia coli. Treatment of pyelonephritic rats was initiated at 72 hours post-infection. Treatment groups included no treatment, or a five day regimen of antibiotic only, antibiotic plus vitamins A and E, or antibiotic, vitamins and either of two NSAIDs. Kidneys were harvested at six weeks post-infection and assessed for histopathologic inflammation. Antibiotic treatment of pyelonephritic rats with vitamins A and E alone or in combination with NSAIDs resulted in significantly less kidney inflammation, as compared with untreated rats or rats treated with antibiotic alone. There was no significant difference in inflammation between animals treated with vitamins alone or vitamins plus NSAIDs. Antibiotic therapy and diet supplementation with vitamins A and E can significantly reduce the inflammation associated with ascending pyelonephritis, suggesting a potential use in the medical management of reflux nephropathy in children.

Inhibitory effect of bacterial attachment on candidal growth due to adherence with mannose-sensitive pili.

A bacterial strain with affinity to Candida albicans was successfully obtained from a natural environment. An uncovered Petri dish containing a suspension of heat-killed C. albicans cells was allowed to stand in a laboratory for several days. Some bacteria which had adhered to the candidal cells were tested for their ability to agglutinate the cells. A bacterial strain, designated later as CAB-1, was found to agglutinate candidal cells through bridging by mannose-sensitive pili. CAB-1 showed similar bacteriological characteristics to those of Citrobacter freundii by ID test. The adherence of CAB-1 to candidal cell was precisely presented by scanning electron microscopy. The inhibitory effect of CAB-1 attachment to candidal cells on the growth of Candida was also preliminarily confirmed.

Preventive Effect of Dapsone on Renal Scarring following Mannose-Sensitive Piliated Bacterial Infection

Renal scarring has been thought to occur in the later stages of chronic pyelonephritis. We previously reported that mannose-sensitive (MS) piliated bacteria promoted renal scarring, which was prevented by antioxidants. The preventive effect of diaphenylsulfone (dapsone), which has a scavenging activity on active oxygen species, on renal scarring was examined. Female Sprague-Dawley rats were inoculated with clinical isolates of Serratia marcescens which had both MS and mannose-resistant pili or with recombinant strains which had MS pili on their surface; they were then administered 20 mg/kg of dapsone or not. Dapsone significantly suppressed scarring following infection of the kidney. The bacterial counts in the kidneys were not different in dapsone-treated and nontreated rats. We conclude that dapsone is effective in preventing renal scarring, and it is suggested that the clinical use of this drug may prevent renal scar formation following pyelonephritis.

Virulence-associated characteristics and phage lysogenicity of two morphologically distinct colonies of Vibrio cholerae O139 serogroup

The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient. Spontaneous variants with translucent colonies had lost this phage. The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule. These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili. Furthermore, excision of the phage made the strain dependent on purines for growth.

Virulence-associated characteristics and phage lysogenicity of two morphologically distinct colonies of Vibrio cholerae O139 serogroup.

The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient. Spontaneous variants with translucent colonies had lost this phage. The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule. These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili. Furthermore, excision of the phage made the strain dependent on purines for growth.

Renal Scarring Is Enhanced by Phorbol Myristate Acetate following Infection with Bacteria with Mannose-Sensitive Pili

Renal scarring is considered to develop in the later stages of chronic pyelonephritis. In our experimental model of pyelonephritis, bacteria with mannose-sensitive (MS) pili on their surface promoted renal scarring following inoculation into the renal parenchyma. The administration of cyclophosphamide to induce leukopenia and of superoxide dismutase to inactivate superoxide released from polymorphonuclear leukocytes (PMN) at the infection site suppressed any renal scarring following the infection. Conversely, the administration of phorbol myristate acetate at an early stage of infection significantly enhanced the renal scarring. These findings suggest that the PMNs which infiltrate the infection site and the superoxide they release play an important role in any renal scarring following infection with MS-piliated bacteria.

Characteristics of Aerobic Gram-negative Bacteria Colonizing Critically III Patients

Adherence of gram-negative bacteria (GNB) to epithelial surfaces is important for GNB colonization to occur. Pili, rodlike structures projecting from the outer membrane of GNB, and GNB surface hydrophobicity have been shown to enhance GNB adherence. We investigated the types of pili and the hydrophobicity of aerobic GNB colonizing the stomach, oropharynx, and trachea of critically ill patients. Piliation and hydrophobicity of oral, tracheal, and gastric GNB were compared with that of commensal GNB isolated from patients' rectums. Significantly more oropharyngeal than rectal GNB were piliated, and the most common type of pili present was type 1, or mannose-sensitive pili. Mannose-resistant and P pili were present less often, and no colonizing GNB had S pili. Colonizing GNB were hydrophilic rather than hydrophobic, and no differences in hydrophobicity were noted between colonizing GNB and rectal isolates. Our results suggest that pili may be important for oropharyngeal GNB colonization.

Increased renal scarring by bacteria with mannose-sensitive pili

Renal scars are thought to be the end stage of chronic pyelonephritis and one of the most important causes of renal insufficiency and renal hypertension. The role of bacterial pili was examined in scar formation after an infection of newly constructed bacterial strains using the recombinant DNA technique, which possessed either mannose resistant (MR) or mannose sensitive (MS) pili of Serratia marcescens. Strains that differed in only a single virulence factor, namely, MR or MS pili, were used in a rat model of chronic pyelonephritis. In this model, MS-piliated bacteria stimulated renal scarring more severely than non-piliated or MR-piliated bacteria.

Suitability of Colchicine and Superoxide Dismutase for the Suppression of Renal Scarring following an Infection with Bacteria Showing Mannose-Sensitive Pili

Two new strains of Serratia marcescens were constructed by the gene manipulation method from the clinical isolate US 46, which has two kinds of pili--mannose-sensitive (MS) and mannose-resistant (MR) ones--on the cell surface. After cloning the genes of the MS and MR pili, either the MS or the MR gene was transferred to the nonpiliated Escherichia coli, and MS- or MR-piliated strains were obtained. In the experimental pyelonephritis model of rats, MS- or MR-piliated bacteria were inoculated directly to the renal parenchyma, and the following results were obtained. MS-piliated rather than MR-piliated strains stimulated severe scarring of the kidney, and this scarring was suppressed by treatment with colchicine or superoxide dismutase (SOD) during an early stage of the infection. These findings suggest that MS-piliated bacteria stimulated polymorphonuclear leukocytes, which released large amounts of superoxide resulting in renal scarring. SOD was hoped to be a drug capable of preventing renal scarring, and such a result was successfully obtained.

Heterologous protection against invasive Escherichia coli K1 disease in newborn rats by maternal immunization with purified mannose-sensitive pili

Heterologous protection against Escherichia coli K1 bacteremia with antibody to purified mannose-sensitive (MS) pili was demonstrated in a neonatal rat model. The serological relatedness of purified MS pili from 17 E. coli K1 clinical isolates was examined by an enzyme-linked immunosorbent assay. Five pilus serogroups were identified, with the pili in each group showing 50% or greater cross-reactivity with the typing serum of the group. The MS pili from 12 of 17 (70%) strains belonged to just two serogroups. Pregnant Sprague-Dawley rats (dams) were immunized with purified pili, and their newborns (pups) were challenged with heterologous E. coli. Bacteremia was significantly reduced when the pili used for immunization were from the same serogroup as the pili expressed by the challenge bacteria. Thus, immunization with C94 pili and challenge with E03 (71% cross-reactivity) or E04 (50% cross-reactivity) resulted in bacteremia rates of 12 of 17 (17%) versus 51 of 79 (65%) in controls and 0 of 75 (0%) versus 28 of 70 (40%) in controls, respectively (P less than 0.001 for each comparison). With lower cross-reactivity, less protection was observed (P less than 0.05 for 22 to 37% pilus serological relatedness). No protection was seen in pups suckled by dams immunized with MS pili having only 5% serological relatedness to the pili on the challenge strain.

Purification and characterization of Haemophilus influenzae pili, and their structural and serological relatedness to Escherichia coli P and mannose-sensitive pili.

Haemophilus influenzae pili were purified, and their physical and serological properties were examined. The solution properties of the pili were determined, and then a purification scheme involving repeated cycles of precipitation and solubilization was developed. The purified pili from one type b isolate (A02) were found to consist of multiple copies of a 25,000 mol wt subunit. Amino-terminal sequence analysis of A02 pili was carried out to 40 amino acid residues, and a remarkable degree of sequence homology was found with E. coli P and mannose-sensitive (MS) pili (27.5 and 25% homology, respectively). Purified A02 pili were found to be highly immunogenic, and serological analysis by enzyme-linked immunosorbent assay and whole piliated cell agglutination revealed significant cross-reactivity between A02 pilus antiserum and the pili of seven other H. influenzae strains tested (heterologous titers = 2-100% of the homologous titer). Cross-reactivity was also observed between the H. influenzae pili (five of eight strains tested) and the P pili from E. coli strains HU849 and 3669; no cross-reactivity was detected with MS pili from E. coli strain H10407 and C94. The structural similarities between H. influenzae and E. coli P and MS pili suggest a common gene ancestry.

Interaction of bacterial pili and leukocytes.

Since Duguid and Guilles first described the ability of piliated bacteria to bind to leukocytes, much has been learned about the nature of this interaction. Mannose-sensitive (MS) pili bind to specific mannose-containing receptors on the leukocyte surface. While MS pili are responsible for attachment, the relative hydrophobicity of the bacterial surface determines whether the organism is internalized. Both binding and ingestion trigger the leukocyte to respond with degranulation and enhanced oxidative activity. The response to piliated bacteria, however, is delayed as compared to bacteria opsonized with serum, which may account for the reduced bactericidal activity associated with pili-mediated phagocytosis. A number of factors appear to influence the significance of pili-mediated phagocytosis in vivo. These include natural selective pressures in the host tissue, the ability of the organism to undergo pili phase transition and the presence of serum or other host opsonic factors. Antipili antibody does not enhance leukocyte killing of MS + Escherichia coli, but does stimulate leukocyte metabolic activity. Antipili antibody may, therefore, have an adverse effect on the infectious process by promoting the extracellular release of inflammatory material from the granulocyte.

Antibacterial mechanisms of antibody to mannose-sensitive pili of Escherichia coli.

To identify mechanisms whereby antibody to mannose-sensitive pili of Escherichia coli might enhance host defenses, we evaluated the activity of antibody to pili in four antibacterial immune processes. Antiserum to pili and Fab' fragments of IgG antibody to pili inhibited the ability of homologous piliated organisms to adhere to buccal epithelial cells. However, this antiserum did not enhance intravascular clearance, complement-dependent bacteriolysis, or opsonophagocytosis. The addition of antiserum to pili to polymorphonuclear leukocytes and piliated E. coli reduced the rate of killing from 52% to 5%. The addition of complement restored the rate to 52%, but this was much less than the 99% rate achieved with polymorphonuclear leukocytes and either fresh serum or antibody to the whole bacteria. These observations suggest that the principal anti-bacterial property of antibody to mannose-sensitive pili of E. coli is inhibition of bacterial attachment. Whether the anti-opsonic effect of antibody to pili is detrimental to host defense remains to be determined.