Roles of Clostridium difficile cell wall and flagellar proteins in pathogenicity and innate immunity
Dehlawi, Saied Waheed
The number of cases of Clostridium difficile infection (CDI) has been increasing globally. CDI is the main cause of nosocomial diarrhoea, which may be life-threatening in complicated cases, and also costs the health care societies millions of pounds annually. The predominant types and their resistance to antibiotics have been changing and one of the major selective pressures which causes this is antimicrobial use. Although much is known about the role of the toxins in pathogenesis of CDI, the role of immunogenic cell wall components is unclear. They may play a role in colonisation and pathology and a study of these could clarify the infection process. It is therefore important to study the immune responses against these bacterial wall components from different strains and their effects on stimulation of leukocytes to produce cytokines and chemokines. This study was divided into four parts: 1. An epidemiological study to determine frequencies of the predominant types of C. difficile, thus 140 C. difficile isolates from surgical patients and their environment during 2009 were investigated to define their PCR ribotype. This utilised capillary sequencing gel electrophoresis for their analysis. 2. The determination of antimicrobial susceptibility to six antibiotics (ampicillin, erythromycin, tetracycline, metronidazole, moxifloxacin and vancomycin) was assessed and MIC determination by agar dilutions. 3. Investigation of host immunity to molecules with conserved molecular patterns. Surface-layer proteins (SLPs), lipocarbohydrate (LC) and flagellar proteins were separated and purified from five ribotypes of C. difficile (001, 002, 027, 078 and106) predominant in Scotland. a) The immune responses to these molecules were assessed by ELISA by exposing serum of patients and healthy donors and measuring specific IgG levels. b) Innate immunity was investigated by distinguishing responses of a macrophage cell line (THP1) to the above molecules. Induction of interleukins (IL)-1β, IL-6, IL- 8, IL-10 and IL-12 interleukins and TNF-α was detected by ELISA. In this study 15 different ribotypes were identified. The most frequent were 001, 020, 106 ribotypes (52.8%, 7.4% and 5.7%), respectively, while 13 isolates could not be assigned a ribotype. However, all isolates were sensitive to vancomycin, metronidazole and moxifloxacin, but 74.28% of isolates were resistant to erythromycin. The IgG level against bacterial antigens (SLPs, LC and flagella proteins) in donors’ serum showed almost normal distribution to all antigens from the different ribotypes and the sensitivity of the assays was increased by raising the concentration of antigens. Levels to SLPs were generally the highest, but the flagellar protein exceeded the SLPs of the 027 ribotype. The donors, controls, patients and carrier sera gave similar results. The greatest induction of interleukins was obtained using 50μg of antigen with the THP-1 cells activated with 50ng of PMA. The highest induction of all antigens was for IL-10. The highest values for the control LPS was with IL-12. But the best effect for SLPs of 027 was for IL-10 (109.1ng/ml), while the weakest for TNF for SLPs of 027 (4.7ng/ml). In general the IL-1β, IL-6, IL-8 and TNF concentrations ranged from 4.7-60ng/ml for all antigens and in contrast IL-12 and IL-10 average ranged 11- 109.1ng/ml. To conclude, the prevalence of C. difficile and their antibiotic susceptibility are constantly changing. IgG antibodies to SLPs and flagellar proteins from the hypervirulent ribotype 027 were highest in the community and hospitalized individuals. The molecules of conserved molecular patterns are immunogenic with various levels of response in the monocytic THP1 cells. SLPs were best in inducing interleukins. Flagellar proteins from 027 ribotypes accompanied SLPs in IL-10 induction levels. Consequently SLPs and flagellar proteins from 027 ribotypes appeared the best immunogenic bacterial molecules.