Antibody responses to virulence factors in patients infected with

CHINEN I; CREPIN V; GARMENDIA J; FERNÁNDEZ BRANDO, RJ; PALERMO MS; FRANKEL G; RIVAS M

Buenos Aires

Simposio; : 7th International Symposium on Shiga Toxin (Verocytotoxin) Producing E.coli Infections.; 2009

Asociación Argentina de Microbiología

Enterohemorrhagic Escherichia coli (EHEC) mainly serotype O157:H7, are important human pathogens. The ability of EHEC strains to colonise the gut mucosa and to cause disease is associated with a number of virulence factors including expression of Shiga toxin (Stx) and the capacity to cause attaching/effacing (A/E) lesions. The genes of different proteins that are required for formation of A/E lesions in vitro by EHEC O157 are carried on two separate pathogenicity islands: the locus of enterocyte effacement (LEE) and prophage CP-933U. Patients infected with EHEC O157:H7 are known to produce serum and salivary antibodies to LPS and it could be use for the serodiagnosis of the infection. Moreover, it was described that patients infected with O157 and non-O157 EHEC may produce antibodies to intimin, EspA and EspB and to the effector protein Tir. In the present study, 24 patients with E. coli O157:H7 infection (16 HUS, 5 bloody diarrhoeas, and 4 non-bloody diarrhoeas) admitted in different paediatric hospitals between 2001 and 2006 and 12 healthy control children [median children age was 24 months; mean 39.2 ± 38.3 months (range, 7 to 166 months)] were investigated for serum antibody responses against LPSO157, Shiga toxin (Stx), the adhesin intimin (Int280g), the type III secretion system (T3SS) tanslocator protein EspA and the T3SS effector proteins Tir, TccP and Map. Western blot assays (WBA) were used to determine antibody responses against Stx, Int280g, EspA, Tir, TccP and Map using nitrocellulose membranes containing 0.5 mg of each of the purified proteins, following 12% SDS-PAGE and semi-dry transfer (BioRad). ELISA assay was used to determine serum LPSO157 antibodies. The frequency of antibody response in the 24 serum samples of patients with E. coli O157:H7 infection against each of the proteins studied was: 24 (100%) for Tir; 23 (95.8%) for EspA; 22 (91.7%) for LPSO157; 21 (87.5%) for Stx2; 19 (79.2%) for Map; 18 (75%) for TccP; 16 (66.7%) for Int280g. Among control sera the frequency was: 12 (100%) for Tir; 12 (100%) for EspA; 9 (75%) for Int280g; 7 (58.3%) for Stx2; 6 (50%) for TccP; 5 (41.9%) for Map; and 4 (33%) for LPSO157. Tir (n=36, 100%) and EspA (n=35, 97%) were the dominant antigens recognized by the diseased and control sera. 11 (46%) out of the 24 diseased serum samples reacted against all of studied proteins and LPSO157, compared to only one (0.08%) out of 12 control serum samples. We observed variable intensity of antibody responses. While similarly strong response against Tir and EspA was seen in sera form infected and control children, the responses against the other antigens in the control sera were less intense compared to the STEC-infected children group. Further studies are needed to enhance the knowledge about immunoresponse of the population to select which of the proteins (or protein combinations) described could be used as indirect marker for STEC infections.