RESUMO
AIM: Evaluate influence of mutation of Listeria monocytogenes genes coding murein-tetrapeptide L,D-carboxypeptidase Lmo0028 and Lmo1638 on dynamics of infectious process and interaction of purified muropeptides with NOD1 receptor. MATERIALS AND METHODS: Wild type EGDe strain and recombinant strains GIMins1638 H GIMins0028 obtained on its basis by site-specific mutagenesis were used. Infectious process dynamics was studied on the model of intravenous infection of BALB/c mice. Ligand-receptor interaction activity of muropeptides isolated from recombinant and parent strains were assayed on HEK293-hNOD1 cell line expressing NOD1 receptor and containing in their genome beta-galactosidase reporter gene under the control of NF-kappaB dependent promoter expression. RESULTS: Lack of Lmo0028 decelerates reproduction of listerias in animal liver starting from 24 hours and at later terms after the infection whereas lack of Lmo1638 leads to increase of microbial load 6 and 24 hours after the infection with no influence on further infection. Differences in activation of NOD1 receptor by muropeptides isolated from recombinant and parent strains were not detected. CONCLUSION: Despite high homology murein-tetrapeptide L,D-carboxypeptidase Lmo0028 and Lmo1638 make a different contribution to the development of infectious process caused by L. monocytogenes in BALB/c line mice. Lack of differences in NOD1 receptor activation may be associated with compensation of enzymatic functions in strains with mutation in each of the genes owing to the presence of homologous protein.
Assuntos
Proteínas de Bactérias/genética , Carboxipeptidases/genética , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Proteína Adaptadora de Sinalização NOD1/agonistas , Animais , Carga Bacteriana , Proteínas de Bactérias/metabolismo , Carboxipeptidases/metabolismo , Genes Reporter , Células HEK293 , Humanos , Injeções Intravenosas , Isoenzimas/genética , Isoenzimas/metabolismo , Listeria monocytogenes/genética , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , NF-kappa B/genética , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Peptídeos/genética , Peptídeos/farmacologia , Peptidoglicano/metabolismo , Regiões Promotoras Genéticas , Transfecção , Virulência , beta-Galactosidase/análiseRESUMO
AIM: To study the progress of infection caused by mutant Listeria monocytogenes with deletion of Imo0028 gene coding L,D-carboxypeptidase (protein involved in metabolism of peptidoglycan). MATERIALS AND METHODS: Deletion of Imo0028 gene was performed with site-specific mutagenesis method using wild-type strain EGDe. Virulence was studied on mice of BALB/c line. RESULTS: Strain GIMd0028 with deletion of Imo0028 gene did not differ from parent strain on in vitro growth rate, cytotoxicity and production of listeriolysin O and phospholipase PlcA pathogenicity factors. Effective LD50 for wild-type strain EGDe was 1x10(4) CFU/ mouse and 2x10(5) CFU/mouse for intravenous and intraperitoneal inoculation respectively. Deaths of animals were observed after 3 - 7 days. LD50 for mutant strain GIMd0028 was 1x10(5) CFU/mouse for intravenous inoculation. It was not possible to determine LD50 for intraperitoneal inoculation, and infection progressed atypically slowly. For example, deaths of animals were observed during 22 days (time of experiment) starting from day 4 when strain GIMd0028 in dose 10(6) CFU/mouse was used for inoculation. In average, death of 1 - 2 mice out of 75 inoculated was observed. Presence of L. monocytogenes was confirmed by its isolation from liver and brain of dead mice. CONCLUSION: Disruption of function of Imo0028 gene coding L,D-carboxypeptidase protein, which takes part in metabolism of peptidoglycan, changes dynamics of listeriosis.
