RESUMEN

The enteropathogen Shigella flexneri invades epithelial cells, leading to inflammation and tissue destruction. We report that Shigella infection of epithelial cells induces an early genotoxic stress, but the resulting p53 response and cell death are impaired due to the bacterium's ability to promote p53 degradation, mainly through calpain protease activation. Calpain activation is promoted by the Shigella virulence effector VirA and dependent on calcium flux and the depletion of the endogenous calpain inhibitor calpastatin. Further, although VirA-induced calpain activity is critical for regulating cytoskeletal events driving bacterial uptake, calpain activation ultimately leads to necrotic cell death, thereby restricting Shigella intracellular growth. Therefore, calpains work at multiple steps in regulating Shigella pathogenesis by disrupting the p53-dependent DNA repair response early during infection and regulating both formation and ultimate death of the Shigella epithelial replicative niche.

Asunto(s)

Calpaína/metabolismo , Activación Enzimática , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Shigella flexneri/fisiología , Factores de Virulencia/fisiología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fibroblastos/microbiología , Células HeLa , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ratones , FN-kappa B/metabolismo , Necrosis , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Shigella flexneri/crecimiento & desarrollo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

This study was designed to investigate the role of hypermutability of Staphylococcus aureus on bacterial fitness and antibiotic resistance in a model of chronic bone infection. An isogenic pair of strains, S. aureus RN4220 and its mutator counterpart inactivated in the mutL gene were used in a rat model of osteomyelitis of the tibia. The effect of the mutator phenotype in the emergence of antibiotic resistance was assessed in rats infected by each strain separately and treated with rifampicin for 5 days. No difference between the two strains was found in bacterial growth in vitro and in bacterial survival in the animal model, indicating no fitness defect in the mutator strain. In competition studies performed in rats coinfected with the two strains at a same ratio and sacrificed at different times from day 3 to day 42 postinoculation, the mutator strain was clearly disadvantaged because it was found in all rats and at all study times at lower counts (P<0.05 from day 14 to day 42). Two of the 16 rats infected by the mutator strain and none of the 14 rats infected by the wild-type strain had acquired rifampicin-resistant mutants (P=0.4). Data suggest that the S. aureus mutator phenotype was associated with a decreased bacterial fitness in vivo and did not confer significant advantage in the acquisition of antibiotic resistance in a model of chronic bone infection.

Asunto(s)

Farmacorresistencia Bacteriana/genética , Osteomielitis/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/fisiología , Adenosina Trifosfatasas/genética , Animales , Proteínas Bacterianas/genética , Huesos/microbiología , Recuento de Colonia Microbiana , Eliminación de Gen , Pruebas de Sensibilidad Microbiana , Mutagénesis Insercional , Osteomielitis/tratamiento farmacológico , Ratas , Rifampin/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Análisis de Supervivencia

RESUMEN

Comparison of nadA and nadB in 14 Shigella strains and enteroinvasive Escherichia coli versus E. coli showed that at least one locus is altered in all strains. These observations explain the characteristic nicotinic acid auxotrophy of Shigella organisms and are consistent with the previously identified antivirulence nature of these genes for these pathogens.

Asunto(s)

Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Proteínas Bacterianas/metabolismo , Complejos Multienzimáticos/genética , Shigella flexneri/genética , Shigella flexneri/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas de Escherichia coli , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Shigella flexneri/fisiología , Virulencia/genética

RESUMEN

The evolution of bacterial pathogens from commensal organisms involves virulence gene acquisition followed by pathoadaptation to the new host, including inactivation of antivirulence loci (AVL). AVL are core ancestral genes whose expression is incompatible with the pathogenic lifestyle. Previous studies identified cadA (encoding lysine decarboxylase) as an AVL of Shigella spp. In this study, AVL of Shigella were identified by examining a phenotypic difference from its non-pathogenic ancestor, Escherichia coli. Unlike most E. coli strains, Shigella spp. are nicotinic acid auxotrophs, the pathway for the de novo synthesis of NAD being uniformly defective. In Shigella flexneri, this defect is due to alterations in the nadA and/or nadB genes encoding the enzyme complex that converts L-aspartate to quinolinate, a precursor to NAD synthesis. Quinolinate was found to inhibit invasion and cell-to-cell spread of Sh. flexneri 5a and its ability to induce polymorphonuclear neutrophil transepithelial migration. Virulence of other Shigella species was also inhibited by quinolinate. Introduction of functional nadA and nadB genes from E. coli K-12 into Sh. flexneri 5a restored its ability to synthesize quinolinate but also resulted in strong attenuation of virulence in this strain. The results define nadA and nadB as AVL in Shigella and validate the concept of pathoadaptive evolution of bacteria from commensal ancestors by inactivation of AVL. They also suggest that studies focusing on this form of bacterial evolution can identify novel inhibitors of virulence in other bacterial pathogens.

Asunto(s)

NAD/biosíntesis , NAD/genética , Ácidos Quinolínicos/farmacología , Shigella flexneri/patogenicidad , Virulencia/genética , Genes Bacterianos , Células HeLa , Humanos , Ácidos Quinolínicos/metabolismo , Shigella flexneri/genética , Virulencia/fisiología

RESUMEN

The mutator phenotype has been linked in several bacterial genera to a defect in the methyl-mismatch repair system, in which the major components are MutS and MutL. This system is involved both in mismatch repair and in prevention of recombination between homeologous fragments in Escherichia coli and has been shown to play an important role in the adaptation of bacterial populations in changing and stressful environments. In this report we describe the molecular analysis of the mutS and mutL genes of Staphylococcus aureus. A genetic analysis of the mutSL region was performed in S. aureus RN4220. Reverse transcriptase PCR experiments confirmed the operon structure already reported in other gram-positive organisms. Insertional inactivation of mutS and mutL genes and complementation showed the role of both genes in hypermutability in this species. We also designed an in vitro model to study the role of MutS and MutL in homeologous recombination in S. aureus. For this purpose, we constructed a bank of S. aureus RN4220 and mutS and mutL mutants containing the integrative thermosensitive vector pBT1 in which fragments with various levels of identity (74% to 100%) to the S. aureus sodA gene were cloned. MutS and MutL proteins seemed to have a limited effect on the control of homeologous recombination. Sequence of mutS and mutL genes was analyzed in 11 hypermutable S. aureus clinical isolates. In four of five isolates with mutated or deleted mutS or mutL genes, a relationship between alterations and mutator phenotypes could be established by negative complementation of the mutS or mutL mutants.

Asunto(s)

Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Recombinación Genética/genética , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Clonación Molecular , Proteínas de Escherichia coli , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mutación/genética , Operón/genética , Fenotipo , Staphylococcus aureus/clasificación

RESUMEN

Over 124 methicillin-susceptible Staphylococcus aureus 0/74 fluoroquinolone-susceptible versus 5/50 fluoroquinolone-resistant isolates were hypermutable. Hypermutable isolates combined mutations in gyrA, parC, and/or parE genes. One strain had a large deletion of the mutator mutS and mutL genes. No relevant mutation in mutS and mutL genes was found in the other isolates.

Asunto(s)

Antiinfecciosos/farmacología , Fluoroquinolonas/farmacología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Cartilla de ADN , Replicación del ADN , Farmacorresistencia Bacteriana , Genes Bacterianos/genética , Resistencia a la Meticilina , Mutación/genética , Mutación/fisiología

RESUMEN

The aim of this study was to characterize the resistance mechanisms of two strains of Staphylococcus aureus UCN42 and UCN43 isolated from cystic fibrosis patients that displayed an unusual phenotype of resistance to macrolides. The strains were resistant to erythromycin and the 16-membered macrolide spiramycin but susceptible to clindamycin. None of the strains contained erm or msr(A) genes by PCR. Sequencing of genes encoding ribosomal targets of macrolides revealed mutations in conserved regions of the L4 ribosomal protein, a R168S mutation for S. aureus UCN43 and mutations at positions G69A and T70P for both strains. This observation extends previous reports of similar mutations in streptococci and mycoplasma to S. aureus.

Asunto(s)

Farmacorresistencia Bacteriana/genética , Macrólidos/farmacología , Proteínas Ribosómicas/genética , Staphylococcus aureus/efectos de los fármacos , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación

RESUMEN

Incidence of resistance to erythromycin at our institution reached 53% in 122 Staphylococcus aureus isolates obtained from patients with cystic fibrosis (CF) from 1997 to 1999. Macrolide-resistance genes were sought for in 20 erythromycin-resistant isolates from 9 patients with CF by use of polymerase chain reaction; 13 strains did not contain any known macrolide-resistance genes. Sequence of ribosomal genes rrl (23S rRNA), rplD (L4 protein), and rplV (L22 protein) revealed the presence of mutations in the target site of macrolides in 15 of the 20 isolates. A higher proportion of hypermutator strains was observed in a group of 89 CF staphylococcal isolates, compared with that in the 74 non-CF control isolates (13/89 vs. 1/74 with resistance to rifampin [P=.0045]; 9/89 vs. 1/74 with resistance to streptomycin [P=.04]). Various mutations or deletions of the mutator mutS gene were found not only in 5 of 11 hypermutable strains but also in 3 nonhypermutable strains harboring a large number of ribosomal mutations. The presence of a high proportion of hypermutable strains might explain the adaptation of certain strains in the patients, as well as the emergence of macrolide resistance as a result of antibiotic selective pressure in CF.

Asunto(s)

Antibacterianos/farmacología , Proteínas Bacterianas , Fibrosis Quística/microbiología , Proteínas de Unión al ADN , Farmacorresistencia Bacteriana Múltiple , Mutación/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Adenosina Trifosfatasas/genética , Adulto , ADN Ribosómico/genética , Proteínas de Escherichia coli/genética , Femenino , Humanos , Macrólidos , Masculino , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Fenotipo , Staphylococcus aureus/clasificación

RESUMEN

Six strains of Staphylococcus aureus isolated from cystic fibrosis patients after treatment with azithromycin were cross-resistant to azithromycin and erythromycin. None of the isolates contained erm or msr(A) genes, but they all carried either A2058G/U or A2059G mutations within the rrl genes, with a majority of the rRNA copies bearing the mutation. One strain displayed an additional mutation in the rplV gene, encoding the L22 ribosomal protein.

Asunto(s)

Antibacterianos/farmacología , Proteínas de Transporte de Membrana , Metiltransferasas , Mutación/genética , Ribosomas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Azitromicina/farmacología , Proteínas Bacterianas/genética , Fibrosis Quística/microbiología , ADN Bacteriano/genética , Resistencia a Medicamentos , Eritromicina/farmacología , Genes Bacterianos/genética , Humanos , Pruebas de Sensibilidad Microbiana , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa