RESUMEN
Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second-messengers - cGMP mediated by the parasite's cGMP-dependent protein kinase (PKG), and Ca2+ , mediated by the parasite's calcium-dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Hepatocitos/parasitología , Plasmodium berghei/metabolismo , Proteínas Quinasas/metabolismo , Animales , Anopheles/parasitología , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , GMP Cíclico/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Plasmodium berghei/enzimología , Plasmodium berghei/genética , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Esporozoítos/metabolismoRESUMEN
AIMS: In this study, multidrug-resistant bacteria previously recovered from the indoor air of a large-scale swine-feeding operation were tested for the presence of five macrolide, lincosamide and streptogramin (MLS) resistance genes and five tetracycline (tet) resistance genes. METHODS AND RESULTS: Enterococcus spp. (n = 16) and Streptococcus spp. (n =16) were analysed using DNA-DNA hybridization, polymerase chain reaction (PCR) and oligoprobing of PCR products. All isolates carried multiple MLS resistance genes, while 50% of the Enterococcus spp. and 44% of the Streptococcus spp. also carried multiple tet resistance genes. All Enterococcus spp. carried erm(A) and erm(B), 69% carried erm(F), 44% carried mef(A), 75% carried tet(M), 69% carried tet(L) and 19% carried tet(K). All Streptococcus spp. carried erm(B), 94% carried erm(F), 75% carried erm(A), 38% carried mef(A), 50% carried tet(M), 81% carried tet(L) and 13% carried tet(K). CONCLUSIONS: Multidrug resistance among airborne bacteria recovered from a swine operation is encoded by multiple MLS and tet resistance genes. These are the first data regarding resistance gene carriage among airborne bacteria from swine-feeding operations. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence of multiple resistance genes reported here suggests that airborne Gram-positive bacteria from swine operations may be important contributors to environmental reservoirs of resistance genes.
Asunto(s)
Microbiología del Aire , Contaminación del Aire Interior , Crianza de Animales Domésticos , Farmacorresistencia Bacteriana Múltiple/genética , Enterococcus/efectos de los fármacos , Genes Bacterianos , Streptococcus/efectos de los fármacos , Animales , Enterococcus/genética , Streptococcus/genética , PorcinosAsunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Plásmidos/efectos de los fármacos , Resistencia a la Tetraciclina/genética , Tetraciclina/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Humanos , NigeriaRESUMEN
Of the 176 randomly selected, commensal, gram-negative bacteria isolated from healthy children with low exposure to antibiotics, 138 (78%) carried one or more of the seven macrolide resistance genes tested in this study. These isolates included 79 (91%) isolates from the oral cavity and 59 (66%) isolates from urine samples. The mef(A) gene, coding for an efflux protein, was found in 73 isolates (41%) and was the most frequently carried gene. The mef(A) gene could be transferred from the donors into a gram-positive E. faecalis recipient and a gram-negative Escherichia coli recipient. The erm(B) gene transferred and was maintained in the E. coli transconjugants but was found in 0 to 100% of the E. faecalis transconjugants tested, while the other five genes could be transferred only into the E. coli recipient. The individual macrolide resistance genes were identified in 3 to 12 new genera. Eight (10%) of the oral isolates and 30 (34%) of the urine isolates for which the MICs were 2 to >500 microg of erythromycin per ml did not hybridize with any of the seven genes and may carry novel macrolide resistance genes.
