RESUMO
The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.
Assuntos
Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Fosfolipases A2/biossíntese , Rickettsia typhi/enzimologia , Rickettsia typhi/patogenicidade , Tifo Endêmico Transmitido por Pulgas/enzimologia , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Domínio Catalítico , Chlorocebus aethiops , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Mutagênese Sítio-Dirigida , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/genética , Rickettsia typhi/genética , Tifo Endêmico Transmitido por Pulgas/genética , Tifo Endêmico Transmitido por Pulgas/microbiologia , Tifo Endêmico Transmitido por Pulgas/patologia , Células VeroRESUMO
Management of chronic wound has an immense impact on social and economic conditions in the world. Healthcare costs, aging population, physical trauma, and comorbidities of diabetes and obesity seem to be the major factors of this increasing incidence of chronic wounds. Conditions of chronic wound could not restore functional epidermis; thus, delaying the closure of the wound opening in an expected manner. Failures in restoration of skin integrity delay healing due to changes in skin pathology, such as chronic ulceration or nonhealing. The role of different traditional medicines has been explored for use in the healing of cutaneous wounds, where several phytochemicals, such as flavonoids, alkaloids, phenolic acids, tannins are known to provide potential wound healing properties. However, the delivery of plant-based therapeutics could be improved by the novel platform of nanotechnology. Thus, the objectives of novel delivery strategies of principal bioactive from plant sources are to accelerate the wound healing process, avoid wound complications and enhance patient compliance. Therefore, the opportunities of nanotechnology-based drug delivery of natural wound healing therapeutics have been included in the present discussion with special emphasis on nanofibers, vesicular structures, nanoparticles, nanoemulsion, and nanogels.
Assuntos
Nanofibras , Cicatrização , Idoso , Sistemas de Liberação de Medicamentos , Humanos , Nanotecnologia , Pele/patologiaRESUMO
Streptococcus pyogenes expresses the LPXTG motif-containing cell envelope serine protease SpyCep (also called ScpC, PrtS) that degrades and inactivates the major chemoattractant interleukin 8 (IL-8), thereby impairing host neutrophil recruitment. In this study, we identified a novel function of SpyCep: the ability to mediate uptake into primary human endothelial cells. SpyCep triggered its uptake into endothelial cells but not into human epithelial cells originating from pharynx or lung, indicating an endothelial cell-specific uptake mechanism. SpyCep mediated cellular invasion by an endosomal/lysosomal pathway distinct from the caveolae-mediated invasion pathway of S. pyogenes. Recombinant expression and purification of proteolytically active SpyCep and a series of subfragments allowed functional dissection of the domains responsible for endothelial cell invasion and IL-8 degradation. The N-terminal PR domain was sufficient to mediate endothelial cell invasion, whereas for IL-8-degrading activity, the protease domain and the flanking A domain were required. A polyclonal rabbit serum raised against the recombinant protease efficiently blocked the invasion-mediating activity of SpyCep but not its proteolytic function, further indicating that SpyCep-mediated internalization is independent from its enzymatic activity. SpyCep may thus specifically mediate its own uptake as secreted protein into human endothelial cells.
Assuntos
Células Endoteliais/metabolismo , Interleucina-8/metabolismo , Peptídeo Hidrolases/metabolismo , Streptococcus pyogenes/enzimologia , Animais , Anticorpos/imunologia , Linhagem Celular , Clonagem Molecular , Endocitose , Endossomos/metabolismo , Células Endoteliais/citologia , Humanos , Lisossomos/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/imunologia , Estrutura Terciária de Proteína , Transporte Proteico , Streptococcus pyogenes/genéticaRESUMO
Strains of Rickettsia rickettsii, the tick-borne agent of Rocky Mountain spotted fever, vary considerably in virulence. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia ankyrin repeat protein 2 (RARP-2). Homologs of RARP-2 are present in all strains of R. rickettsii, but the protein in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system-dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith colocalizes with multilamellar membranous structures bearing markers of the endoplasmic reticulum (ER), whereas the Iowa protein shows no colocalization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith RARP-2 in R. rickettsii Iowa converts this avirulent strain's typically nonlytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith RARP-2 did not, however, restore the virulence of the Iowa strain in a guinea pig model, likely due to the multifactorial nature of rickettsial virulence.IMPORTANCE Members of the genus Rickettsia are obligate intracellular bacteria that exhibit a range of virulence from harmless endosymbionts of arthropods to the etiologic agents of severe disease. Despite the growing number of available genomes, little is known regarding virulence determinants of rickettsiae. Here, we have characterized an ankyrin repeat-containing protein, RARP-2, which differs between a highly virulent and an avirulent strain of R. rickettsii, the agent of Rocky Mountain spotted fever. RARP-2 is secreted by a type IV secretion system into the cytosol of the host cell, where it interacts with and manipulates the structure of the endoplasmic reticulum. RARP-2 from the avirulent strain is truncated by the loss of seven of 10 ankyrin repeat units but, although secreted, fails to alter ER structure. Recognition of those rickettsial factors associated with virulence will facilitate understanding of regional and strain-specific variation in severity of disease.