RESUMO
Previous studies have shown that the administration of antibiotics at subinhibitory concentrations stimulates biofilm formation by the majority of multidrug-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated the effect of subinhibitory concentrations of mupirocin on biofilm formation by the community-associated (CA) mupirocin-sensitive MRSA strain USA300 and the highly mupirocin-resistant clinical S. aureus SA01 to SA05 isolates. We found that mupirocin increased the ability of MRSA cells to attach to surfaces and form biofilms. Confocal laser scanning microscopy (CLSM) demonstrated that mupirocin treatment promoted thicker biofilm formation, which also correlated with the production of extracellular DNA (eDNA). Furthermore, quantitative real-time PCR (RT-qPCR) results revealed that this effect was largely due to the involvement of holin-like and antiholin-like proteins (encoded by the cidA gene), which are responsible for modulating cell death and lysis during biofilm development. We found that cidA expression levels significantly increased by 6.05- to 35.52-fold (P < 0.01) after mupirocin administration. We generated a cidA-deficient mutant of the USA300 S. aureus strain. Exposure of the ΔcidA mutant to mupirocin did not result in thicker biofilm formation than that in the parent strain. We therefore hypothesize that the mupirocin-induced stimulation of S. aureus biofilm formation may involve the upregulation of cidA.
Assuntos
Antibacterianos/farmacologia , Mupirocina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Microscopia Confocal , Staphylococcus aureus/genéticaRESUMO
Endosymbiotic Wolbachia bacteria are, to date, considered the most widespread symbionts in arthropods and are the cornerstone of major biological control strategies. Such a high prevalence is based on the ability of Wolbachia to manipulate their hosts' reproduction. One manipulation called cytoplasmic incompatibility (CI) is based on the death of the embryos generated by crosses between infected males and uninfected females or between individuals infected with incompatible Wolbachia strains. CI can be seen as a modification-rescue system (or mod-resc) in which paternal Wolbachia produce mod factors, inducing embryonic defects, unless the maternal Wolbachia produce compatible resc factors. Transgenic experiments in Drosophila melanogaster and Saccharomyces cerevisiae converged towards a model where the cidB Wolbachia gene is involved in the mod function while cidA is involved in the resc function. However, as cidA expression in Drosophila males was required to observe CI, it has been proposed that cidA could be involved in both resc and mod functions. A recent correlative study in natural Culex pipiens mosquito populations has revealed an association between specific cidA and cidB variations and changes in mod phenotype, also suggesting a role for both these genes in mod diversity. Here, by studying cidA and cidB genomic repertoires of individuals from newly sampled natural C. pipiens populations harbouring wPipIV strains from North Italy, we reinforce the link between cidB variation and mod phenotype variation fostering the involvement of cidB in the mod phenotype diversity. However, no association between any cidA variants or combination of cidA variants and mod phenotype variation was observed. Taken together our results in natural C. pipiens populations do not support the involvement of cidA in mod phenotype variation.
Assuntos
Culex/genética , Culex/microbiologia , Citoplasma/genética , Citoplasma/microbiologia , Genes Bacterianos/genética , Wolbachia/genética , Animais , Animais Geneticamente Modificados/genética , Cruzamentos Genéticos , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Feminino , Itália , Masculino , Fenótipo , Reprodução/genética , Simbiose/genéticaRESUMO
Wolbachia-mediated cytoplasmic incompatibility (CI) is a conditional embryonic lethality induced when Wolbachia-modified sperm fertilizes an uninfected egg. The Wolbachia proteins, CidA and CidB control CI. CidA is a rescue factor that reverses lethality. CidA binds to CidB. CidB contains a deubiquitinating enzyme and induces CI. Precisely how CidB induces CI and what it targets are unknown. Likewise, how CidA prevents sterilization by CidB is not clear. To identify CidB substrates in mosquitos we conducted pull-down assays using recombinant CidA and CidB mixed with Aedes aegypti lysates to identify the protein interactomes of CidB and the CidB/CidA protein complex. Our data allow us to cross compare CidB interactomes across taxa for Aedes and Drosophila. Our data replicate several convergent interactions, suggesting that CI targets conserved substrates across insects. Our data support a hypothesis that CidA rescues CI by tethering CidB away from its substrates. Specifically, we identify ten convergent candidate substrates including P32 (protamine-histone exchange factor), karyopherin alpha, ubiquitin-conjugating enzyme, and bicoid stabilizing factor. Future analysis on how these candidates contribute to CI will clarify mechanisms.
Assuntos
Aedes , Wolbachia , Animais , Masculino , Drosophila , Drosophila melanogaster , Sêmen , Citoplasma/metabolismoRESUMO
Wolbachia are widespread endosymbiotic bacteria that manipulate the reproduction of arthropods through a diversity of cellular mechanisms. In cytoplasmic incompatibility (CI), a sterility syndrome originally discovered in the mosquito Culex pipiens, uninfected eggs fertilized by sperm from infected males are selectively killed during embryo development following the abortive segregation of paternal chromosomes in the zygote. Despite the recent discovery of Wolbachia CI factor (cif) genes, the mechanism by which they control the fate of paternal chromosomes at fertilization remains unknown. Here, we have analyzed the cytological distribution and cellular impact of CidA and CidB, a pair of Cif proteins from the Culex-infecting Wolbachia strain wPip. We show that expression of CidB in Drosophila S2R+ cells induces apoptosis unless CidA is co-expressed and associated with its partner. In transgenic Drosophila testes, both effectors colocalize in germ cells until the histone-to-protamine transition in which only CidB is retained in maturing spermatid nuclei. We further show that CidB is similarly targeted to maturing sperm of naturally infected Culex mosquitoes. At fertilization, CidB associates with paternal DNA regions exhibiting DNA replication stress, as a likely cause of incomplete replication of paternal chromosomes at the onset of the first mitosis. Importantly, we demonstrate that inactivation of the deubiquitylase activity of CidB does not abolish its cell toxicity or its ability to induce CI in Drosophila. Our study thus demonstrates that CI functions as a transgenerational toxin-antidote system and suggests that CidB acts by poisoning paternal DNA replication in incompatible crosses.
Assuntos
Culex , Wolbachia , Animais , Culex/genética , Citoplasma , Citosol , Drosophila , Masculino , Wolbachia/genéticaRESUMO
Extracellular DNA (eDNA) is an important component of the extracellular polymeric substance matrix and is important in the establishment and persistence of Staphylococcus aureus UAMS-1 biofilms. The aim of the study was to determine the temporal expression of genes involved in early biofilm formation and eDNA production. We used qPCR to investigate expression of agrB, which is associated with secreted virulence factors and biofilm dispersal, cidA, which is associated with biofilm adherence and genomic DNA release, and alsS, which is associated with cell lysis, eDNA release and acid tolerance. The contribution of eDNA to the stability of the biofilm matrix was assessed by digesting with DNase I (Pulmozyme) and quantifying structure by confocal microscopy and comstat image analysis. AgrB expression initially increased at 24 h but then dramatically decreased at 72 h in an inverse relationship to biomass, supporting its role in regulating biofilm dispersal. cidA and alsS expression steadily increased over 72 h, suggesting that eDNA was an important component of early biofilm development. DNase I had no effect on biomass, but did cause the biofilms to become more heterogeneous. Carbohydrates in the matrix appeared to play an important role in structural stability.
Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Carboidratos , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/fisiologia , Desoxirribonuclease I/metabolismo , Espaço Extracelular/metabolismo , Humanos , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Objective To investigate the relationships between the expressions of cidA and lrgA and the biofilm formation of Staphylococcus epidermidis clinical isolates. Methods Thirty-nine S. epidermidis clinical isolates were divided into biofilm formation group and non-biofilm formation group according to adhesion assays and icaA amplification, 20 strains for biofilm formation group and 19 strains for another group respectively. Amplify cidA and lrgA fragment with PCR, detect mRNA levels of cidA and lrgA with RT-PCR and then calculate the ratios of cid/lrg mRNA. Results All of the 39 isolates of S. epidermidis existed cidA and lrgA gene.Select 2 strains of S. epidermidis, Y36 and Y26, at random for detecting the cidA and lrgA mRNA levels of different phases, the highest level of cidA mRNA shows on 4 h's culture while lrgA shows on6 h. The relative expression levels of cidA of 39 isolates of S. epidermidis were 0.340 + 0.250 in biofilm formation group and 0. 406 +0. 408( P =0. 541 ) in non-biofilm formation group for 4 h culture, while the relative expression levels of lrgA were 0.325 ± 0.218 and 0.253 ± 0.211 respectively ( P = 0. 299). There were no significant differences between these two groups. The ratios of cid/lrg mRNA were 1.067 ±0.529 and 1.958 ±1.877 respectively, the difference of population distribution was significant( P = 0.001 ). Conclusion The expressions of cidA and lrgA of S. epidermidis clinical isolates may be time-limited. There were no significant differeces of expressions of cidA and lrgA between biofilm formation group and non-biofilm formation group. The ratios of cid/lrg mRNA maybe have some biological significance.