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1.
PLoS Genet ; 20(6): e1011317, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38843312

RESUMO

Evaluation of the apportionment of genetic diversity of human bacterial commensals within and between human populations is an important step in the characterization of their evolutionary potential. Recent studies showed a correlation between the genomic diversity of human commensal strains and that of their host, but the strength of this correlation and of the geographic structure among human populations is a matter of debate. Here, we studied the genomic diversity and evolution of the phylogenetically related oro-nasopharyngeal healthy-carriage Streptococcus mitis and Streptococcus pneumoniae, whose lifestyles range from stricter commensalism to high pathogenic potential. A total of 119 S. mitis genomes showed higher within- and among-host variation than 810 S. pneumoniae genomes in European, East Asian and African populations. Summary statistics of the site-frequency spectrum for synonymous and non-synonymous variation and ABC modelling showed this difference to be due to higher ancestral bacterial population effective size (Ne) in S. mitis, whose genomic variation has been maintained close to mutation-drift equilibrium across (at least many) generations, whereas S. pneumoniae has been expanding from a smaller ancestral bacterial population. Strikingly, both species show limited differentiation among human populations. As genetic differentiation is inversely proportional to the product of effective population size and migration rate (Nem), we argue that large Ne have led to similar differentiation patterns, even if m is very low for S. mitis. We conclude that more diversity within than among human populations and limited population differentiation must be common features of the human microbiome due to large Ne.


Assuntos
Evolução Molecular , Variação Genética , Genoma Bacteriano , Streptococcus mitis , Streptococcus pneumoniae , Streptococcus mitis/genética , Humanos , Streptococcus pneumoniae/genética , Filogenia , Genética Populacional
2.
Nucleic Acids Res ; 51(19): 10375-10394, 2023 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-37757859

RESUMO

Despite enabling Streptococcus pneumoniae to acquire antibiotic resistance and evade vaccine-induced immunity, transformation occurs at variable rates across pneumococci. Phase variants of isolate RMV7, distinguished by altered methylation patterns driven by the translocating variable restriction-modification (tvr) locus, differed significantly in their transformation efficiencies and biofilm thicknesses. These differences were replicated when the corresponding tvr alleles were introduced into an RMV7 derivative lacking the locus. RNA-seq identified differential expression of the type 1 pilus, causing the variation in biofilm formation, and inhibition of competence induction in the less transformable variant, RMV7domi. This was partly attributable to RMV7domi's lower expression of ManLMN, which promoted competence induction through importing N-acetylglucosamine. This effect was potentiated by analogues of some proteobacterial competence regulatory machinery. Additionally, one of RMV7domi's phage-related chromosomal island was relatively active, which inhibited transformation by increasing expression of the stress response proteins ClpP and HrcA. However, HrcA increased competence induction in the other variant, with its effects depending on Ca2+ supplementation and heat shock. Hence the heterogeneity in transformation efficiency likely reflects the diverse signalling pathways by which it is affected. This regulatory complexity will modulate population-wide responses to synchronising quorum sensing signals to produce co-ordinated yet stochastic bet hedging behaviour.


Assuntos
Proteínas de Bactérias , Streptococcus pneumoniae , Proteínas de Bactérias/metabolismo , Biofilmes , Proteínas de Choque Térmico/metabolismo , Percepção de Quorum , Streptococcus pneumoniae/metabolismo
3.
Microbiology (Reading) ; 168(12)2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36748691

RESUMO

Streptococcus pneumoniae may inhabit the upper respiratory tract of humans without causing harm but it also causes diseases with high morbidity and mortality. It has excellent adaptive capabilities thanks to its ability to shuffle its genetic content by acquiring and incorporating DNA from other bacteria and is highly competent for genetic transformation. Sugar sensing, cleavage and transport ensure its fitness and survival in the host, and intracellular survival in macrophages has been linked to virulence. The polysaccharide capsule and toxin pneumolysin are the most important virulence determinants. Polysaccharide-based vaccines provide protection against the serotypes represented in vaccine formulations.


Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Humanos , Streptococcus pneumoniae/genética , Infecções Pneumocócicas/microbiologia , Estresse Financeiro , Fatores de Virulência , Virulência
4.
Nucleic Acids Res ; 48(20): 11468-11485, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33119758

RESUMO

Type I restriction-modification (R-M) systems consist of a DNA endonuclease (HsdR, HsdM and HsdS subunits) and methyltransferase (HsdM and HsdS subunits). The hsdS sequences flanked by inverted repeats (referred to as epigenetic invertons) in certain Type I R-M systems undergo invertase-catalyzed inversions. Previous studies in Streptococcus pneumoniae have shown that hsdS inversions within clonal populations produce subpopulations with profound differences in the methylome, cellular physiology and virulence. In this study, we bioinformatically identified six major clades of the tyrosine and serine family invertases homologs from 16 bacterial phyla, which potentially catalyze hsdS inversions in the epigenetic invertons. In particular, the epigenetic invertons are highly enriched in host-associated bacteria. We further verified hsdS inversions in the Type I R-M systems of four representative host-associated bacteria and found that each of the resultant hsdS allelic variants specifies methylation of a unique DNA sequence. In addition, transcriptome analysis revealed that hsdS allelic variations in Enterococcus faecalis exert significant impact on gene expression. These findings indicate that epigenetic switches driven by invertases in the epigenetic invertons broadly operate in the host-associated bacteria, which may broadly contribute to bacterial host adaptation and virulence beyond the role of the Type I R-M systems against phage infection.


Assuntos
Proteínas de Bactérias/genética , Enzimas de Restrição-Modificação do DNA/genética , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Bacteroides fragilis/genética , Metilação de DNA , DNA Bacteriano/química , Enterococcus faecalis/genética , Sequências Repetidas Invertidas , Streptococcus agalactiae/genética , Treponema denticola/genética
5.
Mol Microbiol ; 113(3): 672-681, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32185830

RESUMO

Bottlenecks reduce the size of the gene pool within populations of all life forms with implications for their subsequent survival. Here, we examine the effects of bottlenecks on bacterial commensal-pathogens during transmission between, and dissemination within, hosts. By reducing genetic diversity, bottlenecks may alter individual or population-wide adaptive potential. A diverse range of hypermutable mechanisms have evolved in infectious agents that allow for rapid generation of genetic diversity in specific genomic loci as opposed to the variability arising from increased genome-wide mutation rates. These localised hypermutable mechanisms include multi-gene phase variation (PV) of outer membrane components, multi-allele PV of restriction systems and recombination-driven antigenic variation. We review selected experimental and theoretical (mathematical) models pertaining to the hypothesis that localised hypermutation (LH) compensates for fitness losses caused by bottlenecks and discuss whether bottlenecks have driven the evolution of hypermutable loci.


Assuntos
Bactérias/genética , Variação Genética/genética , Interações Hospedeiro-Patógeno/genética , Variação Antigênica , Evolução Biológica , Evolução Molecular , Pool Gênico , Modelos Genéticos , Mutação , Recombinação Genética/genética , Seleção Genética/genética
6.
Brain Behav Immun ; 93: 299-311, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33444732

RESUMO

C1 esterase inhibitor (C1INH) is known to exert its inhibitory effect by binding to several target proteases of the contact and complement systems. One of C1INH's targets comprise mannose-binding lectin (MBL), a critical player in post-stroke pathophysiology. We therefore explored the effects of recombinant human (rh) and plasma derived (pd) C1INH in C57BL/6J mice subjected to transient occlusion of the middle cerebral artery (tMCAo), receiving 15U/mouse of pd or rhC1INH intravenously, at reperfusion. We analyzed the compounds' (i)neuroprotective effects, (ii) plasma presence, (iii)effects on circulating and brain MBL, (iv)time course of endothelial deposition, and (v) effects on the formation of active complement products. rhC1INH-treated mice had neuroprotective effects, including reduced behavioral deficits and neuronal loss, associated with decreased MBL brain deposition and decreased formation of complement C4b active fragments. In contrast, pdC1INH did not show these neuroprotective effects despite its longer plasma residence time. We also analyzed the response to tMCAo in C1INH-deficient mice, observing a poorer ischemic outcome compared to the wild type mice, which could be partially prevented by rhC1INH administration. In conclusion, we show that rhC1INH exhibits stronger neuroprotective effects than the corresponding plasma-derived protein after experimental ischemia/reperfusion injury in the brain, placing it as a promising drug for stroke. Differential effects are likely related to more effective MBL inhibition which further confirms it as a useful pharmacological target for stroke.


Assuntos
Preparações Farmacêuticas , Traumatismo por Reperfusão , Animais , Encéfalo/metabolismo , Proteína Inibidora do Complemento C1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle
7.
Am J Respir Crit Care Med ; 201(3): 335-347, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31626559

RESUMO

Rationale: Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with Streptococcus pneumoniae (Spn), although a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited.Objectives: Using a controlled human infection model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells.Methods: We collected BAL from healthy pneumococcal-challenged participants aged 18-49 years. Confocal microscopy and molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays in vitro, whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations.Measurements and Main Results: AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized individuals were positively correlated with nasal pneumococcal density (r = 0.71; P = 0.029). Similarly, AM-heightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density (r = 0.61, P = 0.025).Conclusions: Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines.


Assuntos
Macrófagos Alveolares/imunologia , Nasofaringe/microbiologia , Nariz/microbiologia , Streptococcus pneumoniae/isolamento & purificação , Adolescente , Adulto , Bactérias/imunologia , Humanos , Pessoa de Meia-Idade , Aspiração Respiratória , Adulto Jovem
8.
Thorax ; 75(1): 8-16, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31699806

RESUMO

BACKGROUND: Antibiotic resistance is a major global threat. We hypothesised that the chronic obstructive pulmonary disease (COPD) airway is a reservoir of antimicrobial resistance genes (ARGs) that associate with microbiome-specific COPD subgroups. OBJECTIVE: To determine the resistance gene profiles in respiratory samples from COPD patients and healthy volunteers. METHODS: Quantitative PCR targeting 279 specific ARGs was used to profile the resistomes in sputum from subjects with COPD at stable, exacerbation and recovery visits (n=55; COPD-BEAT study), healthy controls with (n=7) or without (n=22) exposure to antibiotics in the preceding 12 months (EXCEED study) and in bronchial brush samples from COPD (n=8) and healthy controls (n=7) (EvA study). RESULTS: ARG mean (SEM) prevalence was greater in stable COPD samples (35.2 (1.6)) than in healthy controls (27.6 (1.7); p=0.004) and correlated with total bacterial abundance (r2=0.23; p<0.001). Prevalence of ARG positive signals in individuals was not related to COPD symptoms, lung function or their changes at exacerbation. In the COPD subgroups designated High γProteobacteria and High Firmicutes, ARG prevalence was not different at stable state but significantly declined from stable through exacerbation to recovery in the former (p=0.011) without changes in total bacterial abundance. The ARG patterns were similar in COPD versus health, COPD microbiome-subgroups and between sputum and bronchoscopic samples independent of antibiotic exposure in the last 12 months. CONCLUSIONS: ARGs are highly prevalent in sputum, broadly in proportion to bacterial abundance in both healthy and COPD subjects. Thus, COPD appears to be an ARG reservoir due to high levels of bacterial colonisation.


Assuntos
Farmacorresistência Bacteriana/genética , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Escarro/microbiologia , Idoso , Carga Bacteriana , Feminino , Genes Bacterianos , Humanos , Masculino , Microbiota , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
9.
Environ Microbiol ; 22(12): 5058-5072, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32483914

RESUMO

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.


Assuntos
Bacteriófagos/fisiologia , Evolução Molecular , Fluxo Gênico , Listeria monocytogenes/genética , Transferência Genética Horizontal , Variação Genética , Genoma Bacteriano/genética , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/virologia , Listeriose/epidemiologia , Listeriose/microbiologia , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Recombinação Genética
10.
Mol Cell Probes ; 54: 101671, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33160071

RESUMO

Traumatic brain injury (TBI) is the leading cause of death in the global population. Disturbed inflammatory processes after TBI exacerbate secondary brain injury and contribute to unfavorable outcomes. Multiple inflammatory events that accompany brain trauma, such as glial activation, chemokine release, or the initiation of the complement system cascade, have been identified as potential targets for TBI treatment. However, the participation of chemokines in the complement activation remains unknown. Our studies sought to determine the changes in the expression of the molecules involved in the CCL2/CCL7/CCL12/CCR2 pathway in the injured brain and the effect of CCL2, CCL7, and CCL12 (10, 100, and 500 ng/mL) on the classic and lectin complement pathways and inflammatory factors in microglial cell cultures. Brain injury in mice was modeled by controlled cortical impact (CCI). Our findings indicate a time-dependent upregulation of CCL2, CCL7, and CCL12 at the mRNA and protein levels within the cortex, striatum, and/or thalamus beginning 24 h after the trauma. The analysis of the expression of the receptor of the tested chemokines, CCR2, revealed its substantial upregulation within the injured brain areas mainly on the mRNA level. Using primary cortical microglial cell cultures, we observed a substantial increase in the expression of CCL2, CCL7, and CCL12 after 24 h of LPS (100 ng/mL) treatment. CCL2 stimulation of microglia increased the level of IL-1ß mRNA but did not influence the expression of IL-18, IL-6, and IL-10. Moreover, CCL2 significantly increased the expression of Iba1, a marker of microglia activation. CCL2 and CCL12 upregulated the expression of C1qa but did not influence the expression of C1ra and C1s1 (classical pathway); moreover, CCL2 increased ficolin A expression and reduced collectin 11 expression (lectin pathway). Additionally, we observed the downregulation of pentraxin 3, a modulator of the complement cascade, after CCL2 and CCL12 treatment. We did not detect the expression of ficolin B, Mbl1, and Mbl2 in microglial cells. Our data identify CCL2 as a modulator of the classical and lectin complement pathways suggesting that CCL2 may be a promising target for pharmacological intervention after brain injury. Moreover, our study provides evidence that CCL2 and two other CCR2 ligands may play a role in the development of changes in TBI.


Assuntos
Lesões Encefálicas Traumáticas/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Proteínas do Sistema Complemento/metabolismo , Microglia/metabolismo , Proteínas Quimioatraentes de Monócitos/metabolismo , Receptores CCR2/metabolismo , Regulação para Cima , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL7/genética , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR2/genética , Transdução de Sinais , Fatores de Tempo
11.
BMC Infect Dis ; 20(1): 505, 2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32660552

RESUMO

BACKGROUND: Meningococcal meningitis (MM) is a life-threatening disease associated with approximately 10% case fatality rates and neurological sequelae in 10-20% of the cases. Recently, we have shown that the matrix metalloproteinase (MMP) inhibitor BB-94 reduced brain injury in a mouse model of MM. The present study aimed to assess whether doxycycline (DOX), a tetracycline that showed a neuroprotective effect as adjuvant therapy in experimental pneumococcal meningitis (PM), would also exert a beneficial effect when given as adjunctive therapy to ceftriaxone (CRO) in experimental MM. METHODS: BALB/c mice were infected by the intracisternal route with a group C Neisseria meningitidis strain. Eighteen h post infection (hpi), animals were randomised for treatment with CRO [100 mg/kg subcutaneously (s.c.)], CRO plus DOX (30 mg/kg s.c.) or saline (control s.c.). Antibiotic treatment was repeated 24 and 40 hpi. Mouse survival and clinical signs, bacterial counts in cerebella, brain damage, MMP-9 and cyto/chemokine levels were assessed 48 hpi. RESULTS: Analysis of bacterial load in cerebella indicated that CRO and CRO + DOX were equally effective at controlling meningococcal replication. No differences in survival were observed between mice treated with CRO (94.4%) or CRO + DOX (95.5%), (p > 0.05). Treatment with CRO + DOX significantly diminished both the number of cerebral hemorrhages (p = 0.029) and the amount of MMP-9 in the brain (p = 0.046) compared to untreated controls, but not to CRO-treated animals (p > 0.05). Levels of inflammatory markers in the brain of mice that received CRO or CRO + DOX were not significantly different (p > 0.05). Overall, there were no significant differences in the parameters assessed between the groups treated with CRO alone or CRO + DOX. CONCLUSIONS: Treatment with CRO + DOX showed similar bactericidal activity to CRO in vivo, suggesting no antagonist effect of DOX on CRO. Combined therapy significantly improved mouse survival and disease severity compared to untreated animals, but addition of DOX to CRO did not offer significant benefits over CRO monotherapy. In contrast to experimental PM, DOX has no adjunctive activity in experimental MM.


Assuntos
Antibacterianos/uso terapêutico , Ceftriaxona/uso terapêutico , Doxiciclina/uso terapêutico , Meningite Meningocócica/tratamento farmacológico , Neisseria meningitidis Sorogrupo C , Animais , Antibacterianos/administração & dosagem , Carga Bacteriana/efeitos dos fármacos , Ceftriaxona/administração & dosagem , Hemorragia Cerebral/tratamento farmacológico , Quimiocinas/análise , Quimiocinas/metabolismo , Modelos Animais de Doenças , Doxiciclina/administração & dosagem , Quimioterapia Combinada , Feminino , Humanos , Estimativa de Kaplan-Meier , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/metabolismo , Meningite Meningocócica/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Resultado do Tratamento
12.
Nucleic Acids Res ; 46(21): 11438-11453, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30321375

RESUMO

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.


Assuntos
Epigênese Genética , Genoma Bacteriano , Ilhas Genômicas , Streptococcus pneumoniae/genética , Motivos de Aminoácidos , Antitoxinas/genética , Proteínas de Bactérias/genética , Metilação de DNA , Enzimas de Restrição-Modificação do DNA/genética , DNA Bacteriano/genética , Escherichia coli , Mutação , RNA Bacteriano/genética
13.
Int J Mol Sci ; 22(1)2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33375205

RESUMO

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.


Assuntos
Lesões Encefálicas Traumáticas/genética , Ativação do Complemento/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Lectinas/genética , Animais , Lesões Encefálicas Traumáticas/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Complemento C1/genética , Complemento C1/metabolismo , Complemento C1q/genética , Complemento C1q/metabolismo , Complemento C1r/genética , Complemento C1r/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Hipocampo/metabolismo , Humanos , Lectinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Neostriado/metabolismo , Tálamo/metabolismo , Fatores de Tempo
14.
J Bacteriol ; 201(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31285240

RESUMO

Virus-host interactions are regulated by complex coevolutionary dynamics. In Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of the nrdR nucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogen S. pneumoniaeIMPORTANCE With antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogen Streptococcus pneumoniae and explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.


Assuntos
Proteínas de Bactérias/genética , Bacteriófagos/fisiologia , Metilação de DNA , Streptococcus pneumoniae/virologia , Proteínas Virais/genética , Bacteriófagos/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Lisogenia , Boca/microbiologia , Análise de Sequência de RNA , Streptococcus pneumoniae/genética
15.
BMC Genomics ; 17(1): 1020, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27964731

RESUMO

BACKGROUND: How the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome). RESULTS: Chromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM)  and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for m4C methylation, but has one additional spacer  when compared to the clinical isolate M120. CONCLUSIONS: These findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile.


Assuntos
Clostridioides difficile/classificação , Clostridioides difficile/genética , Microbiologia Ambiental , Genoma Bacteriano , Genômica , Ribotipagem , Análise de Sequência de DNA , Sistemas CRISPR-Cas , Clostridioides difficile/isolamento & purificação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Biologia Computacional/métodos , Metilação de DNA , Elementos de DNA Transponíveis , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
16.
BMC Genomics ; 17: 491, 2016 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-27411385

RESUMO

BACKGROUND: Biocides and antibiotics are used to eradicate or prevent the growth of microbial species on surfaces (occasionally on catheters), or infected sites, either in combination or sequentially, raising concerns about the development of co-resistance to both antimicrobial types. The effect of such compounds on Salmonella enterica, a major food-borne and zoonotic pathogen, has been analysed in different studies, but only few works evaluated its biological cost, and the overall effects at the genomic and transcriptomic levels associated with diverse phenotypes resulting from biocide exposure, which was the aim of this work. RESULTS: Exposure to triclosan, clorhexidine, benzalkonium, (but not to hypochlorite) resulted in mutants with different phenotypes to a wide range of antimicrobials even unrelated to the selective agent. Most biocide-resistant mutants showed increased susceptibility to compounds acting on the cell wall (ß-lactams) or the cell membranes (poly-L-lysine, polymyxin B, colistin or toxic anions). Mutations (SNPs) were found in three intergenic regions and nine genes, which have a role in energy production, amino acids, carbohydrates or lipids metabolism, some of them involved in membrane transport and pathogenicity. Comparative transcriptomics of biocide-resistant mutants showed over-expression of genes encoding efflux pumps (sugE), ribosomal and transcription-related proteins, cold-shock response (cpeE) and enzymes of microaerobic metabolism including those of the phosphotransferase system. Mainly ribosomal, metabolic and pathogenicity-related genes had affected expression in both in vitro-selected biocide mutants and field Salmonella isolates with reduced biocide susceptibility. CONCLUSIONS: Multiple pathways can be involved in the adaptation of Salmonella to biocides, mainly related with global stress, or involving metabolic and membrane alterations, and eventually causing "collateral sensitivity" to other antimicrobials. These changes might impact the bacterial-environment interaction, imposing significant bacterial fitness costs which may reduce the chances of fixation and spread of biocide resistant mutants.


Assuntos
Adaptação Biológica , Antibacterianos/farmacologia , Desinfetantes/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Análise por Conglomerados , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Genes Bacterianos , Aptidão Genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Testes de Sensibilidade Microbiana , Mutação
17.
PLoS Pathog ; 10(3): e1004026, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24651834

RESUMO

The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.


Assuntos
Bacteriemia/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/patogenicidade , Animais , Bacteriemia/genética , Bacteriemia/imunologia , Feminino , Citometria de Fluxo , Técnicas de Inativação de Genes , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Virulência
18.
BMC Genomics ; 16: 345, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25924916

RESUMO

BACKGROUND: The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. RESULTS: Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. CONCLUSIONS: In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.


Assuntos
Anti-Infecciosos Locais/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Staphylococcus aureus/efeitos dos fármacos , Triclosan/farmacologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação para Baixo/efeitos dos fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Genótipo , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Regulação para Cima/efeitos dos fármacos
19.
Antimicrob Agents Chemother ; 59(6): 3413-23, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25824225

RESUMO

Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRI(r)) and triclosan-hypersusceptible (TRI(hs)) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRI(r) mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRI(r) mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRI(r) mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRI(r) mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRI(r) mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits.


Assuntos
Antibacterianos/farmacologia , Desinfetantes/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/metabolismo , Triclosan/farmacologia , Proteínas de Bactérias/metabolismo , Compostos de Benzalcônio/farmacologia , Clorexidina/farmacologia , Escherichia coli/genética , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Hipoclorito de Sódio/farmacologia
20.
Infect Immun ; 82(9): 3790-801, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24958712

RESUMO

The polysaccharide capsule surrounding Streptococcus pneumoniae is essential for virulence. Recently, Streptococcus mitis, a human commensal and a close relative of S. pneumoniae, was also shown to have a capsule. In this study, the S. mitis type strain switched capsule by acquisition of the serotype 4 capsule locus of S. pneumoniae TIGR4, following induction of competence for natural transformation. Comparison of the wild type with the capsule-switching mutant and with a capsule deletion mutant showed that the capsule protected S. mitis against phagocytosis by RAW 264.7 macrophages. This effect was enhanced in the S. mitis strain expressing the S. pneumoniae capsule, which showed, in addition, increased resistance against early clearance in a mouse model of lung infection. Expression of both capsules also favored survival in human blood, and the effect was again more pronounced for the capsule-switching mutant. S. mitis survival in horse blood or in a mouse model of bacteremia was not significantly different between the wild type and the mutant strains. In all models, S. pneumoniae TIGR4 showed higher rates of survival than the S. mitis type strain or the capsule-switching mutant, except in the lung model, in which significant differences between S. pneumoniae TIGR4 and the capsule-switching mutant were not observed. Thus, we identified conditions that showed a protective function for the capsule in S. mitis. Under such conditions, S. mitis resistance to clearance could be enhanced by capsule switching to serotype 4, but it was enhanced to levels lower than those for the virulent strain S. pneumoniae TIGR4.


Assuntos
Cápsulas Bacterianas/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus mitis/imunologia , Animais , Bacteriemia/imunologia , Bacteriemia/microbiologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Cavalos/imunologia , Cavalos/microbiologia , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Sorotipagem , Infecções Estreptocócicas/microbiologia , Streptococcus pneumoniae/imunologia , Virulência/imunologia
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