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1.
Nat Commun ; 10(1): 2763, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235751

RESUMO

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/patogenicidade , Infecções Relacionadas a Cateter/microbiologia , Plasmídeos/genética , Pneumonia Bacteriana/microbiologia , Infecções Urinárias/microbiologia , Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções Relacionadas a Cateter/epidemiologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Pneumonia Bacteriana/epidemiologia , Proteômica , Estudos Retrospectivos , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Cateteres Urinários/efeitos adversos , Cateteres Urinários/microbiologia , Sistema Urinário/microbiologia , Infecções Urinárias/epidemiologia , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
Proc Natl Acad Sci U S A ; 116(4): 1378-1383, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30626645

RESUMO

Acinetobacter baumannii (Ab) is a nosocomial pathogen with one of the highest rates of multidrug resistance (MDR). This is partially due to transmissible plasmids. Many Ab strains harbor a constitutively active type VI secretion system (T6SS) that is employed to kill nonkin bacteria. T6SS and plasmid conjugation both involve cell-to-cell contact. Paradoxically, successful conjugation requires the survival of the recipient, which is the target of the T6SS. Thus, an active T6SS in either the donor or the recipient poses a challenge to plasmid conjugation. Here, we show that large conjugative MDR plasmids heavily rely on their distinctive ability to repress the T6SS of their hosts to enable their own dissemination and the conjugation of other plasmids, contributing to the propagation of MDR among Acinetobacter isolates.


Assuntos
Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/fisiologia , Farmacorresistência Bacteriana Múltipla/fisiologia , Sistemas de Secreção Tipo VI/fisiologia , Infecções por Acinetobacter/microbiologia , Proteínas de Bactérias/metabolismo , Plasmídeos/metabolismo
3.
J Biol Chem ; 292(22): 9075-9087, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28373284

RESUMO

Pathogenic Acinetobacter species, including Acinetobacter baumannii and Acinetobacter nosocomialis, are opportunistic human pathogens of increasing relevance worldwide. Although their mechanisms of drug resistance are well studied, the virulence factors that govern Acinetobacter pathogenesis are incompletely characterized. Here we define the complete secretome of A. nosocomialis strain M2 in minimal medium and demonstrate that pathogenic Acinetobacter species produce both a functional type I secretion system (T1SS) and a contact-dependent inhibition (CDI) system. Using bioinformatics, quantitative proteomics, and mutational analyses, we show that Acinetobacter uses its T1SS for exporting two putative T1SS effectors, an Repeats-in-Toxin (RTX)-serralysin-like toxin, and the biofilm-associated protein (Bap). Moreover, we found that mutation of any component of the T1SS system abrogated type VI secretion activity under nutrient-limited conditions, indicating a previously unrecognized cross-talk between these two systems. We also demonstrate that the Acinetobacter T1SS is required for biofilm formation. Last, we show that both A. nosocomialis and A. baumannii produce functioning CDI systems that mediate growth inhibition of sister cells lacking the cognate immunity protein. The Acinetobacter CDI systems are widely distributed across pathogenic Acinetobacter species, with many A. baumannii isolates harboring two distinct CDI systems. Collectively, these data demonstrate the power of differential, quantitative proteomics approaches to study secreted proteins, define the role of previously uncharacterized protein export systems, and observe cross-talk between secretion systems in the pathobiology of medically relevant Acinetobacter species.


Assuntos
Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Humanos
4.
Cell Microbiol ; 19(2)2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27532510

RESUMO

Several pathogens co-opt host intracellular compartments to survive and replicate, and they thereafter disperse progeny to prosper in a new niche. Little is known about strategies displayed by Serratia marcescens to defeat immune responses and disseminate afterwards. Upon invasion of nonphagocytic cells, Serratia multiplies within autophagosome-like vacuoles. These Serratia-containing vacuoles (SeCV) circumvent progression into acidic/degradative compartments, avoiding elimination. In this work, we show that ShlA pore-forming toxin (PFT) commands Serratia escape from invaded cells. While ShlA-dependent, Ca2+ local increase was shown in SeCVs tight proximity, intracellular Ca2+ sequestration prevented Serratia exit. Accordingly, a Ca2+ surge rescued a ShlA-deficient strain exit capacity, demonstrating that Ca2+ mobilization is essential for egress. As opposed to wild-type-SeCV, the mutant strain-vacuole was wrapped by actin filaments, showing that ShlA expression rearranges host actin. Moreover, alteration of actin polymerization hindered wild-type Serratia escape, while increased intracellular Ca2+ reorganized the mutant strain-SeCV actin distribution, restoring wild-type-SeCV phenotype. Our results demonstrate that, by ShlA expression, Serratia triggers a Ca2+ signal that reshapes cytoskeleton dynamics and ends up pushing the SeCV load out of the cell, in an exocytic-like process. These results disclose that PFTs can be engaged in allowing bacteria to exit without compromising host cell integrity.


Assuntos
Proteínas de Bactérias/metabolismo , Exocitose , Proteínas Hemolisinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Serratia marcescens/fisiologia , Vacúolos/microbiologia , Animais , Células CHO , Cálcio/metabolismo , Sinalização do Cálcio , Cátions Bivalentes/metabolismo , Cricetinae , Cricetulus , Citoesqueleto/metabolismo , Serratia marcescens/metabolismo
5.
Infect Immun ; 82(9): 3542-54, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24914224

RESUMO

Serratia marcescens is a Gram-negative bacterium that thrives in a wide variety of ambient niches and interacts with an ample range of hosts. As an opportunistic human pathogen, it has increased its clinical incidence in recent years, being responsible for life-threatening nosocomial infections. S. marcescens produces numerous exoproteins with toxic effects, including the ShlA pore-forming toxin, which has been catalogued as its most potent cytotoxin. However, the regulatory mechanisms that govern ShlA expression, as well as its action toward the host, have remained unclear. We have shown that S. marcescens elicits an autophagic response in host nonphagocytic cells. In this work, we determine that the expression of ShlA is responsible for the autophagic response that is promoted prior to bacterial internalization in epithelial cells. We show that a strain unable to express ShlA is no longer able to induce this autophagic mechanism, while heterologous expression of ShlA/ShlB suffices to confer on noninvasive Escherichia coli the capacity to trigger autophagy. We also demonstrate that shlBA harbors a binding motif for the RcsB regulator in its promoter region. RcsB-dependent control of shlBA constitutes a feed-forward regulatory mechanism that allows interplay with flagellar-biogenesis regulation. At the top of the circuit, activated RcsB downregulates expression of flagella by binding to the flhDC promoter region, preventing FliA-activated transcription of shlBA. Simultaneously, RcsB interaction within the shlBA promoter represses ShlA expression. This circuit offers multiple access points to fine-tune ShlA production. These findings also strengthen the case for an RcsB role in orchestrating the expression of Serratia virulence factors.


Assuntos
Autofagia/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Proteínas Hemolisinas/genética , Serratia marcescens/genética , Transcrição Genética/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Células Epiteliais/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/genética , Flagelos/metabolismo , Proteínas Hemolisinas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Serratia marcescens/metabolismo
6.
J Bacteriol ; 194(11): 2949-61, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22467788

RESUMO

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Serratia/microbiologia , Serratia marcescens/metabolismo , Serratia marcescens/patogenicidade , Ácidos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , Magnésio/metabolismo , Viabilidade Microbiana , Dados de Sequência Molecular , Alinhamento de Sequência , Serratia marcescens/genética , Serratia marcescens/crescimento & desenvolvimento , Virulência
7.
PLoS One ; 6(8): e24054, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21901159

RESUMO

Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell.


Assuntos
Autofagia , Serratia marcescens/fisiologia , Vacúolos/microbiologia , Cloreto de Amônio/farmacologia , Androstadienos/farmacologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Células Epiteliais/microbiologia , Técnica Indireta de Fluorescência para Anticorpo , Gentamicinas/farmacologia , Humanos , Macrolídeos/farmacologia , Microscopia Confocal , Serratia marcescens/efeitos dos fármacos , Wortmanina
8.
PLoS One ; 5(10): e13501, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20976072

RESUMO

BACKGROUND: Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro. PRINCIPAL FINDINGS: Cos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300-600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels. However, FNR and Fld did not exhibit any protection at shorter incubation times (2 h and 4 h) to 4 mM hydrogen peroxide or to a 48 h exposure to 300 µM methyl viologen. We found enhanced methyl viologen damage exerted by FNR that may be due to depletion of NADPH pools through NADPH-MV diaphorase activity as previously observed for other overexpressed enzymes. SIGNIFICANCE: The results presented are a first report of antioxidant function of these heterologous enzymes of vegetal and cyanobacterial origin in mammalian cells.


Assuntos
Ferredoxina-NADP Redutase/metabolismo , Flavodoxina/metabolismo , Estresse Oxidativo , Animais , Sequência de Bases , Células COS , Cercopithecus aethiops , Primers do DNA , Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transgenes
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