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1.
PLoS Pathog ; 20(10): e1012651, 2024 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-39423229

RESUMO

Leukotriene B4 (LTB4) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB4 and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB4 synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB4 in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB4 synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB4 production by neutrophils. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB4 in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB4 synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB4 synthesis and colonize the host.

2.
PLoS Pathog ; 20(1): e1011280, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38271464

RESUMO

Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.


Assuntos
Peste , Yersinia pestis , Humanos , Animais , Camundongos , Yersinia pestis/metabolismo , Peste/microbiologia , Sistemas de Secreção Tipo III/metabolismo , Leucotrieno B4/metabolismo , Leucócitos/metabolismo , Inflamação , Proteínas de Bactérias/metabolismo
3.
EMBO Rep ; 24(10): e57369, 2023 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-37501563

RESUMO

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn-seq (dTn-seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans-complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn-seq to identify genes with secreted functions. We then applied dTn-seq to identify an AcrAB efflux system as required for growth in metal-limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over 30 years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition.


Assuntos
Peste , Yersinia pestis , Humanos , Yersinia pestis/genética , Peste/genética , Peste/microbiologia , Fenóis , Tiazóis/farmacologia , Metais , Proteínas de Bactérias/genética
4.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34716262

RESUMO

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.


Assuntos
Fenóis/farmacologia , Peste/metabolismo , Tiazóis/farmacologia , Zinco/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Feminino , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Ferro/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Fenóis/metabolismo , Peste/microbiologia , Sideróforos/metabolismo , Tiazóis/metabolismo , Virulência , Fatores de Virulência/metabolismo , Yersinia pestis/patogenicidade
5.
J Biol Chem ; 298(3): 101651, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35101443

RESUMO

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.


Assuntos
Proteínas de Bactérias , Corantes Fluorescentes , Bactérias Gram-Negativas Quimiolitotróficas , Sideróforos , Acinetobacter baumannii , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Caulobacter crescentus , Enterobactina/análise , Enterobactina/metabolismo , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Bactérias Gram-Negativas Quimiolitotróficas/química , Bactérias Gram-Negativas Quimiolitotróficas/genética , Bactérias Gram-Negativas Quimiolitotróficas/metabolismo , Humanos , Ferro/metabolismo , Klebsiella pneumoniae , Sideróforos/análise , Sideróforos/metabolismo
6.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31871100

RESUMO

Yersinia pestis causes a rapid, lethal disease referred to as plague. Y. pestis actively inhibits the innate immune system to generate a noninflammatory environment during early stages of infection to promote colonization. The ability of Y. pestis to create this early noninflammatory environment is in part due to the action of seven Yop effector proteins that are directly injected into host cells via a type 3 secretion system (T3SS). While each Yop effector interacts with specific host proteins to inhibit their function, several Yop effectors either target the same host protein or inhibit converging signaling pathways, leading to functional redundancy. Previous work established that Y. pestis uses the T3SS to inhibit neutrophil respiratory burst, phagocytosis, and release of inflammatory cytokines. Here, we show that Y. pestis also inhibits release of granules in a T3SS-dependent manner. Moreover, using a gain-of-function approach, we discovered previously hidden contributions of YpkA and YopJ to inhibition and that cooperative actions by multiple Yop effectors are required to effectively inhibit degranulation. Independent from degranulation, we also show that multiple Yop effectors can inhibit synthesis of leukotriene B4 (LTB4), a potent lipid mediator released by neutrophils early during infection to promote inflammation. Together, inhibition of these two arms of the neutrophil response likely contributes to the noninflammatory environment needed for Y. pestis colonization and proliferation.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Neutrófilos/fisiologia , Fatores de Virulência/metabolismo , Yersinia pestis/patogenicidade , Proteínas de Bactérias/genética , Degranulação Celular , Mutação com Ganho de Função , Humanos , Leucotrieno B4/metabolismo , Neutrófilos/metabolismo , Peste/imunologia , Vesículas Secretórias/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/genética , Yersinia pestis/genética , Yersinia pestis/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-33106262

RESUMO

The U.S. Food and Drug Administration (FDA) hosted a public workshop entitled "Advancing Animal Models for Antibacterial Drug Development" on 5 March 2020. The workshop mainly focused on models of pneumonia caused by Pseudomonas aeruginosa and Acinetobacter baumannii The program included discussions from academic investigators, industry, and U.S. government scientists. The potential use of mouse, rabbit, and pig models for antibacterial drug development was presented and discussed.


Assuntos
Acinetobacter baumannii , Antibacterianos , Animais , Antibacterianos/uso terapêutico , Desenvolvimento de Medicamentos , Camundongos , Modelos Animais , Coelhos , Suínos , Estados Unidos , United States Food and Drug Administration
8.
J Immunol ; 200(10): 3556-3567, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29610142

RESUMO

Silicosis is a lung inflammatory disease caused by chronic exposure to crystalline silica (CS). Leukotriene B4 (LTB4) plays an important role in neutrophilic inflammation, which drives silicosis and promotes lung cancer. In this study, we examined the mechanisms involved in CS-induced inflammatory pathways. Phagocytosis of CS particles is essential for the production of LTB4 and IL-1ß in mouse macrophages, mast cells, and neutrophils. Phagosomes enclosing CS particles trigger the assembly of lipidosome in the cytoplasm, which is likely the primary source of CS-induced LTB4 production. Activation of the JNK pathway is essential for both CS-induced LTB4 and IL-1ß production. Studies with bafilomycin-A1- and NLRP3-deficient mice revealed that LTB4 synthesis in the lipidosome is independent of inflammasome activation. Small interfering RNA knockdown and confocal microscopy studies showed that GTPases Rab5c, Rab40c along with JNK1 are essential for lipidosome formation and LTB4 production. BI-78D3, a JNK inhibitor, abrogated CS-induced neutrophilic inflammation in vivo in an air pouch model. These results highlight an inflammasome-independent and JNK activation-dependent lipidosome pathway as a regulator of LTB4 synthesis and CS-induced sterile inflammation.


Assuntos
Inflamassomos/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Leucotrieno B4/metabolismo , Dióxido de Silício/farmacologia , Animais , Linhagem Celular , Humanos , Interleucina-1beta/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Células RAW 264.7 , Silicose/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
9.
PLoS Pathog ; 11(10): e1005241, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26495854

RESUMO

Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.


Assuntos
Macrófagos/microbiologia , Yersinia pestis/fisiologia , Proteínas rab1 de Ligação ao GTP/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Legionella pneumophila/fisiologia , Lisossomos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fagossomos/fisiologia , Vacúolos/microbiologia , Vacúolos/fisiologia
10.
Mol Microbiol ; 89(2): 276-87, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23701256

RESUMO

Autotransporters, the largest family of secreted proteins in Gram-negative bacteria, perform a variety of functions, including adherence, cytotoxicity and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to disease in the mouse model of bubonic plague. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologues but is not conserved in the Yersinia enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis-specific plasmid pPCP1. Together, these data show that post-translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence.


Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/metabolismo , Yersinia pestis/patogenicidade , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Humanos , Macrófagos , Camundongos , Peste/microbiologia , Peste/patologia , Serina Endopeptidases/genética , Virulência , Yersinia pestis/genética , Yersinia pestis/metabolismo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/patogenicidade
11.
bioRxiv ; 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-39005373

RESUMO

Leukotriene B4 (LTB4) is critical for initiating the inflammatory cascade in response to infection. However, Yersinia pestis colonizes the host by inhibiting the timely synthesis of LTB4 and inflammation. Here, we show that the bacterial type 3 secretion system (T3SS) is the primary pathogen associated molecular pattern (PAMP) responsible for LTB4 production by leukocytes in response to Yersinia and Salmonella, but synthesis is inhibited by the Yop effectors during Yersinia interactions. Moreover, we unexpectedly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require two distinct host signaling pathways. We show that the SKAP2/PLC signaling pathway is essential for LTB4 production by neutrophils but not macrophages. Instead, phagocytosis and the NLRP3/CASP1 inflammasome are needed for LTB4 synthesis by macrophages. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered a second unrelated PAMP-mediated signal independently activates the MAP kinase pathway needed for LTB4 synthesis. Together, these data demonstrate significant differences in the signaling pathways required by macrophages and neutrophils to quickly respond to bacterial infections.

12.
iScience ; 27(1): 108600, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38179062

RESUMO

Fleas transmit Yersinia pestis directly within the dermis of mammals to cause bubonic plague. Syringe-mediated inoculation is widely used to recapitulate bubonic plague and study Y. pestis pathogenesis. However, intradermal needle inoculation is tedious, error prone, and poses a significant safety risk for laboratorians. Microneedle arrays (MNAs) are micron-scale polymeric structures that deliver materials to the dermis, while minimizing the risk of needle sticks. We demonstrated that MNA inoculation is a viable strategy to recapitulate bubonic plague and study bacterial virulence by defining the parameters needed to establish a lethal infection in the mouse model and characterizing the course of infection using live-animal optical imaging. Using MNAs, we also demonstrated that Y. pestis must overcome calprotectin-mediated zinc restriction within the dermis and dermal delivery of an attenuated mutant has vaccine potential. Together, these data demonstrate that MNAs are a safe alternative to study Y. pestis pathogenesis in the laboratory.

13.
Microbiol Spectr ; 10(5): e0269322, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36094219

RESUMO

The rise in infections caused by antibiotic-resistant bacteria is outpacing the development of new antibiotics. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are a group of clinically important bacteria that have developed resistance to multiple antibiotics and are commonly referred to as multidrug resistant (MDR). The medical and research communities have recognized that, without new antimicrobials, infections by MDR bacteria will soon become a leading cause of morbidity and death. Therefore, there is an ever-growing need to expedite the development of novel antimicrobials to combat these infections. Toward this end, we set out to refine an existing mouse model of pulmonary Pseudomonas aeruginosa infection to generate a robust preclinical tool that can be used to rapidly and accurately predict novel antimicrobial efficacy. This refinement was achieved by characterizing the virulence of a panel of genetically diverse MDR P. aeruginosa strains in this model, by both 50% lethal dose (LD50) analysis and natural history studies. Further, we defined two antibiotic regimens (aztreonam and amikacin) that can be used as comparators during the future evaluation of novel antimicrobials, and we confirmed that the model can effectively differentiate between successful and unsuccessful treatments, as predicted by in vitro inhibitory data. This validated model represents an important tool in our arsenal to develop new therapies to combat MDR P. aeruginosa strains, with the ability to provide rapid preclinical evaluation of novel antimicrobials and support data from clinical studies during the investigational drug development process. IMPORTANCE The prevalence of antibiotic resistance among bacterial pathogens is a growing problem that necessitates the development of new antibiotics. Preclinical animal models are important tools to facilitate and speed the development of novel antimicrobials. Successful outcomes in animal models not only justify progression of new drugs into human clinical trials but also can support FDA decisions if clinical trial sizes are small due to a small population of infections with specific drug-resistant strains. However, in both cases the preclinical animal model needs to be well characterized and provide robust and reproducible data. Toward this goal, we have refined an existing mouse model to better predict the efficacy of novel antibiotics. This improved model provides an important tool to better predict the clinical success of new antibiotics.


Assuntos
Amicacina , Pseudomonas aeruginosa , Camundongos , Humanos , Animais , Amicacina/farmacologia , Aztreonam/farmacologia , Testes de Sensibilidade Microbiana , Drogas em Investigação/farmacologia , Farmacorresistência Bacteriana Múltipla , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias
14.
J Bacteriol ; 193(21): 5936-49, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21873491

RESUMO

Yersinia pestis CO92 has 12 open reading frames encoding putative conventional autotransporters (yaps), nine of which appear to produce functional proteins. Here, we demonstrate the ability of the Yap proteins to localize to the cell surface of both Escherichia coli and Yersinia pestis and show that a subset of these proteins undergoes processing by bacterial surface omptins to be released into the supernatant. Numerous autotransporters have been implicated in pathogenesis, suggesting a role for the Yaps as virulence factors in Y. pestis. Using the C57BL/6 mouse models of bubonic and pneumonic plague, we determined that all of these genes are transcribed in the lymph nodes during bubonic infection and in the lungs during pneumonic infection, suggesting a role for the Yaps during mammalian infection. In vitro transcription studies did not identify a particular environmental stimulus responsible for transcriptional induction. The primary sequences of the Yaps reveal little similarity to any characterized autotransporters; however, two of the genes are present in operons, suggesting that the proteins encoded in these operons may function together. Further work aims to elucidate the specific functions of the Yaps and clarify the contributions of these proteins to Y. pestis pathogenesis.


Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/biossíntese , Peste/microbiologia , Yersinia pestis/genética , Yersinia pestis/metabolismo , Animais , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Pulmão/microbiologia , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças dos Roedores/microbiologia , Serina Endopeptidases/metabolismo , Fatores de Virulência/biossíntese
15.
Infect Immun ; 79(2): 644-52, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21115720

RESUMO

Yersinia pestis is a highly pathogenic Gram-negative organism and the causative agent of bubonic and pneumonic plague. Y. pestis is capable of causing major epidemics; thus, there is a need for vaccine targets and a greater understanding of the role of these targets in pathogenesis. Two prime Y. pestis vaccine candidates are the usher-chaperone fimbriae Psa and Caf. Herein we report that Y. pestis requires, in a nonredundant manner, both PsaA and Caf1 to achieve its full pathogenic ability in both pneumonic and bubonic plague in C57BL/6J mice. Deletion of psaA leads to a decrease in the organ bacterial burden and to a significant increase in the 50% lethal dose (LD50) after subcutaneous infection. Deletion of caf1 also leads to a significant decrease in the organ bacterial burden but more importantly leads to a significantly greater increase in the LD50 than was observed for the ΔpsaA mutant strain after subcutaneous infection of C57BL/6J mice. Furthermore, the degree of attenuation of the Δcaf1 mutant strain is mouse background dependent, as the Δcaf1 mutant strain was attenuated to a lesser degree in BALB/cJ mice by the subcutaneous route than in C57BL/6J mice. This observation that the degree of requirement for Caf1 is dependent on the mouse background indicates that the virulence of Y. pestis is dependent on the genetic makeup of its host and provides further support for the hypothesis that PsaA and Caf1 have different targets.


Assuntos
Cápsulas Bacterianas/fisiologia , Peste/microbiologia , Yersinia pestis/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Regulação Bacteriana da Expressão Gênica/fisiologia , Pulmão/microbiologia , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peste/genética , Peste/imunologia
16.
Antibiotics (Basel) ; 10(8)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34438996

RESUMO

BACKGROUND: Pseudomonas aeruginosa (PsA) is a common etiology of bacteria-mediated lower respiratory tract infections, including pneumonia, hospital acquired pneumonia (HAP), and ventilator-associated pneumonia (VAP). Given the paucity of novel antibiotics in our foreseeable pipeline, developing novel non-antibiotic antimicrobial therapies saliently targeting drug resistant PsA isolates remains a priority. Lytic bacteriophages (or phages) have come under scrutiny as a potential antimicrobial for refractory bacterial infections. We evaluated intratracheally and intraperitoneally (IP) administered phage therapy (with/without meropenem) in an acute immunocompromised mouse model of multi-drug resistant (MDR) PsA pulmonary infection. The MDR P. aeruginosa respiratory disease model used in these studies was developed to investigate novel therapies that might have efficacy as either monotherapies or as combination therapy with meropenem. METHODS: We utilized eight-week-old, 18 g BALB/cJ female mice and an MDR strain of PsA (UNC-D). Mice were immunosuppressed with cyclophosphamide. We employed a three-phage cocktail targeting PsA (PaAH2ΦP (103), PaBAP5Φ2 (130), and PaΦ (134)), confirmed to exhibit in vitro suppression of the infecting isolate out to 45 h. Suppression was confirmed with phages acting in isolation and in combination with meropenem. RESULTS: IP administration of phage did not protect mice from death. A one-time delivery of phage directly to the lungs via a single intubation-mediated, intratracheal (IMIT) instillation protected mice from lethal infection. Protection was observed despite delaying therapy out to 6 h. Finally, we observed that, by slowing the progression of infection by treatment with a sub-efficacious dose of meropenem, we could protect the mice from lethal infection via IP phage administration coupled to meropenem, observing partial additive effects of phage-antibiotic combination therapy. CONCLUSIONS: A personalized phage cocktail administered via IMIT exhibits high therapeutic efficacy, despite delayed treatment of 6 h in a lethal MDR PsA pneumonia model. IP phage alone did not forestall mortality, but exhibited efficacy when combined with meropenem and IMIT-administered phage. These additive effects of combined IP phage and meropenem confirm that phage may indeed reach the lung bed via the systemic circulation and protect mice if the infection is not too acute. Therefore, adjunctive phage therapy with concerted attention to identifying optimal phage targeting of the infecting isolate in vitro may exhibit transformative potential for combating the specter of MDR bacterial infections. Phage should serve as an integral component of a four-pronged approach coupled with antibiotics, source control, and immune optimization.

17.
Nat Commun ; 12(1): 7016, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853318

RESUMO

Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.


Assuntos
Enterobacteriaceae/metabolismo , Sideróforos/metabolismo , Zinco/metabolismo , Transportadores de Cassetes de Ligação de ATP , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Transporte , Colo/microbiologia , Colo/patologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Feminino , Complexo Antígeno L1 Leucocitário , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Fenóis , Salmonella typhi , Tiazóis
18.
Open Forum Infect Dis ; 7(8): ofaa236, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32766380

RESUMO

BACKGROUND: Plasma gelsolin (pGSN) is an abundant circulating protein quickly consumed by extensive tissue damage. Marked depletion is associated with later poor outcomes in diverse clinical circumstances. Repletion with recombinant human (rhu)-pGSN in animal models of inflammation lessens mortality and morbidity. METHODS: Neutropenic mice were treated with different meropenem doses ±12 mg of rhu-pGSN commencing 1 day before an intratracheal challenge with multidrug-resistant Pseudomonas aeruginosa. Survival, bacterial counts, and pulmonary pathology were compared between corresponding meropenem groups with and without rhu-pGSN. RESULTS: Overall survival was 35/64 (55%) and 46/64 (72%) in mice given meropenem without and with rhu-pGSN, respectively (Δ = 17%; 95% CI, 1-34). In control mice receiving meropenem 1250 mg/kg/d where the majority died, the addition of rhu-pGSN increased survival from 5/16 (31%) to 12/16 (75%) (Δ = 44%; 95% CI, 13-75). Survival with minor lung injury was found in 26/64 (41%) mice receiving only meropenem, vs 38/64 (59%) in mice given meropenem plus rhu-pGSN (Δ = 19%; 95% CI, 2-36). CONCLUSIONS: In a series of dose-ranging experiments, both mortality and lung injury were reduced by the addition of rhu-pGSN to meropenem against carbapenem-resistant P. aeruginosa. Rhu-pGSN offers a novel candidate therapy for antibiotic-resistant pneumonia.

19.
iScience ; 23(10): 101571, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33083738

RESUMO

Clostridioides difficile (C. diff) is the leading cause of antibiotic-associated colitis. Here, we report that lemon exosome-like nanoparticles (LELNs) manipulated probiotics to inhibit C. diff infection (CDI). LELN-manipulated Lactobacillus rhamnosus GG (LGG) and Streptococcus thermophilus ST-21 (STH) (LELN-LS) decrease CDI mortality via an LELN-mediated increase in bile resistance and gut survivability. LELN-LS treatment increases the AhR ligands indole-3-lactic acid (I3LA) and indole-3-carboxaldehyde (I3Ald), leading to induction of IL-22, and increases lactic acid leading to a decrease of C. diff fecal shedding by inhibiting C. diff growth and indole biosynthesis. A synergistic effect between STH and LGG was identified. The STH metabolites inhibit gluconeogenesis of LGG and allow fructose-1,6-bisphosphate (FBP) to accumulate in LGG; accumulated FBP then activates lactate dehydrogenase of LGG (LGG-LDH) and enhances production of lactic acid and the AhR ligand. Our findings provide a new strategy for CDI prevention and treatment with a new type of prebiotics.

20.
Infect Immun ; 77(1): 317-26, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18936182

RESUMO

Many proteins secreted by the type V secretion system (autotransporters) have been linked to virulence in gram-negative bacteria. Several putative conventional autotransporters are present in the Yersinia pestis genome, but only one, YapE, is conserved in the other pathogenic Yersinia species. Here, we introduce YapE and demonstrate that it is secreted via a type V mechanism. Inactivation of yapE in Y. pestis results in decreased efficiency in colonization of tissues during bubonic infection. Coinfection with wild-type bacteria only partially compensates for this defect. Analysis of the host immune response suggests that YapE is required for either efficient colonization at the inoculation site or dissemination to draining lymph nodes. YapE also demonstrates adhesive properties capable of mediating interactions with bacteria and eukaryotic cells. These findings support a role for YapE in modulating host-pathogen interactions that are important for colonization of the mammalian host.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Peste/microbiologia , Fatores de Virulência/metabolismo , Yersinia pestis/patogenicidade , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Contagem de Colônia Microbiana , Sequência Conservada , Citocinas/biossíntese , Feminino , Deleção de Genes , Ordem dos Genes , Pulmão/microbiologia , Linfonodos/imunologia , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Peste/imunologia , Baço/microbiologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Yersinia pestis/genética , Yersinia pestis/imunologia
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