RESUMO
Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of L. monocytogenes that becomes active upon the entry of the bacterium into the cytosol of infected cells. L. monocytogenes can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by lpd, bkdA1, bkdA2, and bkdB, is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient Listeria strains by in-frame deletion of lpd, bkdA1, bkdA2, and bkdB genes. To determine the role in in vivo and in vitro, mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in ΔbkdA1 strain than those in the wild-type. This study demonstrates that L. monocytogenes strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type prfA may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for L. monocytogenes to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between L. monocytogenes membrane integrity and the function of PrfA. This knowledge will increase our understanding of L. monocytogenes pathogenesis, which may provide insight into the development of antimicrobial agents.
Assuntos
Proteínas de Bactérias , Listeria monocytogenes , Listeriose , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/enzimologia , Listeria monocytogenes/metabolismo , Camundongos , Animais , Virulência , Listeriose/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Feminino , Linhagem CelularRESUMO
Listeria monocytogenes is well recognized for both its broad resistance to stress conditions and its ability to transition from a soil bacterium to an intracellular pathogen of mammalian hosts. The bacterium's impressive ability to adapt to changing environments and conditions requires the rapid sensing of environmental cues and the coordinated response of gene products that enable bacterial growth and survival. Two-component signaling systems (TCSs) have been long recognized for their ability to detect environmental stimuli and transmit those signals into transcriptional responses; however, often the precise nature of the stimulus triggering TCS responses can be challenging to define. L. monocytogenes has up to 16 TCSs that have been recognized based on homology and included in this list are several whose functions remain poorly described. This review highlights the current understanding of the breadth and scope of L. monocytogenes TCS as relates to stress resistance and pathogenesis. Precise signals still often remain elusive, but the gene networks associated with TCSs are providing clues into possible functions.
Assuntos
Listeria monocytogenes , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Mamíferos , Transdução de SinaisRESUMO
Listeria monocytogenes (Lm) is a widespread environmental Gram-positive bacterium that can transition into a pathogen following ingestion by a susceptible host. To cross host barriers and establish infection, Lm is dependent upon the regulated secretion and activity of many proteins including PrsA2, a peptidyl-prolyl cis-trans isomerase with foldase activity. PrsA2 contributes to the stability and activity of a number of secreted virulence factors that are required for Lm invasion, replication, and cell-to-cell spread within the infected host. In contrast, a second related secretion chaperone, PrsA1, has thus far no identified contributions to Lm pathogenesis. Here we describe the characterization of a two-component signal transduction system PieRS that regulates the expression of a regulon that includes the secretion chaperones PrsA1 and PrsA2. PieRS regulated gene products are required for bacterial resistance to ethanol exposure and are important for bacterial survival during transit through the gastrointestinal tract. PrsA1 was also found to make a unique contribution to Lm survival in the GI tract, revealing for the first time a non-overlapping requirement for both secretion chaperones PrsA1 and PrsA2 during the process of intra-gastric infection.
Assuntos
Listeria monocytogenes , Listeriose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Translocação Bacteriana , Humanos , Intestinos , Listeria monocytogenes/genética , Listeriose/microbiologia , Chaperonas Moleculares/metabolismo , Fatores de Virulência/metabolismoRESUMO
The study of T cell memory and the target of vaccine design have focused on memory subsumed by T cells bearing the αß T cell receptor. Alternatively, γδ T cells are thought to provide rapid immunity, particularly at mucosal borders. Here, we have shown that a distinct subset of mucosal γδ T cells mounts an immune response to oral Listeria monocytogenes (Lm) infection and leads to the development of multifunctional memory T cells capable of simultaneously producing interferon-γ and interleukin-17A in the murine intestinal mucosa. Challenge infection with oral Lm, but not oral Salmonella or intravenous Lm, induced rapid expansion of memory γδ T cells, suggesting contextual specificity to the priming pathogen. Importantly, memory γδ T cells were able to provide enhanced protection against infection. These findings illustrate that γδ T cells play a role with hallmarks of adaptive immunity in the intestinal mucosa.
Assuntos
Memória Imunológica/imunologia , Intestinos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa/imunologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Feminino , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Listeria monocytogenes/imunologia , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
The Gram-positive bacterium Listeria monocytogenes survives in environments ranging from the soil to the cytosol of infected host cells. Key to L. monocytogenes intracellular survival is the activation of PrfA, a transcriptional regulator that is required for the expression of multiple bacterial virulence factors. Mutations that constitutively activate prfA (prfA* mutations) result in high-level expression of multiple bacterial virulence factors as well as the physiological adaptation of L. monocytogenes for optimal replication within host cells. Here, we demonstrate that L. monocytogenesprfA* mutants exhibit significantly enhanced resistance to oxidative stress in comparison to that of wild-type strains. Transposon mutagenesis of L. monocytogenesprfA* strains resulted in the identification of three novel gene targets required for full oxidative stress resistance only in the context of PrfA activation. One gene, lmo0779, predicted to encode an uncharacterized protein, and two additional genes known as cbpA and ygbB, encoding a cyclic di-AMP binding protein and a 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, respectively, contribute to the enhanced oxidative stress resistance of prfA* strains while exhibiting no significant contribution in wild-type L. monocytogenes Transposon inactivation of cbpA and lmo0779 in a prfA* background led to reduced virulence in the liver of infected mice. These results indicate that L. monocytogenes calls upon specific bacterial factors for stress resistance in the context of PrfA activation and thus under conditions favorable for bacterial replication within infected mammalian cells.
Assuntos
Interações Hospedeiro-Patógeno , Listeria monocytogenes/genética , Listeriose/metabolismo , Listeriose/microbiologia , Estresse Oxidativo , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Camundongos , Mutagênese Insercional , Especificidade de ÓrgãosRESUMO
Listeria monocytogenes is a facultative Gram-positive intracellular bacterium that is capable of causing serious invasive infections in pregnant women, resulting in abortion, still-birth, and disseminated fetal infection. Previously, a clinical L. monocytogenes isolate, 07PF0776, was identified as having an enhanced ability to target cardiac tissue. This tissue tropism appeared to correlate with amino acid variations found within internalin B (InlB), a bacterial surface protein associated with host cell invasion. Given that the mammalian receptor bound by InlB, Met, is abundantly expressed by placental tissue, we assessed isolate 07PF0776 for its ability to be transmitted from mother to fetus. Pregnant Swiss Webster mice were infected on gestational day E13 via tail vein injection with the standard isolate 10403S, a noncardiotropic strain, or 07PF0776, the cardiac isolate. Pregnant mice infected with 07PF0776 exhibited significantly enhanced transmission of L. monocytogenes to placentas and fetuses compared to 10403S. Both bacterial burdens and the frequency of placental and fetal infection were increased in mice infected with the cardiac isolate. Strain 07PF0776 also exhibited an enhanced ability to invade Jar human trophoblast tissue culture cells in comparison to 10403S, and was found to have increased levels of InlB associated with the bacterial cell surface. Overexpression of surface InlB via genetic manipulation was sufficient to confer enhanced invasion of the placenta and fetus to both 10403S and 07PF0776. These data support a central role for surface InlB in promoting vertical transmission of L. monocytogenes.
Assuntos
Proteínas de Bactérias/fisiologia , Feto/fisiopatologia , Coração/fisiopatologia , Listeria monocytogenes/patogenicidade , Listeriose/transmissão , Proteínas de Membrana/fisiologia , Virulência/fisiologia , Adulto , Feminino , Feto/microbiologia , Coração/microbiologia , Humanos , Transmissão Vertical de Doenças Infecciosas , Masculino , GravidezRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for large numbers of postsurgical nosocomial infections across the United States and worldwide. Propofol anesthesia is widely used in surgery and in intensive care units, and recent evidence indicates that even brief exposure to propofol can substantially increase host susceptibility to microbial infection. Here, we delineate the impact of propofol sedation on MRSA bloodstream infections in mice in the presence and absence of prophylactic antibiotic treatment. Consistent with previous reports, brief periods of anesthesia with propofol were sufficient to significantly increase bacterial burdens and kidney pathology in mice infected with MRSA. Propofol exposure increased neutrophilic infiltrates into the kidney and enhanced bacterial dissemination throughout kidney tissue. Propofol sedation reduced populations of effector phagocytes and mature dendritic cells within the kidney and led to the apparent expansion of myeloid-derived suppressor cell-like populations. When propofol was coadministered with vancomycin prophylaxis, it dramatically increased kidney abscess formation and bacterial dissemination throughout kidney tissue at early times post-S. aureus infection compared to antibiotic-treated but nonsedated animals. Taken together, our data indicate that short-term sedation with propofol significantly increases the severity of bloodstream MRSA infection, even when administered in conjunction with vancomycin prophylaxis.
Assuntos
Bacteriemia/patologia , Suscetibilidade a Doenças/induzido quimicamente , Hipnóticos e Sedativos/efeitos adversos , Nefropatias/induzido quimicamente , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Propofol/efeitos adversos , Infecções Estafilocócicas/patologia , Animais , Carga Bacteriana , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Nefropatias/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologiaRESUMO
Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates.
Assuntos
Listeria monocytogenes/metabolismo , Peptidilprolil Isomerase/metabolismo , Bacillus subtilis/enzimologia , Cristalografia por Raios X , Citosol/enzimologia , Citosol/metabolismo , Isoenzimas , Lipoproteínas/metabolismo , Listeria monocytogenes/enzimologia , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/química , Relação Estrutura-Atividade , Virulência , Fatores de Virulência/metabolismoRESUMO
Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol.
Assuntos
Evasão da Resposta Imune , Lipoproteínas/imunologia , Listeria monocytogenes/imunologia , Peptídeos/imunologia , Feromônios/imunologia , Vacúolos/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Lipoproteínas/genética , Listeria monocytogenes/genética , Camundongos , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/imunologia , Peptídeos/genética , Feromônios/genética , Vacúolos/microbiologiaRESUMO
Infections by pathogenic microorganisms elicit host immune responses, which crucially limit those infections. Pathogens employ various strategies to evade host immunity. We have identified the exploitation of the repertoire of potent immunosuppressive responses elicited normally by apoptotic cells ("Innate Apoptotic Immunity"; IAI) as one of these strategies. In the case of Listeria monocytogenes, an environmentally ubiquitous, foodborne bacterial pathogen capable of causing life-threatening invasive disease in immunocompromised and elderly individuals, the induction of host cell apoptosis appears to play an important role in pathogenesis. Previous studies have documented extensive lymphocyte apoptosis resulting from L. monocytogenes infection and demonstrated paradoxically that lymphocyte-deficient animals exhibit diminished susceptibility to listerial pathogenicity. We speculated that the triggering of IAI following the induction of host cell apoptosis was responsible for enhanced pathogenesis, and that the administration of exogenous apoptotic cells would serve to exert this effect. Importantly, apoptotic cells, which are not susceptible to L. monocytogenes infection, do not provide a niche for bacterial replication. Our experiments confirm that apoptotic cells, including exogenous apoptotic cells induced to die independently of the pathogen, specifically enhance pathogenesis. The recognition of a role of apoptotic cells and Innate Apoptotic Immunity in microbial pathogenesis provides an intriguing and novel insight for therapeutic approaches for the control of pathogenic infections.
Assuntos
Apoptose/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/patologia , Animais , Linhagem Celular , Células Cultivadas , Citocinas/imunologia , Modelos Animais de Doenças , Imunidade Inata , Inflamação/imunologia , Inflamação/patologia , Listeria monocytogenes/imunologia , Listeriose/microbiologia , Linfócitos/imunologia , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ΔprsA2 ΔhtrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ΔprsA2 ΔhtrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion.
Assuntos
Proteínas de Choque Térmico/fisiologia , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Chaperonas Moleculares/fisiologia , Peptidilprolil Isomerase/fisiologia , Serina Endopeptidases/fisiologia , Animais , Citoplasma/microbiologia , Células Epiteliais/microbiologia , Glucuronidase/metabolismo , Humanos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/fisiologia , Macrófagos/microbiologia , Camundongos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Fatores de Virulência/metabolismoRESUMO
The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1, Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogen Listeria monocytogenes Within a few hours of systemic infection, the massive proliferation of L. monocytogenes in Perforin-2(-/-)mice leads to a rapid appearance of acute disease symptoms. We go on to show in cultured Perforin-2(-/-)cells that the vacuole-to-cytosol transitioning of L. monocytogenesis greatly accelerated. Unexpectedly, we found that in Perforin-2(-/-)macrophages,Listeria-containing vacuoles quickly (≤ 15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation of L. monocytogenes to its replicative niche in the cytosol. This hypothesis was supported by our finding that aL. monocytogenes strain expressing virulence factors at a constitutively high level replicated equally well in Perforin-2(+/+)and Perforin-2(-/-)macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification of Listeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.
Assuntos
Citosol/fisiologia , Listeria monocytogenes/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Vacúolos/fisiologia , Animais , Células Cultivadas , Citosol/microbiologia , Regulação da Expressão Gênica/fisiologia , Listeria monocytogenes/ultraestrutura , Listeriose/metabolismo , Listeriose/microbiologia , Proteínas de Membrana/genética , Camundongos , Proteínas Citotóxicas Formadoras de Poros/genética , Estrutura Terciária de ProteínaRESUMO
The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis/trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation of L. monocytogenes mutants lacking prsA2. PrsA homologues encoded by Bacillus subtilis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus, and Lactococcus lactis were examined for functional complementation of a variety of L. monocytogenes PrsA2-associated phenotypes central to L. monocytogenes pathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity for L. monocytogenes target proteins required for pathogenesis. The L. monocytogenes PrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation.
Assuntos
Listeria monocytogenes/enzimologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Sequência de Aminoácidos , Sistemas de Secreção Bacterianos , Sequência Conservada , Evolução Molecular , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/genética , Listeria monocytogenes/química , Listeria monocytogenes/genética , Listeriose , Chaperonas Moleculares/química , Dados de Sequência Molecular , Peptidilprolil Isomerase/química , Filogenia , Alinhamento de Sequência , Especificidade da Espécie , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Whether vaccination against a virus can protect against more virulent coinfection with the virus and additional pathogen(s) remains poorly characterized. Overlapping endemicity of human immunodeficiency virus (HIV) and malaria suggests that HIV/malaria coinfection frequently complicates acute and chronic HIV infection. Here we showed that vaccination of macaques with recombinant Listeria ΔactA prfA* expressing simian/human immunodeficiency virus (SHIV) gag and env elicited Gag- and Env-specific T-cell responses, and protected against life-threatening SHIV-related malaria after SHIV/Plasmodium fragile coinfection. SHIV antigen immunization reduced peak viremia, resisted SHIV/malaria-induced lymphoid destruction, and blunted coinfection-accelerated decline of CD4(+) T-cell counts after SHIV/malaria coinfection. SHIV antigen immunization also weakened coinfection-driven overreactive proinflammatory interferon-γ (IFNγ) responses and led to developing T helper cell 17/22 (Th17/Th22) responses after SHIV/malaria coinfection. The findings suggest that vaccination against AIDS virus can alter patterns of immune responses to the SHIV/malaria coinfection and protect against life-threatening SHIV-related malaria.
Assuntos
Antígenos Virais/imunologia , Coinfecção/imunologia , Infecções por HIV/imunologia , Malária/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Coinfecção/microbiologia , Coinfecção/parasitologia , Coinfecção/prevenção & controle , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Infecções por HIV/parasitologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/imunologia , Macaca mulatta/imunologia , Macaca mulatta/microbiologia , Macaca mulatta/virologia , Malária/microbiologia , Malária/prevenção & controle , Plasmodium/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação/métodos , Vacinas Sintéticas/imunologiaRESUMO
Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen Listeria monocytogenes (Lm). Critical to Lm virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since Lm is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection. IMPORTANCE: Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Listeria monocytogenes , Listeriose , Dobramento de Proteína , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/química , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Listeriose/microbiologia , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Peptidilprolil Isomerase/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/química , Concentração de Íons de Hidrogênio , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Estabilidade Proteica , HumanosRESUMO
Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-ß 2 (TGF-ß(2)) in the developing intestine. We hypothesized that low epithelial TGF-ß(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-ß(2) in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-ß(2), Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-ß(2) than term intestine. TGF-ß(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-ß(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-ß(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-ß(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.
Assuntos
Comunicação Autócrina , Colo/metabolismo , Enterocolite Necrosante/metabolismo , Mucosa Intestinal/metabolismo , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Animais , Western Blotting , Linhagem Celular , Colo/patologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Idade Gestacional , Humanos , Imuno-Histoquímica , Mucosa Intestinal/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Papio anubis , Papio cynocephalus , Nascimento Prematuro , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad7/genética , Transfecção , Fator de Crescimento Transformador beta2/genéticaRESUMO
Listeria monocytogenes is a food-borne bacterial pathogen and the causative agent of human and animal listeriosis. Among the three major genetic lineages of L. monocytogenes (i.e., LI, LII, and LIII), LI and LII are predominantly associated with food-borne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor gene, lmo0753, that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains, including a DNA binding domain, with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, we constructed lmo0753 deletion and complementation mutants in two fully sequenced L. monocytogenes LII strains, 10403S and EGDe, and compared the flagellar motility, phospholipase C production, hemolysis, and intracellular growth of the mutants and their respective wild types. Our results suggested that lmo0753 plays a role in hemolytic activity in both EGDe and 10403S. More interestingly, we found that deletion of lmo0753 led to the loss of l-rhamnose utilization in EGDe, but not in 10403S. RNA-seq analysis of EGDe Δ0753 incubated in phenol red medium containing l-rhamnose as the sole carbon source revealed that 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome were up- and downregulated more than 2-fold, respectively, compared to the wild-type strain. Genes related to biotin biosynthesis, general stress response, and rhamnose metabolism were shown to be differentially regulated. Findings from this study collectively suggested varied functional roles of lmo0753 in different LII L. monocytogenes strain backgrounds associated with human listeriosis outbreaks.
Assuntos
Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Ramnose/metabolismo , Fatores de Transcrição/metabolismo , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Humanos , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia , Fatores de Transcrição/genéticaRESUMO
Vertically transmitted infections are a significant cause of fetal morbidity and mortality during pregnancy and pose substantial risks to fetal development. These infections are primarily transmitted to the fetus through two routes: (1) direct invasion and crossing the placenta which separates maternal and fetal circulation, or (2) ascending the maternal genitourinary tact and entering the uterus. Only two bacterial species are commonly found to cross the placenta and infect the fetus: Listeria monocytogenes and Treponema pallidum subsp. pallidum. L. monocytogenes is a Gram-positive, foodborne pathogen found in soil that acutely infects a wide variety of mammalian species. T. pallidum is a sexually transmitted spirochete that causes a chronic infection exclusively in humans. We briefly review the pathogenesis of these two very distinct bacteria that have managed to overcome the placental barrier and the role placental immunity plays in resisting infection. Both organisms share characteristics which contribute to their transplacental transmission. These include the ability to disseminate broadly within the host, evade immune phagocytosis, and the need for a strong T cell response for their elimination.
Assuntos
Listeria monocytogenes , Treponema pallidum , Gravidez , Humanos , Animais , Feminino , Placenta , Treponema , MamíferosRESUMO
The soil-dwelling bacterium Listeria monocytogenes survives a multitude of conditions when residing in the outside environment and as a pathogen within host cells. Key to survival within the infected mammalian host is the expression of bacterial gene products necessary for nutrient acquisition. Similar to many bacteria, L. monocytogenes uses peptide import to acquire amino acids. Peptide transport systems play an important role in nutrient uptake as well as in additional functions that include bacterial quorum sensing and signal transduction, recycling of peptidoglycan fragments, adherence to eukaryotic cells, and alterations in antibiotic susceptibility. It has been previously described that CtaP, encoded by lmo0135, is a multifunctional protein associated with activities that include cysteine transport, resistance to acid, membrane integrity, and bacterial adherence to host cells. ctaP is located next to two genes predicted to encode membrane-bound permeases lmo0136 and lmo0137, termed CtpP1 and CtpP2, respectively. Here, we show that CtpP1 and CtpP2 are required for bacterial growth in the presence of low concentrations of cysteine and for virulence in mouse infection models. Taken together, the data identify distinct nonoverlapping roles for two related permeases that are important for the growth and survival of L. monocytogenes within host cells. IMPORTANCE Bacterial peptide transport systems are important for nutrient uptake and may additionally function in a variety of other roles, including bacterial communication, signal transduction, and bacterial adherence to eukaryotic cells. Peptide transport systems often consist of a substrate-binding protein associated with a membrane-spanning permease. The environmental bacterial pathogen Listeria monocytogenes uses the substrate-binding protein CtaP not only for cysteine transport but also for resistance to acid, maintenance of membrane integrity, and bacterial adherence to host cells. In this study, we demonstrate complementary yet distinct functional roles for two membrane permeases, CtpP1 and CtpP2, that are encoded by genes linked to ctaP and that contribute to bacterial growth, invasion, and pathogenicity.