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1.
PLoS Pathog ; 20(5): e1012214, 2024 May.
Article in English | MEDLINE | ID: mdl-38722857

ABSTRACT

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.


Subject(s)
Bacterial Proteins , Cadherins , Epithelial Cells , Mycobacterium tuberculosis , Serine Proteases , Cadherins/metabolism , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/metabolism , Animals , Humans , Mice , Serine Proteases/metabolism , Serine Proteases/genetics , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , A549 Cells , Tuberculosis/microbiology , Tuberculosis/metabolism , Female
2.
DNA Cell Biol ; 41(12): 1063-1074, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36394437

ABSTRACT

l-Arginine serves as a carbon and nitrogen source and is critical for Mycobacterium tuberculosis (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the argCJBDFGH gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of argC (rv1652), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in Mycobacterium bovis bacille Calmette-Guerin.


Subject(s)
Mycobacterium bovis , Mycobacterium tuberculosis , Mycobacterium bovis/genetics , Mycobacterium bovis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Arginine/genetics , Arginine/metabolism , Multigene Family
3.
Microb Pathog ; 173(Pt B): 105880, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36402348

ABSTRACT

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb can overcome macrophage intracellular killing and lead to persistent infections. The proteases of Mtb are critical virulence factors that participate in immune responses. We determined that Rv3090 is a cell wall-associated protease and a potential pathogenic factor. To characterize the role of Rv3090 in Mtb, recombinant Msg_Rv3090 and Msg_pAIN strains were constructed to infect macrophages and mice. Lactate dehydrogenase assays and flow cytometry results showed that Rv3090 induces late macrophage apoptosis. In vivo infection experiments indicated that Rv3090 could induce hepatocyte and lung cell apoptosis and cause pathological damage to the spleen, livers and lungs. Msg_Rv3090 specifically stimulated the secretion of inflammatory cytokines including TNF-α, IL-6 and IL-1ß. Overexpression of Rv3090 significantly promoted the survival of Msg in livers and lungs. Thus, Rv3090 protease triggered late cell apoptosis and contributed to the pathogenicity and dissemination of Mtb.


Subject(s)
Mycobacterium tuberculosis , Peptide Hydrolases , Animals , Mice , Apoptosis , Endopeptidases , Virulence Factors
4.
Vet Microbiol ; 273: 109529, 2022 Oct.
Article in English | MEDLINE | ID: mdl-35944391

ABSTRACT

Extracellular DNases/nucleases are important virulence factors in many bacteria. However, no DNase/nucleases have been reported in Mycobacterium avium subsp. paratuberculosis (MAP), which is a pathogen of paratuberculosis. Genome analyses of MAP K-10 revealed that the map3916c gene putatively encodes a nuclease. In this study, we show that MAP3916c is an extracellular nonspecific DNase requiring a divalent cation, especially Mg2+. The optimum DNase activity of MAP3916c was exhibited at 41 °C and pH 9.0. Site-directed mutagenesis studies indicated that 125-Histidine is necessary for MAP3916c DNase activity. In addition, MAP3916c DNase could destroy the neutrophil extracellular traps (NETs) induced by Phorbol 12-myristate 13-acetate in vitro and degrade the NETs induced by MAP K-10 upon infection. Furthermore, MAP3916c DNase promoted the colonization of MAP K-10, induced the formation of granulomas in the liver and small intestine and promoted the release of IL-1ß, IL-6 and TNF-α inflammatory cytokines during the infection of mice. These results indicated that MAP3916c is relevant to NETs escape and the pathogenicity of MAP. It also provides a basis for further study of the function of nuclease activity on the MAP immune evasion.


Subject(s)
Deoxyribonucleases , Extracellular Traps , Mycobacterium avium subsp. paratuberculosis , Paratuberculosis , Animals , Deoxyribonucleases/genetics , Deoxyribonucleases/metabolism , Extracellular Traps/metabolism , Macrophages/microbiology , Mice , Mycobacterium avium subsp. paratuberculosis/enzymology , Mycobacterium avium subsp. paratuberculosis/pathogenicity , Paratuberculosis/microbiology , Virulence
5.
Front Microbiol ; 11: 2204, 2020.
Article in English | MEDLINE | ID: mdl-33042041

ABSTRACT

Patatin-like phospholipases (PLPs) are important virulence factors of many pathogens. However, there are no prevailing studies regarding PLPs as a virulence factor of Mycobacterium tuberculosis (Mtb). Analysis of Rv3091, a putative protein of Mtb, shows that it belongs to the PLPs family. Here, we cloned and expressed the rv3091 gene in Mycobacterium smegmatis and, subsequently, conducted protein purification and characterization. We show that it possesses phospholipase A1, phospholipase A2, and lipase activity. We confirm the putative active site residues, namely, Ser214 and Asp407, using site directed mutagenesis. The Rv3091 is an extracellular protein that alters the colony morphology of M. smegmatis. The presence of Rv3091 enhances the intracellular survival capability of M. smegmatis in murine peritoneal macrophages. Additionally, it promotes M. smegmatis phagosomal escape from macrophages. Moreover, Rv3091 significantly increased the survival of M. smegmatis and aggravated lesions in C57BL/6 J murine lungs in vivo. Taken together, our results indicate that Rv3091 as an extracellular PLP that is critical to the pathogenicity of mycobacterium as it allows mycobacterium to utilize phospholipids for its growth and provides resistance to phagosome killing, resulting in its enhanced intracellular survival.

6.
J Biol Chem ; 295(31): 10638-10652, 2020 07 31.
Article in English | MEDLINE | ID: mdl-32518157

ABSTRACT

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(ß1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(ß1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-ß-N-acetylhexosaminidase activities, including exo-(ß1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.


Subject(s)
Bacterial Proteins/chemistry , Epitopes/chemistry , Glycoside Hydrolases/chemistry , Polysaccharides, Bacterial/chemistry , Streptococcus suis/chemistry , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Disaccharides/chemistry , Disaccharides/genetics , Disaccharides/metabolism , Epitopes/genetics , Epitopes/metabolism , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Mice , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/metabolism , Protein Domains , Rabbits , Streptococcus suis/genetics , Streptococcus suis/metabolism , Swine
7.
Front Microbiol ; 11: 812, 2020.
Article in English | MEDLINE | ID: mdl-32390998

ABSTRACT

Vitamins are essential nutrients and key cofactors of enzymes that regulate cellular metabolism, and also activate the immune system. Recent studies have shown that vitamin B1 (VB 1) and vitamin C (Vc) can inhibit Mycobacterium tuberculosis growth, but the precise mechanism is still not well understood. In the present study, we have used RNA-sequencing (RNA-seq), liquid chromatography coupled to mass spectrometry (LC-MS) and single-molecule real-time (SMRT) sequencing to analyze the transcriptional, metabolic and methylation profiles of Mycobacterium bovis BCG when treated with VB 1 and Vc. Our results show that, after vitamin treatment, variant metabolites were mainly clustered in pathways related to amino acid metabolism. Treatment with both vitamins significantly up-regulated the gene encoding cysteine synthase A. Additionally, only BCG that was treated with VC showed m4c modifications. Genes harboring this methylation were up-regulated, suggesting that m4c methylation can promote gene transcription to some extent. Overall, this study contributes to the understanding of the effects of VB 1 and VC, and suggests that these vitamins constitute potential anti-tuberculosis drugs.

8.
Microb Pathog ; 142: 104055, 2020 Feb 11.
Article in English | MEDLINE | ID: mdl-32058021

ABSTRACT

Serine protease is the virulence factor of many pathogens. However, there are no prevailing data available for serine protease as a virulence factor derived from Mycobacterium avium subsp. paratuberculosis (MAP). The MAP3292c gene from MAP, the predicted serine protease, was expressed in Escherichia coli and characterized by biochemical methods. MAP3292c protein efficiently hydrolyzed casein at optimal temperature and pH of 41 °C and 9.0, respectively. Furthermore, divalent metal ions of Ca2+ significantly promoted the protease activity of MAP3292c, and MAP3292c had autocleavage activity between serine 86 and asparagine 87. Site-directed mutagenesis studies showed that the serine 238 residue had catalytic roles in MAP3292c. Furthermore, a BALB/c mouse model confirmed that MAP3292c significantly promoted the survival of Mycobacterium smegmatis in vivo; caused damage to the liver, spleen, and lung; and promoted the release of inflammatory cytokines IL-1ß, IL-6, and TNF-α in mice. Finally, we confirmed that MAP3292c was relevant to mycobacterial pathogenicity.

9.
Tuberculosis (Edinb) ; 119: 101880, 2019 12.
Article in English | MEDLINE | ID: mdl-31731061

ABSTRACT

Mycobacterium tuberculosis (MTB) serine proteases are important pathogen-associated virulence factors that are involved in the invasion, bacterial persistence, and degradation of host defense factors. The current study identified and characterized a novel serine protease, Rv3194c, of MTB. A heterologous Rv3194c protein, purified from Escherichia coli, possessed proteolytic activity that could hydrolyze bovine serum albumin (BSA), milk, casein, and gelatin at an optimal temperature of 40 °C and a pH of 8.0. Furthermore, the divalent metal ions Ca2+ and Mn2+ increased the activity of Rv3194c. Betulinic acid, a Traditional Chinese Medicine (TCM) monomer; PMSF, a chemical inhibitor; and the Roche inhibitor cocktail inhibited proteolytic activity. Site-directed mutagenesis demonstrated that D308 and particularly S309 play a crucial role in the catalytic activity of Rv3194c protease. The cellular assays revealed that Rv3194c inhibits THP1-derived macrophage migration. Moreover, Rv3194c degraded the complement components, C3b and C5a, causing inhibition of phagocytosis and chemotaxis. In mice, Rv3194c enhanced the persistence of Mycobacterium smegmatis (Ms) in the lung, induced lung lesions, and promoted the release of inflammatory cytokines. The results of this study indicate that Rv3194c may play an important role in the pathogenicity of mycobacteria.


Subject(s)
Macrophages/microbiology , Mycobacterium tuberculosis/pathogenicity , Serine Proteases/metabolism , Animals , Disease Models, Animal , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mycobacterium tuberculosis/enzymology , Virulence
10.
BMC Microbiol ; 19(1): 104, 2019 05 22.
Article in English | MEDLINE | ID: mdl-31117936

ABSTRACT

BACKGROUND: Vitamin B1 (VB1) is a crucial dietary nutrient and essential cofactor for several key enzymes in the regulation of cellular and metabolic processes, and more importantly in the activation of immune system. To date, the precise role of VB1 in Mycobacterium tuberculosis remains to be fully understood. RESULTS: In this study, the transcriptional and metabolic profiles of VB1-treated Mycobacterium. bovis BCG were analyzed by RNA-sequencing and LC-MS (Liquid chromatography coupled to mass spectrometry). The selection of BCG strain was based on its common physiological features shared with M. tuberculosis. The results of cell growth assays demonstrated that VB1 inhibited the BCG growth rate in vitro. Transcriptomic analysis revealed that the expression levels of genes related to fatty acid metabolism, cholesterol metabolism, glycolipid catabolism, DNA replication, protein translation, cell division and cell wall formation were significantly downregulated in M. bovis BCG treated with VB1. In addition, the metabolomics LC-MS data indicated that most of the amino acids and adenosine diphosphate (ADP) were decreased in M. bovis BCG strain after VB1 treatment. CONCLUSIONS: This study provides the molecular and metabolic bases to understand the impacts of VB1 on M.bovis BCG.


Subject(s)
Bacterial Proteins/genetics , Metabolome/drug effects , Mycobacterium bovis/growth & development , Thiamine/pharmacology , Chromatography, Liquid , Gene Expression Profiling/methods , Gene Expression Regulation, Bacterial/drug effects , Mass Spectrometry , Metabolomics/methods , Mycobacterium bovis/chemistry , Mycobacterium bovis/drug effects , Mycobacterium bovis/genetics , Sequence Analysis, RNA
11.
Microb Pathog ; 126: 63-73, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30366126

ABSTRACT

The capacity of Mycobacterium tuberculosis to survive and cause disease is strongly correlated with its ability to escape multiple defense strategies in hosts. In particular, M. tuberculosis has the remarkable capacity to survive within the hostile environment of macrophages. Here, we found that the PE17 (Rv1646) protein promoted intracellular survival of M. smegmatis in peritoneal macrophages from mice. Further experiments confirmed that the recombinant PE17 protein was localized in the cell wall of M. smegmatis. Results from the macrophage infection model showed that PE17 significantly downregulated pro-inflammatory cytokines (interleukin-6, interleukin-12, and tumer necrosis factor-α) secretion from macrophages induced by M. smegmatis and promoted macrophage necrosis. Furthermore, a C57BL/6 mouse infection model confirmed that PE17 significantly prolonged the survival of M. smegmatis in vivo and aggravated lesions in organs of infected mice. Moreover, persistent high levels of interferon-γ and interleukin-1ß in infected mice indicated that the bacteria were not easily removed in vivo. Overall, our present results suggested that the PE17 may act as an important pathogenic factor in M. tuberculosis.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Macrophages/immunology , Macrophages/microbiology , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Virulence Factors/metabolism , Animals , Antigens, Bacterial/genetics , Apoptosis , Bacterial Proteins/genetics , Cell Death , Cell Wall , Cytokines/metabolism , Disease Models, Animal , Female , Gene Expression Regulation, Bacterial , Host-Pathogen Interactions , Interferon-gamma/metabolism , Interleukin-12/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Kidney/microbiology , Kidney/pathology , Liver/microbiology , Liver/pathology , Lung/microbiology , Lung/pathology , Macrophages/ultrastructure , Mice , Mice, Inbred C57BL , Microbial Viability , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Recombinant Proteins , Spleen/microbiology , Spleen/pathology , Tumor Necrosis Factor-alpha/metabolism , Virulence , Virulence Factors/genetics
12.
Front Immunol ; 9: 677, 2018.
Article in English | MEDLINE | ID: mdl-29670633

ABSTRACT

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which mainly causes pulmonary injury and tubercles. Although macrophages are generally considered to harbor the main cells of M. tuberculosis, new evidence suggests that neutrophils are rapidly recruited to the infected lung. M. tuberculosis itself, or its early secreted antigenic target protein 6 (ESAT-6), can induce formation of neutrophil extracellular traps (NETs). However, NETs trap mycobacteria but are unable to kill them. The role of NETs' formation in the pathogenesis of mycobacteria remains unclear. Here, we report a new M. tuberculosis extracellular factor, bifunctional enzyme Rv0888, with both nuclease and sphingomyelinase activities. Rv0888 sphingomyelinase activity can induce NETs' formation in vitro and in the lung of the mice and enhance the colonization ability of Mycobacterium smegmatis in the lungs of mice. Mice infected by M. smegmatis harboring Rv0888 sphingomyelinase induced pathological injury and inflammation of the lung, which was mainly mediated by NETs, induced by Rv0888 sphingomyelinase, associated protein (myeloperoxidase) triggered caspase-3. In summary, the study sheds new light on the pathogenesis of mycobacteria and reveals a novel target for TB treatment.


Subject(s)
Bacterial Proteins/metabolism , Lung/immunology , Mycobacterium Infections, Nontuberculous/immunology , Mycobacterium smegmatis/physiology , Mycobacterium tuberculosis/physiology , Neutrophils/immunology , Sphingomyelin Phosphodiesterase/metabolism , Tuberculosis, Pulmonary/immunology , Animals , Bacterial Proteins/genetics , Caspase 3/metabolism , Cells, Cultured , Extracellular Space/metabolism , Extracellular Traps/metabolism , Humans , Lung/microbiology , Mice , Mice, Inbred C57BL , Peroxidase/metabolism , Sphingomyelin Phosphodiesterase/genetics
13.
J Vet Sci ; 17(4): 505-513, 2016 Dec 30.
Article in English | MEDLINE | ID: mdl-27994210

ABSTRACT

Mycobacterium (M.) vaccae is a fast-growing species of saprophytic bacteria that is widely distributed. To understand the host immune responses induced by M. vaccae isolated from bovine submaxillary lymph nodes, C57BL/6 mice were infected with reference strain M. bovis Bacillus Calmette-Guérin (BCG) and isolated M. vaccae using intraperitoneal injections. Comparison of the bacterial replication and organ pathology between M. vaccae and M. bovis BCG revealed that M. vaccae was more malignant than M. bovis in mice. We also demonstrated that serum from the M. vaccae-infected mice contained a higher expression level of gamma-interferon (IFN-γ), tumor necrosis factor alpha, monocyte chemoattractant protein-1, interleukin (IL)-4, IL-12, IL-10 and transforming growth factor beta than did the other groups, especially after week 4. Furthermore, when the numbers of CD3⁺CD4⁺IFN-γ⁺ and CD3⁺CD4⁺IL4⁺cells in the infected mice were observed by flow cytometry, we found that a powerful T helper 1 (Th1) response was induced by M. vaccae infection, which was associated with the emergence of CD3⁺CD4⁺IFN-γ⁺cells. However, the Th1 response declined over time, which was associated with appearance of the CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD152⁺Treg cell reaction. In addition, a strong Th2 response was found. Finally, we found that M. vaccae infection increased the production of type I IFNs, which was associated with a reduced Th1 response.


Subject(s)
Cytokines/genetics , Mycobacterium Infections/immunology , Mycobacterium/immunology , Th1 Cells/immunology , Animals , Cattle , Cytokines/metabolism , Disease Models, Animal , Female , Mice , Mice, Inbred C57BL , Mycobacterium bovis/physiology , Specific Pathogen-Free Organisms , Tuberculosis, Bovine/immunology , Tuberculosis, Bovine/microbiology
14.
Vet Microbiol ; 190: 5-11, 2016 Jul 15.
Article in English | MEDLINE | ID: mdl-27283850

ABSTRACT

Hemorrhagic pneumonia caused by Pseudomonas aeruginosa remains one of the most costly infectious diseases among farmed mink and commonly leads to large economic losses during mink production. The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink. A broad-host-range phage from the Podoviridae family, YH30, was isolated using the mink-originating P. aeruginosa (serotype G) D7 strain as a host. The genome of YH30 was 72,192bp (54.92% G+C), contained 86 open reading frames and lacked regions encoding known virulence factors, integration-related proteins or antibiotic resistance determinants. These characteristics make YH30 eligible for use in phage therapy. The results of a curative treatment experiment demonstrated that a single intranasal administration of YH30 was sufficient to cure hemorrhagic pneumonia in mink. The mean colony count of P. aeruginosa in the blood and lung of YH30-protected mink was less than 10(3) CFU/mL (g) within 24h of bacterial challenge and ultimately became undetectable, whereas that in unprotected mink reached more than 10(8) CFU/mL (g). Additionally, YH30 dramatically improved the pathological manifestations of lung injury in mink with hemorrhagic pneumonia. Our work demonstrates the potential of phages to treat P. aeruginosa-caused hemorrhagic pneumonia in mink.


Subject(s)
Biological Therapy/veterinary , Pneumonia, Bacterial/veterinary , Pseudomonas Infections/veterinary , Pseudomonas Phages/physiology , Pseudomonas aeruginosa/virology , Administration, Intranasal , Animals , Bacterial Load , Biological Therapy/standards , Genome, Viral/genetics , Microscopy, Electron, Transmission , Mink , Pneumonia, Bacterial/therapy , Pseudomonas Infections/therapy , Pseudomonas Phages/genetics , Pseudomonas Phages/isolation & purification , Pseudomonas Phages/ultrastructure , Pseudomonas aeruginosa/physiology , Treatment Outcome
15.
Res Microbiol ; 166(8): 633-43, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26254772

ABSTRACT

The treatment, in farmed mink, of hemorrhagic pneumonia caused by multidrug-resistant Pseudomonas aeruginosa strains has become increasingly difficult. This study investigated the potential use of phages as a therapy against hemorrhagic pneumonia caused by P. aeruginosa in a murine hemorrhagic pneumonia model. An N4-like phage designated YH6 was isolated using P. aeruginosa strain D9. YH6 is a virulent phage with efficient and broad host lytic activity against P. aeruginosa. No bacterial virulence- or lysogenesis-related ORF is present in the YH6 genome, making it eligible for use in phage therapy. In our murine experiments, a single intranasal administration of YH6 (2 × 10(7) PFU) 2 h after D9 intranasal injections at double minimum lethal dose was sufficient to protect mice against hemorrhagic pneumonia. The bacterial load in the lungs of YH6-protected mice was less than 10(3) CFU/g within 24 h after challenge and ultimately became undetectable, whereas the amount of bacteria in the lung tissue derived from unprotected mice was more than 10(8) CFU/g within 24 h after challenge. In view of its protective efficacy in this murine hemorrhagic pneumonia model, YH6 may serve as an alternative treatment strategy for infections caused by multidrug-resistant P. aeruginosa.


Subject(s)
Pneumonia, Bacterial/therapy , Pseudomonas Infections/therapy , Pseudomonas Phages , Pseudomonas aeruginosa/pathogenicity , Administration, Intranasal , Animals , Bacterial Load , Biological Therapy , Disease Models, Animal , Drug Resistance, Multiple, Bacterial , Female , Lung/microbiology , Lung/pathology , Mice , Pneumonia, Bacterial/microbiology , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Pseudomonas Phages/isolation & purification , Pseudomonas Phages/physiology , Pseudomonas aeruginosa/virology
16.
Microbiol Immunol ; 59(9): 533-44, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26249788

ABSTRACT

Pseudomonas aeruginosa poses a major threat to human health and to the mink industry. Thus, development of vaccines that elicit robust humoral and cellular immunity against P. aeruginosa is greatly needed. In this study, a recombinant attenuated Salmonella vaccine (RASV) that expresses the outer membrane proteins fusion OprF190-342 -OprI21-83 (F1I2) from P. aeruginosa was constructed and the potency of this vaccine candidate assessed by measuring F1I2-specific humoral immune responses upon vaccination through s.c. or oral routes. S.C. administration achieved higher serum IgG titers and IgA titers in the intestine and induced stronger F1I2-specific IgG and IgA titers in lung homogenate than did oral administration, which resulted in low IgG titers and no local IgA production. High titers of IFN-γ, IL-4, and T-lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized s.c., indicating elicitation of cellular immunity. Importantly, when immunized mice were challenged with P. aeruginosa by the intranasal route 30 days after the initial immunization, s.c. vaccination achieved 77.78% protection, in contrast to 41.18% via oral administration and 66.67% via Escherichia coli-expressed F1I2 (His-F1I2) vaccination. These results indicate that s.c. vaccination provides a better protective response against P. aeruginosa infection than do oral administration and the His-F1I2 vaccine.


Subject(s)
Bacterial Proteins/immunology , Drug Carriers , Lipoproteins/immunology , Pseudomonas Infections/prevention & control , Pseudomonas Vaccines/immunology , Pseudomonas aeruginosa/immunology , Salmonella typhimurium/genetics , Administration, Oral , Animals , Antibodies, Bacterial/analysis , Antibodies, Bacterial/blood , Bacterial Proteins/genetics , Disease Models, Animal , Escherichia coli , Female , Immunoglobulin A/analysis , Immunoglobulin G/blood , Injections, Subcutaneous , Interferon-gamma/metabolism , Interleukin-4/metabolism , Intestinal Mucosa/immunology , Lipoproteins/genetics , Lung/immunology , Mice, Inbred BALB C , Mink , Pseudomonas Vaccines/administration & dosage , Pseudomonas Vaccines/genetics , Pseudomonas aeruginosa/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Salmonella Vaccines/administration & dosage , Salmonella Vaccines/genetics , Salmonella typhimurium/growth & development , Serum/immunology , Survival Analysis , T-Lymphocyte Subsets/immunology , Treatment Outcome , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology
17.
Appl Microbiol Biotechnol ; 99(2): 667-80, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25381907

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen that localizes to and colonizes mucosal tissue. Thus, vaccines that elicit a strong mucosal response against P. aeruginosa should be superior to other vaccination strategies. In this study, to stimulate rapid and enhanced mucosal immune responses, mannose-modified chitosan microspheres loaded with the recombinant outer membrane protein OprF190-342-OprI21-83 (FI) (FI-MCS-MPs) of P. aeruginosa were developed as a potent subunit vaccine for mucosal delivery. FI-MCS-MPs were successfully obtained via the tripolyphosphate ionic crosslinking method. Confocal and immunohistochemical analyses indicated that FI-MCS-MPs exhibited the ability to bind the macrophage mannose receptor (MMR, CD206) in vitro and in vivo. After intranasal immunization of mice with FI-MCS-MPs, FI-specific humoral immune responses were detected, measured as local IgM antibody titers in lung tissue slurry; IgA antibody titers in nasal washes, bronchoalveolar lavage (BAL), and intestinal lavage; and systemic IgA and IgG antibody titers in serum. FI-MCS-MPs induced early and high mucosal and systemic humoral antibody responses comparable to those in the group vaccinated with unmodified mannose. High levels of IFN-γ and IL-4 in addition to T lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized with FI-MCS-MPs, resulting in the establishment of cellular immunity. Additionally, when immunized mice were challenged with P. aeruginosa via the nasal cavity, FI-MCS-MPs demonstrated 75 % protective efficacy. Together, these data indicate that mannose-modified chitosan microspheres are a promising subunit delivery system for vaccines against P. aeruginosa infection.


Subject(s)
Chitosan/pharmacology , Immunity, Mucosal , Mannose/pharmacology , Pseudomonas Infections/immunology , Pseudomonas Vaccines/immunology , Pseudomonas aeruginosa , Administration, Intranasal , Animals , Antibodies, Bacterial/blood , Antibody Formation , Bacterial Proteins/immunology , Base Sequence , Cell Line , Chitosan/chemistry , Female , Immunoglobulin A/blood , Immunoglobulin A/chemistry , Immunoglobulin G/blood , Interferon-gamma/immunology , Interleukin-4/immunology , Lipoproteins/immunology , Macrophages/chemistry , Macrophages/immunology , Mannose/chemistry , Mice , Mice, Inbred BALB C , Microspheres , Molecular Sequence Data , Pseudomonas Infections/prevention & control , Pseudomonas Vaccines/chemistry , Recombinant Proteins/immunology , T-Lymphocytes/immunology
18.
PLoS Pathog ; 10(5): e1004109, 2014 May.
Article in English | MEDLINE | ID: mdl-24831957

ABSTRACT

The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.


Subject(s)
Calcium/metabolism , Mucoproteins/chemistry , Mucoproteins/metabolism , Protein Interaction Domains and Motifs , Staphylococcus Phages/enzymology , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Catalytic Domain , Cell Wall/drug effects , Cell Wall/metabolism , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/growth & development , Methicillin-Resistant Staphylococcus aureus/ultrastructure , Microbial Sensitivity Tests , Models, Molecular , Molecular Sequence Data , Mucoproteins/pharmacology , Sequence Homology, Amino Acid
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