ABSTRACT
The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.
Subject(s)
Bacterial Proteins , Lysosomes , Macrophages , Streptococcus pyogenes , Streptolysins , Streptococcus pyogenes/immunology , Humans , Macrophages/microbiology , Macrophages/immunology , Macrophages/metabolism , Lysosomes/metabolism , Lysosomes/microbiology , Streptolysins/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Phagosomes/microbiology , Phagosomes/metabolism , THP-1 Cells , Phagocytosis , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcal Infections/metabolism , Cathepsin B/metabolism , Hydrogen-Ion ConcentrationABSTRACT
The pro-inflammatory cytokine IL-6 regulates antimicrobial responses that are broadly crucial in the defense against infection. Our prior work shows that IL-6 promotes the killing of the M4 serotype group A Streptococcus (GAS) but does not impact the globally disseminated M1T1 serotype associated with invasive infections. Using in vitro and in vivo infection models, we show that IL-6 induces phagocyte reactive oxygen species (ROS) that are responsible for the differential susceptibility of M4 and M1T1 GAS to IL-6-mediated defenses. Clinical isolates naturally deficient in capsule, or M1T1 strains deficient in capsule production, are sensitive to this ROS killing. The GAS capsule is made of hyaluronic acid, an antioxidant that detoxifies ROS and can protect acapsular M4 GAS when added exogenously. During in vitro interactions with macrophages and neutrophils, acapsular GAS can also be rescued with the antioxidant N-acetylcysteine, suggesting this is a major virulence contribution of the capsule. In an intradermal infection model with gp91phox -/- (chronic granulomatous disease [CGD]) mice, phagocyte ROS production had a modest effect on bacterial proliferation and the cytokine response but significantly limited the size of the bacterial lesion in the skin. These data suggest that the capsule broadly provides enhanced resistance to phagocyte ROS but is not essential for invasive infection. Since capsule-deficient strains are observed across several GAS serotypes and are competent for transmission and both mild and invasive infections, additional host or microbe factors may contribute to ROS detoxification during GAS infections.
Subject(s)
Hyaluronic Acid , Streptococcal Infections , Animals , Mice , Reactive Oxygen Species , Antioxidants , Interleukin-6 , Neutrophils/microbiology , Streptococcus pyogenes , Streptococcal Infections/microbiology , Bacterial ProteinsABSTRACT
[This corrects the article DOI: 10.1371/journal.pgen.1003007.].
ABSTRACT
The development of a severe invasive bacterial infection in an otherwise healthy individual is one of the most striking and fascinating aspects of human medicine. A small cadre of gram-positive pathogens of the genera Streptococcus and Staphylococcus stand out for their unique invasive disease potential and sophisticated ability to counteract the multifaceted components of human innate defense. This review illustrates how these leading human disease agents evade host complement deposition and activation, impede phagocyte recruitment and activation, resist the microbicidal activities of host antimicrobial peptides and reactive oxygen species, escape neutrophil extracellular traps, and promote and accelerate phagocyte cell death through the action of pore-forming cytolysins. Understanding the molecular basis of bacterial innate immune resistance can open new avenues for therapeutic intervention geared to disabling specific virulence factors and resensitizing the pathogen to host innate immune clearance.
Subject(s)
Gram-Positive Bacteria/immunology , Gram-Positive Bacterial Infections/immunology , Gram-Positive Bacterial Infections/microbiology , Immunity, Innate , Animals , Gram-Positive Bacteria/genetics , Gram-Positive Bacteria/pathogenicity , Humans , Immune Evasion , VirulenceABSTRACT
Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.
Subject(s)
Autophagy , Bacterial Infections/metabolism , Cysteine Endopeptidases/metabolism , Membrane Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Bacterial Infections/genetics , Bacterial Infections/mortality , Caenorhabditis elegans/metabolism , Cell Line , Cell Membrane/metabolism , Enzyme Stability , Genetic Predisposition to Disease , Humans , Membrane Proteins/genetics , Mice , Mice, Knockout , Microtubule-Associated Proteins/metabolism , Phagosomes/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Transport , Proteolysis , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Bacillus anthracis, the causative agent of anthrax, relies on multiple virulence factors to subvert the host immune defense. Using Caenorhabditis elegans as an infection model, we screened approximately 5,000 transposon mutants of B. anthracis Sterne for decreased virulence. One of the attenuated mutants resulted in loss of expression of yceG and yceH, the last two genes in a six-gene cluster of tellurite resistance genes. We generated an analogous insertional mutant to confirm the phenotype and characterize the role of yceGH in resistance to host defenses. Loss of yceGH rendered the mutants more sensitive to tellurite toxicity as well as to host defenses such as reactive oxygen species and the cathelicidin family of antimicrobial peptides. Additionally, we see decreased survival in mammalian models of infection, including human whole blood and in mice. We identify a novel role for the yceGH genes in B. anthracis Sterne virulence and suggest that C. elegans is a useful infection model to study anthrax pathogenesis.
Subject(s)
Anthrax/immunology , Bacillus anthracis/genetics , Bacillus anthracis/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Tellurium/immunology , Animals , Anthrax/microbiology , Caenorhabditis elegans/immunology , Caenorhabditis elegans/microbiology , Immunity, Innate/genetics , Immunity, Innate/immunology , Mutation/genetics , Mutation/immunology , Virulence/genetics , Virulence/immunology , Virulence Factors/genetics , Virulence Factors/immunologyABSTRACT
Streptococcal collagen-like protein 1 (Scl-1) is one of the most highly expressed proteins in the invasive M1T1 serotype group A Streptococcus (GAS), a globally disseminated clone associated with higher risk of severe invasive infections. Previous studies using recombinant Scl-1 protein suggested a role in cell attachment and binding and inhibition of serum proteins. Here, we studied the contribution of Scl-1 to the virulence of the M1T1 clone in the physiological context of the live bacterium by generating an isogenic strain lacking the scl-1 gene. Upon subcutaneous infection in mice, wild-type bacteria induced larger lesions than the Δscl mutant. However, loss of Scl-1 did not alter bacterial adherence to or invasion of skin keratinocytes. We found instead that Scl-1 plays a critical role in GAS resistance to human and murine phagocytic cells, allowing the bacteria to persist at the site of infection. Phenotypic analyses demonstrated that Scl-1 mediates bacterial survival in neutrophil extracellular traps (NETs) and protects GAS from antimicrobial peptides found within the NETs. Additionally, Scl-1 interferes with myeloperoxidase (MPO) release, a prerequisite for NET production, thereby suppressing NET formation. We conclude that Scl-1 is a virulence determinant in the M1T1 GAS clone, allowing GAS to subvert innate immune functions that are critical in clearing bacterial infections.
Subject(s)
Immune Evasion , Neutrophils/immunology , Neutrophils/microbiology , Streptococcus pyogenes/immunology , Streptococcus pyogenes/metabolism , Virulence Factors/metabolism , Animals , Bacterial Adhesion , Cells, Cultured , Gene Deletion , Humans , Keratinocytes/microbiology , Mice , Mice, Inbred C57BL , Microbial Viability , Phagocytes/immunology , Phagocytes/microbiology , Streptococcal Infections/microbiology , Streptococcal Infections/pathology , Streptococcus pyogenes/genetics , Virulence Factors/geneticsABSTRACT
Staphylococcus aureus causes a wide range of human disease ranging from localized skin and soft tissue infections to potentially lethal systemic infections. S. aureus has the biosynthetic ability to generate numerous virulence factors that assist in circumventing the innate immune system during disease pathogenesis. Recent studies have uncovered a set of extracellular peptides produced by community-associated methicillin-resistant S. aureus (CA-MRSA) with homology to the phenol-soluble modulins (PSMs) from Staphylococcus epidermidis. CA-MRSA PSMs contribute to skin infection and recruit and lyse neutrophils, and truncated versions of these peptides possess antimicrobial activity. In this study, novel CA-MRSA PSM derivatives were discovered by the use of microbial imaging mass spectrometry. The novel PSM derivatives are compared with their parent full-length peptides for changes in hemolytic, cytolytic, and neutrophil-stimulating activity. A potential contribution of the major S. aureus secreted protease aureolysin in processing PSMs is demonstrated. Finally, we show that PSM processing occurs in multiple CA-MRSA strains by structural confirmation of additional novel derivatives. This work demonstrates that IMS can serve as a useful tool to go beyond genome predictions and expand our understanding of the important family of small peptide virulence factors.
Subject(s)
Mass Spectrometry/methods , Methicillin-Resistant Staphylococcus aureus/metabolism , Phenol/chemistry , Amino Acid Sequence , Animals , Anti-Infective Agents/pharmacology , Bacterial Infections/metabolism , Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Erythrocytes/cytology , Hemolysis , Humans , Immunosuppressive Agents/pharmacology , Metalloendopeptidases/chemistry , Mice , Molecular Sequence Data , Neutrophils/cytology , Neutrophils/metabolism , Sequence Homology, Amino Acid , Sheep , Skin/metabolism , Skin/microbiology , Staphylococcal Skin Infections/microbiology , Virulence Factors/chemistryABSTRACT
MCs (mast cells) are critical components of the host innate immune defence against bacterial pathogens, providing a variety of intra- and extra-cellular antimicrobial functions. In the present study we show, for the first time, that the transcriptional regulator HIF-1α (hypoxia-inducible factor-1α) mediates the extracellular antimicrobial activity of human and murine MCs by increasing the formation of MCETs (MC extracellular traps).
Subject(s)
Extracellular Space/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Mast Cells/physiology , Animals , Anti-Infective Agents/pharmacology , Cells, Cultured , Echinomycin/pharmacology , Humans , Interleukin-6/genetics , Interleukin-8/genetics , Mast Cells/drug effects , Mast Cells/microbiology , Mice , Mice, Inbred C57BL , Piperazines/pharmacology , Pyridones/pharmacology , Staphylococcus aureus/pathogenicityABSTRACT
The ClpXP protease is a critical bacterial intracellular protease that regulates protein turnover in many bacterial species. Here we identified a pharmacological inhibitor of the ClpXP protease, F2, and evaluated its action in Bacillus anthracis and Staphylococcus aureus. We found that F2 exhibited synergistic antimicrobial activity with cathelicidin antimicrobial peptides and antibiotics that target the cell well and/or cell membrane, such as penicillin and daptomycin, in B. anthracis and drug-resistant strains of S. aureus. ClpXP inhibition represents a novel therapeutic strategy to simultaneously sensitize pathogenic bacteria to host defenses and pharmaceutical antibiotics.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Bacteria/drug effects , Endopeptidase Clp/antagonists & inhibitors , Escherichia coli Proteins/antagonists & inhibitors , Protease Inhibitors/pharmacology , Amino Acid Sequence , Bacillus anthracis/drug effects , Bacillus anthracis/genetics , Cell Membrane/metabolism , Drug Resistance, Bacterial , Drug Synergism , Methicillin-Resistant Staphylococcus aureus/drug effects , Molecular Sequence Data , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Tetrazoles/pharmacology , CathelicidinsABSTRACT
BACKGROUND: The secreted enzyme EndoS, an endoglycosidase from Streptococcus pyogenes, hydrolyzes the N-linked glycan of the constant region of immunoglobulin G (IgG) heavy chain and renders the antibody unable to interact with Fc receptors and elicit effector functions. In this study we couple targeted allelic replacement mutagenesis and heterologous expression to elucidate the contribution of EndoS to group A Streptococcus (GAS) phagocyte resistance and pathogenicity in vitro and in vivo. RESULTS: Knocking out the EndoS gene in GAS M1T1 background revealed no significant differences in bacterial survival in immune cell killing assays or in a systemic mouse model of infection. However, exogenous addition and heterologous expression of EndoS was found to increase GAS resistance to killing by neutrophils and monocytes in vitro. Additionally, heterologous expression of EndoS in M49 GAS increased mouse virulence in vivo. CONCLUSIONS: We conclude that in a highly virulent M1T1 background, EndoS has no significant impact on GAS phagocyte resistance and pathogenicity. However, local accumulation or high levels of expression of EndoS in certain GAS strains may contribute to virulence.
Subject(s)
Glycoside Hydrolases/metabolism , Immunoglobulin G/metabolism , Phagocytes/immunology , Phagocytes/microbiology , Streptococcus pyogenes/enzymology , Streptococcus pyogenes/pathogenicity , Animals , Disease Models, Animal , Gene Deletion , Glycoside Hydrolases/genetics , Humans , Mice , Microbial Viability , Phagocytosis , Streptococcal Infections/microbiology , Streptococcal Infections/pathology , Streptococcus pyogenes/immunology , VirulenceABSTRACT
The extracellular protozoan parasite Trichomonas vaginalis causes the most prevalent non-viral sexually transmitted human infection, yet the pathogenesis of infection is poorly understood, and host cell receptors have not been described. The surface of T. vaginalis is covered with a glycoconjugate called lipophosphoglycan (LPG), which plays a role in the adherence and cytotoxicity of parasites to human cells. T. vaginalis LPG contains high amounts of galactose, making this polysaccharide a candidate for recognition by the galactose-binding galectin family of lectins. Here we show that galectin-1 (gal-1) is expressed by human cervical epithelial cells and binds T. vaginalis LPG. Gal-1 binds to parasites in a carbohydrate-dependent manner that is inhibited in the presence of T. vaginalis LPG. Addition of purified gal-1 to cervical epithelial cells also enhances parasite binding, while a decrease in gal-1 expression by small interfering RNA (siRNA) transfection decreases parasite binding. In contrast, the related galectin-7 (gal-7) does not bind T. vaginalis in a carbohydrate-dependent manner, and is unable to mediate attachment of parasites to host cells. Our data are consistent with the presence of multiple host cell receptors for T. vaginalis of which gal-1 is the first to be identified and highlight the importance of glycoconjugates in host-pathogen interactions.
Subject(s)
Epithelial Cells/parasitology , Galectin 1/metabolism , Glycosphingolipids/metabolism , Receptors, Cell Surface/metabolism , Trichomonas vaginalis/physiology , Animals , Cell Adhesion , Cell Line , Cervix Uteri/parasitology , Female , Gene Silencing , HumansABSTRACT
The resurgence of Group A Streptococcus (GAS) infections in the past two decades has been a rising major public health concern. Due to a large number of GAS infections occurring in the skin, mast cells (MCs), innate immune cells known to localize to the dermis, could play an important role in controlling infection. MCs can exert their antimicrobial activities either early during infection, by degranulation and release of antimicrobial proteases and the cathelicidin-derived antimicrobial peptide LL-37, or by forming antibacterial MC extracellular traps (MCETs) in later stages of infection. We demonstrate that MCs do not directly degranulate in response to GAS, reducing their ability to control bacterial growth in early stages of infection. However, MC granule components are highly cytotoxic to GAS due to the pore-forming activity of LL-37, while MC granule proteases do not significantly affect GAS viability. We therefore confirmed the importance of MCETs by demonstrating their capacity to reduce GAS survival. The data therefore suggests that LL-37 from MC granules become embedded in MCETs, and are the primary effector molecule by which MCs control GAS infection. Our work underscores the importance of a non-traditional immune effector cell, utilizing a non-conventional mechanism, in the defense against an important human pathogen.
Subject(s)
Cell Degranulation/immunology , Extracellular Traps/immunology , Mast Cells/immunology , Skin Diseases, Bacterial/immunology , Streptococcal Infections/immunology , Streptococcus pyogenes/immunology , Antimicrobial Cationic Peptides/immunology , Cell Line , Extracellular Traps/microbiology , Humans , Mast Cells/microbiology , Mast Cells/pathology , Secretory Vesicles/immunology , Secretory Vesicles/microbiology , Secretory Vesicles/pathology , Skin Diseases, Bacterial/microbiology , Skin Diseases, Bacterial/pathology , Streptococcal Infections/microbiology , Streptococcal Infections/pathology , CathelicidinsABSTRACT
UNLABELLED: Based on in vitro synergy studies, the addition of nafcillin to daptomycin was used to treat refractory methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Daptomycin is a de facto cationic antimicrobial peptide in vivo, with antistaphylococcal mechanisms reminiscent of innate host defense peptides (HDPs). In this study, the effects of nafcillin on HDP activity against MRSA were examined in vitro and in vivo. Exposures to ß-lactam antimicrobials in general, and nafcillin in particular, significantly increased killing of S. aureus by selected HDPs from keratinocytes, neutrophils, and platelets. This finding correlated with enhanced killing of MRSA by whole blood, neutrophils, and keratinocytes after growth in nafcillin. Finally, nafcillin pretreatment ex vivo reduced MRSA virulence in a murine subcutaneous infection model. Despite the lack of direct activity against MRSA, these studies show potent, consistent, and generalized nafcillin-mediated "sensitization" to increased killing of MRSA by various components of the innate host response. The use of nafcillin as adjunctive therapy in MRSA bacteremia merits further study and should be considered in cases refractory to standard therapy. KEY MESSAGES: Nafcillin has been used as adjunctive therapy to clear persistent MRSA bacteremia. Nafcillin enhances killing of MRSA by a cadre of innate host defense peptides. Nafcillin increases binding of human cathelicidin LL-37 to the MRSA membrane. Nafcillin enhances killing of MRSA by neutrophils. Nafcillin reduces virulence of MRSA in a murine subcutaneous infection model.
Subject(s)
Immunity, Innate/drug effects , Methicillin-Resistant Staphylococcus aureus/drug effects , Nafcillin/pharmacology , Staphylococcal Infections/drug therapy , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Antimicrobial Cationic Peptides/metabolism , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Cationic Peptides/therapeutic use , Bacteremia/drug therapy , Bacteremia/microbiology , Cell Line , Cells, Cultured , Daptomycin/metabolism , Daptomycin/pharmacology , Daptomycin/therapeutic use , Dose-Response Relationship, Drug , Drug Synergism , Drug Therapy, Combination , Female , Humans , Keratinocytes/metabolism , Male , Methicillin-Resistant Staphylococcus aureus/physiology , Mice , Microbial Viability/drug effects , Middle Aged , Nafcillin/metabolism , Nafcillin/therapeutic use , Neutrophils/metabolism , Neutrophils/physiology , Phagocytosis/drug effects , Phagocytosis/physiology , Staphylococcal Infections/microbiology , CathelicidinsABSTRACT
PURPOSE: Guidelines recommend daptomycin combination therapy as an option for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin failure. Recent data suggest that combining daptomycin with a ß-lactam may have unique benefits; however, there are very limited clinical data regarding the use of ceftaroline with daptomycin. METHODS: All 26 cases from the 10 medical centers in which ceftaroline plus daptomycin was used for treatment of documented refractory staphylococcal bacteremia from March 2011 to November 2012 were included. In vitro (synergy studies, binding assays, cathelicidin LL-37 killing assays), and in vivo (virulence assays using a murine subcutaneous infection model) studies examining the effects of ceftaroline with daptomycin were also performed. FINDINGS: Daptomycin plus ceftaroline was used in 26 cases of staphylococcal bacteremia (20 MRSA, 2 vancomycin-intermediate S aureus, 2 methicillin-susceptible S aureus [MSSA], 2 methicillin-resistant S epidermidis). Bacteremia persisted for a median of 10 days (range, 3-23 days) on previous antimicrobial therapy. After daptomycin plus ceftaroline was started, the median time to bacteremia clearance was 2 days (range, 1-6 days). In vitro studies showed ceftaroline synergy against MRSA and enhanced MRSA killing by cathelicidin LL-37 and neutrophils. Ceftaroline also induced daptomycin binding in MSSA and MRSA to a comparable degree as nafcillin. MRSA grown in subinhibitory concentrations of ceftaroline showed attenuated virulence in a murine subcutaneous infection model. IMPLICATIONS: Ceftaroline plus daptomycin may be an option to hasten clearance of refractory staphylococcal bacteremia. Ceftaroline offers dual benefit via synergy with both daptomycin and sensitization to innate host defense peptide cathelicidin LL37, which could attenuate virulence of the pathogen.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacteremia/drug therapy , Cephalosporins/therapeutic use , Daptomycin/therapeutic use , Staphylococcal Infections/drug therapy , Adult , Aged , Aged, 80 and over , Animals , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/metabolism , Cephalosporins/pharmacology , Daptomycin/pharmacology , Drug Therapy, Combination , Female , Humans , Male , Mice , Middle Aged , Salvage Therapy , Staphylococcus/drug effects , Staphylococcus/growth & development , Cathelicidins , CeftarolineABSTRACT
UNLABELLED: The Mac/IdeS protein of group A Streptococcus (GAS) is a secreted cysteine protease with cleavage specificity for IgG and is highly expressed in the GAS serotype M1T1 clone, which is the serotype most frequently isolated from patients with life-threatening invasive infections. While studies of Mac/IdeS with recombinant protein have shown that the protein can potentially prevent opsonophagocytosis of GAS by neutrophils, the role of the protein in immune evasion as physiologically produced by the living organism has not been studied. Here we examined the contribution of Mac/IdeS to invasive GAS disease by generating a mutant lacking Mac/IdeS in the hyperinvasive M1T1 background. While Mac/IdeS was highly expressed and proteolytically active in the hyperinvasive strain, elimination of the bacterial protease did not significantly influence GAS phagocytic uptake, oxidative-burst induction, cathelicidin sensitivity, resistance to neutrophil or macrophage killing, or pathogenicity in pre- or postimmune mouse infectious challenges. We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence. Given the conservation of Mac/IdeS and homologues across GAS strains, it is possible that Mac/IdeS serves another important function in GAS ecology or contributes to virulence in other strain backgrounds. IMPORTANCE: Group A Streptococcus (GAS) causes human infections ranging from strep throat to life-threatening conditions such as flesh-eating disease and toxic shock syndrome. Common disease-associated clones of GAS can cause both mild and severe infections because of a characteristic mutation and subsequent change in the expression of several genes that develops under host immune selection. One of these genes encodes Mac/IdeS, a protease that has been shown to cleave antibodies important to the immune defense system. In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse. These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.
Subject(s)
Bacterial Proteins/metabolism , Immunoglobulin G/metabolism , Phagocytes/microbiology , Streptococcus pyogenes/immunology , Streptococcus pyogenes/pathogenicity , Virulence Factors/metabolism , Animals , Bacterial Proteins/genetics , Cell Line , Coculture Techniques , Disease Models, Animal , Gene Deletion , Humans , Male , Mice , Microbial Viability , Phagocytosis , Proteolysis , Streptococcal Infections/microbiology , Streptococcal Infections/pathology , Streptococcus pyogenes/genetics , Virulence , Virulence Factors/geneticsABSTRACT
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 increases HIF-1 levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinetobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Hypoxia-Inducible Factor 1/immunology , Immunity, Innate/drug effects , Piperazines/therapeutic use , Pyridones/therapeutic use , Skin Diseases, Bacterial/prevention & control , Skin/microbiology , Animals , Cell Line , Female , Humans , Hypoxia-Inducible Factor 1/agonists , Keratinocytes/drug effects , Keratinocytes/immunology , Keratinocytes/microbiology , Mice , Phagocytes/drug effects , Phagocytes/immunology , Phagocytes/microbiology , Piperazines/pharmacology , Pyridones/pharmacology , Skin/drug effects , Skin/immunology , Skin Diseases, Bacterial/immunology , Staphylococcal Infections/immunology , Staphylococcal Infections/prevention & control , Staphylococcus aureus/drug effects , Staphylococcus aureus/immunologyABSTRACT
We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.
Subject(s)
Genome, Protozoan , Sequence Analysis, DNA , Trichomonas vaginalis/genetics , Animals , Biological Transport/genetics , DNA Transposable Elements , DNA, Protozoan/genetics , Gene Transfer, Horizontal , Genes, Protozoan , Humans , Hydrogen/metabolism , Metabolic Networks and Pathways/genetics , Molecular Sequence Data , Multigene Family , Organelles/metabolism , Oxidative Stress/genetics , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/physiology , RNA Processing, Post-Transcriptional , Repetitive Sequences, Nucleic Acid , Sexually Transmitted Diseases/parasitology , Trichomonas Infections/parasitology , Trichomonas Infections/transmission , Trichomonas vaginalis/cytology , Trichomonas vaginalis/metabolism , Trichomonas vaginalis/pathogenicityABSTRACT
The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.