Biomedical subjects

Victor Nizet

Publications and source records attributed to Victor Nizet.

At least 37 records · Page 2Linked to original sources

PubMed2005

Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity.

Golden color imparted by carotenoid pigments is the eponymous feature of the human pathogen Staphylococcus aureus. Here we demonstrate a role of this hallmark phenotype in virulence. Compared with the wild-type (WT) bacterium, a S. aureus mutant with disrupted carotenoid biosynthesis is more susceptible to oxidant killing, has impaired neutrophil survival, and is less pathogenic in a mouse subcutaneous abscess model. The survival advantage of WT S. aureus over the carotenoid-deficient mutant is lost upon inhibition of neutrophil oxidative burst or in human or murine nicotinamide adenine dinucleotide phosphate oxidase-deficient hosts. Conversely, heterologous expression of the S. aureus carotenoid in the nonpigmented Streptococcus pyogenes confers enhanced oxidant and neutrophil resistance and increased animal virulence. Blocking S. aureus carotenogenesis increases oxidant sensitivity and decreases whole-blood survival, suggesting a novel target for antibiotic therapy.

Abscess↗

PubMed2005

IKKalpha limits macrophage NF-kappaB activation and contributes to the resolution of inflammation.

Inflammation and innate immunity involve signalling pathways leading to the production of inflammatory mediators. Usually such responses are self-limiting, but aberrant resolution of inflammation results in chronic diseases. Much attention has focused on pro-inflammatory signalling but little is known about the mechanisms that resolve inflammation. The IkappaB kinase (IKK) complex contains two catalytic subunits, IKKalpha and IKKbeta, and controls the activation of NF-kappaB transcription factors, which play a pivotal role in inflammation. Ample evidence indicates that IKKbeta mediates NF-kappaB activation in response to pro-inflammatory cytokines and microbial products. IKKalpha regulates an alternative pathway important for lymphoid organogenesis, but the role of IKKalpha in inflammation is unknown. Here we describe a new role for IKKalpha in the negative regulation of macrophage activation and inflammation. IKKalpha contributes to suppression of NF-kappaB activity by accelerating both the turnover of the NF-kappaB subunits RelA and c-Rel, and their removal from pro-inflammatory gene promoters. Inactivation of IKKalpha in mice enhances inflammation and bacterial clearance. Hence, the two IKK catalytic subunits have evolved opposing but complimentary roles needed for the intricate control of inflammation and innate immunity.

Animals↗

PubMed2005

Virulence role of group B Streptococcus beta-hemolysin/cytolysin in a neonatal rabbit model of early-onset pulmonary infection.

We examined the virulence role of group B Streptococcus (GBS) beta-hemolysin/cytolysin (beta h/c) in a neonatal-rabbit model of GBS pulmonary infection. Rabbits infected intratracheally with wild-type (wt) GBS developed focal pneumonia and, by 18 h after infection, had 100-fold more bacteria in lung tissue than did rabbits infected with a delta beta h/c mutant. Mortality (40% vs. 0%), development of bacteremia, and mean bacterial blood counts were all significantly higher in the rabbits challenged with wt GBS than in those challenged with the delta beta h/c mutant. Lung compliance during mechanical ventilation was impaired after injection of wt GBS but not after injection of the Delta beta h/c mutant strain. This work, to our knowledge, provides the first in vivo evidence for a critical role of the beta h/c toxin in GBS neonatal pneumonia and in the breakdown of the pulmonary barrier to systemic infection.

Age Factors↗

PubMed2005

Cutaneous defense mechanisms by antimicrobial peptides.

The skin actively contributes to host defense by mounting an innate immune response that includes the production of antimicrobial peptides. These peptides, which include but are not limited to the cathelicidin and defensin gene families, provide rapid, broad-spectrum defense against infection by acting as natural antibiotics and by participating in host cell processes involved in immune defense. This review discusses the biology and clinical relevance of antimicrobial peptides expressed in the skin. The importance of the epithelial contribution to host immunity is evident, as alterations in antimicrobial peptide expression have been associated with various pathologic processes.

Antimicrobial Cationic Peptides↗

PubMed2005

Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection.

The pathogen group A Streptococcus (GAS) produces a wide spectrum of infections including necrotizing fasciitis (NF). Streptolysin S (SLS) produces the hallmark beta-haemolytic phenotype produced by GAS. The nine-gene GAS locus (sagA-sagI) resembling a bacteriocin biosynthetic operon is necessary and sufficient for SLS production. Using precise, in-frame allelic exchange mutagenesis and single-gene complementation, we show sagA, sagB, sagC, sagD, sagE, sagF and sagG are each individually required for SLS production, and that sagE may further serve an immunity function. Limited site-directed mutagenesis of specific amino acids in the SagA prepropeptide supports the designation of SLS as a bacteriocin-like toxin. No significant pleotrophic effects of sagA deletion were observed on M protein, capsule or cysteine protease production. In a murine model of NF, the SLS-negative M1T1 GAS mutant was markedly diminished in its ability to produce necrotic skin ulcers and spread to the systemic circulation. The SLS toxin impaired phagocytic clearance and promoted epithelial cell cytotoxicity, the latter phenotype being enhanced by the effects of M protein and streptolysin O. We conclude that all genetic components of the sag operon are required for expression of functional SLS, an important virulence factor in the pathogenesis of invasive M1T1 GAS infection.

Amino Acid Sequence↗

PubMed2005

Keratinocyte production of cathelicidin provides direct activity against bacterial skin pathogens.

Immune defense at an interface with the external environment reflects the functions of physical and chemical barriers provided by epithelial and immune cells. Resident epithelial cells, such as keratinocytes, produce numerous peptides with direct antimicrobial activity but also provide a physical barrier against invading pathogens and signal the recruitment of circulating immune cells, such as neutrophils. Antimicrobial peptides such as cathelicidin are produced constitutively by neutrophils and are inducible in keratinocytes in response to infection. The multiplicity of antimicrobial peptides and their cellular sources has resulted in an incomplete understanding of the role of cathelicidin production by epithelial cells in cutaneous immune defense. Therefore, this study sought to evaluate keratinocyte antimicrobial activity and the potential contribution of keratinocyte cathelicidin to host protection against two leading human skin pathogens. Wild-type mice and those with a targeted deletion of the cathelicidin gene, Cnlp, were rendered neutropenic prior to cutaneous infection. Interestingly, Cnlp-deficient mice remained more susceptible to group A streptococcus infection than mice with Cnlp intact, suggesting the involvement of epithelial cell-derived cathelicidin in host immune defense. Keratinocytes were then isolated in culture and found to inhibit the growth of Staphylococcus aureus, an effect that was partially dependent on their ability to synthesize and activate cathelicidin. Further, lentivirus-mediated delivery of activated human cathelicidin enhanced keratinocyte antimicrobial activity. Combined, these data illustrate the potential contribution of keratinocyte cathelicidin to the innate immune defense of skin against bacterial pathogens and highlight the need to consider epithelial antimicrobial function in the diagnosis and therapy of skin infection.

Animals↗

PubMed2005

Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development.

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with DeltaPGM mutant cells reaching sizes approximately 3-fold larger than those of the wild type (WT). The DeltaPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the DeltaPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the DeltaPGM mutant. In analysis of the aborted infectious process, we found that DeltaPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the DeltaPGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae DeltaPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.

Amino Acid Sequence↗

PubMed2005

A toll-like receptor 2-responsive lipid effector pathway protects mammals against skin infections with gram-positive bacteria.

flake (flk), an N-ethyl-N-nitrosourea-induced recessive germ line mutation of C57BL/6 mice, impairs the clearance of skin infections by Streptococcus pyogenes and Staphylococcus aureus, gram-positive pathogens that elicit innate immune responses by activating Toll-like receptor 2 (TLR2). Positional cloning and sequencing revealed that flk is a novel allele of the stearoyl coenzyme A desaturase 1 gene (Scd1). flake homozygotes show reduced sebum production and are unable to synthesize the monounsaturated fatty acids (MUFA) palmitoleate (C(16:1)) and oleate (C(18:1)), both of which are bactericidal against gram-positive (but not gram-negative) organisms in vitro. However, intradermal MUFA administration to S. aureus-infected mice partially rescues the flake phenotype, which indicates that an additional component of the sebum may be required to improve bacterial clearance. In normal mice, transcription of Scd1-a gene with numerous NF-kappaB elements in its promoter--is strongly and specifically induced by TLR2 signaling. Similarly, the SCD1 gene is induced by TLR2 signaling in a human sebocyte cell line. These observations reveal the existence of a regulated, lipid-based antimicrobial effector pathway in mammals and suggest new approaches to the treatment or prevention of infections with gram-positive bacteria.

Animals↗

PubMed2005

D-alanylation of teichoic acids promotes group a streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion.

Group A streptococcus (GAS) is a leading cause of severe, invasive human infections, including necrotizing fasciitis and toxic shock syndrome. An important element of the mammalian innate defense system against invasive bacterial infections such as GAS is the production of antimicrobial peptides (AMPs) such as cathelicidins. In this study, we identify a specific GAS phenotype that confers resistance to host AMPs. Allelic replacement of the dltA gene encoding d-alanine-d-alanyl carrier protein ligase in an invasive serotype M1 GAS isolate led to loss of teichoic acid d-alanylation and an increase in net negative charge on the bacterial surface. Compared to the wild-type (WT) parent strain, the GAS DeltadltA mutant exhibited increased susceptibility to AMP and lysozyme killing and to acidic pH. While phagocytic uptake of WT and DeltadltA mutants by human neutrophils was equivalent, neutrophil-mediated killing of the DeltadltA strain was greatly accelerated. Furthermore, we observed the DeltadltA mutant to be diminished in its ability to adhere to and invade cultured human pharyngeal epithelial cells, a likely proximal step in the pathogenesis of invasive infection. Thus, teichoic acid d-alanylation may contribute in multiple ways to the propensity of invasive GAS to bypass mucosal defenses and produce systemic infection.

Alanine↗

PubMed2005

Blood-brain barrier invasion by group B Streptococcus depends upon proper cell-surface anchoring of lipoteichoic acid.

Group B streptococci (GBSs) are the leading cause of neonatal meningitis. GBSs enter the CNS by penetrating the blood-brain barrier (BBB), which consists of specialized human brain microvascular endothelial cells (hBMECs). To identify GBS factors required for BBB penetration, we generated random mutant libraries of a virulent strain and screened for loss of hBMEC invasion in vitro. Two independent hypo-invasive mutants possessed disruptions in the same gene, invasion associated gene (iagA), which encodes a glycosyltransferase homolog. Allelic replacement of iagA in the GBS chromosome produced a 4-fold decrease in hBMEC invasiveness. Mice challenged with the GBS DeltaiagA mutant developed bacteremia comparably to WT mice, yet mortality was significantly lower (20% vs. 90%), as was the incidence of meningitis. The glycolipid diglucosyldiacylglycerol, a cell membrane anchor for lipoteichoic acid (LTA) and predicted product of the IagA glycosyltransferase, was absent in the DeltaiagA mutant, which consequently shed LTA into the media. Attenuation of virulence of the DeltaiagA mutant was found to be independent of TLR2-mediated signaling, but bacterial supernatants from the DeltaiagA mutant containing released LTA inhibited hBMEC invasion by WT GBS. Our data suggest that LTA expression on the GBS surface plays a role in bacterial interaction with BBB endothelium and the pathogenesis of neonatal meningitis.

Animals↗

PubMed2005

HIF-1alpha expression regulates the bactericidal capacity of phagocytes.

Hypoxia is a characteristic feature of the tissue microenvironment during bacterial infection. Here we report on our use of conditional gene targeting to examine the contribution of hypoxia-inducible factor 1, alpha subunit (HIF-1alpha) to myeloid cell innate immune function. HIF-1alpha was induced by bacterial infection, even under normoxia, and regulated the production of key immune effector molecules, including granule proteases, antimicrobial peptides, nitric oxide, and TNF-alpha. Mice lacking HIF-1alpha in their myeloid cell lineage showed decreased bactericidal activity and failed to restrict systemic spread of infection from an initial tissue focus. Conversely, activation of the HIF-1alpha pathway through deletion of von Hippel-Lindau tumor-suppressor protein or pharmacologic inducers supported myeloid cell production of defense factors and improved bactericidal capacity. HIF-1alpha control of myeloid cell activity in infected tissues could represent a novel therapeutic target for enhancing host defense.

Animals↗

PubMed2004

Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections.

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.

Animals↗

PubMed2004

Sword and shield: linked group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense.

Group B Streptococcus (GBS) is a major cause of pneumonia, bacteremia, and meningitis in neonates and has been found to persist inside host phagocytic cells. The pore-forming GBS beta-hemolysin/cytolysin (betaH/C) encoded by cylE is an important virulence factor as demonstrated in several in vivo models. Interestingly, cylE deletion results not only in the loss of betaH/C activity, but also in the loss of a carotenoid pigment of unknown function. In this study, we sought to define the mechanism(s) by which cylE may contribute to GBS phagocyte resistance and increased virulence potential. We found that cylE-deficient GBS was more readily cleared from a mouse's bloodstream, human whole blood, and isolated macrophage and neutrophil cultures. Survival was linked to the ability of betaH/C to induce cytolysis and apoptosis of the phagocytes. At a lower bacterial inoculum, cylE also contributed to enhanced survival within phagocytes that was attributed to the ability of carotenoid to shield GBS from oxidative damage. In oxidant killing assays, cylE mutants were shown to be more susceptible to hydrogen peroxide, hypochlorite, superoxide, and singlet oxygen. Together, these data suggest a mechanism by which the linked cylE-encoded phenotypes, betaH/C (sword) and carotenoid (shield), act in partnership to thwart the immune phagocytic defenses.

Animals↗

PubMed2004

Discovery and characterization of sialic acid O-acetylation in group B Streptococcus.

Group B Streptococcus (GBS) is the leading cause of human neonatal sepsis and meningitis. The GBS capsular polysaccharide is a major virulence factor and the active principle of vaccines in phase II trials. All GBS capsules have a terminal alpha 2-3-linked sialic acid [N-acetylneuraminic acid (Neu5Ac)], which interferes with complement-mediated killing. We show here that some of the Neu5Ac residues of the GBS type III capsule are O-acetylated at carbon position 7, 8, or 9, a major modification evidently missed in previous studies. Data are consistent with initial O-acetylation at position 7, and subsequent migration of the O-acetyl ester at positions 8 and 9. O-acetylation was also present on several other GBS serotypes (Ia, Ib, II, V, and VI). Deletion of the CMP-Neu5Ac synthase gene neuA by precise, in-frame allelic replacement gave intracellular accumulation of O-acetylated Neu5Ac, whereas overexpression markedly decreased O-acetylation. Given the known GBS Neu5Ac biosynthesis pathway, these data indicate that O-acetylation occurs on free Neu5Ac, competing with the CMP-Neu5Ac synthase. O-acetylation often generates immunogenic epitopes on bacterial capsular polysaccharides and can modulate human alternate pathway complement activation. Thus, our discovery has important implications for GBS pathogenicity, immunogenicity, and vaccine design.

Acetic Acid↗

PubMed2004

Extracellular virulence factors of group B Streptococci.

Group B Streptococcus (GBS) is the leading cause of severe bacterial infections in human newborn infants. Advances in streptococcal molecular genetics and refinement of in vitro and in vivo model systems of GBS disease have led to the discovery and characterization of several extracellular virulence factors elaborated by this pathogen. This review summarizes our current understanding of GBS extracellular virulence factors including the beta-hemolysin/cytolysin, C5a-peptidase, hyaluronate lyase, CAMP factor, oligopeptidase, and carbohydrate exotoxin CM101. The molecular basis and potential pathogenic role(s) of each factor are considered in the context of neonatal infection.

Humans↗

PubMed2004

Effect of a bacterial pheromone peptide on host chemokine degradation in group A streptococcal necrotising soft-tissue infections.

BACKGROUND: Necrotising soft-tissue infections due to group A streptococcus (GAS) are rare (about 0.2 cases per 100000 people). The disease progresses rapidly, causing severe necrosis and hydrolysis of soft tissues. Histopathological analysis of necrotic tissue debrided from two patients (one with necrotising fasciitis and one with myonecrosis) showed large quantities of bacteria but no infiltrating neutrophils. We aimed to investigate whether the poor neutrophil chemotaxis was linked with the ability of group A streptococcus (GAS) to degrade host chemokines. METHODS: We did RT-PCR, ELISA, and dot-blot assays to establish whether GAS induces synthesis of interleukin 8 mRNA, but subsequently degrades the released chemokine protein. Class-specific protease inhibitors were used to characterise the protease that degraded the chemokine. We used a mouse model of human soft-tissue infections to investigate the pathogenic relevance of GAS chemokine degradation, and to test the therapeutic effect of a GAS pheromone peptide (SilCR) that downregulates activity of chemokine protease. FINDINGS: The only isolates from the necrotic tissue were two beta-haemolytic GAS strains of an M14 serotype. A trypsin-like protease released by these strains degraded human interleukin 8 and its mouse homologue MIP2. When innoculated subcutaneously in mice, these strains produced a fatal necrotic soft-tissue infection that had reduced neutrophil recruitment to the site of injection. The M14 GAS strains have a missense mutation in the start codon of silCR, which encodes a predicted 17 aminoacid pheromone peptide, SilCR. Growth of the M14 strain in the presence of SilCR abrogated chemokine proteolysis. When SilCR was injected together with the bacteria, abundant neutrophils were recruited to the site of infection, bacteria were cleared without systemic spread, and the mice survived. The therapeutic effect of SilCR was also obtained in mice challenged with M1 and M3 GAS strains, a leading cause of invasive infections. INTERPRETATION: The unusual reduction in neutrophils in necrotic tissue of people with GAS soft-tissue infections is partly caused by a GAS protease that degrades interleukin 8. In mice, degradation can be controlled by administration of SilCR, which downregulates GAS chemokine protease activity. This downregulation increases neutrophil migration to the site of infection, preventing bacterial spread and development of a fulminant lethal systemic infection.

Adult↗

PubMed2004

Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB.

A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2-D gel analysis of the secreted M1T1 proteome, coupled with MALDI-TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA- M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase-shift to a SpeB-/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte-stimulating capacity. The proteome of the SpeB-in vivo phase-shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage-encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB- phase-shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.

Animals↗

PubMed2004

Molecular pathogenesis of neonatal group B streptococcal infection: no longer in its infancy.

The process of human infection by group B Streptococcus (GBS) is complex and multifactorial. While this bacterium has adapted well to asymptomatic colonization of adult humans, it remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Sequencing of two complete GBS genomes has provided additional context for interpretation of experimental data and comparison to other well-studied pathogens. Here we review recent discoveries regarding GBS virulence mechanisms, many of which are revealed or magnified by the unique circumstances of the birthing process and the deficiencies of neonatal immune defence. Appreciation of the formidable array of GBS virulence factors underscores why this bacterium remains at the forefront of neonatal pathogens.

Adult↗