RESUMO
Staphylococcus aureus is a Gram-positive pathogen that resists many facets of innate immunity including nitric oxide (NO·). Staphylococcus aureus NO-resistance stems from its ability to evoke a metabolic state that circumvents the negative effects of reactive nitrogen species. The combination of l-lactate and peptides promotes S. aureus growth at moderate NO-levels, however, neither nutrient alone suffices. Here, we investigate the staphylococcal malate-quinone and l-lactate-quinone oxidoreductases (Mqo and Lqo), both of which are critical during NO-stress for the combined utilization of peptides and l-lactate. We address the specific contributions of Lqo-mediated l-lactate utilization and Mqo-dependent amino acid consumption during NO-stress. We show that Lqo conversion of l-lactate to pyruvate is required for the formation of ATP, an essential energy source for peptide utilization. Thus, both Lqo and Mqo are essential for growth under these conditions making them attractive candidates for targeted therapeutics. Accordingly, we exploited a modelled Mqo/Lqo structure to define the catalytic and substrate-binding residues.We also compare the S. aureus Mqo/Lqo enzymes to their close relatives throughout the staphylococci and explore the substrate specificities of each enzyme. This study provides the initial characterization of the mechanism of action and the immunometabolic roles for a newly defined staphylococcal enzyme family.
Assuntos
Ácido Láctico/química , Óxido Nítrico/metabolismo , Oxirredutases/química , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Trifosfato de Adenosina/biossíntese , Aminoácidos/metabolismo , Catálise , Ácido Láctico/metabolismo , Oxirredutases/imunologia , Oxirredutases/metabolismo , Peptídeos/metabolismo , Ácido Pirúvico/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Especificidade por Substrato , VirulênciaRESUMO
UNLABELLED: Staphylococcus aureus is a prolific human pathogen capable of causing severe invasive disease with a myriad of presentations. The ability of S. aureus to cause infection is strongly linked with its capacity to overcome the effects of innate immunity, whether by directly killing immune cells or expressing factors that diminish the impact of immune effectors. One such scenario is the induction of lactic acid fermentation by S. aureus in response to host nitric oxide (NO·). This fermentative activity allows S. aureus to balance redox during NO·-induced respiration inhibition. However, little is known about the metabolic substrates and pathways that support this activity. Here, we identify glycolytic hexose catabolism as being essential for S. aureus growth in the presence of high levels of NO·. We determine that glycolysis supports S. aureus NO· resistance by allowing for ATP and precursor metabolite production in a redox-balanced and respiration-independent manner. We further demonstrate that glycolysis is required for NO· resistance during phagocytosis and that increased levels of extracellular glucose limit the effectiveness of phagocytic killing by enhancing NO· resistance. Finally, we demonstrate that S. aureus glycolysis is essential for virulence in both sepsis and skin/soft tissue models of infection in a time frame consistent with the induction of innate immunity and host NO· production. IMPORTANCE: Staphylococcus aureus is a leading human bacterial pathogen capable of causing a wide variety of diseases that, as a result of antibiotic resistance, are very difficult to treat. The frequency of S. aureus tissue invasion suggests that this bacterium has evolved to resist innate immunity and grow using the nutrients present in otherwise sterile host tissue. We have identified glycolysis as an essential component of S. aureus virulence and attribute its importance to promoting nitric oxide resistance and growth under low oxygen conditions. Our data suggest that diabetics, a patient population characterized by excess serum glucose, may be more susceptible to S. aureus as a result of increased glucose availability. Furthermore, the essential nature of S. aureus glycolysis indicates that a newly developed glycolysis inhibitor may be a highly effective treatment for S. aureus infections.
Assuntos
Tolerância a Medicamentos , Glicólise , Óxido Nítrico/toxicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Fermentação , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Fagócitos/imunologia , Fagócitos/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , VirulênciaRESUMO
Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction.
Assuntos
Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Haemophilus influenzae/fisiologia , Domínios e Motivos de Interação entre Proteínas , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
Nontypeable Haemophilus influenzae is a major cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Colonization requires adherence to host epithelial cells, which is mediated by surface proteins such as the Hap adhesin. In this study, we identified a relationship between Hap levels in the outer membrane and lipopolysaccharide (LPS) biosynthesis enzymes. We found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB, or lsgD genes, which are involved in the synthesis of the LPS oligosaccharide core in H. influenzae strain Rd/HapS243A, resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript levels. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 or IgA1 protease or levels of p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of hap transcript levels. Consistently, in lgtC phase-off bacteria, Hap was absent from the outer membrane, and hap transcript levels were reduced. Hap localization and hap transcript levels were not related to LPS size but to the functions of the LPS biosynthesis enzymes themselves. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript levels. Together, these data highlight a novel interplay between Hap and LPS biosynthesis that can influence H. influenzae interactions with the host.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Inativação Gênica , Haemophilus influenzae/metabolismo , Lipopolissacarídeos/biossíntese , Serina Endopeptidases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Membrana Celular/genética , Células Epiteliais/microbiologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Humanos , Transporte Proteico , Serina Endopeptidases/genéticaRESUMO
Autotransporters are a large class of proteins that are found in the outer membrane of Gram-negative bacteria and are almost universally implicated in virulence. These proteins consist of a C-terminal ß-domain that is embedded in the outer membrane and an N-terminal domain that is exposed on the bacterial surface and is endowed with effector function. In this article, we review and compare the structural and functional characteristics of the Haemophilus influenzae IgA1 protease and Hap monomeric autotransporters and the H. influenzae Hia and Hsf trimeric autotransporters. All of these proteins play a role in colonization of the upper respiratory tract and in the pathogenesis of H. influenzae disease.