Glycolytic Dependency of High-Level Nitric Oxide Resistance and Virulence in Staphylococcus aureus

ABSTRACTStaphylococcus 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.IMPORTANCEStaphylococcusaureus 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

A Tale of Two Proteins: Insights into the Haemophilus influenzae Hap and Hia Autotransporters

<p>Nontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx that can cause localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the process of colonization enabled by bacterial surface adhesive structures called adhesins. One group of NTHi adhesins are autotransporters, proteins that have an N-terminal signal sequence, a C-terminal &#946;-barrel domain, and an internal passenger domain with effector function. The goal of this work was to increase our understanding of two NTHi autotransporters, Hap and Hia.</p><p>Hap is a monomeric autotransporter that mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap also self-associates with protein on neighboring bacteria, resulting in bacterial aggregation and microcolony formation. The Hap passenger domain contains the regions responsible for adhesive activity. To define the molecular mechanism of Hap adhesive activity, we crystallized the Hap passenger domain. Characterization of the crystal structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions occurred in the passenger domain. Progressive deletions of the &#946;-loops that comprise the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial settling. To further support that the C-terminal domain was responsible for Hap-Hap interactions,</p><p>7</p><p>we purified the wild type and truncated passenger domains and conjugated the proteins to latex beads. By light microscopy we visualized bead aggregation when the wild type passenger domain was conjugated to the beads, but not when the truncated passenger domain was conjugated. These results show that the C-terminal portion of the Hap passenger domain is responsible for Hap-Hap interactions leading to multimerization. Hap multimerization could be important in microcolony formation that leads to biofilm formation in vivo.</p><p>The ECM binding domain in located in the final 511 amino acids of the Hap passenger domain. To pin-point the region of the ECM protein fibronectin that is recognized by Hap, we spotted small fragments of fibronectin onto nitrocellulose membranes and incubated the membrane with purified Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest fibronectin region necessary for binding was comprised of the first two type III repeats, FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin, we mutated motifs in the Hap passenger domain that are important for fibronectin binding in other bacterial proteins. Based on assessment by ELISA, many of the mutations located between amino acids 525-725 caused reduced bacterial binding to fibronectin. However, no mutation totally ablated binding, suggesting that a larger Hap region is involved in fibronectin binding.</p><p>8</p><p>In an additional study, we identified a relationship between Hap levels in the outer membrane and the expression of lipopolysaccharide (LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis of 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. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the 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 wild type levels of hap transcript. 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. lgtC is one of several phase variable LPS biosynthesis genes. Using an antibody against the epitope formed in part by the lgtC gene product, we identified lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous observations, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that Hap localization in the outer membrane and hap transcript levels were not related to LPS size but instead to the functions of the LPS synthesis enzymes themselves. This relationship could be beneficial to bacteria in vivo as a way to regulate Hap expression.</p><p>9</p><p>Early models suggested that autotransporters do not require accessory factors for folding and OM insertion. However, mounting recent evidence has suggested that the Bam complex is required for OM localization of most &#946;-barrel proteins, including autotransporters. We studied the role of the Bam complex in OM localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains with mutations in the Bam complex and found that BamA and BamD were needed for Hia localization, while BamB, BamC, and BamE were not necessary. In further studies, we mutated the C-terminus of Hia and found that the final and third-to-last amino acids were the most important for outer membrane localization.</p><p>In summary, this work provides insights into the regulation and adhesive activity of Hap and the outer membrane localization of Hia. We have learned important details about these factors that shed light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.</p>Dissertatio

Glycolytic Dependency of High-Level Nitric Oxide Resistance and Virulence in Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a prolific human pathogen capable of causing severe invasive disease with a myriad of pre-sentations. The ability of S. aureus to cause infection is strongly linked with its capacity to overcome the effects of innate immu-nity, whether by directly killing immune cells or expressing factors that diminish the impact of immune effectors. One such sce-nario 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 sub-strates 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. aureusNO · 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 effective-ness 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 identifie

Structure and function of the Haemophilus influenzae autotransporters

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 β-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