Divergent Responses of Different Endothelial Cell Types to Infection with Candida albicans and Staphylococcus aureus

Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types

RpiR Homologues May Link Staphylococcus aureus RNAIII Synthesis and Pentose Phosphate Pathway Regulation ▿ †

Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant synthesis, the rpiR homologues were inactivated, and the effects on PPP activity and virulence factor synthesis were assessed. Two of the three homologues (RpiRB and RpiRC) positively influence the transcription of the PPP genes rpiA and zwf, while the third homologue (RpiRA) is slightly antagonistic to the other homologues. In addition, inactivation of RpiRC altered the temporal transcription of RNAIII, the effector molecule of the agr quorum-sensing system. These data confirm the close linkage of central metabolism and virulence determinant synthesis, and they establish a metabolic override for quorum-sensing-dependent regulation of RNAIII transcription

Influence of a Functional sigB Operon on the Global Regulators sar and agr in Staphylococcus aureus

The growth phase-dependent activity profile of the alternate transcription factor ς(B) and its effects on the expression of sar and agr were examined in three different Staphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant ς(B) activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective in rsbU. ς(B) activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3 (sarP1-P2-P3)-driven firefly luciferase (luc+) reporter gene fusion demonstrated a strong ς(B) activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or ΔrsbUVWsigB mutants. In contrast, expression of the agr locus, as measured by RNAIII levels and by an hldp::luc+ fusion, was found to be higher in the absence of ς(B) activity, such as in rsbU or ΔrsbUVWsigB mutants, than in wild-type strains. Overexpression of ς(B) in BB255 derivatives resulted in a clear increase in sarP1-P2-P3::luc+ expression as well as a strong decrease in hldp::luc+ expression. The data presented here suggest that ς(B) increases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner

Structure Analysis of the Staphylococcus aureus UDP-N-acetyl-mannosamine Dehydrogenase Cap5O Involved in Capsular Polysaccharide Biosynthesis*

Bacterial UDP-sugar dehydrogenases are part of the biosynthesis pathway of extracellular polysaccharides. These compounds act as important virulence factors by protecting the cell from opsonophagocytosis and complement-mediated killing. In Staphylococcus aureus, the protein Cap5O catalyzes the oxidation of UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid. Cap5O is crucial for the production of serotype 5 capsular polysaccharide that prevents the interaction of bacteria with both phagocytic and nonphagocytic eukaryotic cells. However, details of its catalytic mechanism remain unknown. We thus crystallized Cap5O and solved the first structure of an UDP-N-acetyl-mannosamine dehydrogenase. This study revealed that the catalytic cysteine makes a disulfide bond that has never been observed in other structurally characterized members of the NDP-sugar dehydrogenase family. Biochemical and mutagenesis experiments demonstrated that the formation of this disulfide bridge regulates the activity of Cap5O. We also identified two arginine residues essential for Cap5O activity. Previous data suggested that Cap5O is activated by tyrosine phosphorylation, so we characterized the phosphorylation site and examined the underlying regulatory mechanism