Effect of Lipidation on the Localization and Activity of a Lysozyme Inhibitor in Neisseria gonorrhoeae.

The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme.IMPORTANCENeisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme.

Ceramide-enriched membrane domains in infectious biology and development.

Ceramide has been shown to be critically involved in multiple biological processes, for instance induction of apoptosis after ligation of death receptors or application of gamma-irradiation or UV-A light, respectively, regulation of cell differentiation, control of tumor cell growth, infection of mammalian cells with pathogenic bacteria and viruses or the control of embryo and organ development to name a few examples. Ceramide molecules form distinct large domains in the cell membrane, which may serve to re-organize cellular receptors and signalling molecules. Thus, in many conditions, ceramide may be involved in the spatial and temporal organisation of specific signalling pathways explaining the pleiotrophic effects of this lipid. Here, we focus on the role of ceramide and ceramide-enriched membrane domains, respectively, in bacterial infections, in particular of the lung, and sepsis. We describe the role of ceramide for infections with Neisseriae gonorhoeae, Staphylococcus aureus and Pseudomonas aeruginosa. Finally, we discuss newly emerging aspects of the cellular function of ceramide, i.e. its role in germ line and embryo development.

Neisseria gonorrhoeae PLD directly interacts with Akt kinase upon infection of primary, human, cervical epithelial cells.

Neisseria gonorrhoeae secrets a phospholipase D (NgPLD), which augments complement receptor 3 (CR3)-mediated invasion of cervical epithelial cells. To elucidate the signalling pathways triggered with gonococcus CR3-engagement and the putative function of NgPLD in these events, we analysed the contribution of the phosphoinositide-Akt pathway to cervical infection. Our data indicated that Akt plays a critical role in cervical infection. Inhibition of myosin light chain kinase, PtdIns(4,5)P2, and Akt functions resulted in decreased gonococcus invasion of primary, human, cervical epithelial cells as well as Akt kinase activity. Akt activity was similarly impaired when cervical cells were challenged with NgPLD-mutant gonococci. Conversely, the PI3-kinase inhibitor, LY294002, enhanced gonococcal invasion of, and Akt activity within, primary cervical cells. We demonstrated that NgPLD directly binds to the Akt PH domain and can compete with a natural Akt ligand, PtdIns(3,4,5)P3, for Akt binding. Collectively, our data suggested that NgPLD augments gonococcus invasion of cervical epithelia by interacting with Akt kinase in a PI3-kinase-independent manner, which results in subversion of normal cervical cell signalling.

[Ultracytochemistry of phosphohydrolase of the microorganisms and phagocytes in gonococcal infection]. / Ul'tratsitokimiia fosfogidrolaz mikroorganizmov i fagotsitov pri gonokokkovoi infektsii.

The complex study of phosphohydrolases (Mg2+ATPase, Na+,K+ATPase) and adenylate cyclase in gonococci, both in vivo and in vitro, and in phagocytes in gonococcal infection has been carried out with the use of ultracytochemical techniques. The enzymatic activity in the cellular structures of micro- and macroorganisms under study has been visualized. The prospects of the ultracytochemical approach to the study of the enzymes of nucleotide metabolism, their role in the life of eu- and prokaryotes, as well as the ways for affecting the enzymatic systems of cells are discussed.

Hyperamylasemia associated with gonococcal salpingitis and perihepatitis.

A case of hyperamylasemia associated with gonococcal salpingitis and perihepatitis (Fitzhugh-Curtis syndrome) is reported. Other potential causes of hyperamylasemia such as pancreatic, parotic, biliary, or intestinal disease were carefully excluded. In addition, the amylase to creatinine clearance ratio was within normal limits, suggesting tubal rather than pancreatic source of hyperamylasemia. This finding is of clinical importance since acute salpingitis is a prevalent disease and its initial presentation may mimic acute pancreatitis or other causes of surgical abdomen. The amylase to creatinine clearance ratio is of value in differentiating such cases from those with hyperamylasemia due to pancreatitis.