Transcriptomic determinants of the response of ST-111 Pseudomonas aeruginosa AG1 to ciprofloxacin identified by a top-down systems biology approach.

Pseudomonas aeruginosa is an opportunistic pathogen that thrives in diverse environments and causes a variety of human infections. Pseudomonas aeruginosa AG1 (PaeAG1) is a high-risk sequence type 111 (ST-111) strain isolated from a Costa Rican hospital in 2010. PaeAG1 has both blaVIM-2 and blaIMP-18 genes encoding for metallo-ß-lactamases, and it is resistant to ß-lactams (including carbapenems), aminoglycosides, and fluoroquinolones. Ciprofloxacin (CIP) is an antibiotic commonly used to treat P. aeruginosa infections, and it is known to produce DNA damage, triggering a complex molecular response. In order to evaluate the effects of a sub-inhibitory CIP concentration on PaeAG1, growth curves using increasing CIP concentrations were compared. We then measured gene expression using RNA-Seq at three time points (0, 2.5 and 5 h) after CIP exposure to identify the transcriptomic determinants of the response (i.e. hub genes, gene clusters and enriched pathways). Changes in expression were determined using differential expression analysis and network analysis using a top-down systems biology approach. A hybrid model using database-based and co-expression analysis approaches was implemented to predict gene-gene interactions. We observed a reduction of the growth curve rate as the sub-inhibitory CIP concentrations were increased. In the transcriptomic analysis, we detected that over time CIP treatment resulted in the differential expression of 518 genes, showing a complex impact at the molecular level. The transcriptomic determinants were 14 hub genes, multiple gene clusters at different levels (associated to hub genes or as co-expression modules) and 15 enriched pathways. Down-regulation of genes implicated in several metabolism pathways, virulence elements and ribosomal activity was observed. In contrast, amino acid catabolism, RpoS factor, proteases, and phenazines genes were up-regulated. Remarkably, > 80 resident-phage genes were up-regulated after CIP treatment, which was validated at phenomic level using a phage plaque assay. Thus, reduction of the growth curve rate and increasing phage induction was evidenced as the CIP concentrations were increased. In summary, transcriptomic and network analyses, as well as the growth curves and phage plaque assays provide evidence that PaeAG1 presents a complex, concentration-dependent response to sub-inhibitory CIP exposure, showing pleiotropic effects at the systems level. Manipulation of these determinants, such as phage genes, could be used to gain more insights about the regulation of responses in PaeAG1 as well as the identification of possible therapeutic targets. To our knowledge, this is the first report of the transcriptomic analysis of CIP response in a ST-111 high-risk P. aeruginosa strain, in particular using a top-down systems biology approach.

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox.

Staphylococcus aureus is a common commensal and frequent opportunistic pathogen that causes invasive infections that often recur. Co-evolution with the host has led to the development of toxins that affect diverse immune cell types. Recent reports have highlighted the contributions of staphylococcal protein A (SpA). This small oligomeric secreted protein contains 4-5 homologous domains with two distinct immunoglobulin-binding sites; one for IgG Fc domains, while a separate site binds an evolutionarily conserved surface on Fab encoded by VHIII clan related genes. The Fab-binding site has been implicated in in vivo supraclonal VHIII-BCR targeted B-cell depletion by an activation induced death pathway. Yet the concept of a superantigen for B lymphocytes poses a seeming paradox. Unlike TCR that are expressed only in a membrane-associated form, BCR are expressed in both a membrane BCR form and in secreted Ig forms, which permeate virtually every part of the body at high levels. We therefore asked, why circulating immunoglobulin do not block the superantigen properties of SpA? Herein, we show that soluble IgG molecules are not in vivo inhibitors of these B-cell superantigen effects but are instead essential for potentiating these properties. We also show that the Fc subclass of circulating IgG is an indirect critical determinant of the B-cell superantigen effect. In contrast, host FcγR and complement are not required for SpA mediated in vivo B-cell depletion. Unexpectedly, after VHIII-IgG2a pretreatment SpA challenge resulted in fatal anaphylactic reactions, which we speculate may have involved FcγR interactions with mast cells and basophils. Cumulatively, our findings illuminate a cunning and potent molecular strategy by which a bacterial toxin effectively confounds the contributions of host B-lymphocytes to immune defenses.