Forecasting Staphylococcus aureus Infections Using Genome-Wide Association Studies, Machine Learning, and Transcriptomic Approaches.

Staphylococcus aureus is a major human and animal pathogen, colonizing diverse ecological niches within its hosts. Predicting whether an isolate will infect a specific host and its subsequent clinical fate remains unknown. In this study, we investigated the S. aureus pangenome using a curated set of 356 strains, spanning a wide range of hosts, origins, and clinical display and antibiotic resistance profiles. We used genome-wide association study (GWAS) and random forest (RF) algorithms to discriminate strains based on their origins and clinical sources. Here, we show that the presence of sak and scn can discriminate strains based on their host specificity, while other genes such as mecA are often associated with virulent outcomes. Both GWAS and RF indicated the importance of intergenic regions (IGRs) and coding DNA sequence (CDS) but not sRNAs in forecasting an outcome. Additional transcriptomic analyses performed on the most prevalent clonal complex 8 (CC8) clonal types, in media mimicking nasal colonization or bacteremia, indicated three RNAs as potential RNA markers to forecast infection, followed by 30 others that could serve as infection severity predictors. Our report shows that genetic association and transcriptomics are complementary approaches that will be combined in a single analytical framework to improve our understanding of bacterial pathogenesis and ultimately identify potential predictive molecular markers. IMPORTANCE Predicting the outcome of bacterial colonization and infections, based on extensive genomic and transcriptomic data from a given pathogen, would be of substantial help for clinicians in treating and curing patients. In this report, genome-wide association studies and random forest algorithms have defined gene combinations that differentiate human from animal strains, colonization from diseases, and nonsevere from severe diseases, while it revealed the importance of IGRs and CDS, but not small RNAs (sRNAs), in anticipating an outcome. In addition, transcriptomic analyses performed on the most prevalent clonal types, in media mimicking either nasal colonization or bacteremia, revealed significant differences and therefore potent RNA markers. Overall, the use of both genomic and transcriptomic data in a single analytical framework can enhance our understanding of bacterial pathogenesis.

A small regulatory RNA alters Staphylococcus aureus virulence by titrating RNAIII activity.

Staphylococcus aureus is an opportunistic human and animal pathogen with an arsenal of virulence factors that are tightly regulated during bacterial infection. The latter is achieved through a sophisticated network of regulatory proteins and regulatory RNAs. Here, we describe the involvement of a novel prophage-carried small regulatory S. aureus RNA, SprY, in the control of virulence genes. An MS2-affinity purification assay reveals that SprY forms a complex in vivo with RNAIII, a major regulator of S. aureus virulence genes. SprY binds to the 13th stem-loop of RNAIII, a key functional region involved in the repression of multiple mRNA targets. mRNAs encoding the repressor of toxins Rot and the extracellular complement binding protein Ecb are among the targets whose expression is increased by SprY binding to RNAIII. Moreover, SprY decreases S. aureus hemolytic activity and virulence. Our results indicate that SprY titrates RNAIII activity by targeting a specific stem loop. Thus, we demonstrate that a prophage-encoded sRNA reduces the pathogenicity of S. aureus through RNA sponge activity.

Functionality and cross-regulation of the four SprG/SprF type I toxin-antitoxin systems in Staphylococcus aureus.

Toxin-antitoxin (TA) systems are ubiquitous among bacteria, frequently expressed in multiple copies, and important for functions such as antibiotic resistance and persistence. Type I TA systems are composed of a stable toxic peptide whose expression is repressed by an unstable RNA antitoxin. Here, we investigated the functionalities, regulation, and possible cross-talk between three core genome copies of the pathogenicity island-encoded 'sprG1/sprF1' type I TA system in the human pathogen Staphylococcus aureus. Except for SprG4, all RNA from these pairs, sprG2/sprF2, sprG3/sprF3, sprG4/sprF4, are expressed in the HG003 strain. SprG2 and SprG3 RNAs encode toxic peptides whose overexpression triggers bacteriostasis, which is counteracted at the RNA level by the overexpression of SprF2 and SprF3 antitoxins. Complex formation between each toxin and its cognate antitoxin involves their overlapping 3' ends, and each SprF antitoxin specifically neutralizes the toxicity of its cognate SprG toxin without cross-talk. However, overexpression studies suggest cross-regulations occur at the RNA level between the SprG/SprF TA systems during growth. When subjected to H2O2-induced oxidative stress, almost all antitoxin levels dropped, while only SprG1 and SprF1 were reduced during phagocytosis-induced oxidative stress. SprG1, SprF1, SprF2, SprG3 and SprF3 levels also decrease during hyperosmotic stress. This suggests that novel SprG/SprF TA systems are involved in S. aureus persistence.

sRNA and cis-antisense sRNA identification in Staphylococcus aureus highlights an unusual sRNA gene cluster with one encoding a secreted peptide.

The human pathogen Staphylococcus aureus expresses a set of transcriptional factors and small RNAs (sRNAs) to adapt to environmental variations. Recent harmonization of staphylococcal sRNA data allowed us to search for novel sRNAs using DETR'PROK, a computational pipeline for identifying sRNA in prokaryotes. We performed RNA-Seq on Newman strain and identified a set of 48 sRNA candidates. To avoid bioinformatic artefacts, we applied a series of cut-offs and tested experimentally each selected intergenic region. This narrowed the field to 24 expressed sRNAs, of which 21 were new and designated with Srn identifiers. Further examination of these loci revealed that one exhibited an unusual condensed sRNA cluster of about 650 nucleotides. We determined the transcriptional start sites within this region and demonstrated the presence of three contiguous sRNA genes (srn_9342, srn_9344 and srn_9345) expressed from the positive strand, and two others (srn_9343 and srn_9346) transcribed from the opposite one. Using comparative genomics, we showed that genetic organization of the srn_9342-9346 locus is specific to Newman and that its expression is growth-phase dependent and subjected to nutrient deprivation and oxidative stress. Finally, we demonstrated that srn_9343 encodes a secreted peptide that could belong to a novel S. aureus toxin-antitoxin system.

Centrosomal localization of cyclins E and A: structural similarities and functional differences.

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G1/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.

The cyclin A centrosomal localization sequence recruits MCM5 and Orc1 to regulate centrosome reduplication.

Centrosomes are the major microtubule-organizing centers in animal cells and regulate formation of a bipolar mitotic spindle. Aberrant centrosome number causes chromosome mis-segregation, and has been implicated in genomic instability and tumor development. Previous studies have demonstrated a role for the DNA replication factors MCM5 and Orc1 in preventing centrosome reduplication. Cyclin A-Cdk2 localizes on centrosomes by means of a modular centrosomal localization sequence (CLS) that is distinct from that of cyclin E. Here, we show that cyclin A interacts with both MCM5 and Orc1 in a CLS-dependent but Cdk-independent manner. Although the MRAIL hydrophobic patch is contained within the cyclin A CLS, binding of both MCM5 and Orc1 to cyclin A does not require a wild-type hydrophobic patch. The same domain in MCM5 that mediates interaction with cyclin E also binds cyclin A, resulting in centrosomal localization of MCM5. Finally, unlike its function in DNA synthesis, MCM5-mediated inhibition of centrosome reduplication in S-phase-arrested CHO cells does not require binding to other MCM family members. These results suggest that cyclins E and A sequentially prevent centrosome reduplication throughout interphase by recruitment of DNA replication factors such as MCM5 and Orc1.

Discovery of a distinct domain in cyclin A sufficient for centrosomal localization independently of Cdk binding.

Centrosomes have recently emerged as key regulators of the cell cycle. The G1/S transition requires a functional centrosome, and centrosomal localization of numerous proteins, including cyclin/Cdk complexes, is important for the G2/M transition. Here we identify a modular centrosomal localization signal (CLS) localizing cyclin A to centrosomes independently of Cdk binding. The cyclin A CLS is located in a distinct part of the molecule compared with the cyclin E CLS and includes the MRAIL hydrophobic patch involved in substrate recognition. The cyclin A CLS interacts with p27(KIP1), and expression of p27(KIP1) removes cyclin A but not cyclin E from centrosomes. Expression of the cyclin A CLS displaces both endogenous cyclin A and E from centrosomes and inhibits DNA replication, supporting an emerging concept that DNA replication is linked to centrosomal events. Structural analysis indicates that differences in surface charge and length of the C-terminal helix explain why the MRAIL region in cyclin E is not a functional CLS. These results indicate that the cyclin A CLS may contribute to targeting and recognition of centrosomal Cdk substrates and is required for specific effects of p27(KIP1) on cyclin A-Cdk2.

Phosphorylation of TPX2 by Plx1 enhances activation of Aurora A.

Entry into mitosis requires the activation of mitotic kinases, including Aurora A and Polo-like kinase 1 (Plk1). Increased levels of these kinases are frequently found associated with human cancers, and therefore it is imperative to understand the processes leading to their activation. We demonstrate that TPX2, but neither Ajuba nor Inhibitor-2, can activate Aurora A directly. Moreover, Plx1 can induce Aurora A T-loop phosphorylation indirectly in vivo during oocyte maturation. We identify Ser204 in TPX2 as a Plx1 phosphorylation site. Mutating Ser204 to alanine decreases activation of Aurora A, whereas a phosphomimetic Asp mutant exhibits enhanced activating ability. Finally, we show that phosphorylation of TPX2 with Plx1 increases its ability to activate Aurora A. Taken together, our data indicate that Plx1 promotes activation of Aurora A, most likely through TPX2. In light of the current literature, we propose a model in which Plx1 and Aurora A activate each other in a positive feedback loop.

Phosphorylation of p53 is regulated by TPX2-Aurora A in xenopus oocytes.

p53 is an important tumor suppressor regulating the cell cycle at multiple stages in higher vertebrates. The p53 gene is frequently deleted or mutated in human cancers, resulting in loss of p53 activity. This leads to centrosome amplification, aneuploidy, and tumorigenesis, three phenotypes also observed after overexpression of the oncogenic kinase Aurora A. Accordingly, recent studies have focused on the relationship between these two proteins. p53 and Aurora A have been reported to interact in mammalian cells, but the function of this interaction remains unclear. We recently reported that Xenopus p53 can inhibit Aurora A activity in vitro but only in the absence of TPX2. Here we investigate the interplay between Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2 is required for nearly all Aurora A activation and for full p53 synthesis and phosphorylation in vivo during oocyte maturation. In vitro, phosphorylation mediated by Aurora A targets serines 129 and 190 within the DNA binding domain of p53. Glutathione S-transferase pull-down studies indicate that the interaction occurs via the p53 transactivation domain and the Aurora A catalytic domain around the T-loop. Our studies suggest that targeting of TPX2 might be an effective strategy for specifically inhibiting the phosphorylation of Aurora A substrates, including p53.

Aurora-A kinase Ser349 phosphorylation is required during Xenopus laevis oocyte maturation.

Xenopus laevis Aurora-A is phosphorylated in vivo onto three amino acids: Ser53, Thr295 and Ser349. The activation of the kinase depends on its autophosphorylation on Thr295 within the T-loop. The phosphorylation of Ser53 by still unknown kinase(s) prevents its degradation. The present work focused on the regulation of Aurora-A function via Ser349 phosphorylation. Mutagenesis of Ser349 to alanine (S349A) had few impact in vitro on the capability of the kinase to autophosphorylate as well as on its activity. These data in addition to in gel kinase assays and site-specific proteolytic digestion experiments prove that Ser349 is clearly neither a primary autophosphorylation site, nor an autophosphorylation site depending on the priming phosphorylation of Thr295. Using specific antibodies, we also show that the phosphorylation of Aurora-A Ser349 is a physiological event during Xenopus oocyte maturation triggered by progesterone. A peak of phosphorylation paralleled the decrease of Aurora activity observed between meiosis I and II. In response to progesterone, X. laevis stage VI oocytes microinjected with the Aurora-A S349A mutant proceeded normally to germinal vesicle breakdown (GVBD), but degenerated rapidly soon after. Since phosphorylation of Ser349 is responsible for a decrease in kinase activity, our results suggest that a down-regulation of Aurora-A activity involving Ser349 phosphorylation is required in the process of maturation.

Phosphorylation of maskin by Aurora-A participates in the control of sequential protein synthesis during Xenopus laevis oocyte maturation.

At the end of oogenesis, Xenopus laevis stage VI oocytes are arrested at the G2/M transition (prophase) waiting for progesterone to release the block and begin maturation. Progesterone triggers a cascade of phosphorylation events such as a decrease of pK(a) and an increase of maturating-promoting factor activity. Progression through meiosis was controlled by the sequential synthesis of several proteins. For instance, the MAPK kinase kinase c-Mos is the very first protein to be produced, whereas cyclin B1 appears only after meiosis I. After the meiotic cycles, the oocyte arrests at metaphase of meiosis II with an elevated c-Mos kinase activity (cytostatic factor). By using a two-hybrid screen, we have identified maskin, a protein involved in the control of mRNA sequential translation, as a binding partner of Aurora-A, a protein kinase necessary for oocyte maturation. Here we showed that, in vitro, Aurora-A directly binds to maskin and that both proteins can be co-immunoprecipitated from oocyte extracts, suggesting that they do associate in vivo. We also demonstrated that Aurora-A phosphorylates maskin on a Ser residue conserved in transforming acidic coiled coil proteins from Drosophila to human. When the phosphorylation of this Ser was inhibited in vivo by microinjection of synthetic peptides that mimic the maskin-phosphorylated sequence, we observed a premature maturation. Under these conditions, proteins such as cyclin B1 and Cdc6, which are normally detected only in meiosis II, were massively produced in meiosis I before the occurrence of the nuclear envelope breakdown. This result strongly suggests that phosphorylation of maskin by Aurora-A prevents meiosis II proteins from being produced during meiosis I.

Phosphorylation of histone and histone-like proteins by aurora kinases during mitosis.

Successful cell division requires that daughter cells inherit not only a complete set of chromosomes, but also only one centrosome, and similar amounts of organelles and cytoplasmic components. The different mitotic processes are driven by cell cycle-regulated protein kinases and phosphatases and their fidelity is closely monitored by a number of checkpoint mechanisms. Histone H3 is phosphorylated during mitosis, but the kinases involved were not known until recently. Recent work has revealed that Aurora kinases are required for mitotic phosphorylation of histone H3 and of its centromeric variant CENP-A. This finding has stimulated functional studies of the role(s) of Aurora kinases and H3 phosphorylation during mitosis, which are reviewed in this chapter.

Regulation of EDEN-dependent deadenylation of Aurora A/Eg2-derived mRNA via phosphorylation and dephosphorylation in Xenopus laevis egg extracts.

Deadenylation is an intimate part of the post-transcriptional regulation of maternal mRNAs in embryos. EDEN-BP is so far the only known member of a complex regulating the deadenylation of maternal mRNA in Xenopus laevis embryos in a manner that is dependent on the 3'-untranslated region called EDEN (embryo deadenylation element). In this report, we show that calcium activation of cell-free extracts triggers EDEN binding protein (EDEN-BP) dephosphorylation and concomitant deadenylation of a chimeric RNA bearing Aurora A/Eg2 EDEN sequence. Deadenylation of mRNA deprived of EDEN sequence (default deadenylation) does not change with egg activation. Kinase and phosphatase inhibitors downregulate EDEN-dependent deadenylation but they do not substantially influence default deadenylation. Using indestructible Delta90 cyclin B to revert interphase extracts to the M-phase, we show that modulation of EDEN-dependent deadenylation is independent of M-phase promoting factor (MPF) activity. These results suggest that the increase in EDEN-dependent deadenylation following egg activation is achieved, at least partially, via dephosphorylation and/or phosphorylation of regulatory proteins, including EDEN-BP dephosphorylation. This regulation proceeds in a manner independent from MPF inactivation.

A Method for Analyzing the Ubiquitination and Degradation of Aurora-A.

The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts from xenopus eggs or somatic cell lines.