Differential survival of Staphylococcal species in macrophages.

Staphylococcus aureus is considered an extracellular pathogen, yet the bacterium is able to survive within and escape from host cells. An agr/sae mutant of strain USA300 is unable to escape from macrophages but can replicate and survive within. We questioned whether such "non-toxic" S. aureus resembles the less pathogenic coagulase-negative Staphylococcal (CoNS) species like S. epidermidis, S. carnosus, S. lugdunensis, S. capitis, S. warneri, or S. pettenkoferi. We show that the CoNS are more efficiently killed in macrophage-like THP-1 cells or in human primary macrophages. Mutations in katA, copL, the regulatory system graRS, or sigB did not impact bacterial survival in THP-1 cells. Deletion of the superoxide dismutases impaired S. aureus survival in primary macrophages but not in THP-1 cells. However, expression of the S. aureus-specific sodM in S. epidermidis was not sufficient to protect this species from being killed. Thus, at least in those cells, better bacterial survival of S. aureus could not be linked to higher protection from ROS. However, "non-toxic" S. aureus was found to be insensitive to pH, whereas most CoNS were protected when phagosomal acidification was inhibited. Thus, species differences are at least partially linked to differences in sensitivity to acidification.

Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

The 5' NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates.

Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5' ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium's physiology, was found to be 5' NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the -1 position of the P3 promoter. An increase in RNAIII's NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII's secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII's secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria.IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5' NAD cap in specific RNAs. While the presence of the 5' NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5' NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium's virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

Oxidative stress drives the selection of quorum sensing mutants in the Staphylococcus aureus population.

Quorum sensing (QS) is the central mechanism by which social interactions within the bacterial community control bacterial behavior. QS-negative cells benefit by exploiting public goods produced by the QS-proficient population. Mechanisms to keep the balance between producers and nonproducers within the population are expected but have not been elucidated for peptide-based QS systems in gram-positive pathogens. The Agr system of Staphylococcus aureus comprises the secretion and sensing of an autoinducing peptide to activate its own expression via the response regulator AgrA as well as the expression of a regulatory RNAIII and psmα/psmß coding for phenol-soluble modulins (PSMs). Agr mutants can be monitored on blood agar due to their nonhemolytic phenotype. In vitro evolution and competition experiments show that they readily accumulate in a process that is accelerated by ciprofloxacin, while the wild type (WT) is retained in the population at low numbers. However, agr mutants possess a fitness advantage only under aerobic conditions. Under hypoxia, Agr activity is increased but without the expected fitness cost. The Agr-imposed oxygen-dependent fitness cost is not due to a metabolic burden but due to the reactive oxygen species (ROS)-inducing capacity of the PSMs and RNAIII-regulated factors. Thus, selection of mutants is dictated by the QS system itself. Under aerobic conditions, emergence of agr-negative mutants may provide the population with a fitness advantage while hypoxia favors QS maintenance and even affords increased toxin production. The oxygen-driven tuning of the Agr system might be of importance to provide the pathogen with capabilities crucial for disease progression.