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.

The capsular polysaccharide obstructs wall teichoic acid functions in Staphylococcus aureus.

BACKGROUND: The cell envelope of Staphylococcus aureus contains two major secondary cell wall glycopolymers: capsular polysaccharide (CP) and wall teichoic acid (WTA). Both the CP and the WTA are attached to the cell wall and play distinct roles in S. aureus colonization, pathogenesis, and bacterial evasion of host immune defenses. OBJECTIVE: We aimed to investigate whether CP interferes with WTA-mediated properties. METHODS: Strains with natural heterogeneous expression of CP, strains with homogeneous high CP expression and CP-deficient strains were compared to WTA deficient controls regarding WTA dependent phage binding, cell adhesion, IgG deposition, and virulence in vivo. RESULTS: WTA-mediated phage adsorption, specific antibody deposition and cell adhesion were negatively correlated with CP expression. WTA, but not CP, enhanced the bacterial burden in a mouse abscess model, while CP overexpression resulted in intermediate virulence in vivo. CONCLUSIONS: CP protects the bacteria from WTA-dependent opsonization and phage binding. This protection comes at the cost of diminished adhesion to host cells. The highly complex regulation and mostly heterogeneous expression of CP has probably evolved to ensure the survival and optimal physiological adaptation of the bacterial population as a whole.

Methicillin-resistant Staphylococcus aureus alters cell wall glycosylation to evade immunity.

Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.

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.

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.

Exotoxins from Staphylococcus aureus activate 5-lipoxygenase and induce leukotriene biosynthesis.

Massive neutrophil infiltration is an early key event in infectious inflammation, accompanied by chemotactic leukotriene (LT)B4 generation. LTB4 biosynthesis is mediated by 5-lipoxygenase (5-LOX), but which pathogenic factors cause 5-LOX activation during bacterial infections is elusive. Here, we reveal staphylococcal exotoxins as 5-LOX activators. Conditioned medium of wild-type Staphylococcus aureus but not of exotoxin-deficient strains induced 5-LOX activation in transfected HEK293 cells. Two different staphylococcal exotoxins mimicked the effects of S. aureus-conditioned medium: (1) the pore-forming toxin α-hemolysin and (2) amphipathic α-helical phenol-soluble modulin (PSM) peptides. Interestingly, in human neutrophils, 5-LOX activation was exclusively evoked by PSMs, which was prevented by the selective FPR2/ALX receptor antagonist WRW4. 5-LOX activation by PSMs was confirmed in vivo as LT formation in infected paws of mice was impaired in response to PSM-deficient S. aureus. Conclusively, exotoxins from S. aureus are potent pathogenic factors that activate 5-LOX and induce LT formation in neutrophils.

Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus.

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

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.

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus.

Capsular polysaccharide (CP) biosynthesis in Staphylococcus aureus is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in nongrowing cells. Expression of the corresponding biosynthesis gene cluster is driven by one promoter element (Pcap ). Here, we demonstrate that Pcap contains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat (IR) that is crucial for cap expression. The essentiality of the IR is derived from this region acting as a SigB binding site rather than as an operator site for the proposed cap activators RbsR and MsaB. Furthermore, Pcap contains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for cap repressors such as SaeR, CodY and Rot. Heterogeneous CP synthesis is determined by SigB activity and repressor binding to the upstream region. SigB dependency and regulation by the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even when transcription is rendered constitutive, suggesting additional posttranscriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis.

Function and regulation of Staphylococcus aureus wall teichoic acids and capsular polysaccharides.

Staphylococcus aureus produces different secondary cell wall glycopolymers such as wall teichoic acids (WTA) and capsular polysaccharides (CP). These structures play an important role in S. aureus colonization, pathogenesis and bacterial evasion of the host immune defences. To fulfil their diverse functions, biosynthesis of both glycopolymers has to be tightly controlled. Regulation of WTA biosynthesis and modification is only partially understood. The transcription factor MgrA and the two-component systems (TCS) Agr, GraRS, and ArlRS control WTA export, chain-length and modification. CP synthesis is determined by transcriptional and post-transcriptional regulatory circuits. On the transcriptional level expression of the capA-P operon is mainly driven by the alternative Sigma factor B and modulated by several transcriptional factors and TCS. Post-transcriptional mechanisms are in place to avoid conflict between precursor usage by the CP synthesis machinery and the synthesis machinery of other cell wall glycopolymers. The complex interplay of these regulatory systems determines the peculiar, strictly temporal expression of CP in the late growth phase and the high degree of phenotypic heterogeneity. Differential expression of CP, WTA and its modification systems during infection and colonisation are likely important for disease development, immune escape and survival within the host.

Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus.

In gram-positive bacteria, RNase J1, RNase J2 and RNase Y are thought to be major contributors to mRNA degradation and maturation. In Staphylococcus aureus, RNase Y activity is restricted to regulating the mRNA decay of only certain transcripts. Here the saePQRS operon was used as a model to analyze RNase Y specificity in living cells. A RNase Y cleavage site is located in an intergenic region between saeP and saeQ. This cleavage resulted in rapid degradation of the upstream fragment and stabilization of the downstream fragment. Thereby, the expression ratio of the different components of the operon was shifted towards saeRS, emphasizing the regulatory role of RNase Y activity. To assess cleavage specificity different regions surrounding the sae CS were cloned upstream of truncated gfp, and processing was analyzed in vivo using probes up- and downstream of CS. RNase Y cleavage was not determined by the cleavage site sequence. Instead a 24-bp double-stranded recognition structure was identified that was required to initiate cleavage 6 nt upstream. The results indicate that RNase Y activity is determined by secondary structure recognition determinants, which guide cleavage from a distance.

Long Noncoding RNA SSR42 Controls Staphylococcus aureus Alpha-Toxin Transcription in Response to Environmental Stimuli.

Staphylococcus aureus is a human pathogen causing a variety of diseases by versatile expression of a large set of virulence factors that most prominently features the cytotoxic and hemolytic pore-forming alpha-toxin. Expression of alpha-toxin is regulated by an intricate network of transcription factors. These include two-component systems sensing quorum and environmental signals as well as regulators reacting to the nutritional status of the pathogen. We previously identified the repressor of surface proteins (Rsp) as a virulence regulator. Acute cytotoxicity and hemolysis are strongly decreased in rsp mutants, which are characterized by decreased transcription of toxin genes as well as loss of transcription of a 1,232-nucleotide (nt)-long noncoding RNA (ncRNA), SSR42. Here, we show that SSR42 is the effector of Rsp in transcription regulation of the alpha-toxin gene, hla SSR42 transcription is enhanced after exposure of S. aureus to subinhibitory concentrations of oxacillin which thus leads to an SSR42-dependent increase in hemolysis. Aside from Rsp, SSR42 transcription is under the control of additional global regulators, such as CodY, AgrA, CcpE, and σB, but is positioned upstream of the two-component system SaeRS in the regulatory cascade leading to alpha-toxin production. Thus, alpha-toxin expression depends on two long ncRNAs, SSR42 and RNAIII, which control production of the cytolytic toxin on the transcriptional and translational levels, respectively, with SSR42 as an important regulator of SaeRS-dependent S. aureus toxin production in response to environmental and metabolic signals.IMPORTANCEStaphylococcus aureus is a major cause of life-threatening infections. The bacterium expresses alpha-toxin, a hemolysin and cytotoxin responsible for many of the pathologies of S. aureus Alpha-toxin production is enhanced by subinhibitory concentrations of antibiotics. Here, we show that this process is dependent on the long noncoding RNA, SSR42. Further, SSR42 itself is regulated by several global regulators, thereby integrating environmental and nutritional signals that modulate hemolysis of the pathogen.

European external quality assessments for identification, molecular typing and characterization of Staphylococcus aureus.

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols. Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed. Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes. Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16.

In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutrophaIMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

Influence of Sae-regulated and Agr-regulated factors on the escape of Staphylococcus aureus from human macrophages.

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild-type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol-soluble modulins (PSMs) for phagosomal escape. Thus, Sae-regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis-related caspase 3, 7 or 8 nor to NLRP3-dependent inflammasome activation.

Phenotypic heterogeneity and temporal expression of the capsular polysaccharide in Staphylococcus aureus.

Bacteria respond to ever-changing environments through several adaptive strategies. This includes mechanisms leading to a high degree of phenotypic variability within a genetically homogeneous population. In Staphylococcus aureus, the capsular polysaccharide (CP) protects against phagocytosis, but also impedes adherence to endothelial cells and/or matrix proteins. We analysed the regulation of core biosynthesis genes (capA-P) necessary for CP synthesis using single-cell assays (immunofluorescence and promoter-activity). In persistent human carriers, we found a distinct subpopulation of nasal S. aureus to be CP positive. In vitro, cap expression is also heterogeneous and strongly growth-phase dependent. We asked whether this peculiar expression pattern (earlyOff/lateHeterogen) is orchestrated by the quorum system Agr. We show that the Agr-driven effector molecule RNAIII promotes cap expression largely via inactivation of the repressor Rot. High NaCl, deletion of CodY or Sae also resulted in higher cap expression but did not change the earlyOFF/lateHeterogen expression pattern. Activity of the quorum system itself is largely homogenous and does not account for the observed heterogeneity of cap expression or the strictly growth phase dependent expression. Our findings are in contrast to the prevailing view that quorum sensing is the main driving force for virulence gene expression when bacterial cell densities increase.

The catabolite control protein E (CcpE) affects virulence determinant production and pathogenesis of Staphylococcus aureus.

Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus.

rRNA regulation during growth and under stringent conditions in Staphylococcus aureus.

The control of rRNA synthesis and, thereby, translation is vital for adapting to changing environmental conditions. The decrease of rRNA is a common feature of the stringent response, which is elicited by the rapid synthesis of (p)ppGpp. Here we analysed the properties and regulation of one representative rRNA operon of Staphylococcus aureus under stringent conditions and during growth. The promoters, P1 and P2, are severely downregulated at low intracellular guanosine triphosphate (GTP) concentrations either imposed by stringent conditions or in a guanine auxotroph guaBA mutant. In a (p)ppGpp(0) strain, the GTP level increased under stringent conditions, and rRNA transcription was upregulated. The correlation of the intracellular GTP levels and rRNA promoter activity could be linked to GTP nucleotides in the initiation region of both promoters at positions between +1 and +4. This indicates that not only transcriptional initiation, but also the first steps of elongation, requires high concentrations of free nucleotides. However, the severe downregulation of rRNA in post-exponential growth phase is independent of (p)ppGpp, the composition of the initiation region and the intracellular nucleotide pool. In summary, rRNA transcription in S. aureus is only partially and presumably indirectly controlled by (p)ppGpp.

Methionine biosynthesis in Staphylococcus aureus is tightly controlled by a hierarchical network involving an initiator tRNA-specific T-box riboswitch.

In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5'-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA (tRNAi(fMet)) while binding of elongator tRNA(Met) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5' met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence tRNAi(fMet) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.