PepO,a CovRS‐controlled endopeptidase,disrupts Streptococcus pyogenes quorum sensing

Group A Streptococcus (GAS, Streptococcus pyogenes) is a human‐restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two‐component system and the Rgg2/3 quorum‐sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg²⁺ and a fragment of the antimicrobial peptide LL‐37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP‐type quorum‐sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non‐virulent GAS lifestyles.     

Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions

Streptococcus pyogenes (group A streptococcus, GAS) is a very important human pathogen with remarkable adaptation capabilities. Survival within the harsh host surroundings requires sensing potential on the bacterial side, which leads in particular to coordinately regulated virulence factor expression. GAS 'stand-alone' response regulators (RRs) and two-component signal transduction systems (TCSs) link the signals from the host environment with adaptive responses of the bacterial cell. Numerous putative regulatory systems emerged from GAS genome sequences. Only three RRs [Mga, RofA-like protein (RALP) and Rgg/RopB] and three TCSs (CsrRS/CovRS, FasBCAX and Ihk/Irr) have been studied in some detail with respect to their growth-phase-dependent activity and their influence on GAS-host cell interaction. In particular, the Mga-, RALP- and Rgg/RopB-regulated pathways display interconnected activities that appear to influence GAS colonization, persistence and spreading mechanisms, in a growth-phase-related fashion. Here, we have summarized our current knowledge about these RRs and TCSs to highlight the questions that should be addressed in future research on GAS pathogenicity.     

Induction of a quorum sensing pathway by environmental signals enhances group A streptococcal resistance to lysozyme

The human‐restricted pathogen Streptococcus pyogenes (Group A Streptococcus, GAS) is responsible for wide‐ranging pathologies at numerous sites in the body but has the proclivity to proliferate in individuals asymptomatically. The ability to survive in diverse tissues is undoubtedly benefited by sensory pathways that recognize environmental cues corresponding to stress and nutrient availability and thereby trigger adaptive responses. We investigated the impact that environmental signals contribute to cell‐to‐cell chemical communication [quorum sensing (QS)] by monitoring activity of the Rgg2/Rgg3 and SHP‐pheromone system in GAS. We identified metal limitation and the alternate carbon source mannose as two environmental indicators likely to be encountered by GAS in the host that significantly induced the Rgg‐SHP system. Disruption of the metal regulator MtsR partially accounted for the response to metal depletion, whereas ptsABCD was primarily responsible for QS induction due to mannose, but each sensory system induced Rgg‐SHP signaling apparently by different mechanisms. Significantly, we found that induction of QS, regardless of the GAS serotype tested, led to enhanced resistance to the antimicrobial agent lysozyme. These results indicate the benefits for GAS to integrate environmental signals with intercellular communication pathways in protection from host defenses.     

Molecular basis of H2O2 resistance mediated by Streptococcal Dpr. Demonstration of the functional involvement of the putative ferroxidase center by site-directed mutagenesis in Streptococcus suis

H(2)O(2) is an unavoidable cytotoxic by-product of aerobic life. Dpr, a recently discovered member of the Dps protein family, provides a means for catalase-negative bacteria to tolerate H(2)O(2). Potentially, Dpr could bind free intracellular iron and thus inhibit the Fenton chemistry-catalyzed formation of toxic hydroxyl radicals (H(2)O(2) + Fe(2+) --> (.)OH + (-)OH + Fe(3+)). We explored the in vivo function of Dpr in the catalase- and NADH peroxidase-negative pig and human pathogen Streptococcus suis. We show that: (i) a Dpr allelic exchange knockout mutant was hypersensitive ( approximately 10(6)-fold) to H(2)O(2), (ii) Dpr incorporated iron in vivo, (iii) a putative ferroxidase center was present in Dpr, (iv) single amino acid substitutions D74A or E78A to the putative ferroxidase center abolished the in vivo iron incorporation, and (v) the H(2)O(2) hypersensitive phenotype was complemented by wild-type Dpr or by a membrane-permeating iron chelator, but not by the site-mutated forms of Dpr. These results demonstrate that the putative ferroxidase center of Dpr is functionally active in iron incorporation and that the H(2)O(2) resistance is mediated by Dpr in vivo by its iron binding activity.     

NADH-peroxidase activity and H2O2 decomposition in a peroxidogenic strain of Streptococcus durans

Streptococcus durans S-76 can accumulate hydrogen peroxide to high concentrations under aerobic conditions when it is previously grown anaerobically. An NADH-peroxidase enzyme protects this bacterium from the bactericidal effect of H2O2. The relationship between oxygen uptake and H2O2 excretion into the medium has been investigated in cultures containing or lacking glucose under various conditions of incubation. The results obtained suggest that neither oxygen nor H2O2 regulate the cellular levels of NADH-peroxidase whose activity seems to be controlled exclusively by the availability of reduced NADH.     

Heat-induced cytotoxicity in H2O2-resistant Chinese hamster fibroblasts

Hydrogen-peroxide-resistant Chinese hamster fibroblasts, derived from the HA-1 cell line, were isolated following continuous culturing in the presence of progressively increasing concentrations of hydrogen peroxide. The hydrogen-peroxide-resistant phenotype has been stable for over 360 days following removal from H2O2 stress. These H2O2-resistant cell lines demonstrate increased resistance to hyperthermic cell killing mediated by continuous heating at 43 degrees C but not 45 degrees C. The relationship between mammalian cellular adaptation to oxidative stress mediated by H2O2 and resistance to 43 degrees C hyperthermia is discussed.     

Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2.

The manganese peroxidase produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+), is easily inactivated by the hydrogen peroxide (H2O2) presented in the reaction. We attempted to increase H2O2 resistance by the conformational stabilization around the H2O2-binding pocket. Based on its structural model, engineering of oxidizable Met273 located near the pocket to a non-oxidizable Leu showed a great improvement. Furthermore, after treatment at 1 mM H2O2 where the wild-type is completely inactivated, full activity can be retained by engineering the Asn81, which might have conformational changes due to the environment of the pocket, to a non-bulky and non-oxidizable Ser.     

Insulin signaling is inhibited by micromolar concentrations of H(2)O(2). Evidence for a role of H(2)O(2) in tumor necrosis factor alpha-mediated insulin resistance.

Both hyperglycemia and tumor necrosis factor alpha (TNFalpha) were found to induce insulin resistance at the level of the insulin receptor (IR). How this effect is mediated is, however, not understood. We investigated whether oxidative stress and production of hydrogen peroxide could be a common mediator of the inhibitory effect. We report here that micromolar concentrations of H(2)O(2) dramatically inhibit insulin-induced IR tyrosine phosphorylation (pretreatment with 500 microM H(2)O(2) for 5 min inhibits insulin-induced IR tyrosine phosphorylation to 8%), insulin receptor substrate 1 phosphorylation, as well as insulin downstream signaling such as activation of phosphatidylinositol 3-kinase (inhibited to 57%), glucose transport (inhibited to 36%), and mitogen-activated protein kinase activation (inhibited to 7.2%). Both sodium orthovanadate, a selective inhibitor of tyrosine-specific phosphatases, as well as the protein kinase C inhibitor G?6976 reduced the inhibitory effect of hydrogen peroxide on IR tyrosine phosphorylation. To investigate whether H(2)O(2) is involved in hyperglycemia- and/or TNFalpha-induced insulin resistance, we preincubated the cells with the H(2)O(2) scavenger catalase prior to incubation with 25 mM glucose, 25 mM 2-deoxyglucose, 5.7 nM TNFalpha, or 500 microM H(2)O(2), respectively, and subsequent insulin stimulation. Whereas catalase treatment completely abolished the inhibitory effect of H(2)O(2) and TNFalpha on insulin receptor autophosphorylation, it did not reverse the inhibitory effect of hyperglycemia. In conclusion, these results demonstrate that hydrogen peroxide at low concentrations is a potent inhibitor of insulin signaling and may be involved in the development of insulin resistance in response to TNFalpha.     

The Mga virulence regulon: infection where the grass is greener

Co-ordinate regulation of virulence gene expression in response to different host environments is central to the success of the group A streptococcus (GAS, Streptococcus pyogenes) as an important human pathogen. Mga represents a ubiquitous stand-alone virulence regulator that controls genes (Mga regulon) whose products are necessary for adherence, internalization and host immune evasion. Mga highly activates a core set of virulence genes, including its own gene, by directly binding to their promoters. Yet, Mga also influences expression of over 10% of the GAS genome, primarily genes and operons involved in metabolism and sugar utilization. Expression of the Mga regulon is influenced by conditions that signify favourable growth conditions, presumably allowing GAS to take advantage of promising new niches in the host. The ability of Mga to respond to growth signals clearly involves regulation of mga expression via global regulatory networks such as RALPs, Rgg/RopB and the catabolite control protein CcpA. However, the presence of predicted PTS regulatory domains (PRDs) within Mga suggests an intriguing model whereby phosphorylation of Mga by the PTS phosphorelay might link growth and sugar utilization with virulence in GAS. As Mga homologues have been found in several important Gram-positive pathogens, the Mga regulon could provide a valuable paradigm for increasing our understanding of global virulence networks in bacteria.     

Stable H2O2-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity

Hydrogen peroxide (H2O2)-resistant variants of the Chinese hamster ovary HA-1 line have been derived by culturing cells in progressively higher concentrations of H2O2 (greater than 200 days, in 50-800 microM H2O2). The H2O2-resistant phenotype has been stable for over 60 passages (240 days) following removal from the H2O2 stress. The resistant cells demonstrate both increased capacity to deplete exogenously added H2O2 from the growth medium and increased catalase activity. H2O2 resistance correlates well with catalase activity. An increase in chromosome number occurred in the cells adapted to 200-800 microM H2O2, but increases in aneuploidy and tetraploidy were not necessary for resistance. These results suggest that adaptation to chronic oxidative stress mediated by H2O2 in mammalian cells is accompanied by a stable heritable change in expression of catalase activity.     

Decrease of H2O2 plasma membrane permeability during adaptation to H2O2 in Saccharomyces cerevisiae

Contrary to what is widely believed, recent published results show that H2O2 does not freely diffuse across biomembranes. The fast removal of H2O2 by antioxidant enzymes is able to generate a gradient if H2O2 is produced in a different compartment from that containing the enzymes (Antunes, F., and Cadenas, E. (2000) FEBS Lett. 475, 121-126). In this work, we extended these studies and tested whether an active regulation of biomembranes permeability characteristics is part of the cell response to oxidative stress. Using Saccharomyces cerevisiae as a model, we showed that: (a) H2O2 gradients across the plasma membrane are formed upon exposure to external H2O2; (b) there is a correlation between the magnitude of the gradients and the resistance to H2O2; (c) there is not a correlation between the intracellular capacity to remove H2O2 and the resistance to H2O2; (d) the plasma membrane permeability to H2O2 decreases by a factor of two upon acquisition of resistance to this agent by pre-exposing cells either to nonlethal doses of H2O2 or to cycloheximide, an inhibitor of protein synthesis; and (e) erg3Delta and erg6Delta mutants, which have impaired ergosterol biosynthesis pathways, show higher plasma membrane permeability to H2O2 and are more sensitive to H2O2. Altogether, the regulation of the plasma membrane permeability to H2O2 emerged as a new mechanism by which cells respond and adapt to H2O2. The consequences of the results to cellular redox compartmentalization and to the origin and evolution of the eukaryotic cell are discussed.