Prophage terminase with tRNase activity sensitizes Salmonella enterica to oxidative stress.

Phage viruses shape the evolution and virulence of their bacterial hosts. The Salmonella enterica genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNALeu. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. S. enterica adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.

Anaerobic respiration of host-derived methionine sulfoxide protects intracellular Salmonella from the phagocyte NADPH oxidase.

Intracellular Salmonella experiencing oxidative stress downregulates aerobic respiration. To maintain cellular energetics during periods of oxidative stress, intracellular Salmonella must utilize terminal electron acceptors of lower energetic value than molecular oxygen. We show here that intracellular Salmonella undergoes anaerobic respiration during adaptation to the respiratory burst of the phagocyte NADPH oxidase in macrophages and in mice. Reactive oxygen species generated by phagocytes oxidize methionine, generating methionine sulfoxide. Anaerobic Salmonella uses the molybdenum cofactor-containing DmsABC enzymatic complex to reduce methionine sulfoxide. The enzymatic activity of the methionine sulfoxide reductase DmsABC helps Salmonella maintain an alkaline cytoplasm that supports the synthesis of the antioxidant hydrogen sulfide via cysteine desulfuration while providing a source of methionine and fostering redox balancing by associated dehydrogenases. Our investigations demonstrate that nontyphoidal Salmonella responding to oxidative stress exploits the anaerobic metabolism associated with dmsABC gene products, a pathway that has accrued inactivating mutations in human-adapted typhoidal serovars.

The methylglyoxal pathway is a sink for glutathione in Salmonella experiencing oxidative stress.

Salmonella suffer the cytotoxicity of reactive oxygen species generated by the phagocyte NADPH oxidase in the innate host response. Periplasmic superoxide dismutases, catalases and hydroperoxidases detoxify superoxide and hydrogen peroxide (H2O2) synthesized in the respiratory burst of phagocytic cells. Glutathione also helps Salmonella combat the phagocyte NADPH oxidase; however, the molecular mechanisms by which this low-molecular-weight thiol promotes resistance of Salmonella to oxidative stress are currently unknown. We report herein that Salmonella undergoing oxidative stress transcriptionally and functionally activate the methylglyoxal pathway that branches off from glycolysis. Activation of the methylglyoxal pathway consumes a substantial proportion of the glutathione reducing power in Salmonella following exposure to H2O2. The methylglyoxal pathway enables Salmonella to balance glucose utilization with aerobic respiratory outputs. Salmonella take advantage of the metabolic flexibility associated with the glutathione-consuming methylglyoxal pathway to resist reactive oxygen species generated by the enzymatic activity of the phagocyte NADPH oxidase in macrophages and mice. Taken together, glutathione fosters oxidative stress resistance in Salmonella against the antimicrobial actions of the phagocyte NADPH oxidase by promoting the methylglyoxal pathway, an offshoot metabolic adaptation of glycolysis.

Gre factors help Salmonella adapt to oxidative stress by improving transcription elongation and fidelity of metabolic genes.

Detoxification, scavenging, and repair systems embody the archetypical antioxidant defenses of prokaryotic and eukaryotic cells. Metabolic rewiring also aids with the adaptation of bacteria to oxidative stress. Evolutionarily diverse bacteria combat the toxicity of reactive oxygen species (ROS) by actively engaging the stringent response, a stress program that controls many metabolic pathways at the level of transcription initiation via guanosine tetraphosphate and the α-helical DksA protein. Studies herein with Salmonella demonstrate that the interactions of structurally related, but functionally unique, α-helical Gre factors with the secondary channel of RNA polymerase elicit the expression of metabolic signatures that are associated with resistance to oxidative killing. Gre proteins both improve transcriptional fidelity of metabolic genes and resolve pauses in ternary elongation complexes of Embden-Meyerhof-Parnas (EMP) glycolysis and aerobic respiration genes. The Gre-directed utilization of glucose in overflow and aerobic metabolism satisfies the energetic and redox demands of Salmonella, while preventing the occurrence of amino acid bradytrophies. The resolution of transcriptional pauses in EMP glycolysis and aerobic respiration genes by Gre factors safeguards Salmonella from the cytotoxicity of phagocyte NADPH oxidase in the innate host response. In particular, the activation of cytochrome bd protects Salmonella from phagocyte NADPH oxidase-dependent killing by promoting glucose utilization, redox balancing, and energy production. Control of transcription fidelity and elongation by Gre factors represent important points in the regulation of metabolic programs supporting bacterial pathogenesis.

StkP- and PhpP-Mediated Posttranslational Modifications Modulate the S. pneumoniae Metabolism, Polysaccharide Capsule, and Virulence.

Pneumococcal Ser/Thr kinase (StkP) and its cognate phosphatase (PhpP) play a crucial role in bacterial cytokinesis. However, their individual and reciprocal metabolic and virulence regulation-related functions have yet to be adequately investigated in encapsulated pneumococci. Here, we demonstrate that the encapsulated pneumococcal strain D39-derived D39ΔPhpP and D39ΔStkP mutants displayed differential cell division defects and growth patterns when grown in chemically defined media supplemented with glucose or nonglucose sugars as the sole carbon source. Microscopic and biochemical analyses supported by RNA-seq-based global transcriptomic analyses of these mutants revealed significantly down- and upregulated polysaccharide capsule formation and cps2 genes in D39ΔPhpP and D39ΔStkP mutants, respectively. While StkP and PhpP individually regulated several unique genes, they also participated in sharing the regulation of the same set of differentially regulated genes. Cps2 genes were reciprocally regulated in part by the StkP/PhpP-mediated reversible phosphorylation but independent of the MapZ-regulated cell division process. StkP-mediated dose-dependent phosphorylation of CcpA proportionately inhibited CcpA-binding to Pcps2A, supporting increased cps2 gene expression and capsule formation in D39ΔStkP. While the attenuation of the D39ΔPhpP mutant in two mouse infection models corroborated with several downregulated capsules-, virulence-, and phosphotransferase systems (PTS)-related genes, the D39ΔStkP mutant with increased amounts of polysaccharide capsules displayed significantly decreased virulence in mice compared to the D39 wild-type, but more virulence compared to D39ΔPhpP. NanoString technology-based inflammation-related gene expression and Meso Scale Discovery-based multiplex chemokine analysis of human lung cells cocultured with these mutants confirmed their distinct virulence phenotypes. StkP and PhpP may, therefore, serve as critical therapeutic targets.

Promiscuity of response regulators for thioredoxin steers bacterial virulence.

The exquisite specificity between a sensor kinase and its cognate response regulator ensures faithful partner selectivity within two-component pairs concurrently firing in a single bacterium, minimizing crosstalk with other members of this conserved family of paralogous proteins. We show that conserved hydrophobic and charged residues on the surface of thioredoxin serve as a docking station for structurally diverse response regulators. Using the OmpR protein, we identify residues in the flexible linker and the C-terminal ß-hairpin that enable associations of this archetypical response regulator with thioredoxin, but are dispensable for interactions of this transcription factor to its cognate sensor kinase EnvZ, DNA or RNA polymerase. Here we show that the promiscuous interactions of response regulators with thioredoxin foster the flow of information through otherwise highly dedicated two-component signaling systems, thereby enabling both the transcription of Salmonella pathogenicity island-2 genes as well as growth of this intracellular bacterium in macrophages and mice.

Oxidative stress activates transcription of Salmonella pathogenicity island-2 genes in macrophages.

The type III secretion system encoded in the Salmonella pathogenicity island-2 (SPI-2) gene cluster facilitates intracellular growth of nontyphoidal Salmonella by interfering with the maturation of Salmonella-containing vacuoles along the degradative pathway. SPI-2 gene products also protect Salmonella against the antimicrobial activity of reactive oxygen species (ROS) synthesized by the phagocyte NADPH oxidase 2 (NOX2). However, a potential relationship between inflammatory ROS and the activation of transcription of SPI-2 genes by intracellular Salmonella is unclear. Here, we show that ROS engendered in the innate host response stimulate SPI-2 gene transcription. We found that the expression of SPI-2 genes in Salmonella-sustaining oxidative stress conditions involves DksA, a protein otherwise known to regulate the stringent response of bacteria to nutritional stress. We also demonstrate that the J and zinc-2-oxidoreductase domains of DnaJ as well as the ATPase activity of the DnaK chaperone facilitate loading of DksA onto RNA polymerase complexed with SPI-2 promoters. Furthermore, the DksA-driven transcription of SPI-2 genes in Salmonella experiencing oxidative stress is contingent on upstream OmpR, PhoP, and SsrB signaling events that participate in the removal of nucleoid proteins while simultaneously recruiting RNA polymerase to SPI-2 promoter regions. Taken together, our results suggest the activation of SPI-2 gene transcription in Salmonella subjected to ROS produced by the respiratory burst of macrophages protects this intracellular pathogen against NOX2-mediated killing. We propose that Salmonella have co-opted inflammatory ROS to induce SPI-2-mediated protective responses against NOX2 host defenses.

Lipopolysaccharide from the commensal microbiota of the breast enhances cancer growth: role of S100A7 and TLR4.

The role of commensal bacterial microbiota in the pathogenesis of human malignancies has been a research field of incomparable progress in recent years. Although breast tissue is commonly assumed to be sterile, recent studies suggest that human breast tissue may contain a bacterial microbiota. In this study, we used an immune-competent orthotopic breast cancer mouse model to explore the existence of a unique and independent bacterial microbiota in breast tumors. We observed some similarities in breast cancer microbiota with skin; however, breast tumor microbiota was mainly enriched with Gram-negative bacteria, serving as a primary source of lipopolysaccharide (LPS). In addition, dextran sulfate sodium (DSS) treatment in late-stage tumor lesions increased LPS levels in the breast tissue environment. We also discovered an increased expression of S100A7 and low level of TLR4 in late-stage tumors with or without DSS as compared to early-stage tumor lesions. The treatment of breast cancer cells with LPS increased the expression of S100A7 in breast cancer cells in vitro. Furthermore, S100A7 overexpression downregulated TLR4 and upregulated RAGE expression in breast cancer cells. Analysis of human breast cancer samples also highlighted the inverse correlation between S100A7 and TLR4 expression. Overall, these findings suggest that the commensal microbiota of breast tissue may enhance breast tumor burden through a novel LPS/S100A7/TLR4/RAGE signaling axis.

Macrophages in Microbial Pathogenesis: Commonalities of Defense Evasion Mechanisms.

Macrophages are key arsenals of the immune system against invaders. After compartmental isolation of a pathogen in phagosomes, the host immune response attempts to neutralize the pathogen. However, pathogens possess the ability to subvert these assaults and can also convert macrophages into their replicative niche. The multiple host defense evasion mechanisms employed by these pathogens include phagosome maturation arrest, molecular mimicry through secretory antigens, interference with host signaling, active radical neutralization, inhibition of phagosome acidification, alteration of programmed cell death, and other mechanisms. Macrophage biology as a part of the host-pathogen interaction has expanded rapidly in the past decade. The present review aims to shed some light upon the macrophage defense evasion strategies employed by pathogens. We have also incorporated recent knowledge in the field of macrophage dynamics during infection and evolutionary perspectives of macrophage dynamics.

M.tb-Rv2462c of Mycobacterium tuberculosis Shows Chaperone-like Activity and Plays a Role in Stress Adaptation and Immunomodulation.

Mycobacterium tuberculosis (M.tb)-encoded factors protect it against host-generated stresses and support its survival in the hostile host environment. M.tb possesses two peptidyl-prolyl cis-trans isomerases and a probable trigger factor encoded by Rv2462c which has an FKBP-like PPIase domain. PPIases are known to assist the folding of peptidyl-prolyl bonds and are involved in various cellular processes important for bacterial survival in host-generated stresses. In this study, we aim to functionally characterize Rv2462c of M.tb. Our data suggest that the trigger factor of M.tb exhibits chaperone activity both in vitro and in vivo. Heterologous expression of M.tb-Rv2462c locus into Mycobacterium smegmatis enhanced its survival within macrophages, adaptation to oxidative stress and biofilm formation. M.tb-trigger factor has strong immunomodulatory potential and modifies the cytokine profile of the host towards the proinflammatory axis.

Novel Tyrosine Kinase-Mediated Phosphorylation With Dual Specificity Plays a Key Role in the Modulation of Streptococcus pyogenes Physiology and Virulence.

Streptococcus pyogenes (Group A Streptococcus, GAS) genomes do not contain a gene encoding a typical bacterial-type tyrosine kinase (BY-kinase) but contain an orphan gene-encoding protein Tyr-phosphatase (SP-PTP). Hence, the importance of Tyr-phosphorylation is underappreciated and not recognized for its role in GAS pathophysiology and pathogenesis. The fact that SP-PTP dephosphorylates Abl-tyrosine kinase-phosphorylated myelin basic protein (MBP), and SP-STK (S. pyogenes Ser/Thr kinase) also autophosphorylates its Tyr101-residue prompted us to identify a putative tyrosine kinase and Tyr-phosphorylation in GAS. Upon a genome-wide search of kinases possessing a classical Walker motif, we identified a non-canonical tyrosine kinase M5005_Spy_1476, a ∼17 kDa protein (153 aa) (SP-TyK). The purified recombinant SP-TyK autophosphorylated in the presence of ATP. In vitro and in vivo phosphoproteomic analyses revealed two key phosphorylated tyrosine residues located within the catalytic domain of SP-TyK. An isogenic mutant lacking SP-TyK derived from the M1T1 strain showed a retarded growth pattern. It displayed defective cell division and long chains with multiple parallel septa, often resulting in aggregates. Transcriptomic analysis of the mutant revealed 287 differentially expressed genes responsible for GAS pathophysiology and pathogenesis. SP-TyK also phosphorylated GAS CovR, WalR, SP-STP, and SDH/GAPDH proteins with dual specificity targeting their Tyr/Ser/Thr residues as revealed by biochemical and mass-spectrometric-based phosphoproteomic analyses. SP-TyK-phosphorylated CovR bound to PcovR efficiently. The mutant displayed sustained release of IL-6 compared to TNF-α during co-culturing with A549 lung cell lines, attenuation in mice sepsis model, and significantly reduced ability to adhere to and invade A549 lung cells and form biofilms on abiotic surfaces. SP-TyK, thus, plays a critical role in fine-tuning the regulation of key cellular functions essential for GAS pathophysiology and pathogenesis through post-translational modifications and hence, may serve as a promising target for future therapeutic developments.

Activated Protein Phosphatase 2A Disrupts Nutrient Sensing Balance Between Mechanistic Target of Rapamycin Complex 1 and Adenosine Monophosphate-Activated Protein Kinase, Causing Sarcopenia in Alcohol-Associated Liver Disease.

BACKGROUND AND AIMS: Despite the high clinical significance of sarcopenia in alcohol-associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate-activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown). APPROACH AND RESULTS: Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol-associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a "pseudofed" state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild-type mice were observed in PI3Kγ-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations. CONCLUSIONS: Our study describes the mechanistic underpinnings for ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.

Lessons from SARS-CoV-2 Pandemic: Evolution, Disease Dynamics and Future.

The COVID-19 pandemic is rising at an unprecedented rate. The surging number of deaths every day, global lockdown and travel restrictions have resulted in huge losses to society. The impact is massive and will leave a historical footprint. The Spanish Flu of 1918, which was the last pandemic that had a similar impact, was shadowed under the consequences of World War I. All the brilliance, strength and economies of countries worldwide are aimed at fighting the COVID-19 pandemic. The knowledge about coronavirus dynamics, its nature and epidemiology are expanding every day. The present review aims to summarize the structure, epidemiology, symptoms, statistical status of the disease status, intervention strategies and deliberates the lessons learnt during the pandemic. The intervention approaches, antiviral drug repurposing and vaccine trials are intensified now. Statistical interpretations of disease dynamics and their projections may help the decision-makers.

Resistin in metabolism, inflammation, and disease.

Resistin is a small secretory protein that has a pleiotropic role in rodents and humans. Both rodent resistin and human resistin have an extremely stable and high-order multimeric structure. Moreover, there is significant variation in the source of secretion and the diversity of functions of resistin. Mouse resistin resists insulin action and contributes to type 2 diabetes mellitus, while human resistin plays a role in inflammation and also functions as a small accessory chaperone. Currently, active research in the area identified a significant role for resistin in stress biology and as a biomarker in diagnostics to evaluate disease status and treatment outcome. This review summarizes recent developments within resistin biology including their association with obesity, inflammation, stress response mechanisms, and its role in clinical diagnostics.

SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.

Impaired Ribosomal Biogenesis by Noncanonical Degradation of ß-Catenin during Hyperammonemia.

Increased ribosomal biogenesis occurs during tissue hypertrophy, but whether ribosomal biogenesis is impaired during atrophy is not known. We show that hyperammonemia, which occurs in diverse chronic disorders, impairs protein synthesis as a result of decreased ribosomal content and translational capacity. Transcriptome analyses, real-time PCR, and immunoblotting showed consistent reductions in the expression of the large and small ribosomal protein subunits (RPL and RPS, respectively) in hyperammonemic murine skeletal myotubes, HEK cells, and skeletal muscle from hyperammonemic rats and human cirrhotics. Decreased ribosomal content was accompanied by decreased expression of cMYC, a positive regulator of ribosomal biogenesis, as well as reduced expression and activity of ß-catenin, a transcriptional activator of cMYC. However, unlike the canonical regulation of ß-catenin via glycogen synthase kinase 3ß (GSK3ß)-dependent degradation, GSK3ß expression and phosphorylation were unaltered during hyperammonemia, and depletion of GSK3ß did not prevent ammonia-induced degradation of ß-catenin. Overexpression of GSK3ß-resistant variants, genetic depletion of IκB kinase ß (IKKß) (activated during hyperammonemia), protein interactions, and in vitro kinase assays showed that IKKß phosphorylated ß-catenin directly. Overexpressing ß-catenin restored hyperammonemia-induced perturbations in signaling responses that regulate ribosomal biogenesis. Our data show that decreased protein synthesis during hyperammonemia is mediated via a novel GSK3ß-independent, IKKß-dependent impairment of the ß-catenin-cMYC axis.

Ethanol sensitizes skeletal muscle to ammonia-induced molecular perturbations.

Ethanol causes dysregulated muscle protein homeostasis while simultaneously causing hepatocyte injury. Because hepatocytes are the primary site for physiological disposal of ammonia, a cytotoxic cellular metabolite generated during a number of metabolic processes, we determined whether hyperammonemia aggravates ethanol-induced muscle loss. Differentiated murine C2C12 myotubes, skeletal muscle from pair-fed or ethanol-treated mice, and human patients with alcoholic cirrhosis and healthy controls were used to quantify protein synthesis, mammalian target of rapamycin complex 1 (mTORC1) signaling, and autophagy markers. Alcohol-metabolizing enzyme expression and activity in mouse muscle and myotubes and ureagenesis in hepatocytes were quantified. Expression and regulation of the ammonia transporters, RhBG and RhCG, were quantified by real-time PCR, immunoblots, reporter assays, biotin-tagged promoter pulldown with proteomics, and loss-of-function studies. Alcohol and aldehyde dehydrogenases were expressed and active in myotubes. Ethanol exposure impaired hepatocyte ureagenesis, induced muscle RhBG expression, and elevated muscle ammonia concentrations. Simultaneous ethanol and ammonia treatment impaired protein synthesis and mTORC1 signaling and increased autophagy with a consequent decreased myotube diameter to a greater extent than either treatment alone. Ethanol treatment and withdrawal followed by ammonia exposure resulted in greater impairment in muscle signaling and protein synthesis than ammonia treatment in ethanol-naive myotubes. Of the three transcription factors that were bound to the RhBG promoter in response to ethanol and ammonia, DR1/NC2 indirectly regulated transcription of RhBG during ethanol and ammonia treatment. Direct effects of ethanol were synergistic with increased ammonia uptake in causing dysregulated skeletal muscle proteostasis and signaling perturbations with a more severe sarcopenic phenotype.

A novel STK1-targeted small-molecule as an "antibiotic resistance breaker" against multidrug-resistant Staphylococcus aureus.

Ser/Thr protein kinase (STK1) plays a critical role in cell wall biosynthesis of and drug resistance in methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains lacking STK1 become susceptible to failing cephalosporins, such as Ceftriaxone and Cefotaxime. STK1, despite being nonessential protein for MRSA survival, it can serve as an important therapeutic agent for combination therapy. Here, we report a novel small molecule quinazoline compound, Inh2-B1, which specifically inhibits STK1 activity by directly binding to its ATP-binding catalytic domain. Functional analyses encompassing in vitro growth inhibition of MRSA, and in vivo protection studies in mice against the lethal MRSA challenge indicated that at high concentration neither Inh2-B1 nor Ceftriaxone or Cefotaxime alone was able to inhibit the growth of bacteria or protect the challenged mice. However, the growth of MRSA was inhibited, and a significant protection in mice against the bacterial challenge was observed at a micromolar concentration of Ceftriaxone or Cefotaxime in the presence of Inh2-B1. Cell-dependent minimal to no toxicity of Inh2-B1, and its abilities to down-regulate cell wall hydrolase genes and disrupt the biofilm formation of MRSA clearly indicated that Inh2-B1 serves as a therapeutically important "antibiotic-resistance-breaker," which enhances the bactericidal activity of Ceftriaxone/Cefotaxime against highly pathogenic MRSA infection.

SpyB, a Small Heme-Binding Protein, Affects the Composition of the Cell Wall in Streptococcus pyogenes.

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to ß-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.

The Streptococcus pyogenes orphan protein tyrosine phosphatase, SP-PTP, possesses dual specificity and essential virulence regulatory functions.

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP-PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP-PTP is, therefore, questionable. Here, we demonstrate that SP-PTP possesses dual phosphatase specificity for Tyr- and Ser/Thr-phosphorylated GAS proteins, such as Ser/Thr kinase (SP-STK) and the SP-STK-phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP-PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP-PTP displayed increased Ser-/Thr-/Tyr-phosphorylation. SP-PTP also differentially regulates the expression of ∼50% of the total GAS genes, including several virulence genes potentially through the two-component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP-PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr-phosphorylation in GAS and the relevance of SP-PTP as an important therapeutic target.