The Convergence of Metabolism and Virulence Factor Regulation on Streptococcus pyogenes Pathogenesis

Nutrient acquisition is a strong driving force in the evolution of pathogenesis. For many pathogens such as Streptococcus pyogenes, the coordination of metabolism with the expression of virulence factors is necessary for colonization. Previous research examining the regulation of virulence factors in S. pyogenes identified a unique catabolite sensing regulatory pathway composed of the tagatose bis-phosphate aldolase LacD.1. Examination of LacD.1 has determined that while it still maintains its ancestral enzymatic activity its regulation occurs via a mechanism independent of catalysis. Our approach to understanding how S. pyogenes\u27 metabolism contributes to pathogenesis involves both understanding the adaptation of LacD.1 as a novel metabolic regulator as well as investigating the role of LacD.1-regulated genes in pathogenesis. Analysis of LacD.1\u27s ancestral enzymatic activity and comparison to a paralogous protein encoded in the genome, LacD.2, suggested that the adaptation of LacD.1 required a decrease in enzymatic activity as compared to LacD.2. This decrease in activity is largely due to an increase in Km, and suggests an optimization in affinity for the signaling substrate(s) necessary for LacD.1\u27s regulation. Additionally the regulatory function of LacD.1 was found to be essential for the fitness of bacteria when cultured in media that mimicked a soft tissue environment. The selective advantage observed for bacteria in this environment can likely be attributed to LacD.1\u27s positive regulation of the arc operon, which encodes genes necessary for arginine catabolism and contributes to both the production of ATP and protection from acid stress. To further investigate the arc operon\u27s contribution to the fitness of bacteria in a soft tissue environment we examined the influence of arginine catabolism on pathogenesis. Analysis of several mutants defective in catabolism revealed that both the utilization of arginine as well as the novel utilization of citrulline aids in S. pyogenes pathogenesis. This influence on pathogenesis included contributions to both the cellular metabolism of S. pyogenes as well as the modulation of the host immune response. Taken together this work demonstrates that LacD.1\u27s adaptation to a regulator and its coordination of metabolism and virulence factor expression was essential to increase the fitness of S. pyogenes as a pathogen

Adaptive evolution of the Streptococcus pyogenes regulatory aldolase LacD.1

In the human-pathogenic bacterium Streptococcus pyogenes, the tagatose bisphosphate aldolase LacD.1 likely originated through a gene duplication event and was adapted to a role as a metabolic sensor for regulation of virulence gene transcription. Although LacD.1 retains enzymatic activity, its ancestral metabolic function resides in the LacD.2 aldolase, which is required for the catabolism of galactose. In this study, we compared these paralogous proteins to identify characteristics correlated with divergence and novel function. Surprisingly, despite the fact that these proteins have identical active sites and 82% similarity in amino acid sequence, LacD.1 was less efficient at cleaving both fructose and tagatose bisphosphates. Analysis of kinetic properties revealed that LacD.1's adaptation was associated with a decrease in k(cat) and an increase in K(m). Construction and analysis of enzyme chimeras indicated that non-active-site residues previously associated with the variable activities of human aldolase isoenzymes modulated LacD.1's affinity for substrate. Mutant LacD.1 proteins engineered to have LacD.2-like levels of enzymatic efficiency lost the ability to function as regulators, suggesting that an alteration in efficiency was required for adaptation. In competition under growth conditions that mimic a deep-tissue environment, LacD.1 conferred a significant gain in fitness that was associated with its regulatory activity. Taken together, these data suggest that LacD.1's adaptation represents a form of neofunctionalization in which duplication facilitated the gain of regulatory function important for growth in tissue and pathogenesis

Citrulline protects Streptococcus pyogenes from acid stress using the arginine deiminase pathway and the F1Fo-ATPase

A common stress encountered by both pathogenic and environmental bacteria is exposure to a low-pH environment, which can inhibit cell growth and lead to cell death. One major defense mechanism against this stress is the arginine deiminase (ADI) pathway, which catabolizes arginine to generate two ammonia molecules and one molecule of ATP. While this pathway typically relies on the utilization of arginine, citrulline has also been shown to enter into the pathway and contribute to protection against acid stress. In the pathogenic bacterium Streptococcus pyogenes, the utilization of citrulline has been demonstrated to contribute to pathogenesis in a murine model of soft tissue infection, although the mechanism underlying its role in infection is unknown. To gain insight into this question, we analyzed a panel of mutants defective in different steps in the ADI pathway to dissect how arginine and citrulline protect S. pyogenes in a low-pH environment. While protection provided by arginine utilization occurred through the buffering of the extracellular environment, citrulline catabolism protection was pH independent, requiring the generation of ATP via the ADI pathway and a functional F(1)F(o)-ATP synthase. This work demonstrates that arginine and citrulline catabolism protect against acid stress through distinct mechanisms and have unique contributions to virulence during an infection. IMPORTANCE An important aspect of bacterial pathogenesis is the utilization of host-derived nutrients during an infection for growth and virulence. Previously published work from our lab identified a unique role for citrulline catabolism in Streptococcus pyogenes during a soft tissue infection. The present article probes the role of citrulline utilization during this infection and its contribution to protection against acid stress. This work reveals a unique and concerted action between the catabolism of citrulline and the F(1)F(o)-ATPase that function together to provide protection for bacteria in a low-pH environment. Dissection of these collaborative pathways highlights the complexity of bacterial infections and the contribution of atypical nutrients, such as citrulline, to pathogenesis

SpxA1 and SpxA2 act coordinately to fine-tune stress responses and virulence in Streptococcus pyogenes

SpxA is a unique transcriptional regulator highly conserved among members of the phylum Firmicutes that binds RNA polymerase and can act as an antiactivator. Why some Firmicutes members have two highly similar SpxA paralogs is not understood. Here, we show that the SpxA paralogs of the pathogen Streptococcus pyogenes, SpxA1 and SpxA2, act coordinately to regulate virulence by fine-tuning toxin expression and stress resistance. Construction and analysis of mutants revealed that SpxA1− mutants were defective for growth under aerobic conditions, while SpxA2− mutants had severely attenuated responses to multiple stresses, including thermal and oxidative stresses. SpxA1− mutants had enhanced resistance to the cationic antimicrobial molecule polymyxin B, while SpxA2− mutants were more sensitive. In a murine model of soft tissue infection, a SpxA1− mutant was highly attenuated. In contrast, the highly stress-sensitive SpxA2− mutant was hypervirulent, exhibiting more extensive tissue damage and a greater bacterial burden than the wild-type strain. SpxA1− attenuation was associated with reduced expression of several toxins, including the SpeB cysteine protease. In contrast, SpxA2− hypervirulence correlated with toxin overexpression and could be suppressed to wild-type levels by deletion of speB. These data show that SpxA1 and SpxA2 have opposing roles in virulence and stress resistance, suggesting that they act coordinately to fine-tune toxin expression in response to stress. SpxA2− hypervirulence also shows that stress resistance is not always essential for S. pyogenes pathogenesis in soft tissue

Host and bacterial proteases influence biofilm formation and virulence in a murine model of enterococcal catheter-associated urinary tract infection

Urinary tract infections: targeting enzymes might help Identifying bacterial and host enzymes that support biofilm formation may help prevent urinary tract infections caused by catheters. Enterococcus faecalis bacteria is a leading cause of catheter-associated urinary tract infections, the most common type of hospital-acquired infections. Michael Caparon and colleagues at Washington University School of Medicine in Missouri, USA, studied these infections in mice. They examined the effects of two protein-degrading enzymes, both from the bacterium and one can be activated by urine trypsin-like protease from the animals. Mutations that impaired either one of the enzymes had no effect on the infection, but when both the bacterial enzymes were impaired by mutation the formation of biofilms was significantly reduced. Treating the mice with chemicals that inhibited both bacterial and host enzymes dramatically reduced catheter-induced inflammation and related problems. This suggests drugs targeting these enzymes could be useful in clinical care

Structure-function analysis of the curli accessory protein CsgE defines surfaces essential for coordinating amyloid fiber formation

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal “tails” were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections.Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson’s disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis

NC410 is a novel immunomedicine for the treatment of solid tumors

Background Abnormalities in the extracellular matrix of tumor microenvironments support tumor progression, lead to immune dysfunction, and provide a target for cancer therapeutics. Collagens are a primary component of the extracellular matrix. Abnormal levels of collagen and of the collagen-domain containing complement component 1q (C1q) in tumor microenvironments has been proposed to disrupt anti-tumor immunity. LAIR-1 is an adhesion molecule and inhibitory receptor expressed on the cell surface of several immune cell subsets. LAIR-1 binding to collagen-like domains present in collagens and C1q inhibit immune cell function. LAIR-2 is a soluble homolog of LAIR-1 that binds to and outcompetes LAIR-1 binding to collagens and C1q and serves as a natural decoy to promote immune function.Methods Taking advantage of a natural decoy system, we designed a protein biologic, NC410, composed of LAIR-2 fused with a functional IgG1 Fc domain to target collagen-rich tumors and promote immune activation, infiltration and effector function.Results NC410 has increased avidity due to Fc mediated dimerization, and blocks LAIR-1 interactions with ligands, and LAIR-1 signaling. In vivo administration of NC410 in humanized tumor models reduced tumor growth in a dose dependent fashion. NC410 increased the numbers of infiltrating human CD8+ and CD4+ T cells in the tumor, which is associated with increased levels of chemokines in the local tumor environment. Effector function was also enhanced, as denoted by increased levels of IFN-gamma and Granzyme B in the local tumor environment. In addition, NC410 increased specific collagen degradative products in the serum of humanized tumor-bearing mice, suggesting NC410 may promote tumor microenvironment remodeling and immune accessibility to further promote anti-tumor immunity.Conclusions These data support NC410 as a novel therapeutic for targeting collagen-rich tumors and enabling normalization of the tumor-immune microenvironment. FIH studies have recently been initiated with NC410

Ir-Catalyzed Asymmetric Allylic Alkylation of Dialkyl Malo-nates Enabling the Construction of Enantioenriched All-Carbon Quaternary Centers

ABSTRACT: An enantioselective iridium-catalyzed allylic alkylation of malonates with trisubstituted allylic electro-philes to form all-carbon quaternary stereocenters is reported. This cross-coupling reaction features unprecedented reactivity at ambient temperature, particularly for challenging fully alkyl-substituted allylic electrophiles, and ena-bles the preparation of a wide range of enantioenriched products in up to 93% yield and 97% ee. The products of this transformation can be readily converted to a number of valuable building blocks including vicinal quaternary stereodiads and β-quaternary acids. This method was also used to prepare an enantioenriched intermediate facilitat-ing the asymmetric formal synthesis of the sporochnol family of natural products

Mannose-derived FimH antagonists: a promising anti-virulence therapeutic strategy for urinary tract infections and Crohn’s disease

<p><b>Introduction</b>: Type 1 pili are utilized by Gram-negative bacteria to adhere to host tissue and thus are a key virulence factor in urinary tract infections (UTIs) and Crohn’s disease (CD). This adhesion is mediated through specific binding of the terminal adhesin, FimH, to mannosylated host glycoproteins. FimH is essential for UTI pathogenesis and thus is a promising therapeutic target.</p> <p><b>Areas Covered</b>: Herein, we review the structural frameworks of FimH antagonists disclosed in the patent literature. X-ray crystallographic binding studies of D-mannose and early FimH antagonists have uncovered key molecular interactions. Exploiting this knowledge, mannosides with extraordinarily high binding affinities have been designed. Structure-activity relationships (SAR) and structure-property relationship (SPR) studies have resulted in the rapid development of orally bioavailable FimH antagonists with promising therapeutic potential for UTI and CD.</p> <p><b>Expert opinion</b>: It is our opinion that biaryl or ‘two-ring’ mannosides, which represent the largest and most thoroughly tested class of FimH antagonists, also hold the most promise as a novel treatment for UTIs. These antagonists have also been shown to have efficacy in treating CD. Judging from the strong preclinical data, we predict that one or more FimH antagonists will be entering the clinic within the next 1–2 years.</p