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

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