Clostridioides difficile superoxide reductase mitigates oxygen sensitivity.

Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, the mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide, indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to the parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate SOR's broader impacts on the C. difficile vegetative cell proteome.IMPORTANCEClostridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in the mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome encodes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of SOR's enzymatic activity, including confirmation of its role in protecting C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.

Clostridium scindens exacerbates experimental necrotizing enterocolitis via upregulation of the apical sodium-dependent bile acid transporter.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Evidence indicates that bile acid homeostasis is disrupted during NEC: ileal bile acid levels are elevated in animals with experimental NEC, as is expression of the apical sodium-dependent bile acid transporter (Asbt). In addition, bile acids, which are synthesized in the liver, are extensively modified by the gut microbiome, including via the conversion of primary bile acids to more cytotoxic secondary forms. We hypothesized that the addition of bile acid-modifying bacteria would increase susceptibility to NEC in a neonatal rat model of the disease. The secondary bile acid-producing species Clostridium scindens exacerbated both incidence and severity of NEC. C. scindens upregulated the bile acid transporter Asbt and increased levels of intraenterocyte bile acids. Treatment with C. scindens also altered bile acid profiles and increased hydrophobicity of the ileal intracellular bile acid pool. The ability of C. scindens to enhance NEC requires bile acids, as pharmacological sequestration of ileal bile acids protects animals from developing disease. These findings indicate that bile acid-modifying bacteria can contribute to NEC pathology and provide additional evidence for the role of bile acids in the pathophysiology of experimental NEC.NEW & NOTEWORTHY Necrotizing enterocolitis (NEC), a life-threatening gastrointestinal emergency in premature infants, is characterized by dysregulation of bile acid homeostasis. We demonstrate that administering the secondary bile acid-producing bacterium Clostridium scindens enhances NEC in a neonatal rat model of the disease. C. scindens-enhanced NEC is dependent on bile acids and driven by upregulation of the ileal bile acid transporter Asbt. This is the first report of bile acid-modifying bacteria exacerbating experimental NEC pathology.

The Alternative Sigma Factor SigL Influences Clostridioides difficile Toxin Production, Sporulation, and Cell Surface Properties.

The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen Clostridioides difficile. To explore the contribution of SigL to C. difficile biology, we generated sigL-disruption mutants (sigL::erm) in strains belonging to two phylogenetically distinct lineages-the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleiotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 sigL::erm relative to the isogenic parent strain but remained unchanged in BI-1 sigL::erm. In contrast, secreted toxin levels were significantly elevated only in the BI-1 sigL::erm mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences C. difficile's virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes.

The Role of Bacterial Proteases in Microbe and Host-microbe Interactions.

BACKGROUND: Secreted proteases are an important class of factors used by bacterial to modulate their extracellular environment through the cleavage of peptides and proteins. These proteases can range from broad, general proteolytic activity to high degrees of substrate specificity. They are often involved in interactions between bacteria and other species, even across kingdoms, allowing bacteria to survive and compete within their niche. As a result, many bacterial proteases are of clinical importance. The immune system is a common target for these enzymes, and bacteria have evolved ways to use these proteases to alter immune responses for their benefit. In addition to the wide variety of human proteins that can be targeted by bacterial proteases, bacteria also use these secreted factors to disrupt competing microbes, ranging from outright antimicrobial activity to disrupting processes like biofilm formation. OBJECTIVE: In this review, we address how bacterial proteases modulate host mechanisms of protection from infection and injury, including immune factors and cell barriers. We also discuss the contributions of bacterial proteases to microbe-microbe interactions, including antimicrobial and anti- biofilm dynamics. CONCLUSION: Bacterial secreted proteases represent an incredibly diverse group of factors that bacteria use to shape and thrive in their microenvironment. Due to the range of activities and targets of these proteases, some have been noted for having potential as therapeutics. The vast array of bacterial proteases and their targets remains an expanding field of research, and this field has many important implications for human health.

Synthetic Peptide Libraries Designed From a Minimal Alpha-Helical Domain of AS-48-Bacteriocin Homologs Exhibit Potent Antibacterial Activity.

The circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits antibacterial activity through membrane disruption. The membrane-penetrating activity of enterocin AS-48 has been attributed to a specific alpha-helical region on the circular peptide. Truncated, linearized forms containing these domains have been shown to preserve limited bactericidal activity. We utilized the amino acid sequence of the active helical domain of enterocin AS-48 to perform a homology-based search of similar sequences in other bacterial genomes. We identified similar domains in three previously uncharacterized AS-48-like bacteriocin genes in Clostridium sordellii, Paenibacillus larvae, and Bacillus xiamenensis. Enterocin AS-48 and homologs from these bacterial species were used as scaffolds for the design of a minimal peptide library based on the active helical domain of each bacteriocin sequence. 95 synthetic peptide variants of each scaffold peptide, designated Syn-enterocin, Syn-sordellicin, Syn-larvacin, and Syn-xiamensin, were designed and synthesized from each scaffold sequence based on defined biophysical parameters. A total of 384 total peptides were assessed for antibacterial activity against Gram-negative and Gram-positive bacteria. Minimal Inhibitory Concentrations (MICs) as low as 15.6 nM could be observed for the most potent peptide candidate tested, with no significant cytotoxicity to eukaryotic cells. Our work demonstrates for the first time a general workflow of using minimal domains of natural bacteriocin sequences as scaffolds to design and rapidly synthesize a library of bacteriocin-based antimicrobial peptide variants for evaluation.

The Streptococcal Protease SpeB Antagonizes the Biofilms of the Human Pathogen Staphylococcus aureus USA300 through Cleavage of the Staphylococcal SdrC Protein.

Streptococcus pyogenes, or group A Streptococcus (GAS), is both a pathogen and an asymptomatic colonizer of human hosts and produces a large number of surface-expressed and secreted factors that contribute to a variety of infection outcomes. The GAS-secreted cysteine protease SpeB has been well studied for its effects on the human host; however, despite its broad proteolytic activity, studies on how this factor is utilized in polymicrobial environments are lacking. Here, we utilized various forms of SpeB protease to evaluate its antimicrobial and antibiofilm properties against the clinically important human colonizer Staphylococcus aureus, which occupies niches similar to those of GAS. For our investigation, we used a skin-tropic GAS strain, AP53CovS+, and its isogenic ΔspeB mutant to compare the production and activity of native SpeB protease. We also generated active and inactive forms of recombinant purified SpeB for functional studies. We demonstrate that SpeB exhibits potent biofilm disruption activity at multiple stages of S. aureus biofilm formation. We hypothesized that the surface-expressed adhesin SdrC in S. aureus was cleaved by SpeB, which contributed to the observed biofilm disruption. Indeed, we found that SpeB cleaved recombinant SdrC in vitro and in the context of the full S. aureus biofilm. Our results suggest an understudied role for the broadly proteolytic SpeB as an important factor for GAS colonization and competition with other microorganisms in its niche.IMPORTANCEStreptococcus pyogenes (GAS) causes a range of diseases in humans, ranging from mild to severe, and produces many virulence factors in order to be a successful pathogen. One factor produced by many GAS strains is the protease SpeB, which has been studied for its ability to cleave and degrade human proteins, an important factor in GAS pathogenesis. An understudied aspect of SpeB is the manner in which its broad proteolytic activity affects other microorganisms that co-occupy niches similar to that of GAS. The significance of the research reported herein is the demonstration that SpeB can degrade the biofilms of the human pathogen Staphylococcus aureus, which has important implications for how SpeB may be utilized by GAS to successfully compete in a polymicrobial environment.

The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene.

Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a ß-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala-l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 ΔsrtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface.IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

Novel antimicrobial peptide discovery using machine learning and biophysical selection of minimal bacteriocin domains.

Bacteriocins, the ribosomally produced antimicrobial peptides of bacteria, represent an untapped source of promising antibiotic alternatives. However, bacteriocins display diverse mechanisms of action, a narrow spectrum of activity, and inherent challenges in natural product isolation making in vitro verification of putative bacteriocins difficult. A subset of bacteriocins exert their antimicrobial effects through favorable biophysical interactions with the bacterial membrane mediated by the charge, hydrophobicity, and conformation of the peptide. We have developed a pipeline for bacteriocin-derived compound design and testing that combines sequence-free prediction of bacteriocins using machine learning and a simple biophysical trait filter to generate 20 amino acid peptides that can be synthesized and evaluated for activity. We generated 28,895 total 20-mer candidate peptides and scored them for charge, α-helicity, and hydrophobic moment. Of those, we selected 16 sequences for synthesis and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. Peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Our combined method incorporates machine learning and biophysical-based minimal region determination to create an original approach to swiftly discover bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use.

Stable genetic integration of a red fluorescent protein in a virulent Group A Streptococcus strain.

There are several advantages, both in vitro and in vivo, in utilizing bacteria that express a fluorescent protein. Such a protein can be transiently incorporated into the bacteria or integrated within the bacterial genome. The most widely utilized fluorescent protein is green fluorescent protein (GFP), but limitations exist on its use. Additional fluorescent proteins have been designed that have many advantages over GFP and technologies for their incorporation into bacteria have been optimized. In the current study, we report the successful integration and expression of a stable fluorescent reporter, mCherry (red fluorescent protein, RFP), into the genome of a human pathogen, Group A Streptococcus pyogenes (GAS) isolate AP53(S-). RFP was targeted at the atg codon of the fcR pseudogene that is present in the mga regulon of AP53(S-). Transcription of critical bacterial genes was not functionally altered by the genomic integration of mCherry. Host virulence both in vitro (keratinocyte infection and cytotoxicity) and in vivo (skin infection) was maintained in AP53(S-)-RFP. Additionally, survival of mice infected with either AP53(S-) or AP53(S-)-RFP was similar, demonstrating that overall pathogenicity of the AP53(S-) strain was not altered by the expression of mCherry. These studies demonstrate the feasibility of integrating a fluorescent reporter into the bacterial genome of a naturally virulent isolate of Group A S. pyogenes for comparative experimental studies.

Phage-mimicking antibacterial core-shell nanoparticles.

The increasing frequency of nosocomial infections caused by antibiotic-resistant microorganisms concurrent with the stagnant discovery of new classes of antibiotics has made the development of new antibacterial agents a critical priority. Our approach is an antibiotic-free strategy drawing inspiration from bacteriophages to combat antibiotic-resistant bacteria. We developed a nanoparticle-based antibacterial system that structurally mimics the protein-turret distribution on the head structure of certain bacteriophages and explored a combination of different materials arranged hierarchically to inhibit bacterial growth and ultimately kill pathogenic bacteria. Here, we describe the synthesis of phage-mimicking antibacterial nanoparticles (ANPs) consisting of silver-coated gold nanospheres distributed randomly on a silica core. The silver-coating was deposited in an anisotropic fashion on the gold nanospheres. Structurally, our nanoparticles mimicked the bacteriophages of the family Microviridae by up to 88%. These phage-mimicking ANPs were tested for bactericidal efficacy against four clinically relevant nosocomial pathogens (Staphylococcus aureus USA300, Pseudomonas aeruginosa FRD1, Enterococcus faecalis, and Corynebacterium striatum) and for biocompatibility with skin cells. Bacterial growth of all four bacteria was inhibited (21% to 90%) as well as delayed (by up to 5 h). The Gram-positive organisms were shown to be more sensitive to the nanoparticle treatment. Importantly, the phage-mimicking ANPs did not show any significant cytotoxic effects against human skin keratinocytes. Our results indicate the potential for phage-mimicking antimicrobial nanoparticles as a highly effective, alternative antibacterial agent, which may be suitable for co-administration with existing available formulations.

Neutralization of Streptolysin S-Dependent and Independent Inflammatory Cytokine IL-1ß Activity Reduces Pathology During Early Group A Streptococcal Skin Infection.

The bacterial pathogen Group A Streptococcus (GAS) has been shown to induce a variety of human diseases ranging in severity from pharyngitis to toxic shock syndrome and necrotizing fasciitis. GAS produces a powerful peptide toxin known as Streptolysin S (SLS). Though long recognized as a potent cytolysin, recent evidence from our lab has shown that SLS-dependent cytotoxicity is mediated through activation of the pro-inflammatory mediators p38 MAPK and NFκB. These findings led us to hypothesize that activation of p38 MAPK and NFκB signaling drive the production of pro-inflammatory cytokines which, in turn, serve as positive feedback signals to initiate cytotoxicity in infected host cells. To address this hypothesis, we utilized a cytokine array to characterize the SLS-dependent pro-inflammatory cytokine response to GAS infection in human keratinocytes. From these studies, IL-1ß was found to be markedly upregulated in the presence of SLS, and further investigation revealed that this cytokine contributes to cytotoxicity in human keratinocytes during infection. Subcutaneous infection studies were performed in mice to address the physiological impact of increased IL-1ß production. These studies demonstrated that IL-1ß is produced during GAS skin infection in an SLS-dependent manner. Furthermore, inhibition of this cytokine and the upstream kinases and other signaling mediators that drive its production reduced SLS-mediated lesion formation early in the infection process. Together, our findings indicate that pharmacological inhibition of this inflammatory axis holds promise as a therapeutic strategy to reduce tissue destruction during severe invasive Group A Streptococcal infections.

Rational design of syn-safencin, a novel linear antimicrobial peptide derived from the circular bacteriocin safencin AS-48.

Bacteriocins hold unprecedented promise as a largely untapped source of antibiotic alternatives in the age of multidrug resistance. Here, we describe the first approach to systematically design variants of a novel AS-48 bacteriocin homologue, which we have termed safencin AS-48, from Bacillus safensis, to gain insights into engineering improved activity of bacteriocins. A library of synthetic peptides in which systematic amino acid substitutions to vary the periodicity and abundance of polar, acidic, aliphatic, and hydrophobic residues were generated for a total of 96 novel peptide variants of a single bacteriocin candidate. Using this method, we identified nine synthetic safencin (syn-safencin) variants with broad and potent antimicrobial activities with minimal inhibitory concentrations (MIC) as low as 250 nM against E. coli, P. aeruginosa, X. axonopodis, and S. pyogenes with minimal cytotoxicity to mammalian cells. It is anticipated that the strategies we have developed will serve as general guides for tuning the specificity of a given natural bacteriocin compound for therapeutic specificity.

Virulence Factor Targeting of the Bacterial Pathogen Staphylococcus aureus for Vaccine and Therapeutics.

BACKGROUND: Staphylococcus aureus is a major bacterial pathogen capable of causing a range of infections in humans from gastrointestinal disease, skin and soft tissue infections, to severe outcomes such as sepsis. Staphylococcal infections in humans can be frequent and recurring, with treatments becoming less effective due to the growing persistence of antibiotic resistant S. aureus strains. Due to the prevalence of antibiotic resistance, and the current limitations on antibiotic development, an active and highly promising avenue of research has been to develop strategies to specifically inhibit the activity of virulence factors produced S. aureus as an alternative means to treat disease. OBJECTIVE: In this review we specifically highlight several major virulence factors produced by S. aureus for which recent advances in antivirulence approaches may hold promise as an alternative means to treating diseases caused by this pathogen. Strategies to inhibit virulence factors can range from small molecule inhibitors, to antibodies, to mutant and toxoid forms of the virulence proteins. CONCLUSION: The major prevalence of antibiotic resistant strains of S. aureus combined with the lack of new antibiotic discoveries highlight the need for vigorous research into alternative strategies to combat diseases caused by this highly successful pathogen. Current efforts to develop specific antivirulence strategies, vaccine approaches, and alternative therapies for treating severe disease caused by S. aureus have the potential to stem the tide against the limitations that we face in the post-antibiotic era.

Genomic Characterization of a Pattern D Streptococcus pyogenes emm53 Isolate Reveals a Genetic Rationale for Invasive Skin Tropicity.

UNLABELLED: The genome of an invasive skin-tropic strain (AP53) of serotype M53 group A Streptococcus pyogenes (GAS) is composed of a circular chromosome of 1,860,554 bp and carries genetic markers for infection at skin locales, viz, emm gene family pattern D and FCT type 3. Through genome-scale comparisons of AP53 with other GAS genomes, we identified 596 candidate single-nucleotide polymorphisms (SNPs) that reveal a potential genetic basis for skin tropism. The genome of AP53 differed by ∼30 point mutations from a noninvasive pattern D serotype M53 strain (Alab49), 4 of which are located in virulence genes. One pseudogene, yielding an inactive sensor kinase (CovS(-)) of the two-component transcriptional regulator CovRS, a major determinant for invasiveness, severely attenuated the expression of the secreted cysteine protease SpeB and enhanced the expression of the hyaluronic acid capsule compared to the isogenic noninvasive AP53/CovS(+) strain. The collagen-binding protein transcript sclB differed in the number of 5'-pentanucleotide repeats in the signal peptides of AP53 and Alab49 (9 versus 15), translating into different lengths of their signal peptides, which nonetheless maintained a full-length translatable coding frame. Furthermore, GAS strain AP53 acquired two phages that are absent in Alab49. One such phage (ΦAP53.2) contains the known virulence factor superantigen exotoxin gene tandem speK-slaA Overall, we conclude that this bacterium has evolved in multiple ways, including mutational variations of regulatory genes, short-tandem-repeat polymorphisms, large-scale genomic alterations, and acquisition of phages, all of which may be involved in shaping the adaptation of GAS in specific infectious environments and contribute to its enhanced virulence. IMPORTANCE: Infectious strains of S. pyogenes (GAS) are classified by their serotypes, relating to the surface M protein, the emm-like subfamily pattern, and their tropicity toward the nasopharynx and/or skin. It is generally agreed that M proteins from pattern D strains, which also directly bind human host plasminogen, are skin tropic. We have sequenced and characterized the genome of an invasive pattern D GAS strain (AP53) in comparison to a very similar strain (Alab49) that is noninvasive and developed a genomic rationale as to possible reasons for the skin tropicity of these two strains and the greater invasiveness of AP53.