Regulons and protein-protein interactions of PRD-containing Bacillus anthracis virulence regulators reveal overlapping but distinct functions.

Bacillus anthracis produces three regulators, AtxA, AcpA and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed 'PCVRs' (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA level or activity result in capBCADE transcription in strains lacking acpA and acpB. We used RNA-Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Plasmid and chromosome-borne genes were PCVR controlled, with AtxA, AcpA and AcpB having a ≥ 4-fold effect on transcript levels of 145, 130 and 49 genes respectively. Several genes were coregulated by two or three PCVRs. We determined that AcpA and AcpB form homomultimers, as shown previously for AtxA, and we detected AtxA-AcpA heteromultimers. In co-expression experiments, AcpA activity was reduced by increased levels of AtxA. Our data show that the PCVRs have specific and overlapping activity and that PCVR stoichiometry and potential heteromultimerization can influence target gene expression.

The roles of AtxA orthologs in virulence of anthrax-like Bacillus cereus G9241.

AtxA is a critical transcriptional regulator of plasmid-encoded virulence genes in Bacillus anthracis. Bacillus cereus G9241, which caused an anthrax-like infection, has two virulence plasmids, pBCXO1 and pBC210, that each harbor toxin genes and a capsule locus. G9241 also produces two orthologs of AtxA: AtxA1, encoded on pBCXO1, and AtxA2, encoded on pBC210. The amino acid sequence of AtxA1 is identical to that of AtxA from B. anthracis, while the sequences of AtxA1 and AtxA2 are 79% identical and 91% similar to one another. We found by qRT-PCR that AtxA1 and AtxA2 function as positive regulators of toxin (AtxA1) and capsule operon (both) transcription in G9241 and that a ΔatxA1 mutant produced lower levels of the anthrax toxins and no hyaluronic acid capsule. Deletion of atxA1 or atxA2 decreased the virulence of spores administered intranasally or subcutaneously to C57BL/6 mice but not to A/J mice, and deletion of both genes rendered spores avirulent in A/J mice. In addition, unlike AtxA1, AtxA2 did not form stable homomultimers in vitro, although AtxA1 and AtxA2 formed heterodimers. Our data show that AtxA1 is the primary regulator of G9241 virulence factor expression and that AtxA1 and AtxA2 are both required for full virulence.

Amidine-Based Cationic Conjugated Oligoelectrolytes with Antimicrobial Activity against Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that can cause high-morbidity infections. Due to its robust, flexible genome and ability to form biofilms, it can evade and rapidly develop resistance to antibiotics. Cationic conjugated oligoelectrolytes (COEs) have emerged as a promising class of antimicrobials. Herein, we report a series of amidine-containing COEs with high selectivity for bacteria. From this series, we identified 1b as the most active compound against P. aeruginosa (minimum inhibitory concentration (MIC) = 2 µg/mL) with low cytotoxicity (IC50 (HepG2) = 1024 µg/mL). The activity of 1b was not affected by known drug-resistant phenotypes of 100 diverse P. aeruginosa isolates. Moreover, 1b is bactericidal with a low propensity for P. aeruginosa to develop resistance. Furthermore, 1b is also able to inhibit biofilm formation at subinhibitory concentrations and kills P. aeruginosa in established biofilms. The in vivo efficacy of 1b was demonstrated in biofilm-associated murine wound infection models.

A Review of Omadacycline for Potential Utility in the Military Health System for the Treatment of Wound Infections.

INTRODUCTION: Combat-related wound infections complicate the recovery of wounded military personnel, contributing to overall morbidity and mortality. Wound infections in combat settings present unique challenges because of the size and depth of the wounds, the need to administer emergency care in the field, and the need for subsequent treatment in military facilities. Given the increase in multidrug-resistant pathogens, a novel, broad-spectrum antibiotic is desired across this continuum of care when the standard of care fails. Omadacycline was FDA-approved in 2018 for treatment of adults with acute bacterial skin and skin structure infections (ABSSSI), as well as community-acquired bacterial pneumonia (CABP). It is a broad-spectrum antibiotic with activity against gram-positive, gram-negative, and atypical bacterial pathogens, including multidrug-resistant species. Omadacycline can overcome commonly reported tetracycline resistance mechanisms, ribosomal protection proteins, and efflux pumps, and is available in once-daily intravenous or oral formulations. In this review, we discuss the potential role of omadacycline, which is included in the Department of Defense Formulary, in the context of combat wound infections. MATERIALS AND METHODS: A literature review was undertaken for manuscripts published before July 21, 2023. This included a series of publications found via PubMed and a bibliography made publicly available on the Paratek Pharmaceuticals, Inc. website. Publications presenting primary data published in English on omadacycline in relation to ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter species) pathogens and Clostridioides difficile, including in vitro, in vivo, and clinical data were included. RESULTS: Of 260 identified records, 66 were included for evidence review. Omadacycline has in vitro activity against almost all the ESKAPEE pathogens, apart from P. aeruginosa. Importantly, it has activity against the four most prevalent bacterial pathogens that cause wound infections in the military healthcare system: S. aureus, including methicillin-resistant S. aureus, A. baumannii, K. pneumoniae, and E. coli. In vivo studies in rats have shown that omadacycline is rapidly distributed in most tissues, with the highest tissue-to-blood concentration ratios in bone mineral. The clinical efficacy of omadacycline has been assessed in three separate Phase 3 studies in patients with ABSSSI (OASIS-1 and OASIS-2) and with CABP (OPTIC). Overall, omadacycline has an established safety profile in the treatment of both ABSSSI and CABP. CONCLUSIONS: Omadacycline has broad-spectrum activity, the option to be orally administered and an established safety profile, making it a potentially attractive replacement for moxifloxacin in the military individual first aid kit, especially when accounting for the increasing resistance to fluoroquinolones. Further studies and clinical evaluation are warranted to support broader use of omadacycline to treat combat wound infections in the military healthcare system.

cis-Acting elements that control expression of the master virulence regulatory gene atxA in Bacillus anthracis.

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5' and 3' deletion analysis and site-directed mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A+T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between -13 and +36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax.

An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections.

Genomic studies and experiments with permeability-deficient strains have revealed a variety of biological targets that can be engaged to kill Gram-negative bacteria. However, the formidable outer membrane and promiscuous efflux pumps of these pathogens prevent many candidate antibiotics from reaching these targets. One such promising target is the enzyme FabI, which catalyzes the rate-determining step in bacterial fatty acid biosynthesis. Notably, FabI inhibitors have advanced to clinical trials for Staphylococcus aureus infections but not for infections caused by Gram-negative bacteria. Here, we synthesize a suite of FabI inhibitors whose structures fit permeation rules for Gram-negative bacteria and leverage activity against a challenging panel of Gram-negative clinical isolates as a filter for advancement. The compound to emerge, called fabimycin, has impressive activity against >200 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and does not kill commensal bacteria. X-ray structures of fabimycin in complex with FabI provide molecular insights into the inhibition. Fabimycin demonstrates activity in multiple mouse models of infection caused by Gram-negative bacteria, including a challenging urinary tract infection model. Fabimycin has translational promise, and its discovery provides additional evidence that antibiotics can be systematically modified to accumulate in Gram-negative bacteria and kill these problematic pathogens.

Correction to "An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections".

[This corrects the article DOI: 10.1021/acscentsci.2c00598.].

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production.

Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075, an atxA-regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 "skiA" for "sporulation kinase inhibitor." Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.

Use of Enterococcus faecalis and Bacillus atrophaeus as surrogates to establish and maintain laboratory proficiency for concentration of water samples using ultrafiltration.

The U.S. Environmental Protection Agency's (EPA) Water Laboratory Alliance (WLA) currently uses ultrafiltration (UF) for concentration of biosafety level 3 (BSL-3) agents from large volumes (up to 100-L) of drinking water prior to analysis. Most UF procedures require comprehensive training and practice to achieve and maintain proficiency. As a result, there was a critical need to develop quality control (QC) criteria. Because select agents are difficult to work with and pose a significant safety hazard, QC criteria were developed using surrogates, including Enterococcus faecalis and Bacillus atrophaeus. This article presents the results from the QC criteria development study and results from a subsequent demonstration exercise in which E. faecalis was used to evaluate proficiency using UF to concentrate large volume drinking water samples. Based on preliminary testing EPA Method 1600 and Standard Methods 9218, for E. faecalis and B. atrophaeus respectively, were selected for use during the QC criteria development study. The QC criteria established for Method 1600 were used to assess laboratory performance during the demonstration exercise. Based on the results of the QC criteria study E. faecalis and B. atrophaeus can be used effectively to demonstrate and maintain proficiency using ultrafiltration.