In vitro activity of amixicile against T. vaginalis from clinical isolates.

Trichomoniasis is a sexually transmitted infection in humans caused by the protozoan Trichomonas vaginalis, the leading causative agent of vaginitis in women and urethritis in men worldwide. Metronidazole is the standard treatment for trichomoniasis, with tinidazole as the second line. There are currently no FDA-approved non-nitroimidazole alternative treatments for resistant strains. This study compares the efficacy of a newly synthesized non-nitroimidazole oral drug, amixicile, to that of both metronidazole and the synthetic precursor of amixicile, nitazoxanide with in vitro sensitivity testing. One standard strain from ATCC and three patient-isolated strains of T. vaginalis were used to compare treatments under anaerobic conditions. The minimum inhibitory concentration for metronidazole, nitazoxanide, and amixicile were 12.5 µM, 100 µM, and 6.25 µM, respectively. These results suggest that amixicile may be highly active against T. vaginalis and warrants further investigation as a potential alternative to metronidazole in the treatment of trichomoniasis.

Non-human Primate Macaca mulatta as an Animal Model for Testing Efficacy of Amixicile as a Targeted Anti-periodontitis Therapy.

Periodontitis is an inflammatory condition triggered by selected oral microbiota; thus treatment strategies should be aimed at reducing the abundance of the pathogenic bacteria. An obstacle to preclinical testing of such strategies is the availability of reliable animal models. Here, a non-human primate (NHP), Macaca mulatta, was used to examine the effectiveness of a novel antimicrobial, amixicile, which inhibits pyruvate-ferredoxin oxidoreductase (PFOR) present in anaerobic bacteria. Animals were assessed for their periodontal health, including radiography, clinical attachment loss (CAL), presence of plaque (PI), bleeding on probing (BOP) and pocket depth (PD), and sampled for saliva, gingival crevicular fluid (GCF), and subgingival plaque to determine their baseline clinical status. Amixicile was then administered for 2 weeks (40 mg/kg/day) and the animals were monitored for periodontal health immediately after the antibiotic treatment, then at 1 month-, 3 months-, and 6-months posttreatment. Microbial species present in plaque and saliva were determined through 16S rDNA sequencing. Baseline assessment of the microbiome has shown a significant proportion of bacteria belonging to the Streptococcus, Haemophilus, Porphyromonas, Gemella, and Fusobacterium genera. The abundance of Porphyromonas and Fusobacterium was reduced following treatment with amixicile, whereas that of Escherichia, Haemophilus, and Gemella were elevated. CAL, PD, and BOP were also significantly reduced following the treatment. In conclusion, the NHP model proves useful for preclinical studies of strategies targeting selected members of the oral microbiome. We show that amixicile reduces the levels of anaerobic bacteria under in vivo conditions, correlating with a reduction in CAL, PD, and BOP, thus validating its usefulness as an antimicrobial strategy.

Antibacterial Discovery: 21st Century Challenges.

It has been nearly 50 years since the golden age of antibiotic discovery (1945-1975) ended; yet, we still struggle to identify novel drug targets and to deliver new chemical classes of antibiotics to replace those rendered obsolete by drug resistance. Despite herculean efforts utilizing a wide range of antibiotic discovery platform strategies, including genomics, bioinformatics, systems biology and postgenomic approaches, success has been at best incremental. Obviously, finding new classes of antibiotics is really hard, so repeating the old strategies, while expecting different outcomes, seems to boarder on insanity. The key questions dealt with in this review include: (1) If mutation based drug resistance is the major challenge to any new antibiotic, is it possible to find drug targets and new chemical entities that can escape this outcome; (2) Is the number of novel chemical classes of antibacterials limited by the number of broad spectrum drug targets; and (3) If true, then should we focus efforts on subgroups of pathogens like Gram negative or positive bacteria only, anaerobic bacteria or other group where the range of common essential genes is likely greater?. This review also provides some examples of existing drug targets that appear to escape the specter of mutation based drug resistance, and provides examples of some intermediate spectrum strategies as well as modern molecular and genomic approaches likely to improve the odds of delivering 21st century medicines to combat multidrug resistant pathogens.

Amixicile depletes the ex vivo periodontal microbiome of anaerobic bacteria.

OBJECTIVES: Although periodontal diseases result from overgrowth of anaerobic bacteria, the effect of a specific knockdown of anaerobes on the disease outcome has yet to be examined. We have reported that amixicile, a non-toxic, readily bioavailable, and novel antimicrobial, specifically targets selected oral anaerobes through inhibition of the activity of pyruvate ferredoxin oxidoreductase (PFOR), a major enzyme mediating oxidative decarboxylation of pyruvate. METHODS: Here, we generated an ex vivo microbiome derived from gingival pockets of human subjects with chronic periodontal disease and evaluated the efficacy of amixicile in generating a specific knockdown of anaerobic bacteria present in the microbiome. RESULTS: Our bioinformatics analysis identified PFOR-like coding capacity in over 100 genomes available from the HOMD database. Many of those bacteria were present in our ex vivo microbiome. Significantly, the anaerobic pathogens relying on PFOR for energy generation were specifically reduced in abundance following treatment with amixicile while non-PFOR bacteria were spared. Specifically, Prevotella, Veillonella, Slackia, Porphyromonas, Treponema, Megasphera, and Atobium were reduced in abundance. Such treatment resulted in the conversion of a microbiome resembling a microbiome derived from sites with periodontal disease to one resembling a microbiome present at healthy sites. We also compared the inhibitory spectrum of amixicile to that of metronidazole and showed that the antibiotics have a similar inhibitory spectrum. CONCLUSIONS: This work further demonstrates that amixicile has the potential to reverse and prevent the outgrowth of anaerobic pathogens observed in subjects with periodontal disease.

Amixicile: A Concept Therapeutic for Treatment of Chronic Anaerobic Infections.

Anaerobic microorganisms are often associated with chronic mucosal infections including periodontal disease, inflammatory bowel diseases, and recurrent colitis caused by Clostridioides difficile. Management of these diseases requires a long term strategy, but available antibiotics (e.g., metronidazole) can only be used short term. Conceptually, therapeutics that control chronic inflammation would lessen the risk of associated autoimmune diseases including atherosclerosis, arthritis, type II diabetes, Crohn's and ulcerative colitis and even Alzheimer's disease. To meet this need, an antibiotic must overcome inevitable antibiotic resistance, toxicity to humans or their mitochondria and limit collateral damage (dysbiosis) to gut microbiota. This review describes attributes of amixicile (AMIX), a novel systemic therapeutic, that shows efficacy in animal models for treatment of C. difficile colitis and gastric infections caused by Helicobacter pylori, while limiting collateral damage to gut microflora. Together with the apparent absence of drug resistance, toxicities, and drug metabolism, might qualify amixicile for consideration as a long term therapeutic for management of chronic and acute anaerobic infections.

Amixicile targets anaerobic bacteria within the oral microbiome.

OBJECTIVES: Anaerobic bacteria are the major causative agents of periodontal disease. However, so far, targeted therapy aimed at reducing those pathogens has not been widely implemented. We have previously reported on a novel antimicrobial, amixicile, that targets anaerobic bacteria through inhibition of the function of the major anaerobic metabolic enzyme pyruvate ferredoxin oxidoreductase (PFOR), while not affecting aerotolerant organisms. It effectively inhibited the growth of oral anaerobes both in monocultures as well as in mixed in vitro mixed cultured however, amixicile's activity in in vivo-like conditions remained to be established. METHODS: Here, we expand our study using an ex vivo oral microbiome combined with metagenomic sequencing to determine the effect of amixicile treatment on the composition of the microbiome and compare it to that of metronidazole. RESULTS: Our results show that in the complex microbiomes, anaerobic bacteria are selectively inhibited, while the growth of aerotolerant ones, such as Streptococcus, Klebsiella, Neisseria, and Rothia is unaffected. Veillonella was the most abundant anaerobic genus in our ex vivo microbiome, and we observed complete inhibition of its growth. In addition, growth of other anaerobes, Fusobacterium and Prevotella, was significantly inhibited. It is noteworthy that a change in abundance of bacteriophages, such as Siphoviridae and Myoviridae, associated with the oral microbiome was observed. CONCLUSIONS: Collectively, our data expand on the so far reported inhibitory spectrum of amixicile and demonstrates that it inhibits anaerobic bacteria, including both clinical isolates and laboratory strains.

The small molecule nitazoxanide selectively disrupts BAM-mediated folding of the outer membrane usher protein.

Bacterial pathogens assemble adhesive surface structures termed pili or fimbriae to initiate and sustain infection of host tissues. Uropathogenic Escherichia coli, the primary causative agent of urinary tract infections, expresses type 1 and P pili required for colonization of the bladder and kidney, respectively. These pili are assembled by the conserved chaperone-usher (CU) pathway, in which a periplasmic chaperone works together with an outer membrane (OM) usher protein to build and secrete the pilus fiber. Previously, we found that the small molecule and antiparasitic drug nitazoxanide (NTZ) inhibits CU pathway-mediated pilus biogenesis in E. coli by specifically interfering with proper maturation of the usher protein in the OM. The usher is folded and inserted into the OM by the ß-barrel assembly machine (BAM) complex, which in E. coli comprises five proteins, BamA-E. Here, we show that sensitivity of the usher to NTZ is modulated by BAM expression levels and requires the BamB and BamE lipoproteins. Furthermore, a genetic screen for NTZ-resistant bacterial mutants isolated a mutation in the essential BamD lipoprotein. These findings suggest that NTZ selectively interferes with an usher-specific arm of the BAM complex, revealing new details of the usher folding pathway and BAM complex function. Evaluation of a set of NTZ derivatives identified compounds with increased potency and disclosed that NTZ's nitrothiazole ring is critical for usher inhibition. In summary, our findings indicate highly specific effects of NTZ on the usher folding pathway and have uncovered NTZ analogs that specifically decrease usher levels in the OM.

Amixicile Reduces Severity of Cryptosporidiosis but Does Not Have In Vitro Activity against Cryptosporidium.

Cryptosporidium species cause significant morbidity in malnourished children. Nitazoxanide (NTZ) is the only approved treatment for cryptosporidiosis, but NTZ has diminished effectiveness during malnutrition. Here, we show that amixicile, a highly selective water-soluble derivative of NTZ diminishes Cryptosporidium infection severity in a malnourished mouse model despite a lack of direct anticryptosporidial activity. We suggest that amixicile, by tamping down anaerobes associated with intestinal inflammation, reverses weight loss and indirectly mitigates infection-associated pathology.

Antimicrobial activity of amixicile against Treponema denticola and other oral spirochetes associated with periodontal disease.

BACKGROUND: Periodontal disease is a polymicrobial infection characterized by inflammation of the gingiva, alveolar bone resorption and tooth loss. As periodontal disease progresses, oral treponemes (spirochetes) become dominant bacteria in periodontal pockets. Oral treponemes are anaerobes and all encode the enzyme pyruvate-ferredoxin oxidoreductase (PFOR) which catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. Here we assess the susceptibility of oral treponemes to amixicile (AMIX), a novel inhibitor of PFOR. METHODS: The minimum inhibitory concentration (MIC) of AMIX against several oral treponeme species was determined. The impact of AMIX on processes relevant to virulence including motility, H2 S production, and complement evasion were determined. RESULTS: The growth of all oral treponeme species tested was inhibited by AMIX with MIC concentrations (MIC) ranging from 0.5-1.5 µg/mL. AMIX significantly reduced motility, caused a dose-dependent decrease in hydrogen sulfide production and increased sensitivity to killing by human complement (i.e., serum sensitivity). CONCLUSIONS: AMIX is effective in vitro in inhibiting growth and other processes central to virulence. AMIX could serve could serve as a new selective therapeutic tool for the treatment of periodontal disease.

Small molecule promotes ß-catenin citrullination and inhibits Wnt signaling in cancer.

Wnt (wingless)/ß-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of ß-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of ß-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or ß-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of ß-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.

Amixicile, a novel strategy for targeting oral anaerobic pathogens.

The oral microflora is composed of both health-promoting as well as disease-initiating bacteria. Many of the disease-initiating bacteria are anaerobic and include organisms such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. Here we investigated a novel therapeutic, amixicile, that targets pyruvate:ferredoxin oxidoreductase (PFOR), a major metabolic enzyme involved in energy generation through oxidative decarboxylation of pyruvate. PFOR is present in these anaerobic pathogenic bacteria and thus we hypothesized that amixicile would effectively inhibit their growth. In general, PFOR is present in all obligate anaerobic bacteria, while oral commensal aerobes, including aerotolerant ones, such as Streptococcus gordonii, use pyruvate dehydrogenase to decarboxylate pyruvate. Accordingly, we observed that growth of the PFOR-containing anaerobic periodontal pathogens, grown in both monospecies as well as multispecies broth cultures was inhibited in a dose-dependent manner while that of S. gordonii was unaffected. Furthermore, we also show that amixicile is effective against these pathogens grown as monospecies and multispecies biofilms. Finally, amixicile is the first selective therapeutic agent active against bacteria internalized by host cells. Together, the results show that amixicile is an effective inhibitor of oral anaerobic bacteria and as such, is a good candidate for treatment of periodontal diseases.

Enhanced Klebsiella pneumoniae Carbapenemase Expression from a Novel Tn4401 Deletion.

The Klebsiella pneumoniae carbapenemase gene (blaKPC) is typically located within mobile transposon Tn4401 Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188-bp deletion [between istB and blaKPC]) was present in 14% (39/281) of clinical isolates. MICs showed that Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16 and ≥16, respectively), ertapenem (≥8 and 4, respectively), and cefepime (≥64 and 4, respectively) than E. coli strains with Tn4401b (0.5, ≤0.5, and ≤1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn4401a had a 16-fold increase and Tn4401h a 4-fold increase in blaKPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn4401a and Tn4401h promoter sequences generated higher ß-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. The activity of the isolated P2 promoter was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicated that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.

Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria.

Amixicile is a promising derivative of nitazoxanide (an antiparasitic therapeutic) developed to treat systemic infections caused by anaerobic bacteria, anaerobic parasites, and members of the Epsilonproteobacteria (Campylobacter and Helicobacter). Amixicile selectively inhibits pyruvate-ferredoxin oxidoreductase (PFOR) and related enzymes by inhibiting the function of the vitamin B1 cofactor (thiamine pyrophosphate) by a novel mechanism. Here, we interrogate the amixicile scaffold, guided by docking simulations, direct PFOR inhibition assays, and MIC tests against Clostridium difficile, Campylobacter jejuni, and Helicobacter pylori Docking simulations revealed that the nitro group present in nitazoxanide interacts with the protonated N4'-aminopyrimidine of thiamine pyrophosphate (TPP). The ortho-propylamine on the benzene ring formed an electrostatic interaction with an aspartic acid moiety (B456) of PFOR that correlated with improved PFOR-inhibitory activity and potency by MIC tests. Aryl substitution with electron-withdrawing groups and substitutions of the propylamine with other alkyl amines or nitrogen-containing heterocycles both improved PFOR inhibition and, in many cases, biological activity against C. difficile Docking simulation results correlate well with mechanistic enzymology and nuclear magnetic resonance (NMR) studies that show members of this class of antimicrobials to be specific inhibitors of vitamin B1 function by proton abstraction, which is both novel and likely to limit mutation-based drug resistance.

Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane.

Many bacterial pathogens assemble surface fibers termed pili or fimbriae that facilitate attachment to host cells and colonization of host tissues. The chaperone/usher (CU) pathway is a conserved secretion system that is responsible for the assembly of virulence-associated pili by many different Gram-negative bacteria. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and an integral outer membrane (OM) assembly and secretion platform termed the usher. Nitazoxanide (NTZ), an antiparasitic drug, was previously shown to inhibit the function of aggregative adherence fimbriae and type 1 pili assembled by the CU pathway in enteroaggregativeEscherichia coli, an important causative agent of diarrhea. We show here that NTZ also inhibits the function of type 1 and P pili from uropathogenicE. coli(UPEC). UPEC is the primary causative agent of urinary tract infections, and type 1 and P pili mediate colonization of the bladder and kidneys, respectively. By analysis of the different stages of the CU pilus biogenesis pathway, we show that treatment of bacteria with NTZ causes a reduction in the number of usher molecules in the OM, resulting in a loss of pilus assembly on the bacterial surface. In addition, we determine that NTZ specifically prevents proper folding of the usher ß-barrel domain in the OM. Our findings demonstrate that NTZ is a pilicide with a novel mechanism of action and activity against diverse CU pathways. This suggests that further development of the NTZ scaffold may lead to new antivirulence agents that target the usher to prevent pilus assembly.

Legionella pneumophila utilizes a single-player disulfide-bond oxidoreductase system to manage disulfide bond formation and isomerization.

Legionella pneumophila uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein-disulfide isomerase (PDI) activity. In Escherichia coli, these functions are separated to avoid futile cycling. In L. pneumophila, DsbA2 is maintained as a mixture of disulfides (S-S) and free thiols (SH), but when expressed in E. coli, only the SH form is observed. We provide evidence to suggest that structural differences in DsbB oxidases (LpDsbB1 and LpDsbB2) and DsbD reductases (LpDsbD1 and LpDsbD2) (compared with E. coli) permit bifunctional activities without creating a futile cycle. LpdsbB1 and LpdsbB2 partially complemented an EcdsbB mutant while neither LpdsbD1 nor LpdsbD2 complemented an EcdsbD mutant unless DsbA2 was also expressed. When the dsb genes of E. coli were replaced with those of L. pneumophila, motility was restored and DsbA2 was present as a mixture of redox forms. A dominant-negative approach to interfere with DsbA2 function in L. pneumophila determined that DSB oxidase activity was necessary for intracellular multiplication and assembly/function of the Dot/Icm Type IVb secretion system. Our studies show that a single-player system may escape the futile cycle trap by limiting transfer of reducing equivalents from LpDsbDs to DsbA2.

Preclinical studies of amixicile, a systemic therapeutic developed for treatment of Clostridium difficile infections that also shows efficacy against Helicobacter pylori.

Amixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 µM (NTZ was toxic above 10 µM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · µg/ml (30 mg/kg dose) to 328 h · µg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 µg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.

Response to metronidazole and oxidative stress is mediated through homeostatic regulator HsrA (HP1043) in Helicobacter pylori.

Metronidazole (MTZ) is often used in combination therapies to treat infections caused by the gastric pathogen Helicobacter pylori. Resistance to MTZ results from loss-of-function mutations in genes encoding RdxA and FrxA nitroreductases. MTZ-resistant strains, when cultured at sub-MICs of MTZ (5 to 20 µg/ml), show dose-dependent defects in bacterial growth; depressed activities of many Krebs cycle enzymes, including pyruvate:ferredoxin oxidoreductase (PFOR); and low transcript levels of porGDAB (primer extension), phenotypes consistent with an involvement of a transcriptional regulator. Using a combination of protein purification steps, electrophoretic mobility shift assays (EMSAs), and mass spectrometry analyses of proteins bound to porG promoter sequences, we identified HP1043, an essential homeostatic global regulator (HsrA [for homeostatic stress regulator]). Competition EMSAs and supershift analyses with HsrA-enriched protein fractions confirmed specific binding to porGDAB and hsrA promoter sequences. Exposure to MTZ resulted in >10-fold decreases in levels of HsrA and in levels of the HsrA-regulated gene products PFOR and TlpB. Exposure to paraquat (PQ), hydrogen peroxide, or organic peroxides showed near equivalence with MTZ, revealing a common oxidative stress response pathway. Finally, direct superoxide dismutase (SOD) assays showed an inverse relationship between HsrA levels and SOD activity, suggesting that HsrA may serve as a repressor of sodB. As a homeostatic sentinel, HsrA appears to be ideally positioned to enable rapid shutdown of genes associated with metabolism and growth while activating (directly or indirectly) oxidative defense genes in response to low levels of toxic metabolites (MTZ or oxygen) before they reach DNA-damaging levels.

Improved flavodoxin inhibitors with potential therapeutic effects against Helicobacter pylori infection.

Helicobacter pylori (Hp) infection affects one-half of the human population and produces a variety of diseases from peptic ulcer to cancer. Current eradication therapies achieve modest success rates (around 70%), resistance to the antibiotics of choice is on the rise, and vaccination has not proved to be successful yet. Using an essential Hp protein, flavodoxin, as target, we identified three low-molecular-weight flavodoxin inhibitors with bactericidal anti-Hp properties. To improve their therapeutic indexes, we have now identified and tested 123 related compounds. We have first tested similar compounds available. Then we have designed, synthesized, and tested novel variants for affinity to flavodoxin, MIC for Hp, cytotoxicity, and bactericidal effect. Some are novel bactericidal inhibitors with therapeutic indexes of 9, 38 and 12, significantly higher than those of their corresponding leads. Developing novel Hp-specific antibiotics will help fighting Hp resistance and may have the advantage of not generally perturbing the bacterial flora.

Disulfide bond oxidoreductase DsbA2 of Legionella pneumophila exhibits protein disulfide isomerase activity.

The extracytoplasmic assembly of the Dot/Icm type IVb secretion system (T4SS) of Legionella pneumophila is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential disulfide bond oxidoreductase DsbA2 and not by DsbA1, a second nonessential DSB oxidoreductase. DsbA2, which is widely distributed in the microbial world, is phylogenetically distinct from the canonical DsbA oxidase and the DsbC protein disulfide isomerase (PDI)/reductase of Escherichia coli. Here we show that the extended N-terminal amino acid sequence of DsbA2 (relative to DsbA proteins) contains a highly conserved 27-amino-acid dimerization domain enabling the protein to form a homodimer. Complementation tests with E. coli mutants established that L. pneumophila dsbA1, but not the dsbA2 strain, restored motility to a dsbA mutant. In a protein-folding PDI detector assay, the dsbA2 strain, but not the dsbA1 strain, complemented a dsbC mutant of E. coli. Deletion of the dimerization domain sequences from DsbA2 produced the monomer (DsbA2N), which no longer exhibited PDI activity but complemented the E. coli dsbA mutant. PDI activity was demonstrated in vitro for DsbA2 but not DsbA1 in a nitrocefin-based mutant TEM ß-lactamase folding assay. In an insulin reduction assay, DsbA2N activity was intermediate between those of DsbA2 and DsbA1. In L. pneumophila, DsbA2 was maintained as a mixture of thiol and disulfide forms, while in E. coli, DsbA2 was present as the reduced thiol. Our studies suggest that DsbA2 is a naturally occurring bifunctional disulfide bond oxidoreductase that may be uniquely suited to the majority of intracellular bacterial pathogens expressing T4SSs as well as in many slow-growing soil and aquatic bacteria.