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

Abstract

bMany 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 enteroaggregative Escherichia coli, an important causative agent of diarrhea. We show here that NTZ also inhibits the function of type 1 and P pili from uropathogenic E. 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 ␤-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.

Small Molecules That Sabotage Bacterial Virulence

“…Another compound that inhibits bacterial adherence is the antiparasitic drug nitazoxanide (NTZ) (Figure 2), which is broadly active against the CUP in both enteroaggregative E. coli (EAEC) and UPEC strains [100, 101]. NTZ does not directly target growth in EAEC or UPEC but rather inhibits assembly and biogenesis of aggregative adherence fimbriae, type I and P pili [100].…”

“…To investigate the mechanism for reducing usher protein localization to the outer membrane by NTZ, a series of PapC truncation mutants were examined to identify the precise component of the usher that was disrupted. It was found that NTZ prevents proper folding of the transmembrane β-barrel pore (Figure 3A) [101]. Interestingly, NTZ inihibits growth of several pathogens including Clostridium difficile , Helicobacter pylori , and Campylobacter jejuni through inhibition of pyruvate ferreredoxin oxidoreductase [103, 104].…”

“…Interestingly, NTZ inihibits growth of several pathogens including Clostridium difficile , Helicobacter pylori , and Campylobacter jejuni through inhibition of pyruvate ferreredoxin oxidoreductase [103, 104]. However, the Enterobacteriaceae lack this target and NTZ is effective as a pilicide below the growth inhibitory concentrations observed for growth inhibition in NTZ-susceptible pathogens [100, 101, 105, 106]. Repurposing NTZ as a putative antivirulence therapy may be possible as it is an approved treatment for giardiasis and cryptosporidiosis, potentially expediting the process for entering clinical use.…”

Invasion of Host Cells and Tissues by Uropathogenic Bacteria

“…In the case of UPEC, the formation of long-live intracellular reservoirs may make complete eradication of the pathogen from the urinary tract especially difficult (17, 24). The development of pilicides and mannosides that interfere with the functions of adhesive organelles like type 1 pili may prove useful in hindering bacterial invasion of uroepithelial as well as disrupting IBCs (190–192). Natural products from cranberry and other sources may likewise impede UPEC entry into host cells, either by preventing bacterial attachment or by disrupting signaling through β 1 integrin or other key host receptors (see Fig.…”

Development and Validation of Rp-HPLC Method for the Determination of Nitazoxanide and Ofloxacin in Combined Pharmaceutical Dosage Forms

“…Recent researches showed the activity of this molecule and its active metabolite Tizoxanide against a broad range of obligate and facultative anaerobic gram positive and gram negative bacteria which includes E. coli 19,20 M. tuberculosis 21 -23 , H. pylori 24 -26 C. difficile 26 -29 and C. jejuni 26,30,31 . Furthermore the scientist Rossignol described this molecule as "a first-in-class broad-spectrum antiviral agent" in 2014 32 and now it is found to be active against influenza 32,33 , parainfluenza, Sendai virus (SeV), Respiratory syncytial virus (RSV) A-2, coronavirus 34 ,…”