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.

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.

Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria.

Bacteria assemble a wide range of adhesive proteins, termed adhesins, to mediate binding to receptors and colonization of surfaces. For pathogenic bacteria, adhesins are critical for early stages of infection, allowing the bacteria to initiate contact with host cells, colonize different tissues, and establish a foothold within the host. The adhesins expressed by a pathogen are also critical for bacterial-bacterial interactions and the formation of bacterial communities, including biofilms. The ability to adhere to host tissues is particularly important for bacteria that colonize sites such as the urinary tract, where the flow of urine functions to maintain sterility by washing away non-adherent pathogens. Adhesins vary from monomeric proteins that are directly anchored to the bacterial surface to polymeric, hair-like fibers that extend out from the cell surface. These latter fibers are termed pili or fimbriae, and were among the first identified virulence factors of uropathogenic Escherichia coli. Studies since then have identified a range of both pilus and non-pilus adhesins that contribute to bacterial colonization of the urinary tract, and have revealed molecular details of the structures, assembly pathways, and functions of these adhesive organelles. In this review, we describe the different types of adhesins expressed by both Gram-negative and Gram-positive uropathogens, what is known about their structures, how they are assembled on the bacterial surface, and the functions of specific adhesins in the pathogenesis of urinary tract infections.

A more flexible lipoprotein sorting pathway.

Lipoprotein biogenesis in Gram-negative bacteria occurs by a conserved pathway, each step of which is considered essential. In contrast to this model, LoVullo and colleagues demonstrate that the N-acyl transferase Lnt is not required in Francisella tularensis or Neisseria gonorrhoeae. This suggests the existence of a more flexible lipoprotein pathway, likely due to a modified Lol transporter complex, and raises the possibility that pathogens may regulate lipoprotein processing to modulate interactions with the host.

A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin-resistant and -sensitive Staphylococcus aureus strains.

Staphylococcus aureus is a major human pathogen responsible for a number of serious and sometimes fatal infections. One of its reservoirs on the human body is the skin, which is known to be a source of invasive infection. The potential for an engineered staphylococcus-specific phage lysin (ClyS) to be used for topical decolonization is presented. We formulated ClyS into an ointment and applied it to a mouse model of skin colonization/infection with S. aureus. Unlike the standard topical antibacterial agent mupirocin, ClyS eradicated a significantly greater number of methicillin-susceptible S. aureus (MSSA) and -resistant S. aureus (MRSA) bacteria: a 3-log reduction with ClyS as opposed to a 2-log reduction with mupirocin in our model. The use of ClyS also demonstrated a decreased potential for the development of resistance by MRSA and MSSA organisms compared to that from the use of mupirocin in vitro. Because antibodies may affect enzyme function, we tested antibodies developed after repeated ClyS exposure for their effect on ClyS killing ability. Our results showed no inhibition of ClyS activity at various antibody titers. These data demonstrate the potential of developing ClyS as a novel class of topical antimicrobial agents specific to staphylococcus.

Synergism between a novel chimeric lysin and oxacillin protects against infection by methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is the causative agent of several serious infectious diseases. The emergence of antibiotic-resistant S. aureus strains has resulted in significant treatment difficulties, intensifying the need for new antimicrobial agents. Toward this end, we have developed a novel chimeric bacteriophage (phage) lysin that is active against staphylococci, including methicillin-resistant S. aureus (MRSA). The chimeric lysin (called ClyS) was obtained by fusing the N-terminal catalytic domain of the S. aureus Twort phage lysin with the C-terminal cell wall-targeting domain from another S. aureus phage lysin (phiNM3), which displayed Staphylococcus-specific binding. ClyS was expressed in Escherichia coli, and the purified protein lysed MRSA, vancomycin-intermediate strains of S. aureus (VISA), and methicillin-sensitive (MSSA) strains of S. aureus in vitro. In a mouse nasal decolonization model, a 2-log reduction in the viability of MRSA cells was seen 1 h following a single treatment with ClyS. One intraperitoneal dose of ClyS also protected against death by MRSA in a mouse septicemia model. ClyS showed a typical pattern of synergistic interactions with both vancomycin and oxacillin in vitro. More importantly, ClyS and oxacillin at doses that were not protective individually protected synergistically against MRSA septic death in a mouse model. These results strongly support the development of ClyS as an attractive addition to the current treatment options of multidrug-resistant S. aureus infections and would allow for the reinstatement of antibiotics shelved because of mounting resistance.

PlyC: a multimeric bacteriophage lysin.

Lysins are murein hydrolases produced by bacteriophage that act on the bacterial host cell wall to release progeny phage. When added extrinsically in their purified form, these enzymes produce total lysis of susceptible Gram-positive bacteria within seconds, suggesting a unique antimicrobial strategy. All known Gram-positive lysins are produced as a single polypeptide containing a catalytic activity domain, which cleaves one of the four major peptidoglycan bonds, and a cell-wall-binding domain, which may bind a species-specific carbohydrate epitope in the cell wall. Here, we have cloned and expressed a unique lysin from the streptococcal bacteriophage C(1), termed PlyC. Molecular characterization of the plyC operon reveals that PlyC is, surprisingly, composed of two separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA. Inhibitor studies predicted the presence of an active-site cysteine, and bioinformatic analysis revealed a cysteine, histidine-dependent amidohydrolase/peptidase domain within PlyCA. Point mutagenesis confirmed that PlyCA is responsible for the observed catalytic activity, and Cys-333 and His-420 are the active-site residues. PlyCB was found to self-assemble into an octamer, and this complex alone was able to direct streptococcal cell-wall-specific binding. Similar to no other proteins in sequence databases, PlyC defines a previously uncharacterized structural family of cell-wall hydrolases.