Type IV pili (T4P) are one of the most common forms of bacterial and archaeal surface structures, involved in adherence, motility, competence for DNA uptake, and pathogenesis. Pseudomonas aeruginosa ...has emerged as one of the key model systems for the investigation of T4P structure and function. Although its reputation as a serious nosocomial and opportunistic pathogen is well deserved, its facile growth requirements and the ready availability of molecular tools have allowed for rapid advances in our understanding of how T4P are assembled; their contributions to motility, biofilm formation and virulence; and their complex regulation. This review covers recent findings concerning the three different types of T4P found in P. aeruginosa (type IVa, type IVb, and Tad) and provides details about the modes of translocation mediated by T4aP, the architecture and function of the T4aP assembly system, and the complex regulation of T4aP biogenesis and function.
Active efflux of antimicrobial agents is a primary mechanism by which bacterial pathogens can become multidrug resistant. The combined use of efflux pump inhibitors (EPIs) with pump substrates is ...under exploration to overcome efflux-mediated multidrug resistance. Phenylalanine-arginine β-naphthylamide (PAβN) is a well-studied EPI that is routinely combined with fluoroquinolone antibiotics, but few studies have assessed its utility in combination with β-lactam antibiotics. The initial goal of this study was to assess the efficacy of β-lactams in combination with PAβN against the opportunistic pathogen, Pseudomonas aeruginosa. PAβN reduced the minimal inhibitory concentrations (MICs) of several β-lactam antibiotics against P. aeruginosa; however, the susceptibility changes were not due entirely to efflux inhibition. Upon PAβN treatment, intracellular levels of the chromosomally-encoded AmpC β-lactamase that inactivates β-lactam antibiotics were significantly reduced and AmpC levels in supernatants correspondingly increased, potentially due to permeabilization of the outer membrane. PAβN treatment caused a significant increase in uptake of 8-anilino-1-naphthylenesulfonic acid, a fluorescent hydrophobic probe, and sensitized P. aeruginosa to bulky antibiotics (e.g. vancomycin) that are normally incapable of crossing the outer membrane, as well as to detergent-like bile salts. Supplementation of growth media with magnesium to stabilize the outer membrane increased MICs in the presence of PAβN and restored resistance to vancomycin. Thus, PAβN permeabilizes bacterial membranes in a concentration-dependent manner at levels below those typically used in combination studies, and this additional mode of action should be considered when using PAβN as a control for efflux studies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The coronavirus disease 2019 (COVID-19) pandemic and antimicrobial resistance (AMR) are parallel and interacting health emergencies that provide the opportunity for mutual learning. As their measures ...and consequences are comparable, the COVID-19 pandemic helps to illustrate the potential long-term impact of AMR, which is less acute but not less crucial. They may also impact each other as there is a push to use existing antimicrobials to treat critically ill COVID-19 patients in the absence of specific treatments. Attempts to manage the spread of COVID-19 may also lead to a slowdown in AMR. Understanding how COVID-19 affects AMR trends and what we can expect if these trends remain the same or worsen will help us to plan the next steps for tackling AMR. Researchers should start collecting data to measure the impact of current COVID-19 policies and programs on AMR.
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, designated by the World Health Organization as a critical priority for development of new therapeutics due to high levels of ...intrinsic and acquired antibiotic resistance. Other challenges include its versatility (it can persist in the environment and most strains are capable of causing disease in compromised hosts), robust efflux mechanisms that limit drug penetration, and the propensity to form antimicrobial-tolerant biofilms. Novel therapeutics in development to prevent or treat P. aeruginosa infections include vaccines, biologics such as antimicrobial peptides and therapeutic antibodies, virulence inhibitors, antimicrobials with novel targets, antibody–drug conjugates, resistance inhibitor–antibiotic or antibiotic–potentiator combinations, and bacteriophages or phage-derived lysins.
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IJS, KILJ, NUK, PNG, UL, UM
Iron is essential for many biological functions in bacteria, but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high ...affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria or fungi, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intracellular targets-including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a binding site in an aromatic pocket formed by the plug and barrel domains but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach.
Gram-negative bacteria express a variety of outer membrane transporters to import critical nutrients such as iron. Due to its insolubility, iron is taken up while bound to small-molecule chelators called siderophores. Pseudomonas aeruginosa takes up its own siderophores pyoverdine and pyochelin but can also steal siderophores produced by other bacteria and fungi, giving it a competitive advantage in iron-limited environments. Here, we used whole-cell reporter assays to show that FpvB, originally identified as a secondary transporter for pyoverdine, transports the chemically distinct fungal siderophore ferrichrome and the bacterial siderophore ferrioxamine B with high affinity. FpvB is also used by thiopeptide antibiotic thiostrepton for uptake. We predicted that all of these ligands bind to a common hydrophobic pocket in FpvB and used site-directed mutagenesis coupled with phenotypic assays to identify residues required for uptake. These analyses showed that siderophore and antibiotic uptake could be uncoupled. Our data show that FpvB is a promiscuous transporter of multiple chemically distinct ligands and fills in missing details of ferrichrome transport by P. aeruginosa. A clearer picture of the spectrum of outer membrane transporter substrate specificity is useful for the design of novel siderophore-antibiotic conjugates that can exploit nutrient uptake pathways to kill challenging Gram-negative pathogens.
Thiopeptides are a class of natural product antibiotics with diverse structures and functions. Their complex structures and biosynthesis have intrigued researchers since their discovery in 1948, but ...not a single thiopeptide has been approved for human use. This is mainly due to their poor solubility, challenging synthesis, and low bioavailability. This review summarizes the current research on the biosynthesis and biological activity of thiopeptide antibiotics since 2015. The focus of research since 2015 has been on uncovering biosynthetic routes, developing methods for total synthesis, and understanding the biological activity of thiopeptides. Overall, there is still much to learn about this family of molecules.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward. The motor ATPase ...PilB powers pilus assembly. Here we report the structures of the core ATPase domains of Geobacter metallireducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMP-PNP, at 3.4 and 2.3 Å resolution, respectively. These structures reveal important differences in nucleotide binding between chains. Analysis of these differences reveals the sequential turnover of nucleotide, and the corresponding domain movements. Our data suggest a clockwise rotation of the central sub-pores of PilB, which through interactions with PilC, would support the assembly of a right-handed helical pilus. Our analysis also suggests a counterclockwise rotation of the C2 symmetric PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction.
The mechanisms by which bacteria sense and respond to surface attachment have long been a mystery. Our understanding of the structure and dynamics of bacterial appendages, notably type IV pili (T4P), ...provided new insights into the potential ways that bacteria sense surfaces. T4P are ubiquitous, retractable hair-like adhesins that until recently were difficult to image in the absence of fixation due to their nanoscale size. This review focuses on recent microscopy innovations used to visualize T4P in live cells to reveal the dynamics of their retraction and extension. We discuss recently proposed mechanisms by which T4P facilitate bacterial surface sensing, including the role of surface-exposed PilY1, two-component signal transduction pathways, force-induced structural modifications of the major pilin, and altered dynamics of the T4P motor complex.
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