Single-molecule analysis of the entire perfringolysin O pore formation pathway

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

Changes in toxin production of environmental Pseudomonas aeruginosa isolates exposed to sub-inhibitory concentrations of three common antibiotics

Pseudomonas aeruginosa is an environmental pathogen that can cause severe infections in immunocompromised patients. P. aeruginosa infections are typically treated with multiple antibiotics including tobramycin, ciprofloxacin, and meropenem. However, antibiotics do not always entirely clear the bacteria from the infection site, where they may remain virulent. This is because the effective antibiotic concentration and diffusion in vitro may differ from the in vivo environment in patients. Therefore, it is important to understand the effect of non-lethal sub-inhibitory antibiotic concentrations on bacterial phenotype. Here, we investigate if sub-inhibitory antimicrobial concentrations cause alterations in bacterial virulence factor production using pyocyanin as a model toxin. We tested this using the aforementioned antibiotics on 10 environmental P. aeruginosa strains. Using on-the-spot electrochemical screening, we were able to directly quantify changes in production of pyocyanin in a measurement time of 17 seconds. Upon selecting 3 representative strains to further test the effects of sub-minimum inhibitory concentration (MICs), we found that pyocyanin production changed significantly when the bacteria were exposed to 10-fold MIC of the 3 antibiotics tested, and this was strain specific. A series of biologically relevant measured pyocyanin concentrations were also used to assess the effects of increased virulence on a culture of epithelial cells. We found a decreased viability of the epithelial cells when incubated with biologically relevant pyocyanin concentrations. This suggests that the antibiotic-induced virulence also is a value worth being enclosed in regular testing of pathogens.

ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli

The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic anti-inflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of regulatory elements involved in colibactin expression and in the growth conditions that promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization.

Crystal Structure of Exotoxin A from Aeromonas Pathogenic Species

Aeromonas exotoxin A (AE) is a bacterial virulence factor recently discovered in a clinical case of necrotising fasciitis caused by the flesh-eating Aeromonas hydrophila. Here, database mining shows that AE is present in the genome of several emerging Aeromonas pathogenic species. The X-ray crystal structure of AE was solved at 2.3 Å and presents all the hallmarks common to diphthamide-specific mono-ADP-ribosylating toxins, suggesting AE is a fourth member of this family alongside the diphtheria toxin, Pseudomonas exotoxin A and cholix. Structural homology indicates AE may use a similar mechanism of cytotoxicity that targets eukaryotic elongation factor 2 and thus inhibition of protein synthesis. The structure of AE also highlights unique features including a metal binding site, and a negatively charged cleft that could play a role in interdomain interactions and may affect toxicity. This study raises new opportunities to engineer alternative toxin-based molecules with pharmaceutical potential.

Cytolethal Distending Toxin Subunit B: A Review of Structure–Function Relationship

The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions.