Sander K. Govers

Different Levels of Catabolite Repression Optimize Growth in Stable and Variable Environments

This study uses experimentally evolved brewers yeasts to explore the costs and benefits of different nutrient-switching strategies when energy sources vary or remain constant.

Efficacy of Artilysin Art-175 against Resistant and Persistent Acinetobacter baumannii.

ABSTRACT Bacteriophage-encoded endolysins have shown promise as a novel class of antibacterials with a unique mode of action, i.e., peptidoglycan degradation. However, Gram-negative pathogens are generally not susceptible due to their protective outer membrane. Artilysins overcome this barrier. Artilysins are optimized, engineered fusions of selected endolysins with specific outer membrane-destabilizing peptides. Artilysin Art-175 comprises a modified variant of endolysin KZ144 with an N-terminal fusion to SMAP-29. Previously, we have shown the high susceptibility of Pseudomonas aeruginosa to Art-175. Here, we report that Art-175 is highly bactericidal against stationary-phase cells of multidrug-resistant Acinetobacter baumannii, even resulting in a complete elimination of large inocula (≥108 CFU/ml). Besides actively dividing cells, Art-175 also kills persisters. Instantaneous killing of A. baumannii upon contact with Art-175 could be visualized after immobilization of the bacteria in a microfluidic flow cell. Effective killing of a cell takes place through osmotic lysis after peptidoglycan degradation. The killing rate is enhanced by the addition of 0.5 mM EDTA. No development of resistance to Art-175 under selection pressure and no cross-resistance with existing resistance mechanisms could be observed. In conclusion, Art-175 represents a highly active Artilysin against both A. baumannii and P. aeruginosa, two of the most life-threatening pathogens of the order Pseudomonadales.

Exposure to high hydrostatic pressure rapidly selects for increased RpoS activity and general stress-resistance in Escherichia coli O157:H7.

Exposure to high hydrostatic pressure (HHP) is increasingly being used in food preservation as a non-thermal pasteurization process, and its further implementation necessitates a more thorough understanding of bacterial resistance development and intraspecies variability with regard to inactivation by HHP. In this report, we discovered that exposure to high hydrostatic pressure stress can rapidly select for strongly increased RpoS activity in a hypersensitive Escherichia coli O157:H7 strain (ATCC 43888), leading to a simultaneous increase in HHP and heat resistance. Moreover, the level of RpoS activity correlated well with the original hypersensitivity and the extent of acquired HHP resistance, and extremely HHP-resistant mutants of ATCC 43888 clearly incurred a number of additional RpoS-dependent phenotypes. These findings suggest that implementation of novel processing techniques in the food production chain can readily affect the physiology of food-borne pathogens.

In vivo disassembly and reassembly of protein aggregates in Escherichia coli

Protein misfolding and aggregation are inevitable but detrimental cellular processes. Cells therefore possess protein quality control mechanisms based on chaperones and proteases that (re)fold or hydrolyze unfolded, misfolded, and aggregated proteins. Besides these conserved quality control mechanisms, the spatial organization of protein aggregates (PAs) inside the cell has been proposed as an important additional strategy to deal with their cytotoxicity. In the bacterium Escherichia coli, however, it remained unclear how this spatial organization is established and how this process of assembling PAs in the cell poles affects cellular physiology. In this report, high hydrostatic pressure was used to transiently reverse protein aggregation in living E. coli cells, allowing the subsequent (re)assembly of PAs to be studied in detail. This approach revealed PA assembly to be dependent on intracellular energy and metabolic activity, with the resulting PA structure being confined to the cell pole by nucleoid occlusion. Moreover, a correlation could be observed between the time needed for PA reassembly and the individual lag time of the cells, which might prevent symmetric segregation of cytotoxic PAs among siblings to occur and ensure rapid spatial clearance of molecular damage throughout the emerging population.

‘Artilysation’ of endolysin λSa2lys strongly improves its enzymatic and antibacterial activity against streptococci

Endolysins constitute a promising class of antibacterials against Gram-positive bacteria. Recently, endolysins have been engineered with selected peptides to obtain a new generation of lytic proteins, Artilysins, with specific activity against Gram-negative bacteria. Here, we demonstrate that artilysation can also be used to enhance the antibacterial activity of endolysins against Gram-positive bacteria and to reduce the dependence on external conditions. Art-240, a chimeric protein of the anti-streptococcal endolysin λSa2lys and the polycationic peptide PCNP, shows a similar species specificity as the parental endolysin, but the bactericidal activity against streptococci increases and is less affected by elevated NaCl concentrations and pH variations. Time-kill experiments and time-lapse microscopy demonstrate that the killing rate of Art-240 is approximately two-fold higher compared to wildtype endolysin λSa2lys, with a reduction in viable bacteria of 3 log units after 10 min. In addition, lower doses of Art-240 are required to achieve the same bactericidal effect.

Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association

Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.

Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli

Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image‐based quantitative screen of the single‐gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high‐dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.

Impact of high hydrostatic pressure processing on individual cellular resuscitation times and protein aggregates in Escherichia coli

Live cell biology approaches can contribute to a more comprehensive understanding of heterogeneous injury and resuscitation phenomena in stressed populations of foodborne pathogens and spoilage microorganisms, and in turn lead to better insights in the mechanisms and dynamics of inactivation that can improve food safety and preservation measures. Especially in the context of designing minimal processing strategies, which depend on a synergistic combination of different mild stresses to ensure sufficient microbial reduction, a more profound understanding of the impact of each such stress or hurdle is mandatory. High hydrostatic pressure (HHP) stress is an interesting hurdle in this concept since cells that manage to survive this stress nevertheless tend to be injured and sensitized to subsequent stresses. In this study, populations of Escherichia coli were subjected to different HHP intensities and studied at the single-cell level with time-lapse fluorescence microscopy while monitoring resuscitation times and protein aggregate integrity at the single-cell level. This approach revealed that higher pressure intensities lead to longer and more variable resuscitation times of surviving cells as well as an increased dispersal of intracellular protein aggregates. Interestingly, at mild HHP exposure, cells within the population incurring less dispersion of protein aggregates appeared to have a higher probability of survival.

Intracellular movement of protein aggregates reveals heterogeneous inactivation and resuscitation dynamics in stressed populations of Escherichia coli

Inactivation of bacterial pathogens is of critical importance in fields ranging from antimicrobial therapy to food preservation. The efficacy of an antimicrobial treatment is often experimentally determined through viable plate counts that inherently provide a poor focus on the mechanisms and distribution of (sub)lethal injury and subsequent inactivation or resuscitation behavior of the stressed cells, which are increasingly important features for the proper understanding and design of inactivation strategies. In this report, we employ a live cell biology approach focusing on the energy-dependent motion of intracellular protein aggregates to investigate the heterogeneity within heat stressed Escherichia coli populations. As such, we were able to identify differential dynamics of cellular resuscitation and inactivation that are impossible to distinguish using more traditional approaches. Moreover, our data indicate the existence of late-resuscitating cells that remain physiologically active and are able to persist in the presence of antibiotics before resuscitation.

Severely Heat Injured Survivors of E. coli O157:H7 ATCC 43888 Display Variable and Heterogeneous Stress Resistance Behavior

Although minimal food processing strategies aim to eliminate foodborne pathogens and spoilage microorganisms through a combination of mild preservation techniques, little is actually known on the resistance behavior of the small fraction of microorganisms surviving an inimical treatment. In this study, the conduct of severely heat stressed survivors of E. coli O157:H7 ATCC 43888, as an indicator for the low infectious dose foodborne enterohemorrhagic strains, was examined throughout their resuscitation and outgrowth. Despite the fact that these survivors were initially sublethally injured, they were only marginally more sensitive to a subsequent heat treatment and actually much more resistant to a subsequent high hydrostatic pressure (HHP) shock in comparison with unstressed control cells. Throughout further resuscitation, however, their initial HHP resistance rapidly faded out, while their heat resistance increased and surpassed the initial heat resistance of unstressed control cells. Results also indicated that the population eventually emerging from the severely heat stressed survivors heterogeneously consisted of both growing and non-growing cells. Together, these observations provide deeper insights into the particular behavior and heterogeneity of stressed foodborne pathogens in the context of food preservation.