Abram Aertsen

Comparison of sublethal injury induced in Salmonella enterica serovar Typhimurium by heat and by different nonthermal treatments

We have studied sublethal injury in Salmonella enterica serovar Typhimurium caused by mild heat and by different emerging nonthermal food preservation treatments, i.e., high-pressure homogenization, high hydrostatic pressure, pulsed white light, and pulsed electric field. Sublethal injury was determined by plating on different selective media, i.e., tryptic soy agar (TSA) plus 3% NaCl, TSA adjusted to pH 5.5, and violet red bile glucose agar. For each inactivation technique, at least five treatments using different doses were applied in order to cover an inactivation range of 0 to 5 log units. For all of the treatments performed with a technique, the logarithm of the viability reductions measured on each of the selective plating media was plotted against the logarithm of the viability reduction on TSA as a nonselective medium, and these points were fined by a straight line. Sublethal injury between different techniques was then compared by the slope and the y intercept of these regression lines. The highest levels of sublethal injury were observed for the heat and high hydrostatic pressure treatments. Sublethal injury after those treatments was observed on all selective plating media. For the heat treatment, but not for the high-pressure treatment, sublethal injury occurred at low doses, which were not yet lethal. The other nonthermal techniques resulted in sublethal injury on only some of the selective plating media, and the levels of injury were much lower. The different manifestations of sublethal injury were attributed to different inactivation mechanisms by each of the techniques, and a mechanistic model is proposed to explain these differences.

Biotechnology under high pressure: applications and implications

Pressure is a thermodynamic parameter whose unique effects on biological systems are increasingly being studied in a growing number of scientific fields. As such, the effects of high pressure are currently being investigated at different levels, ranging from proteins, enzymes and viruses to microorganisms, mammalian cells and tissues. Together with the steadily growing knowledge and understanding of high pressure effects on these increasingly complex systems, the purposeful use of high pressure has found several unique applications in bioscience over the past few years, including the disaggregation of proteins, the preparation of viral vaccines and the modulation of food functionality. In this review, recent and emerging applications of high pressure in biotechnology are presented and discussed.

Bacterial interactions in biofilms

It is generally acknowledged that biofilms are the dominant lifestyle of bacteria, both in the natural environment as on manmade settings such as industrial and medical devices. This attached form of cell growth consists of slime matrix embedded bacteria of either a single, but mostly of multiple microbial species that form an interdependent structured community, capable of coordinated and collective behavior. Although research on multispecies biofilms is still in its infancy, this review will focus on these complex communities where cooperation and antagonism are keys to increase the fitness of the different species and where intercellular interactions and communication are means to achieve this goal.

Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

ABSTRACT A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ32, was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.

Stress and How Bacteria Cope with Death and Survival

Bacterial populations that are exposed to rapidly changing and sometimes hostile environments constantly switch between growth, survival, and death. Understanding bacterial survival and death are therefore cornerstones in a full comprehension of microbial life. During the last few years, new insights have emerged regarding the mechanisms of bacterial inactivation under stressful conditions. Particularly under mildly lethal stress, the ultimate cause of inactivation often seems mediated by the cell itself and is subject to additional regulation that integrates information about the global state of the cell and its environmental and social surrounding. This article explores the thin line between bacterial growth and inactivation and focuses on some emerging bacterial survival strategies, both from an individual cell and from a population perspective.

An SOS Response Induced by High Pressure in Escherichia coli

Although pressure is an important environmental parameter in microbial niches such as the deep sea and is furthermore used in food preservation to inactivate microorganisms, the fundamental understanding of its effects on bacteria remains fragmentary. Our group recently initiated differential fluorescence induction screening to search for pressure-induced Escherichia coli promoters and has already reported induction of the heat shock regulon. Here the screening was continued, and we report for the first time that pressure induces a bona fide SOS response in E. coli, characterized by the RecA and LexA-dependent expression of uvrA, recA, and sulA. Moreover, it was shown that pressure is capable of triggering lambda prophage induction in E. coli lysogens. The remnant lambdoid e14 element, however, could not be induced by pressure, as opposed to UV irradiation, indicating subtle differences between the pressure-induced and the classical SOS response. Furthermore, the pressure-induced SOS response seems not to be initiated by DNA damage, since DeltarecA and lexA1 (Ind-) mutants, which are intrinsically hypersensitive to DNA damage, were not sensitized or were only very slightly sensitized for pressure-mediated killing and since pressure treatment was not found to be mutagenic. In light of these findings, the current knowledge of pressure-mediated effects on bacteria is discussed.

Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens

ABSTRACT The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). IMPORTANCE Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens. Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.

A New Family of Lysozyme Inhibitors Contributing to Lysozyme Tolerance in Gram-Negative Bacteria

Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host.

Induction of oxidative stress by high hydrostatic pressure in Escherichia coli

ABSTRACT Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.

Regulation and quality control by Lon-dependent proteolysis.

After their first discovery in Escherichia coli, Lon homologues were found to be widely distributed among prokaryotes to eukaryotes. The ATP-dependent Lon protease belongs to the AAA(+) (ATPases associated with a variety of cellular activities) superfamily, and is involved in both general quality control by degrading abnormal proteins and in the specific control of several regulatory proteins. As such, this enzyme has a pivotal role in quality control and cellular physiology. This review focuses on mechanisms of degradation both from the protease and substrate points of view, and discusses the role of Lon in global regulation, stress response and virulence.