Natalie Verstraeten

Living on a surface: swarming and biofilm formation

Swarming is the fastest known bacterial mode of surface translocation and enables the rapid colonization of a nutrient-rich environment and host tissues. This complex multicellular behavior requires the integration of chemical and physical signals, which leads to the physiological and morphological differentiation of the bacteria into swarmer cells. Here, we provide a review of recent advances in the study of the regulatory pathways that lead to swarming behavior of different model bacteria. It has now become clear that many of these pathways also affect the formation of biofilms, surface-attached bacterial colonies. Decision-making between rapidly colonizing a surface and biofilm formation is central to bacterial survival among competitors. In the second part of this article, we review recent developments in the understanding of the transition between motile and sessile lifestyles of bacteria.

New-found fundamentals of bacterial persistence

Persister cells display tolerance to high doses of bactericidal antibiotics and typically comprise a small fraction of a bacterial population. Recently, evidence was provided for a causal link between therapy failure and the presence of persister cells in chronic infections, underscoring the need for research on bacterial persistence. A series of recent breakthroughs have shed light on the multiplicity of persister genes, the contribution of gene expression noise to persister formation, the importance of active responses to antibiotic tolerance and heterogeneity among persister cells. Moreover, the development of in vivo model systems has highlighted the clinical relevance of persistence. This review discusses these recent advances and how this knowledge fundamentally changes the way in which we will perceive the problem of antibiotic tolerance in years to come.

The Universally Conserved Prokaryotic GTPases

SUMMARY Members of the large superclass of P-loop GTPases share a core domain with a conserved three-dimensional structure. In eukaryotes, these proteins are implicated in various crucial cellular processes, including translation, membrane trafficking, cell cycle progression, and membrane signaling. As targets of mutation and toxins, GTPases are involved in the pathogenesis of cancer and infectious diseases. In prokaryotes also, it is hard to overestimate the importance of GTPases in cell physiology. Numerous papers have shed new light on the role of bacterial GTPases in cell cycle regulation, ribosome assembly, the stress response, and other cellular processes. Moreover, bacterial GTPases have been identified as high-potential drug targets. A key paper published over 2 decades ago stated that, “It may never again be possible to capture [GTPases] in a family portrait” (H. R. Bourne, D. A. Sanders, and F. McCormick, Nature 348:125-132, 1990) and indeed, the last 20 years have seen a tremendous increase in publications on the subject. Sequence analysis identified 13 bacterial GTPases that are conserved in at least 75% of all bacterial species. We here provide an overview of these 13 protein subfamilies, covering their cellular functions as well as cellular localization and expression levels, three-dimensional structures, biochemical properties, and gene organization. Conserved roles in eukaryotic homologs will be discussed as well. A comprehensive overview summarizing current knowledge on prokaryotic GTPases will aid in further elucidating the function of these important proteins.

Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening

Persister cells are phenotypic variants that are extremely tolerant to high concentrations of antibiotics. They constitute a fraction of stationary phase cultures and biofilm populations of numerous bacterial species, such as the opportunistic pathogen Pseudomonas aeruginosa. Even though persisters are believed to be an important cause of incomplete elimination of infectious populations by antibiotics, their nature remains obscure. Most studies on persistence have focused on the model organism Escherichia coli and only a limited number of persistence genes have been identified to date. We performed the first large-scale screening of a P. aeruginosa PA14 mutant library to identify novel genes involved in persistence. A total of 5000 mutants were screened in a high-throughput manner and nine new persistence mutants were identified. Four mutants (with insertions in dinG, spuC, PA14_17880 and PA14_66140) exhibited a low persister phenotype and five mutants (in algR, pilH, ycgM, pheA and PA14_13680) displayed high persistence. These genes may serve as new candidate drug targets in the combat against P. aeruginosa infections.

Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence.

Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence

The evolution of antibiotic resistance is a major threat to society and has been predicted to lead to 10 million casualties annually by 20501. Further aggravating the problem, multidrug tolerance in bacteria not only relies on the build-up of resistance mutations, but also on some cells epigenetically switching to a non–growing antibiotic-tolerant ‘persister’ state2–6. Yet, despite its importance, we know little of how persistence evolves in the face of antibiotic treatment7. Our evolution experiments in Escherichia coli demonstrate that extremely high levels of multidrug tolerance (20–100%) are achieved by single point mutations in one of several genes and readily emerge under conditions approximating clinical, once-daily dosing schemes. In contrast, reversion to low persistence in the absence of antibiotic treatment is relatively slow and only partially effective. Moreover, and in support of previous mathematical models8–10, we show that bacterial persistence quickly adapts to drug treatment frequency and that the observed rates of switching to the persister state can be understood in the context of ‘bet-hedging’ theory. We conclude that persistence is a major component of the evolutionary response to antibiotics that urgently needs to be considered in both diagnostic testing and treatment design in the battle against multidrug tolerance.

Fungal β-1,3-Glucan Increases Ofloxacin Tolerance of Escherichia coli in a Polymicrobial E. coli/Candida albicans Biofilm

ABSTRACT In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.

Bacterial Obg proteins: GTPases at the nexus of protein and DNA synthesis

Abstract Obg proteins (also known as ObgE, YhbZ and CgtA) are conserved P-loop GTPases, essential for growth in bacteria. Like other GTPases, Obg proteins cycle between a GTP-bound ON and a GDP-bound OFF state, thereby controlling cellular processes. Interestingly, the in vitro biochemical properties of Obg proteins suggest that they act as sensors for the cellular GDP/GTP pools and adjust their activity according to the cellular energy status. Obg proteins have been attributed a host of cellular functions, including roles in essential cellular processes (DNA replication, ribosome maturation) and roles in different stress adaptation pathways (stringent response, sporulation, general stress response). This review summarizes the current knowledge on Obg activity and function. Furthermore, we present a model that integrates the different functions of Obg by assigning it a fundamental role in cellular physiology, at the hub of protein and DNA synthesis. In particular, we believe that Obg proteins might provide a connection between different global pathways in order to fine-tune cellular processes in response to a given energy status.

PSEUDOMONAS AERUGINOSA FOSFOMYCIN RESISTANCE MECHANISMS AFFECT NON-INHERITED FLUOROQUINOLONE TOLERANCE

Pseudomonas aeruginosa is an opportunistic pathogen that poses a threat in clinical settings due to its intrinsic and acquired resistance to a wide spectrum of antibiotics. Additionally, the presence of a subpopulation of cells surviving high concentrations of antibiotics, called persisters, makes it virtually impossible to eradicate a chronic infection. The mechanism underlying persistence is still unclear, partly due to the fact that it is a non-inherited phenotype. Based on our findings from a previously performed screening effort for P. aeruginosa persistence genes, we hypothesize that crosstalk can occur between two clinically relevant mechanisms: the persistence phenomenon and antibiotic resistance. This was tested by determining the persistence phenotype of P. aeruginosa strains that are resistant to the antibiotic fosfomycin due to either of two unrelated fosfomycin resistance mechanisms. Overexpression of fosA (PA1129) confers fosfomycin resistance by enzymic modification of the antibiotic, and in addition causes a decrease in the number of persister cells surviving ofloxacin treatment. Both phenotypes require the enzymic function of FosA, as mutation of the Arg119 residue abolishes fosfomycin resistance as well as low persistence. The role for fosfomycin resistance mechanisms in persistence is corroborated by demonstrating a similar phenotype in a strain with a mutation in glpT (PA5235), which encodes a glycerol-3-phosphate transporter essential for fosfomycin uptake. These results indicate that fosfomycin resistance, conferred by glpT mutation or by overexpression of fosA, results in a decrease in the number of persister cells after treatment with ofloxacin and additionally stress that further research into the interplay between fosfomycin resistance and persistence is warranted.

Derivatives of the Mouse Cathelicidin-Related Antimicrobial Peptide (CRAMP) Inhibit Fungal and Bacterial Biofilm Formation

ABSTRACT We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 μM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 μM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.