Fany Reffuveille

Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies.

Bacteria have evolved the ability to form multicellular, surface-adherent communities called biofilms that allow survival in hostile environments. In clinical settings, bacteria are exposed to various sources of stress, including antibiotics, nutrient limitation, anaerobiosis, heat shock, etc., which in turn trigger adaptive responses in bacterial cells. The combination of this and other defense mechanisms results in the formation of highly (adaptively) resistant multicellular structures that are recalcitrant to host immune clearance mechanisms and very difficult to eradicate with the currently available antimicrobial agents, which are generally developed for the eradication of free-swimming (planktonic) bacteria. However, novel strategies that specifically target the biofilm mode of growth have been recently described, thus providing the basis for future anti-biofilm therapy.

Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

D-Enantiomeric Peptides that Eradicate Wild-Type and Multidrug-Resistant Biofilms and Protect against Lethal Pseudomonas aeruginosa Infections

In many infections, bacteria form surface-associated communities known as biofilms that are substantially more resistant to antibiotics than their planktonic counterparts. Based on the design features of active antibiofilm peptides, we made a series of related 12-amino acid L-, D- and retro-inverso derivatives. Specific D-enantiomeric peptides were the most potent at inhibiting biofilm development and eradicating preformed biofilms of seven species of wild-type and multiply antibiotic-resistant Gram-negative pathogens. Moreover, these peptides showed strong synergy with conventional antibiotics, reducing the antibiotic concentrations required for complete biofilm inhibition by up to 64-fold. As shown previously for 1018, these D-amino acid peptides targeted the intracellular stringent response signal (p)ppGpp. The most potent peptides DJK-5 and DJK-6 protected invertebrates from lethal Pseudomonas aeruginosa infections and were considerably more active than a previously described L-amino acid peptide 1018. Thus, the protease-resistant peptides produced here were more effective both in vitro and in vivo.

A Broad-Spectrum Antibiofilm Peptide Enhances Antibiotic Action against Bacterial Biofilms

ABSTRACT Biofilm-related infections account for at least 65% of all human infections, but there are no available antimicrobials that specifically target biofilms. Their elimination by available treatments is inefficient since biofilm cells are between 10- and 1,000-fold more resistant to conventional antibiotics than planktonic cells. Here we describe the synergistic interactions, with different classes of antibiotics, of a recently characterized antibiofilm peptide, 1018, to potently prevent and eradicate bacterial biofilms formed by multidrug-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Combinations of peptide 1018 and the antibiotic ceftazidime, ciprofloxacin, imipenem, or tobramycin were synergistic in 50% of assessments and decreased by 2- to 64-fold the concentration of antibiotic required to treat biofilms formed by Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus. Furthermore, in flow cell biofilm studies, combinations of low, subinhibitory levels of the peptide (0.8 μg/ml) and ciprofloxacin (40 ng/ml) decreased dispersal and triggered cell death in mature P. aeruginosa biofilms. In addition, short-term treatments with the peptide in combination with ciprofloxacin prevented biofilm formation and reduced P. aeruginosa PA14 preexisting biofilms. PCR studies indicated that the peptide suppressed the expression of various antibiotic targets in biofilm cells. Thus, treatment with the peptide represents a novel strategy to potentiate antibiotic activity against biofilms formed by multidrug-resistant pathogens.

Effect of Nitroxides on Swarming Motility and Biofilm Formation, Multicellular Behaviors in Pseudomonas aeruginosa

ABSTRACT The ability of nitric oxide (NO) to induce biofilm dispersion has been well established. Here, we investigated the effect of nitroxides (sterically hindered nitric oxide analogues) on biofilm formation and swarming motility in Pseudomonas aeruginosa. A transposon mutant unable to produce nitric oxide endogenously (nirS) was deficient in swarming motility relative to the wild type and the complemented strain. Moreover, expression of the nirS gene was upregulated by 9.65-fold in wild-type swarming cells compared to planktonic cells. Wild-type swarming levels were substantially restored upon the exogenous addition of nitroxide containing compounds, a finding consistent with the hypothesis that NO is necessary for swarming motility. Here, we showed that nitroxides not only mimicked the dispersal activity of NO but also prevented biofilms from forming in flow cell chambers. In addition, a nirS transposon mutant was deficient in biofilm formation relative to the wild type and the complemented strain, thus implicating NO in the formation of biofilms. Intriguingly, despite its stand-alone action in inhibiting biofilm formation and promoting dispersal, a nitroxide partially restored the ability of a nirS mutant to form biofilms.

Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing.

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed “ruler” protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a “ball-and-chain” architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler proteins N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.

Potentiation of ciprofloxacin action against Gram-negative bacterial biofilms by a nitroxide.

We previously showed that soluble nitroxides (nitric oxide analogues) mimicked the well-established ability of nitric oxide to cause biofilm dispersal and further showed that these compounds could prevent biofilm formation. Here, we investigated the effect of the nitroxide carboxy-TEMPO in combination with sub μg/ml concentrations of ciprofloxacin on pre-formed flow cell biofilms formed by Gram-negative bacteria. Combination therapy led to substantial eradication of existing biofilms formed by Pseudomonas aeruginosa PA14 (99.3%) and Escherichia coli O157 (93%).

Novel roles for two-component regulatory systems in cytotoxicity and virulence-related properties in Pseudomonas aeruginosa

The rapid adaptation of the opportunistic bacterial pathogen Pseudomonas aeruginosa to various growth modes and environmental conditions is controlled in part through diverse two-component regulatory systems. Some of these systems are well studied, but the majority are poorly characterized, even though it is likely that several of these systems contribute to virulence. Here, we screened all available strain PA14 mutants in 50 sensor kinases, 50 response regulators and 5 hybrid sensor/regulators, for contributions to cytotoxicity against cultured human bronchial epithelial cells, as assessed by the release of cytosolic lactate dehydrogenase. This enabled the identification of 8 response regulators and 3 sensor kinases that caused substantial decreases in cytotoxicity, and 5 response regulators and 8 sensor kinases that significantly increased cytotoxicity by 15–58% or more. These regulators were additionally involved in motility, adherence, type 3 secretion, production of cytotoxins, and the development of biofilms. Here we investigated in more detail the roles of FleSR, PilSR and WspR. Not all cognate pairs contributed to cytotoxicity (e.g. PhoPQ, PilSR) in the same way and some differences could be detected between the same mutants in PAO1 and PA14 strain backgrounds (e.g. FleSR, PhoPQ). This study highlights the potential importance of these regulators and their downstream targets on pathogenesis and demonstrates that cytotoxicity can be regulated by several systems and that their contributions are partly dependent on strain background.

Two isoforms of Clp peptidase in Pseudomonas aeruginosa control distinct aspects of cellular physiology

Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogen Pseudomonas aeruginosa, in which there is an archetypical isoform, ClpP1, and a second isoform, ClpP2, about which little is known. Here, we use phenotypic assays to investigate the biological roles of ClpP1 and ClpP2 and biochemical assays to characterize purified ClpP1, ClpP2, ClpX, and ClpA. Interestingly, ClpP1 and ClpP2 have distinct intracellular roles for motility, pigment production, iron scavenging, and biofilm formation. Of particular interest, ClpP2, but not ClpP1, is required for microcolony organization, where multicellular organized structures first form on the pathway to biofilm production. We found that purified ClpP1 with ClpX or ClpA was enzymatically active, yet to our surprise, ClpP2 was inactive and not fully assembled in vitro; attempts to assist ClpP2 assembly and activation by mixing with the other Clp components failed to turn on ClpP2, as did solution conditions that have helped activate other ClpPs in vitro We postulate that the active form of ClpP2 has yet to be discovered, and we present several potential models to explain its activation as well as the unique role ClpP2 plays in the development of the clinically important biofilms in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is responsible for severe infections of immunocompromised patients. Our work demonstrates that two different isoforms of the Clp peptidase, ClpP1 and ClpP2, control distinct aspects of cellular physiology for this organism. In particular, we identify ClpP2 as being necessary for microcolony organization. Pure active forms of ClpP1 and either ClpX or ClpA were characterized as assembled and active, and ClpP2 was incompletely assembled and inactive. By establishing both the unique biological roles of ClpP1 and ClpP2 and their initial biochemical assemblies, we have set the stage for important future work on the structure, function, and biological targets of Clp proteolytic enzymes in this important opportunistic pathogen.