N-acyl homoserine lactone signaling modulates bacterial community associated with human dental plaque.

N-acyl homoserine lactones (AHLs) are small diffusible signaling molecules that mediate a cell density-dependent bacterial communication system known as quorum sensing (QS). AHL-mediated QS regulates gene expression to control many critical bacterial behaviors including biofilm formation, pathogenicity, and antimicrobial resistance. Dental plaque is a complex multispecies oral biofilm formed by successive colonization of the tooth surface by groups of commensal, symbiotic, and pathogenic bacteria, which can contribute to tooth decay and periodontal diseases. While the existence and roles of AHL-mediated QS in oral microbiota have been debated, recent evidence indicates that AHLs play significant roles in oral biofilm development and community dysbiosis. The underlying mechanisms, however, remain poorly characterized. To better understand the importance of AHL signaling in dental plaque formation, we manipulated AHL signaling by adding AHL lactonases or exogenous AHL signaling molecules. We find that AHLs can be detected in dental plaque grown under 5% CO2 conditions, but not when grown under anaerobic conditions, and yet anaerobic cultures are still responsive to AHLs. QS signal disruption using lactonases leads to changes in microbial population structures in both planktonic and biofilm states, changes that are dependent on the substrate preference of the used lactonase but mainly result in the increase in the abundance of commensal and pioneer colonizer species. Remarkably, the opposite manipulation, that is the addition of exogenous AHLs increases the abundance of late colonizer bacterial species. Hence, this work highlights the importance of AHL-mediated QS in dental plaque communities, its potential different roles in anaerobic and aerobic parts of dental plaque, and underscores the potential of QS interference in the control of periodontal diseases.

Evidence for Complex Interplay between Quorum Sensing and Antibiotic Resistance in Pseudomonas aeruginosa.

Quorum sensing (QS) is a cell-density-dependent, intercellular communication system mediated by small diffusible signaling molecules. QS regulates a range of bacterial behaviors, including biofilm formation, virulence, drug resistance mechanisms, and antibiotic tolerance. Enzymes capable of degrading signaling molecules can interfere in QS-a process termed as quorum quenching (QQ). Remarkably, previous work reported some cases where enzymatic interference in QS was synergistic to antibiotics against Pseudomonas aeruginosa. The premise of combination therapy is attractive to fight against multidrug-resistant bacteria, yet comprehensive studies are lacking. Here, we evaluate the effects of QS signal disruption on the antibiotic resistance profile of P. aeruginosa by testing 222 antibiotics and antibacterial compounds from 15 different classes. We found compelling evidence that QS signal disruption does indeed affect antibiotic resistance (40% of all tested compounds; 89/222), albeit not always synergistically (not synergistic for 19% of compounds; 43/222). For some tested antibiotics, such as sulfathiazole and trimethoprim, we were able to relate the changes in resistance caused by QS signal disruption to the modulation of the expression of key genes of the folate biosynthetic pathway. Moreover, using a P. aeruginosa-based Caenorhabditis elegans killing model, we confirmed that enzymatic QQ modulates the effects of antibiotics on P. aeruginosa's pathogenicity in vivo. Altogether, these results show that signal disruption has profound and complex effects on the antibiotic resistance profile of P. aeruginosa. This work suggests that combination therapy including QQ and antibiotics should be discussed not globally but, rather, in case-by-case studies. IMPORTANCE Quorum sensing (QS) is a cell-density-dependent communication system used by a wide range of bacteria to coordinate behaviors. Strategies pertaining to the interference in QS are appealing approaches to control microbial behaviors that depend on QS, including virulence and biofilms. Interference in QS was previously reported to be synergistic with antibiotics, yet no systematic assessment exists. Here, we evaluate the potential of combination treatments using the model opportunistic human pathogen Pseudomonas aeruginosa PA14. In this model, collected data demonstrate that QS largely modulates the antibiotic resistance profile of PA14 (for more than 40% of the tested drugs). However, the outcome of combination treatments is synergistic for only 19% of them. This research demonstrates the complex relationship between QS and antibiotic resistance and suggests that combination therapy including QS inhibitors and antibiotics should be discussed not globally but, rather, in case-by-case studies.

Quorum quenching enzymes and their effects on virulence, biofilm, and microbiomes: a review of recent advances.

INTRODUCTION: Numerous bacterial behaviors are regulated by a cell-density dependent mechanism known as Quorum Sensing (QS). QS relies on communication between bacterial cells using diffusible signaling molecules known as autoinducers. QS regulates physiological processes such as metabolism, virulence, and biofilm formation. Quorum Quenching (QQ) is the inhibition of QS using chemical or enzymatic means to counteract behaviors regulated by QS. AREAS COVERED: We examine the main, diverse QS mechanisms present in bacterial species, with a special emphasis on AHL-mediated QS. We also discuss key in vitro and in vivo systems in which interference in QS was investigated. Additionally, we highlight promising developments, such as the substrate preference of the used enzymatic quencher, in the application of interference in QS to counter bacterial virulence. EXPERT OPINION: Enabled via the recent isolation of highly stable quorum quenching enzymes and/or molecular engineering efforts, the effects of the interference in QS were recently evaluated outside of the traditional model of single species culture. Signal disruption in complex microbial communities was shown to result in the disruption of complex microbial behaviors, and changes in population structures. These new findings, and future studies, may result in significant changes in the traditional views about QS.

Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway.

Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular ("passenger") domain and a short C-terminal segment that contributes four ß strands to a single 12-stranded ß barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to the barrel assembly machinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the ß barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions.IMPORTANCE Trimeric autotransporter adhesins (TAAs) are specialized bacterial outer membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked membrane spanning domain is completed.

Effects of Signal Disruption Depends on the Substrate Preference of the Lactonase.

Many bacteria produce and use extracellular signaling molecules such as acyl homoserine lactones (AHLs) to communicate and coordinate behavior in a cell-density dependent manner, via a communication system called quorum sensing (QS). This system regulates behaviors including but not limited to virulence and biofilm formation. We focused on Pseudomonas aeruginosa, a human opportunistic pathogen that is involved in acute and chronic lung infections and which disproportionately affects people with cystic fibrosis. P. aeruginosa infections are becoming increasingly challenging to treat with the spread of antibiotic resistance. Therefore, QS disruption approaches, known as quorum quenching, are appealing due to their potential to control the virulence of resistant strains. Interestingly, P. aeruginosa is known to simultaneously utilize two main QS circuits, one based on C4-AHL, the other with 3-oxo-C12-AHL. Here, we evaluated the effects of signal disruption on 39 cystic fibrosis clinical isolates of P. aeruginosa, including drug resistant strains. We used two enzymes capable of degrading AHLs, known as lactonases, with distinct substrate preference: one degrading 3-oxo-C12-AHL, the other degrading both C4-AHL and 3-oxo-C12-AHL. Two lactonases were used to determine the effects of signal disruption on the clinical isolates, and to evaluate the importance of the QS circuits by measuring effects on virulence factors (elastase, protease, and pyocyanin) and biofilm formation. Signal disruption results in at least one of these factors being inhibited for most isolates (92%). Virulence factor activity or production were inhibited by up to 100% and biofilm was inhibited by an average of 2.3 fold. Remarkably, the treatments led to distinct inhibition profiles of the isolates; the treatment with the lactonase degrading both signaling molecules resulted in a higher fraction of inhibited isolates (77% vs. 67%), and the simultaneous inhibition of more virulence factors per strain (2 vs. 1.5). This finding suggests that the lactonase AHL preference is key to its inhibitory spectrum and is an essential parameter to improve quorum quenching strategies.

Folding of a bacterial integral outer membrane protein is initiated in the periplasm.

The Bam complex promotes the insertion of ß-barrel proteins into the bacterial outer membrane, but it is unclear whether it threads ß-strands into the lipid bilayer in a stepwise fashion or catalyzes the insertion of pre-folded substrates. Here, to distinguish between these two possibilities, we analyze the biogenesis of UpaG, a trimeric autotransporter adhesin (TAA). TAAs consist of three identical subunits that together form a single ß-barrel domain and an extracellular coiled-coil ("passenger") domain. Using site-specific photocrosslinking to obtain spatial and temporal insights into UpaG assembly, we show that UpaG ß-barrel segments fold into a trimeric structure in the periplasm that persists until the termination of passenger-domain translocation. In addition to obtaining evidence that at least some ß-barrel proteins begin to fold before they interact with the Bam complex, we identify several discrete steps in the assembly of a poorly characterized class of virulence factors.

New functions for the ancient DedA membrane protein family.

The DedA protein family is a highly conserved and ancient family of membrane proteins with representatives in most sequenced genomes, including those of bacteria, archaea, and eukarya. The functions of the DedA family proteins remain obscure. However, recent genetic approaches have revealed important roles for certain bacterial DedA family members in membrane homeostasis. Bacterial DedA family mutants display such intriguing phenotypes as cell division defects, temperature sensitivity, altered membrane lipid composition, elevated envelope-related stress responses, and loss of proton motive force. The DedA family is also essential in at least two species of bacteria: Borrelia burgdorferi and Escherichia coli. Here, we describe the phylogenetic distribution of the family and summarize recent progress toward understanding the functions of the DedA membrane protein family.

Multiple envelope stress response pathways are activated in an Escherichia coli strain with mutations in two members of the DedA membrane protein family.

We have reported that simultaneous deletion of two Escherichia coli genes, yqjA and yghB, encoding related and conserved inner membrane proteins belonging to the DedA protein family results in a number of intriguing phenotypes, including temperature sensitivity at 42°C, altered membrane lipid composition, and cell division defects. We sought to characterize these and other phenotypes in an effort to establish a function for this protein family in E. coli. Here, using reporter assays, we show that the major envelope stress response pathways Cpx, Psp, Bae, and Rcs are activated in strain BC202 (W3110; ΔyqjA ΔyghB) at the permissive growth temperature of 30°C. We previously demonstrated that 10 mM Mg(2+), 400 mM NaCl, and overexpression of tatABC are capable of restoring normal growth to BC202 at elevated growth temperatures. Deletion of the cpxR gene from BC202 results in the loss of the ability of these supplements to restore growth at 42°C. Additionally, we report that the membrane potential of BC202 is significantly reduced and that cell division and growth can be restored either by expression of the multidrug transporter MdfA from a multicopy plasmid or by growth at pH 6.0. Together, these results suggest that the DedA family proteins YqjA and YghB are required for general envelope maintenance and homeostasis of the proton motive force under a variety of growth conditions.

BB0250 of Borrelia burgdorferi is a conserved and essential inner membrane protein required for cell division.

The gene bb0250 of Borrelia burgdorferi is a homolog of the dedA family, encoding integral inner membrane proteins that are present in nearly all species of bacteria. To date, no precise function has been attributed to any dedA gene. Unlike many bacterial species, such as Escherichia coli, which has eight dedA genes, B. burgdorferi possesses only one, annotated bb0250, providing a unique opportunity to investigate the functions of the dedA family. Here, we show that bb0250 is able to restore normal growth and cell division to a temperature-sensitive E. coli mutant with simultaneous deletions of two dedA genes, yqjA and yghB, and encodes a protein that localizes to the inner membrane of E. coli. The bb0250 gene could be deleted from B. burgdorferi only after introduction of a promoterless bb0250 under the control of an inducible lac promoter, indicating that it is an essential gene in this organism. Growth of the mutant in the absence of isopropyl-ß-d-thiogalactopyranoside resulted in cell death, preceded by cell division defects characterized by elongated cells and membrane bulges, demonstrating that bb0250 is required for proper cell division and envelope integrity. Finally, we show that BB0250 depletion leads to imbalanced membrane phospholipid composition in borrelia. These results demonstrate a strong conservation of function of the dedA gene family across diverse species of Gram-negative bacteria and a requirement for this protein family for normal membrane lipid composition and cell division.

Inefficient Tat-dependent export of periplasmic amidases in an Escherichia coli strain with mutations in two DedA family genes.

The DedA family genes are found in most bacterial genomes. Two of these proteins are Escherichia coli YqjA and YghB, predicted inner membrane proteins of unknown function sharing 61% amino acid identity. The E. coli single deletion mutants are largely without phenotype, but the double mutant (BC202; Delta yqjA::Tet(r) Delta yghB::Kan(r)) is characterized by incomplete cell division, temperature sensitivity, and altered phospholipid levels (K. Thompkins et al., J. Bacteriol. 190:4489-4500, 2008). In this report, we have better characterized the cell division chaining defect of BC202. Fluorescence recovery after photobleaching indicates that 58% of the cells in chains are compartmentalized by at least a cytoplasmic membrane. Green fluorescent protein fusions to the cell division proteins FtsZ, ZipA, FtsI, FtsL, and FtsQ are correctly localized to new septation sites in BC202. Periplasmic amidases AmiC and AmiA, secreted by the twin arginine transport (Tat) pathway, are localized to the cytoplasm in BC202. Overexpression of AmiA, AmiC, or AmiB, a periplasmic amidase secreted via the general secretory pathway, restores normal cell division but does not suppress the temperature sensitivity of BC202, indicating that YghB and YqjA may play additional roles in cellular physiology. Strikingly, overexpression of the Tat export machinery (TatABC) results in normal cell division and growth at elevated temperatures. These data collectively suggest that the twin arginine pathway functions inefficiently in BC202, likely due to the altered levels of membrane phospholipids in this mutant. These results underscore the importance of membrane composition in the proper function of the Tat protein export pathway.