Carla Cugini

Farnesol, a common sesquiterpene, inhibits PQS production in Pseudomonas aeruginosa.

Farnesol is a sesquiterpene produced by many organisms, including the fungus Candida albicans. Here, we report that the addition of farnesol to cultures of Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, leads to decreased production of the Pseudomonas quinolone signal (PQS) and the PQS‐controlled virulence factor, pyocyanin. Within 15 min of farnesol addition, decreased transcript levels of pqsA, the first gene in the PQS biosynthetic operon, were observed. Transcript levels of pqsR (mvfR), which encodes the transcription factor that positively regulates pqsA, were unaffected. An Escherichia coli strain producing PqsR and containing the pqsA promoter fused to lacZ similarly showed that farnesol inhibited PQS‐stimulated transcription. Electrophoretic mobility shift assays showed that, like PQS, farnesol stimulated PqsR binding to the pqsA promoter at a previously characterized LysR binding site, suggesting that farnesol promoted a non‐productive interaction between PqsR and the pqsA promoter. Growth with C. albicans leads to decreased production of PQS and pyocyanin by P. aeruginosa, suggesting that the amount of farnesol produced by the fungus is sufficient to impact P. aeruginosa PQS signalling. Related isoprenoid compounds, but not other long‐chain alcohols, also inhibited PQS production at micromolar concen‐trations, suggesting that related compounds may participate in interspecies interactions with P. aeruginosa.

Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin αvβ3

Borrelia burgdorferi, the agent of Lyme disease, expresses several adhesion molecules that are probably required for initial establishment of infection in mammalian hosts, and for colonization of various tissues within the host. The B. burgdorferi outer membrane protein P66 was previously identified as a ligand for β3-chain integrins by using a variety of biochemical approaches. Although the earlier data suggested that P66 is an adhesin that mediates B. burgdorferi attachment to β3-chain integrins, lack of genetic systems in B. burgdorferi precluded definitive demonstration of a role for P66 in β3 integrin attachment by intact borreliae. Recent advances in the genetic manipulation of B. burgdorferi have now made possible the targeted disruption of the p66 gene. Mutants in p66 show dramatically reduced attachment to integrin αvβ3. This is, to our knowledge, the first description of the targeted disruption of a candidate B. burgdorferi virulence factor with a known biochemical function that can be quantified, and demonstrates the importance of B. burgdorferi P66 in the attachment of this pathogenic spirochete to a human cell-surface receptor.

Candida albicans-produced farnesol stimulates Pseudomonas quinolone signal production in LasR-defective Pseudomonas aeruginosa strains

Candida albicans has been previously shown to stimulate the production of Pseudomonas aeruginosa phenazine toxins in dual-species colony biofilms. Here, we report that P. aeruginosa lasR mutants, which lack the master quorum sensing system regulator, regain the ability to produce quorum-sensing-regulated phenazines when cultured with C. albicans. Farnesol, a signalling molecule produced by C. albicans, was sufficient to stimulate phenazine production in LasR− laboratory strains and clinical isolates. P. aeruginosa ΔlasR mutants are defective in production of the Pseudomonas quinolone signal (PQS) due to their inability to properly induce pqsH, which encodes the enzyme necessary for the last step in PQS biosynthesis. We show that expression of pqsH in a ΔlasR strain was sufficient to restore PQS production, and that farnesol restored pqsH expression in ΔlasR mutants. The farnesol-mediated increase in pqsH required RhlR, a transcriptional regulator downstream of LasR, and farnesol led to higher levels of N-butyryl-homoserine lactone, the small molecule activator of RhlR. Farnesol promotes the production of reactive oxygen species (ROS) in a variety of species. Because the antioxidant N-acetylcysteine suppressed farnesol-induced RhlR activity in LasR− strains, and hydrogen peroxide was sufficient to restore PQS production in las mutants, we propose that ROS are responsible for the activation of downstream portions of this quorum sensing pathway. LasR mutants frequently arise in the lungs of patients chronically infected with P. aeruginosa. The finding that C. albicans, farnesol or ROS stimulate virulence factor production in lasR strains provides new insight into the virulence potential of these strains.

Borrelia burgdorferi BBB07 Interaction With Integrin α3β1 Stimulates Production of Pro-Inflammatory Mediators in Primary Human Chondrocytes

Borrelia burgdorferi, the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro‐inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α3β1 on human chondrocytes activates signalling leading to release of several pro‐inflammatory mediators, but the B. burgdorferi protein that binds integrin α3β1 and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg–Gly–Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α3β1 and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro‐inflammatory mediators in primary human chondrocyte cells by interaction with integrin α3β1. This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signalling through α3β1. In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin α3β1 and stimulates production of pro‐inflammatory mediators in primary human chondrocyte cells.

Regulation of Expression of the Borrelia burgdorferi β3-Chain Integrin Ligand, P66, in Ticks and in Culture

ABSTRACT Borrelia burgdorferi is maintained in an infection cycle between mammalian and arthropod hosts. Appropriate gene expression by B. burgdorferi at different stages of this cycle is probably essential for transmission and establishment of infection. The B. burgdorferi β3 integrin ligand P66 is expressed by the bacteria in mammals, laboratory culture, and engorged but not unfed ticks. No in vitro culture conditions in which P66 expression reflected that in the unfed tick were found, suggesting that there are aspects of B. burgdorferi-tick interaction that remain unexplored.

A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development.

Dental plaque formation is a developmental process involving cooperation and competition within a diverse microbial community, approximately 70 % of which is composed of an array of streptococci during the early stages of supragingival plaque formation. In this study, 79 cell-free culture supernatants from a variety of oral streptococci were screened to identify extracellular compounds that inhibit biofilm formation by the oral anaerobe Porphyromonas gingivalis strain 381. The majority of the streptococcal supernatants (61 isolates) resulted in lysis of P. gingivalis cells, and some (17 isolates) had no effect on cell viability, growth or biofilm formation. One strain, however, produced a supernatant that abolished biofilm formation without affecting growth rate. Analysis of this activity led to the discovery that a 48 kDa protein was responsible for the inhibition. Protein sequence identification and enzyme activity assays identified the effector protein as an arginine deiminase. To identify the mechanism(s) by which this protein inhibits biofilm formation, we began by examining the expression levels of genes encoding fimbrial subunits; surface structures known to be involved in biofilm development. Quantitative RT-PCR analysis revealed that exposure of P. gingivalis cells to this protein for 1 h resulted in the downregulation of genes encoding proteins that are the major subunits of two distinct types of thin, single-stranded fimbriae (fimA and mfa1). Furthermore, this downregulation occurred in the absence of arginine deiminase enzymic activity. Hence, our data indicate that P. gingivalis can sense this extracellular protein, produced by an oral streptococcus (Streptococcus intermedius), and respond by downregulating expression of cell-surface appendages required for attachment and biofilm development.

Porphyromonas gingivalis: keeping the pathos out of the biont

The primary goal of the human microbiome initiative has been to increase our understanding of the structure and function of our indigenous microbiota and their effects on human health and predisposition to disease. Because of its clinical importance and accessibility for in vivo study, the oral biofilm is one of the best-understood microbial communities associated with the human body. Studies have shown that there is a succession of select microbial interactions that directs the maturation of a defined community structure, generating the formation of dental plaque. Although the initiating factors that lead to disease development are not clearly defined, in many individuals there is a fundamental shift from a health-associated biofilm community to one that is pathogenic in nature and a central player in the pathogenic potential of this community is the presence of Porphyromonas gingivalis. This anaerobic bacterium is a natural member of the oral microbiome, yet it can become highly destructive (termed pathobiont) and proliferate to high cell numbers in periodontal lesions, which is attributed to its arsenal of specialized virulence factors. Hence, this organism is regarded as a primary etiologic agent of periodontal disease progression. In this review, we summarize some of the latest information regarding what is known about its role in periodontitis, including pathogenic potential as well as ecological and nutritional parameters that may shift this commensal to a virulent state. We also discuss parallels between the development of pathogenic biofilms and the human cellular communities that lead to cancer, specifically we frame our viewpoint in the context of ‘wounds that fail to heal’.

Borrelia burgdorferi and its tropisms for adhesion molecules in the joint.

Borrelia burgdorferi, the spirochete that causes Lyme disease, has evolved elegant strategies for interacting with its mammalian hosts. Among them are several distinct mechanisms of adhesion to cells and extracellular matrix components. The mammalian receptors for B. burgdorferi that have been most thoroughly studied, and for which candidate bacterial ligands have been identified, are decorin, fibronectin, glycosaminoglycans, and beta3-chain integrins. This diversity of adhesion mechanisms allows B. burgdorferi to infect multiple tissues, including the synovial tissues of the joints.

HU protein affects transcription of surface polysaccharide synthesis genes in Porphyromonas gingivalis.

K-antigen capsule synthesis is an important virulence determinant of the oral anaerobe Porphyromonas gingivalis. We previously reported that the locus required for synthesis of this surface polysaccharide in strain W83 (TIGR identification PG0106 to PG0120) is transcribed as a large (∼16.7-kb) polycistronic message. Through sequence analysis, we have now identified a 77-bp inverted repeat located upstream (206 bp) of the start codon of PG0106 that is capable of forming a large hairpin structure. Further sequence analysis just upstream and downstream of the capsule synthesis genes revealed the presence of two genes oriented in the same direction as the operon that are predicted to encode DNA binding proteins: PG0104, which is highly similar (57%) to DNA topoisomerase III, and PG0121, which has high similarity (72%) to DNA binding protein HU (β-subunit). In this report, we show that these two genes, as well as the 77-bp inverted repeat region, are cotranscribed with the capsule synthesis genes, resulting in a large transcript that is ∼19.4 kb (based on annotation). We also show that a PG0121 recombinant protein is a nonspecific DNA binding protein with strong affinity to the hairpin structure, in vitro, and that transcript levels of the capsule synthesis genes are downregulated in a PG0121 deletion mutant. Furthermore, we show that this decrease in transcript levels corresponds to a decrease in the amount of polysaccharide produced. Interestingly, expression analysis of another polysaccharide synthesis locus (PG1136 to PG1143) encoding genes involved in synthesis of a surface-associated phosphorylated branched mannan (APS) indicated that this locus is also downregulated in the PG0121 mutant. Altogether our data indicate that HU protein modulates expression of surface polysaccharides in P. gingivalis strain W83.

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoid-associated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUα (PG1258) and the HUβ (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUβ using comparative expression profiling of the PG0121 (HUβ) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUβ has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake.