Group Maintenance in Aggregative Multicellularity

Aggregative multicellularity occurs when dispersed cells join together to form a highly cooperative unit, in contrast to clonal multicellular organisms formed by cells that remain in contact after descent from a single cell. Because aggregative groups may include non-relatives, aggregative multicellular organisms should be particularly vulnerable to the rise of cheater cells that take advantage of social goods without paying the costs, reducing cooperation, and even threatening extinction. We review the key mechanisms by which aggregative multicellular organisms control cheaters with a focus on the best studied aggregative organisms, Myxococcus xanthus and Dictyostelium discoideum. These include various passive and active mechanisms to maintain high relatedness within aggregates, to enforce cooperation on aggregate members, and the costs of cheating on other key functions. Ultimately, aggregative multicellular organisms are not that different from clonal organisms descended from a single cell.

Circadian rhythms in Per1, PER2 and Ca2+ of a solitary SCN neuron cultured on a microisland

Circadian rhythms in Per1, PER2 expression and intracellular Ca2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100–200 μm in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca2+ under these conditions.

2594. Biofilm-Dispersed Staphylococcus aureus Exhibits a Distinct agr-Independent Host Interaction

Background Staphylococcus aureus biofilms are a common cause of persistent, life-threatening infections. Dispersal of S. aureus cells from established biofilm-based infections is crucial for dissemination within the host, but is poorly understood. We tested the hypothesis that biofilm dispersed S. aureus cells have distinct physiology from planktonic cells and are better equipped to evade host immunity in an agr-dependent manner. Methods Primary murine bone marrow-derived macrophages (BMDMs) were infected with planktonic and biofilm dispersed cells from S. aureus USA300 LAC wild type (WT) and USA300 LAC-agr knockout (KO). Biofilm dispersed cells were collected via glucose deprivation. Gentamicin protection assays were used to enumerate phagocytosed bacteria and fluorescence microscopy to quantify macrophage viability. A 26-plex immunoassay was used to screen for cytokines and chemokines. Reversed phase high-performance liquid chromatography was used to measure relative phenol-soluble modulin (PSM) levels from macrophage co-cultures. Results Compared with planktonic cells, biofilm-dispersed cells in both S. aureus WT and KO backgrounds exhibited: (1) ~10-fold less phagocytosis by BMDMs (p = 0.0003; Figure 1); (2) increased macrophage killing (23% vs. 8%; p = 0.0038; Figure 2); (3) stronger pro- (e.g., IFN-y, IL-2, IL-6, IL-17; Figure 3A) and anti- (e.g., IL-10, IL-4, IL-22; Figure 3B) inflammatory cytokine responses from macrophages (P < 0.05 for all); (4) significantly higher δ toxin PSM production (P = 0.0090; Figure 4) in WT background only. Conclusion S. aureus biofilm dispersed cells are physiologically distinct from planktonic cells and have a unique interaction with the host immune system. Dispersed cells are more resistant to phagocytosis, have a greater propensity to kill macrophages, and mount stronger pro- and anti-inflammatory responses in an agr-independent manner. Dispersed cells also have the ability to produce more δ toxin PSM via well-known agr-dependent pathways. Disclosures All authors: No reported disclosures.

Colonization and immune modulation properties of Klebsiella pneumoniae biofilm-dispersed cells

Biofilm-dispersal is a key determinant for further dissemination of biofilm-embedded bacteria. Recent evidence indicates that biofilm-dispersed bacteria have transcriptional features different from those of both biofilm and planktonic bacteria. In this study, the in vitro and in vivo phenotypic properties of Klebsiella pneumoniae cells spontaneously dispersed from biofilm were compared with those of planktonic and sessile cells. Biofilm-dispersed cells, whose growth rate was the same as that of exponential planktonic bacteria but significantly higher than those of sessile and stationary planktonic forms, colonized both abiotic and biotic surfaces more efficiently than their planktonic counterparts regardless of their initial adhesion capabilities. Microscopy studies suggested that dispersed bacteria initiate formation of microcolonies more rapidly than planktonic bacteria. In addition, dispersed cells have both a higher engulfment rate and better survival/multiplication inside macrophages than planktonic cells and sessile cells. In an in vivo murine pneumonia model, the bacterial load in mice lungs infected with biofilm-dispersed bacteria was similar at 6, 24 and 48 h after infection to that of mice lungs infected with planktonic or sessile bacteria. However, biofilm-dispersed and sessile bacteria trend to elicit innate immune response in lungs to a lesser extent than planktonic bacteria. Collectively, the findings from this study suggest that the greater ability of K. pneumoniae biofilm-dispersed cells to efficiently achieve surface colonization and to subvert the host immune response confers them substantial advantages in the first steps of the infection process over planktonic bacteria.

Fibroadenoma/benign phyllodes: a cytologic diagnostic challenge

Background: To study and compare cytomorphological features of histologically proven cases of benign phyllodes and cellular fibroadenoma.Methods: Smears of histologically-proven cases of benign phyllodes and cellular fibroadenoma in one year, were reviewed. The cellular fibroadenoma had epithelial and/or stromal hypercellularity. The stromal and epithelial components as well as the background cells were qualitatively and quantitatively analyzed.Results: Number, cellularity and type of stromal fragments varied significantly in two groups. Higher number, intermediate to large-sized and hypercellular stromal fragments were commonly seen in phyllodes. Hypercellular (3+ cellularity) fragments were seen in 100% cases of phyllodes against 11.1% cases of fibroadenoma. Large-sized stromal fragments were found in 100% of phyllodes while in only 11.1% cases of fibroadenoma. The ratio of number of epithelial to stromal fragments was significantly high (58.5:1) in fibroadenoma against phyllodes (1.3:1). The epithelial architecture, atypia, apocrine metaplasia and presence of cystic macrophages did not very much in the two groups. The cellularity of the dispersed cells in background did not reveal significant difference though the type of cells varied; the proportion of long and short spindle cells was higher in PT group while proportion of oval cells was higher in FA group.Conclusion: The number, cellularity and nature of stromal fragments, ratio of epithelial to stromal fragments, cellularity and type of background cells are helpful in distinguishing benign phyllodes from cellular fibroadenoma. The identification of these features can improve the pickup rate of phyllodes tumor, thereby assisting proper management.

Comparison of biofilm dispersal approaches in Pseudomonas aeruginosa and evaluation of dispersed cells in acute infection mouse model

ABSTRACTBiofilm dispersal is biologically significant process to fully understand its consequences during biofilm based infections. The mechanism for biofilm dispersal may involve response to environmental cues and changes in intracellular secondary messengers. Considering range of cues for biofilm dispersal, it is significant to study if dispersed cells generated by different methods have similar phenotype. In the present study, we have compared four type of biofilm dispersal in P. aeruginosa based in response to environment cues (starvation and nutrient rich), c-di-GMP knockdown and signaling molecule NO. We have determined their dispersal efficiency, susceptibility of dispersed cells for antibiotics, their transcriptomic changes compared to planktonic and biofilm. We also determined if dispersed cells can cause acute infections in mouse models. In vitro experiments showed that NO and c-di-GMP based dispersal methods had high biofilm reduction efficiency of 50 and 75% respectively, however, the nutrient induced dispersion showed low efficiency (≈30%) and more tolerance to colistin. We also showed that in vitro dispersed cells induced >10-fold transcriptomic expression of genes (at significance level of p < 0.005) related to efflux pumps (mexCD-oprJ), antibiotic resistance (arnDET) in dispersed cells induced by NO and nutrient change, while denitrification pathway and virulence (T3SS, VreI, T2SS) genes in all dispersed cells compared to planktonic and biofilm state. When the ability of the dispersed cells was tested in mouse model of lung infection, c-di-GMP and NO based dispersed cells displayed enhanced infections and haematogenous spread to liver and spleen and higher mortality. Although degree of immune response (cytokines) does not differ based on phenotypes inoculated in our experimental conditions. Based on our data most efficient dispersal methods increase murine mortality. This indicates their capability of making biofilm associated infection more complicated. Our data encourage to be more careful for studies suggesting biofilm dispersal during treatment of biofilm infections.

Pseudomonas aeruginosa Requires the DNA-Specific Endonuclease EndA To Degrade Extracellular Genomic DNA To Disperse from the Biofilm

ABSTRACT The dispersion of biofilms is an active process resulting in the release of planktonic cells from the biofilm structure. While much is known about the process of dispersion cue perception and the subsequent modulation of the c-di-GMP pool, little is known about subsequent events resulting in the release of cells from the biofilm. Given that dispersion coincides with void formation and an overall erosion of the biofilm structure, we asked whether dispersion involves degradation of the biofilm matrix. Here, we focused on extracellular genomic DNA (eDNA) due to its almost universal presence in the matrix of biofilm-forming species. We identified two probable nucleases, endA and eddB, and eddA encoding a phosphatase that were significantly increased in transcript abundance in dispersed cells. However, only inactivation of endA but not eddA or eddB impaired dispersion by Pseudomonas aeruginosa biofilms in response to glutamate and nitric oxide (NO). Heterologously produced EndA was found to be secreted and active in degrading genomic DNA. While endA inactivation had little effect on biofilm formation and the presence of eDNA in biofilms, eDNA degradation upon induction of dispersion was impaired. In contrast, induction of endA expression coincided with eDNA degradation and resulted in biofilm dispersion. Thus, released cells demonstrated a hyperattaching phenotype but remained as resistant to tobramycin as biofilm cells from which they egress, indicating EndA-dispersed cells adopted some but not all of the phenotypes associated with dispersed cells. Our findings indicate for the first time a role of DNase EndA in dispersion and suggest weakening of the biofilm matrix is a requisite for biofilm dispersion. IMPORTANCE The finding that exposure to DNase I impairs biofilm formation or leads to the dispersal of early stage biofilms has led to the realization of extracellular genomic DNA (eDNA) as a structural component of the biofilm matrix. However, little is known about the contribution of intrinsic DNases to the weakening of the biofilm matrix and dispersion of established biofilms. Here, we demonstrate for the first time that nucleases are induced in dispersed Pseudomonas aeruginosa cells and are essential to the dispersion response and that degradation of matrix eDNA by endogenously produced/secreted EndA is required for P. aeruginosa biofilm dispersion. Our findings suggest that dispersing cells mediate their active release from the biofilm matrix via the induction of nucleases.

Candida albicansDispersed Cells Are Developmentally Distinct from Biofilm and Planktonic Cells

ABSTRACTCandida albicanssurface-attached biofilms such as those formed on intravenous catheters with direct access to the bloodstream often serve as a nidus for continuous release of cells capable of initiating new infectious foci. We previously reported that cells dispersed from a biofilm are yeast cells that originate from the top-most hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm dispersal yeast cells displayed enhanced virulence-associated characteristics and drug resistance. However, little is known about their molecular properties. To address that issue, in this study we aimed to define the molecular characteristics of these biofilm dispersal cells. We found that the inducer of dispersal,PES1, genetically interacts with the repressor of filamentation,NRG1, in a manner consistent with the definition of dispersed cells as yeast cells. Further, using a flow biofilm model, we performed comprehensive comparative RNA sequencing on freshly dispersed cells in order to identify unique transcriptomic characteristics. Gene expression analysis demonstrated that dispersed cells largely inherit a biofilm-like mRNA profile. Strikingly, however, dispersed cells seemed transcriptionally reprogrammed to acquire nutrients such as zinc and amino acids and to metabolize alternative carbon sources, while their biofilm-associated parent cells did not induce the same high-affinity transporters or express gluconeogenetic genes, despite exposure to the same nutritional signals. Collectively, the findings from this study characterize cell dispersal as an intrinsic step of biofilm development which generates propagules more adept at colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before the cells experience the associated external signals.IMPORTANCECandida albicanssurface-attached biofilms serve as a reservoir of cells to perpetuate and expand an infection; cells released from biofilms on catheters have direct access to the bloodstream. Biofilm dispersal yeast cells exhibit enhanced adhesion, invasion, and biofilm formation compared to their planktonic counterparts. Here, we show using transcriptome sequencing (RNA-seq) that dispersed yeast cells are developmentally distinct from the cells in their parent biofilms as well as from planktonic yeast cells. Dispersal cells possess an anticipatory expression pattern that primes them to infect new sites in the host, to survive in nutrient-starved niches, and to invade new sites. These studies identified dispersal cells as a unique proliferative cell type of the biofilm and showed that they could serve as targets for antibiofilm drug development in the future.

Candida albicansdispersed cells signify a developmental state distinct from biofilm and planktonic phases

Candida albicanssurface-attached biofilms are sites of amplification of an infection through continuous discharge of cells capable of initiating new infectious foci. Yeast cells released from biofilms on intravenous catheters have direct access to the bloodstream. We previously reported that dispersed cells are largely lateral yeast cells that originate from the hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm-dispersed yeast cells displayed enhanced virulence-associated gene expression and drug resistance. Little is known about the molecular properties of dispersed cells. We found that the inducer of dispersal,PES1, genetically interacts with the repressor of filamentation,NRG1, in a manner that supports a genetic definition of dispersed cells as yeast. We combined a flow biofilm model with RNA sequencing technology, to identify transcriptomic characteristics of freshly dispersed yeast cells versus biofilms or age-matched planktonic yeast cells growing in glucose-rich medium. Dispersed cells largely inherited a biofilm-like mRNA profile but with one stark difference: dispersed cells were transcriptionally reprogrammed to metabolize alternative carbon sources, while their sessile parents expressed glycolytic genes, despite exposure to the same nutritional signals. Our studies hence define dispersal cell production as an intrinsic step of biofilm development which generates propagules capable of colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before experiencing the associated external signals.

Susceptibility of Pseudomonas aeruginosa Dispersed Cells to Antimicrobial Agents Is Dependent on the Dispersion Cue and Class of the Antimicrobial Agent Used

ABSTRACT The biofilm life cycle is characterized by the transition of planktonic cells exhibiting high susceptibly to antimicrobial agents to a biofilm mode of growth characterized by high tolerance to antimicrobials, followed by dispersion of cells from the biofilm back into the environment. Dispersed cells, however, are not identical to planktonic cells but have been characterized as having a unique transitionary phenotype relative to biofilm and planktonic cells, with dispersed cells attaching in a manner similar to exponential-phase cells, but demonstrating gene expression patterns that are distinct from both exponential and stationary-phase planktonic cells. This raised the question whether dispersed cells are as susceptible as planktonic cells and whether the dispersion inducer or the antibiotic class affects the drug susceptibility of dispersed cells. Dispersed cells obtained in response to dispersion cues glutamate and nitric oxide (NO) were thus exposed to tobramycin and colistin. Although NO-induced dispersed cells were as susceptible to colistin and tobramycin as exponential-phase planktonic cells, glutamate-induced dispersed cells were susceptible to tobramycin but resistant to colistin. The difference in colistin susceptibility was independent of cellular c-di-GMP levels, with modulation of c-di-GMP failing to induce dispersion. Instead, drug susceptibility was inversely correlated with LPS modification system and the biofilm-specific transcriptional regulator BrlR. The susceptibility phenotype of glutamate-induced dispersed cells to colistin was found to be reversible, with dispersed cells being rendered as susceptible to colistin within 2 h postdispersion, though additional time was required for dispersed cells to display expression of genes indicative of exponential growth.