Matthew Ramsey

Metabolite Cross-Feeding Enhances Virulence in a Model Polymicrobial Infection

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.

Polymicrobial interactions stimulate resistance to host innate immunity through metabolite perception

Bacteria in the human oral cavity often grow in an attached multispecies biofilm community. Members of this community display defined interactions that have an impact on the physiology of the individual and the group. Here, we show that during coculture growth with streptococci, the oral pathogen Aggregatibacter actinomycetemcomitans displays enhanced resistance to killing by host innate immunity. The mechanism of resistance involves sensing of the streptococcal metabolite hydrogen peroxide by A. actinomycetemcomitans, which stimulates a genetic program resulting in enhanced expression of the complement resistance protein ApiA. The oxidative stress response regulator OxyR mediates induction of apiA transcription, and this induction is required for coculture resistance to killing by human serum. These findings provide evidence that interaction between community members mediates prokaryotic resistance to host innate immunity and reinforce the need to understand how polymicrobial growth affects interaction with the host immune system.

Pseudomonas aeruginosa attachment and biofilm development in dynamic environments

Biofilm formation by Pseudomonas aeruginosa is hypothesized to follow a developmental pattern initiated by attachment to a surface followed by microcolony formation and mature biofilm development. Swimming and twitching motility are important for attachment and biofilm development in P. aeruginosa. However, it is clear that many P. aeruginosa strains lacking swimming motility exist as biofilms in the lungs of cystic fibrosis patients. Consequently, we have developed a dynamic attachment assay to identify motility‐independent attachment‐defective mutants. Using transposon mutagenesis, we identified 14 novel dynamic attachment‐deficient (dad) mutants including four mutants specific to dynamic assay conditions (dad specific). Two of the dad‐specific mutants contain insertions in genes involved in sensing and responding to external stimuli, implying a significant impact of external factors on the biofilm developmental pathway. Observations of initial attachment and long‐term biofilm formation characterized our dad mutants into two distinct classes: biofilm delayed and biofilm impaired. Biofilm‐delayed mutants form wild‐type biofilms but are delayed at least 24 h compared with the wild type, whereas biofilm‐impaired mutants never form wild‐type biofilms in our assays. We propose a dynamic model for attachment and biofilm formation in P. aeruginosa including these two classes.

Real-time mapping of a hydrogen peroxide concentration profile across a polymicrobial bacterial biofilm using scanning electrochemical microscopy

Quantitative detection of hydrogen peroxide in solution above a Streptococcus gordonii (Sg) bacterial biofilm was studied in real time by scanning electrochemical microscopy (SECM). The concentration of hydrogen peroxide was determined to be 0.7 mM to 1.6 mM in the presence of 10 mM glucose over a period of 2 to 8 h. The hydrogen peroxide production measured was higher near the biofilm surface in comparison to Sg grown planktonically. Differential hydrogen peroxide production was observed both by fluorometric as well as by SECM measurements. The interaction between two different species in a bacterial biofilm of Sg and Aggregatibacter actinomycetemcomitans (Aa) in terms of hydrogen peroxide production was also studied by SECM. One-directional y-scan SECM measurements showed the unique spatial mapping of hydrogen peroxide concentration across a mixed species biofilm and revealed that hydrogen peroxide concentration varies greatly dependent upon local species composition.

Clostridium difficile toxin B activates dual caspase‐dependent and caspase‐independent apoptosis in intoxicated cells

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxi‐cation of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase‐dependent and caspase‐independent apoptosis. The apoptotic pathways became evident when caspase‐3‐processed‐vimentin was detected in TcdB‐treated HeLa cells. Caspase‐3 activation was subsequently confirmed in TcdB‐intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB‐induced cell death, but did not alter the time‐course of cytopathic effects. A similar effect was also observed in MCF‐7 cells, which are deficient in caspase‐3 activity. The time‐course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1–556). Unlike TcdB treated cells, intoxication with TcdB1–556 or expression of TcdB1–556 in a transfected cell line, did not stimulate caspase‐3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1–556 treated cells did not demonstrate caspase‐3 activation these cells were apoptotic as determined by differential annexin‐V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase‐independent apoptosis as a result of substrate inactivation and may evoke caspase‐dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.

Discovery of a biofilm electrocline using real-time 3D metabolite analysis

Bacteria are social organisms that possess multiple pathways for sensing and responding to small molecules produced by other microbes. Most bacteria in nature exist in sessile communities called biofilms, and the ability of biofilm bacteria to sense and respond to small molecule signals and cues produced by neighboring biofilm bacteria is particularly important. To understand microbial interactions between biofilms, it is necessary to perform rapid, real-time spatial quantification of small molecules in microenvironments immediately surrounding biofilms; however, such measurements have been elusive. In this study, scanning electrochemical microscopy was used to quantify small molecules surrounding a biofilm in 3D space. Measuring concentrations of the redox-active signaling molecule pyocyanin (PYO) produced by biofilms of the bacterium Pseudomonas aeruginosa revealed a high concentration of PYO that is actively maintained in the reduced state proximal to the biofilm. This gradient results in a reduced layer of PYO that we have termed the PYO “electrocline,” a gradient of redox potential, which extends several hundred microns from the biofilm surface. We also demonstrate that the PYO electrocline is formed under electron acceptor-limiting conditions, and that growth conditions favoring formation of the PYO electrocline correlate to an increase in soluble iron. Additionally, we have taken a “reactive image” of a biofilm surface, demonstrating the rate of bacterial redox activity across a 2D surface. These studies establish methodology for spatially coordinated concentration and redox status measurements of microbe-produced small molecules and provide exciting insights into the roles these molecules play in microbial competition and nutrient acquisition.

Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains

Significance Multidrug-resistant enterococci are leading causes of hospital infection. The antibiotic-perturbed patient gut serves as a staging ground—small numbers of resistant hospital strains colonize and then, greatly amplify in the colon. Little is known of the colonization principles involved—whether hospital strains are competitive or noncompetitive with commensal enterococci or whether mobile elements comprising over 25% of the genome of the former impose significant fitness costs. We unexpectedly found that the prototype vancomycin-resistant Enterococcus faecalis strain V583 was actively killed by fecal organisms, and we traced that to pheromone production by commensal enterococci that trigger lethal mobile element cross-talk. This work highlights the importance of maintaining commensal enterococci in the gut of the hospitalized patient. Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains.

Differential Expression of Desulfovibrio vulgaris Genes in Response to Cu(II) and Hg(II) Toxicity

ABSTRACT The response of Desulfovibrio vulgaris to Cu(II) and Hg(II) was characterized. Both metals increased the lag phase, and Cu(II) reduced cell yield at concentrations as low as 50 μM. mRNA expression was analyzed using random arbitrarily primed PCR, differential display, and quantitative PCR. Both Cu(II) and Hg(II) (50 μM) caused upregulation of mRNA expression for an ATP binding protein (ORF2004) and an ATPase (ORF856) with four- to sixfold increases for Hg(II) and 1.4- to 3-fold increases with Cu(II). These results suggest that D. vulgaris uses an ATP-dependent mechanism for adapting to toxic metals in the environment.

Transcriptional Organization and Regulation of the l-Idonic Acid Pathway (GntII System) in Escherichia coli

The genetic organization of the idn genes that encode the pathway for L-idonate catabolism was characterized. The monocistronic idnK gene is transcribed divergently from the idnDOTR genes, which were shown to form an operon. The 215-bp regulatory region between the idnK and idnD genes contains promoters in opposite orientation with transcription start sites that mapped to positions -26 and -29 with respect to the start codons. The regulatory region also contains a single putative IdnR/GntR binding site centered between the two promoters, a CRP binding site upstream of idnD, and an UP element upstream of idnK. The genes of the L-idonate pathway were shown to be under catabolite repression control. Analysis of idnD- and idnK-lacZ fusions in a nonpolar idnD mutant that is unable to interconvert L-idonate and 5-ketogluconate indicated that either compound could induce the pathway. The L-idonate pathway was first characterized as a subsidiary pathway for D-gluconate catabolism (GntII), which is induced by D-gluconate in a GntI (primary gluconate system) mutant. Here we showed that the idnK and idnD operons are induced by D-gluconate in a GntI system mutant, presumably by endogenous formation of 5-ketogluconate from D-gluconate. Thus, the regulation of the GntII system is appropriate for this pathway, which is primarily involved in L-idonate catabolism; the GntII system can be induced by D-gluconate under conditions that block the GntI system.

Interaction of glyceraldehyde‐3‐phosphate dehydrogenase in the light‐induced rod alpha‐transducin translocation

The light‐dependent subcellular translocation of rod alpha‐transducin (GNAT‐1, or rod Tα) has been well documented. In dark‐adapted animals, rod Tα (rTα) is predominantly located in the rod outer segment (ROS) and translocates into the rod inner segment (RIS) upon exposure to the light. Neither the molecular participants nor the mechanism(s) involved in this protein trafficking are known. We hypothesized that other proteins must interact with rTα to affect the translocations. Using the MBP‐rTα fusion pulldown assay, the yeast two‐hybrid assay and the co‐immunoprecipitation assay, we identified glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and rTα as interacting proteins. Immunoprecipitation also showed β‐actin associates with rTα in the dark but not in the light. To further investigate the involvement of GAPDH in light‐induced rod Tα translocation, GAPDH mRNA was knocked down in vivo by transient expression of siRNAs in rat photoreceptor cells. Under completely dark‐ and light‐adapted conditions, the translocation of rTα was not significantly different within the ‘GAPDH knock‐down photoreceptor cells’ compared to the non‐transfected control cells. However, under partial dark‐adaptation, rTα translocated more slowly in the ‘GAPDH knock‐down cells’ supporting the conclusion that GAPDH is involved in rTα translocation from the RIS to the ROS during dark adaptation.