PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.

Contributions of neutrophils to the adaptive immune response in autoimmune disease.

Neutrophils are granulocytic cytotoxic leukocytes of the innate immune system that activate during acute inflammation. Neutrophils can also persist beyond the acute phase of inflammation to impact the adaptive immune response during chronic inflammation. In the context of the autoimmune disease, neutrophils modulating T and B cell functions by producing cytokines and chemokines, forming neutrophil extracellular traps, and acting as or priming antigen presentation cells. Thus, neutrophils are actively involved in chronic inflammation and tissue damage in autoimmune disease. Using rheumatoid arthritis as an example, this review focuses on functions of neutrophils in adaptive immunity and the therapeutic potential of these cells in the treatment of autoimmune disease and chronic inflammation.

In vitro assays of chemotaxis as a window into mechanisms of toxicant-induced immunomodulation.

Dysregulated cell movement can lead to developmental abnormalities, neoplasia, and immune system disorders, and there are a variety of contexts in which xenobiotics (and biologic) effects on this movement are of interest. Many toxins and toxicants have been shown to disrupt controlled cell movement. Identification of compounds that affect cell movement is crucial to drug discovery. Drug components may have unexpected consequences with respect to cell motility, which would exclude these compounds in drug development. Finally, the development of drugs that target chemotactic pathways may be useful in the treatment of tumors, which often reprogram chemotactic pathways to become metastatic. The effects of these agents on cell movement can be measured using several different in vitro chemotactic assays. This review details the procedures of three in vitro measurements of chemotaxis: the Boyden chamber, the under-agarose assay, and the automated, real-time, ECIS/Taxis assay, and discusses the inferences that can be drawn from the results of such studies.

Measurement of cellular chemotaxis with ECIS/Taxis.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted (1). In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues (2). Another assay system, the under-agarose chemotaxis assay, (3, 4) measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays. The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) (5, 6). In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system (6). In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. Dictyostelium discoidium is known to migrate in the presence of a folate gradient (7, 8) and its chemotactic response can be accurately measured by ECIS/Taxis (9). Leukocyte chemotaxis, in response to SDF1α and to chemotaxis antagonists has also been measured with ECIS/Taxis (10, 11). An example of the leukocyte response to SDF1α is shown in Figure 1.