Microbiology

The human body is colonized by vast communities of microbes, collectively known as microbiota, or microbiome. Although microbes colonize every surface of our bodies that is exposed to the external environment, the biggest collection of microbes colonizing humans and other mammals can be found in the gastrointestinal tract. Given the fact that the human gut is colonized by several hundred microbial species, our group hypothesized that the chemical diversity of this environment should be significant, and that many of the molecules present in that environment would have important signaling roles. Therefore, we devised a protocol to extract these molecules from human feces and test their signaling properties. Potentially bioactive extracts can be tested through addition to culture medium and analyses of bacterial growth and gene expression, among other properties. The protocol described herein provides an easy and rapid method for the extraction and testing of metabolites from fecal samples using Salmonella enterica as a model organism. This protocol can also be adapted to the extraction of small molecules from other matrices, such as cultured mammalian cells, tissues, body fluids, and axenic microbial cultures, and the resulting extracts can be tested against various microbial species.

Here, we describe a protocol for a continuous flow system for C. albicans cultures growing adherent to a plastic surface. The protocol was adapted from a previous method established to simulate blood flow on endothelial cells (Wilson and Hube, 2010). The adapted protocol was used by us for the removal of molecules in C. albicans supernatants, especially farnesol, which accumulate over the time course of incubation and cannot be specifically depleted. The system used, however, allows various applications including the simulation of physiological flow conditions. Several example applications are given on the manufacturer’s website (https://ibidi.com/perfusion-system/112-ibidi-pump-system.html).

The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.

Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important role in bacterial biofilm formation and virulence factor production in many pathogenic strains, QS disruptors have become a hot topic in current antimicrobial research. There are several reporter strains exhibiting QS-regulated phenotypes that have been engineered for the identification of QS inhibitors, including, for example, pigment production (González and Keshavan, 2006; Steindler and Venturi, 2007), gfp, lacZ or lux reporter gene fusions (González and Keshavan, 2006; Steindler and Venturi, 2007), or lethal gene fusions downstream QS-controlled promoters (Weiland-Bräuer et al., 2015). With three parallel QS circuits, the bioluminescent marine bacterium Vibrio campbellii (formerly harveyi, Lin et al., 2010) constitutes a complex Gram-negative model for which an extensive body of knowledge exists, including an array of mutant biosensors. In V. campbellii, bioluminescence is regulated by QS. However, bioluminescence is the result of complex biochemical networks that converge with cell respiration and fatty acid metabolism. It is also an energy-demanding reaction that strongly depends on the overall metabolic state of the bacterium, consuming up to 1/5 of the cell resources (Munn, 2011). Thus, disruption of QS-controlled phenotypes might be the result of toxic side effects or interference with the above-mentioned biochemical pathways rather than QS signaling. Therefore, adequate control experiments should be included. The protocol described herein provides a method and workflow for the identification of putative QS-disrupting compounds in Vibrio. It can also be easily adapted for other QS studies (e.g., detection of AI molecules).

The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we present a purification protocol for molecules excreted by a group of Gram-positive zoonotic pathogen bacteria, the ‘Staphylococcus intermedius group’, that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini (S. delphini). They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. These substances can be isolated and purified from the culture supernatant using this upscalable purification method.

Staphylococcus aureus has a quorum sensing system to regulate the expression of various virulence factors, which is exerted by the agr locus that encodes agrBDCA and a regulatory RNA called RNAIII. AgrB, AgrD, and AgrC proteins are involved in producing and recognizing extracellular quorum sensing molecules and transduce the signal by altering the phosphorylation status of AgrA, which is a positive transcription factor, to regulate cytolysin genes as well as the RNAIII gene. RNAIII regulates the expression of various virulence genes. Expression of the agr locus has been examined in depth at the transcriptional level, but investigations of translational expression are limited, because immunoglobulin G used to detect a specific protein highly reacts to S. aureus protein A. Here, we report a method to detect AgrA that is the transcription factor encoded by the agr regulatory system. Although this is a specific protocol for western blotting of S. aureus AgrA protein, it can also be used for other S. aureus proteins by using the appropriate antibody.