生物化学

Candida albicans is the most common cause of fungal infections worldwide. Infection by C. albicans is closely associated with its ability to form a biofilm, closely packed communities of cells attached to the surfaces of human tissues and implanted devices, in or on the host. When tested for susceptibility to antifungals, such as polyenes, azoles, and allylamines, C. albicanscells in a biofilm are more resistant to antifungal agents than C. albicans cells in the planktonic form. Cyclic Adenosine monophosphate (cAMP) is one of the key elements for triggering hyphal and biofilm formation in C. albicans. It is hard to detect or extract molecular markers (e.g., cAMP) from C. albicans biofilms because the biofilms have a complex three-dimensional architecture with an extracellular matrix surrounding the cell walls of the cells in the biofilm. Here, we present an improved protocol that can effectively measure the level of intracellular cAMP in C. albicans biofilms.

Mycobacterial genomes encode a plethora of genes that are involved in the synthesis, utilization and degradation of cAMP. The genome of M. tuberculosis H37Rv, for example, encodes 16 adenylyl cyclases and 10 genes harbouring the cyclic nucleotide-binding (CNB) domain (Shenoy and Visweswariah, 2006). Cyclic AMP is efficiently secreted by mycobacteria, and cytosolic as well as extracellular levels of cAMP can reach hundreds of micromolar. We have recently reported that an abundantly expressed universal stress protein (USP; Rv1636 in M. tuberculosis H37Rv and MSMEG_3811 in M. smegmatis, respectively) binds cAMP (Banerjee et al., 2015). Given the number of cAMP-binding proteins present in mycobacteria, it is expected that a significant fraction of intracellular cAMP may be bound to protein. The methods typically employed to measure cAMP are radioimmunoassay (RIA) and ELISA. However, these procedures include prior acidification of samples that would dissociate cAMP ‘bound’ to protein, and therefore represent the ‘total’ cAMP present in the sample. In this protocol, we describe a method to separate the fraction of cAMP ‘bound’ to protein from what is ‘free’ or not associated with protein. This is performed by subjecting the cytosolic fraction or the culture supernatant to filtration through a membrane with a 3 kDa cut-off. Only ‘free’ cAMP is able to pass through the membrane. Therefore, cAMP concentrations in the filtrate represent the ‘free’ cAMP in the sample. Cyclic AMP levels in the original cytosolic fraction or the culture supernatant represent the ‘total’ cAMP concentration. Subtracting the ‘free’ from the ‘total’ provides the amount of cAMP bound to protein.

Cyclic AMP (cAMP) is a ubiquitous secondary signaling molecule, commonly associated with many bacterial processes, including the regulation of virulence factors, such as biofilms, pellicles and motility (Wolfgang, 2003). The quantity of available cAMP is controlled by the interplay between the synthesis of adenosine triphosphate (ATP) to cAMP by adenylyl cyclases, and the degradation of cAMP by phosphodiesterase (McDonough et al., 2012). Adequate quantification of cAMP levels within a bacterial cell is an important step in identifying the impact that secondary signaling molecules play on the regulatory pathway within the cell. The principle of this method is to measure total cAMP levels within a bacterial cell, using crude bacterial whole cell lysate. The Cyclic AMP XP^TM Assay kit used in this protocol was originally designed to be used for determining the level of cAMP in eukaryotic cells, however, the antibodies used in coating the wells will react with cAMP from any species and thus can be used for determining levels in bacterial cells. The measurement of cAMP in prokaryotic cells described here is a simple and cost effective method of producing quantifiable results.

Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the merozoite surface. This shedding, which takes place even in the absence of invasion, is therefore a surrogate marker for the degree of egress taking place in a culture, and can be measured using a specific capture ELISA to quantify AMA1 levels in culture supernatants (Collins et al., 2013). The assay uses a monoclonal antibody specific for AMA1 (called 4G2dc1) (Kocken et al., 1998; Collins et al., 2009) to capture and immobilize the protein from culture supernatants, then uses a specific rabbit polyclonal antiserum to detect the immobilized antigen. A phosphatase-conjugated goat anti-rabbit antibody is finally used to quantify the binding of the second antibody. Egress is absolutely dependent upon the activity of a parasite cGMP-dependent protein kinase, PKG, and so is influenced by levels of intracellular cGMP (Collins et al., 2013). This is regulated by the interplay between guanylate cyclases and phosphodiesterases. The latter enzymes may also degrade cAMP, so it may also be informative to measure intracellular cAMP levels.