Microglia, the resident macrophages of the brain, typically react to injuries or chronic diseases with proliferation and expression of differentiated features, such as production of cytokines associated with inflammatory events. Regulation and control of microglial cytokine expression, therefore, is a major focus of scientific interest. It has been shown that GMCSF and Il‐3 are potent mitogens for microglia. Moreover, Il‐3 and other cytokines are products of microglia. It is shown here that interleukin‐1 (Il‐1) as well as tumor necrosis factor (TNFα) increased microglial proliferation in mixed astrocyte‐microglial cultures but had no mitogenic effects on isolated microglia. © Wiley‐Liss, Inc. Lipopolysaccharide (LPS), the bacterial endotoxin, irreversibly inhibited microglial cell division in both mixed astrocyte‐microglial cultures and in isolated microglial cultures. By contrast, the corticosteroids hydrocortisone and aldosterone and the synthetic glucocorticoid dexamethasone reversibly inhibited microglial proliferation. They also antagonized the stimulatory effects of Il‐3 and granulocyte macrophage colony‐stimulating factor (GMCSF). Estradiol and progesterone had no significant effects on mixed cultures but inhibited microglial proliferation in isolated cultures. Conditioned media from mixed cultures, isolated cultures, from the WEHI‐2B cell line, or from fresh (serum‐supplemented) media stimulated microglial proliferation to various extents. In summary, cytokine‐mediated microglial proliferation can be down‐regulated by a variety of steroid hormones. Along with their unimpaired access to brain cells in general, corticosteroids likely maintain an inhibitory tonus on microglial proliferation. It is hypothesized that this inhibition is overcome locally and temporally in brain injury and repair. © Wiley‐Liss, Inc.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30–220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of “elementary bodies” and “not-cell bound antigens”.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis. Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored. IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.