Mycoplasma genitalium is a human pathogen adhering to host target epithelial cells and causing urethritis, cervicitis and pelvic inflammatory disease. Essential for infectivity is a transmembrane adhesion complex called Nap comprising proteins P110 and P140. Here we report the crystal structure of P140 both alone and in complex with the N-terminal domain of P110. By cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) we find closed and open Nap conformations, determined at 9.8 and 15 Å, respectively. Both crystal structures and the cryo-EM structure are found in a closed conformation, where the sialic acid binding site in P110 is occluded. By contrast, the cryo-ET structure shows an open conformation, where the binding site is accessible. Structural information, in combination with functional studies, suggests a mechanism for attachment and release of M. genitalium to and from the host cell receptor, in which Nap conformations alternate to sustain motility and guarantee infectivity.

Bacteria commonly exhibit a high degree of cellular organization and polarity which affect many vital processes such as replication, cell division, and motility. In Shewanella and other bacteria, HubP is a polar marker protein which is involved in proper chromosome segregation, placement of the chemotaxis system, and various aspects of pilus- and flagellum-mediated motility. Here, we show that HubP also recruits a transmembrane multidomain protein, PdeB, to the flagellated cell pole. PdeB is an active phosphodiesterase and degrades the second messenger c-di-GMP. In Shewanella putrefaciens, PdeB affects both the polar and the lateral flagellar systems at the level of function and/or transcription in response to environmental medium conditions. Mutant analysis on fluorescently labeled PdeB indicated that a diguanylate cyclase (GGDEF) domain in PdeB is strictly required for HubP-dependent localization. Bacterial two-hybrid and in vitro interaction studies on purified proteins strongly indicate that this GGDEF domain of PdeB directly interacts with the C-terminal FimV domain of HubP. Polar localization of PdeB occurs late during the cell cycle after cell division and separation and is not dependent on medium conditions. In vitro activity measurements did not reveal a difference in PdeB phosphodiesterase activities in the presence or absence of the HubP FimV domain. We hypothesize that recruitment of PdeB to the flagellated pole by HubP may create an asymmetry of c-di-GMP levels between mother and daughter cells and may assist in organization of c-di-GMP-dependent regulation within the cell. IMPORTANCE c-di-GMP-dependent signaling affects a range of processes in many bacterial species. Most bacteria harbor a plethora of proteins with domains which are potentially involved in synthesis and breakdown of c-di-GMP. A potential mechanism to elicit an appropriate c-di-GMP-dependent response is to organize the corresponding proteins in a spatiotemporal fashion. Here, we show that a major contributor to c-di-GMP levels and flagellum-mediated swimming in Shewanella, PdeB, is recruited to the flagellated cell pole by the polar marker protein HubP. Polar recruitment involves a direct interaction between HubP and a GGDEF domain in PdeB, demonstrating a novel mechanism of polar targeting by the widely conserved HubP/FimV polar marker.

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically extracts lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are extracted. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake.

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically acquires essential lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are transported. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake.

Many human pathogens need to extract lipids from their environment for survival and proliferation. How the lipid uptake is accomplished on a molecular level is largely unknown, and no proteins directly involved in this process have been characterized to date. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213) from Mycoplasma pneumoniae, a human pathogen responsible for approximately 30% of community-acquired human pneumonia. Fluorescence microscopy, using antibodies raised against the ectodomain of P116, shows a ubiquitous distribution of P116 on the cell surface, indicating a direct role in host cell interactions. Single-particle cryo-electron microscopy at 3.3 Å resolution reveals two homodimers connected by a dimerization interface, and a core domain presenting a previously unseen fold. This fold creates a large cavity of ~18,000 Å3 with a fully hydrophobic internal surface that is accessible to solvent. The hydrophobic residues lining the cavity are conserved in P116 orthologues of other Mycoplasma species. We also observed elongated densities with a length of 10-19 Å long and a width of 4 Å within the cavity, which are not accounted for by the structure and which we identified as the essential lipids phosphatidylcholine, sphingomyelin and cholesterol using mass spectrometry. When the cavity is emptied by stringent treatment with detergents, the protein undergoes an extensive conformational change to adopt a closed conformation that no longer allows for the accommodation of lipids. We conducted radioactivity transfer experiment demonstrating a net transfer of cholesterol from high-density lipoproteins (HDLs) to P116, and observed an uptake of cholesterol into previously-emptied P116 from serum. We also found a direct attachment of P116 to HDLs using cryo-electron microscopy. These results reveal the mechanism by which P116 captures essential lipids from the host environment and possibly then delivers them by a wringing movement into the membrane by passive transport.