Sulfide Protects Staphylococcus aureus from Aminoglycoside Antibiotics but Cannot Be Regarded as a General Defense Mechanism against Antibiotics.

Sulfide production has been proposed to be a universal defense mechanism against antibiotics in bacteria (K. Shatalin, E. Shatalina, A. Mironov, and E. Nudler, Science 334:986-990, 2011, doi:10.1126/science.1209855). To gain insight into the mechanism underlying sulfide protection, we systematically and comparatively addressed the interference of sulfide with antibiotic activity against Staphylococcus aureus, as a model organism. The impact of sulfide and sulfide precursors on the antibiotic susceptibility of S. aureus to the most important classes of antibiotics was analyzed using modified disk diffusion assays, killing kinetic assays, and drug uptake studies. In addition, sulfide production and the impact of exogenously added sulfide on the physiology of S. aureus were analyzed. Sulfide protection was found to be limited to aminoglycoside antibiotics, which are known to be taken up by bacterial cells in an energy-dependent process. The protective mechanism was found to rely on an inhibitory effect of sulfide on the bacterial respiratory chain, leading to reduced drug uptake. S. aureus was found to be incapable of producing substantial amounts of sulfide. We propose that bacterial sulfide production should not be regarded as a general defense mechanism against antibiotics, since (i) it is limited to aminoglycosides and (ii) production levels vary considerably among species and, as for S. aureus, may be too low for protection.

Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood.

Matching donor and recipient blood groups based on red blood cell (RBC) surface ABO glycans and antibodies in plasma is crucial to avoid potentially fatal reactions during transfusions. Enzymatic conversion of RBC glycans to the universal group O is an attractive solution to simplify blood logistics and prevent ABO-mismatched transfusions. The gut symbiont Akkermansia muciniphila can degrade mucin O-glycans including ABO epitopes. Here we biochemically evaluated 23 Akkermansia glycosyl hydrolases and identified exoglycosidase combinations which efficiently transformed both A and B antigens and four of their carbohydrate extensions. Enzymatic removal of canonical and extended ABO antigens on RBCs significantly improved compatibility with group O plasmas, compared to conversion of A or B antigens alone. Finally, structural analyses of two B-converting enzymes identified a previously unknown putative carbohydrate-binding module. This study demonstrates the potential utility of mucin-degrading gut bacteria as valuable sources of enzymes for production of universal blood for transfusions.

The extracellular juncture domains in the intimin passenger adopt a constitutively extended conformation inducing restraints to its sphere of action.

Enterohemorrhagic and enteropathogenic Escherichia coli are among the most important food-borne pathogens, posing a global health threat. The virulence factor intimin is essential for the attachment of pathogenic E. coli to the intestinal host cell. Intimin consists of four extracellular bacterial immunoglobulin-like (Big) domains, D00-D2, extending into the fifth lectin subdomain (D3) that binds to the Tir-receptor on the host cell. Here, we present the crystal structures of the elusive D00-D0 domains at 1.5 Å and D0-D1 at 1.8 Å resolution, which confirms that the passenger of intimin has five distinct domains. We describe that D00-D0 exhibits a higher degree of rigidity and D00 likely functions as a juncture domain at the outer membrane-extracellular medium interface. We conclude that D00 is a unique Big domain with a specific topology likely found in a broad range of other inverse autotransporters. The accumulated data allows us to model the complete passenger of intimin and propose functionality to the Big domains, D00-D0-D1, extending directly from the membrane.

Mutational analyses of YqjA, a Tvp38/DedA protein of E. coli.

Membrane proteins of the DedA/Tvp38 protein family are involved in membrane integrity and virulence of pathogenic organisms. However, the structure and exact function of any member of this large protein family are still unclear. In the present study we analyzed the functional and structural properties of a DedA homolog. Purified YqjA variants from Escherichia coli are detectable in different oligomeric states and specific homo-interaction of YqjA monomers in the membrane were confirmed by formation of a disulfide bond in the C-terminal transmembrane helix. Moreover, alanine scanning mutagenesis exhibited different interaction sites crucial for YqjA activity vs. dimer formation.