Structure and Mechanism of the Lincosamide Antibiotic Adenylyltransferase LinB

SummaryLincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate– and binary product–bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve

Dynamic interactions of a conserved enterotoxigenic Escherichia coli adhesin with intestinal mucins govern epithelium engagement and toxin delivery

At present, there is no vaccine for enterotoxigenic Escherichia coli (ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design

Mucus-bacteria interactions in the gut : investigating the role of the mucin Muc2 and its glycosylation in host defense during enteric bacterial infections

The intestinal mucus layer, which is largely composed of the secreted mucin Muc2 provides a first line of defense in the intestine. Muc2 is a heavily O-glycosylated protein with core 1 and core 3 derived O-glycans as primary constituents. It plays an important role in host defense against the attaching/effacing (A/E) pathogen Citrobacter rodentium. However whether it provides protection against the invasive human pathogen Salmonella is still unclear. Furthermore, the role of O-glycosylation in mediating the protective role played by the Muc2 mucin against enteric pathogens has not been investigated. Likewise, although almost all enteric bacterial pathogens must cross the overlying mucus layer to infect the intestinal epithelium, there is very little known about mucus-enteric bacterial interactions and virulence strategies used to accomplish this feat. We began our investigations by comparing Salmonella-induced colitis and mucus dynamics in Muc2-deficient (Muc2 -/-), C3GnT -/-, and C57BL/6 (WT) mice. While absence of core 3 derived O-glycosylation only impacted epithelial barrier integrity, absence of Muc2 resulted in significantly higher barrier disruption, host mortality rates, and increased colonic and systemic Salmonella burdens. Likewise, absence of core 1 derived O-glycans (C1galt1 -/- mice) resulted in heightened susceptibility to C. rodentium, characterized by impaired mucus levels in the lumen, and bacterial aggregation in close proximity to the intestinal epithelial surface, phenotypes not seen in WT or C3GnT -/- counterparts. To understand if the non-motile pathogen C. rodentium used bacterial proteases/mucinases as a mucus degrading strategy to gain access to the underlying epithelium, we investigated the role of a putative mucinase and a class 2 SPATE PicC. While PicC did not affect C. rodentium’s ability to colonize the colon, it appeared to have an unprecedented role in regulating C. rodentium’s activation of the innate receptor TLR2, suggesting that despite its mucinase activity, PicC's major roles in vivo may be to limit C. rodentium aggregation and its recognition by the host's innate immune system. Overall these studies highlight a novel protective role of Muc2 and its O-linked glycosylation in host defense against enteric infections and the importance of Muc2-mediated regulation of pathogen burdens at the intestinal epithelial surface.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Antibiotic Resistance Is Prevalent in an Isolated Cave Microbiome

<div><p>Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.</p> </div

Induction of daptomycin resistance in <i>Paenibacillus lautus</i> LC231.

<p>LC231 was cultured in TSB supplemented with 1.25 mM CaCl₂ and 4 µg/ml daptomycin added from start (zero time point) (1) or early log phase (2). Growth was compared to growth control with no drug (3). Arrow represents the time point at which daptomycin was added during early log phase.</p

Summary of Antibiotic Inactivation Studies for Gram-positive Isolates.

<p>Strains were grown in 50% TSB for 5 days in presence of 20 µg/ml antibiotic. Conditional media was used for setting up disk diffusion assays and LC-MS analyses. Inactivation was defined as the absence of a zone of clearance around the disk. Hydrolytic mechanism of ß-lactam resistance is inferred.</p

Kinetic parameters for MPH (2′)-II from <i>B. faecium</i> DSM 4810.

<p>1- GTP held at 200 µM for antibiotic substrates, 2-erythomycin held at 400 µM.</p

Kinetic parameters for MPH (2′)-II from <i>B. paraconglomeratum.</i>

<p>1- GTP held at 200 µM for antibiotic substrates, 2-erythomycin held at 400 µM.</p

Resistance levels of Lechuguilla cave bacteria at 20 µg/ml against various antibiotics: (top) Gram-positive strains (bottom) Gram-negative strains.

<p>Antibiotics are grouped according to their mode of action/target, where each color represents a different target.</p