The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3␤ (MAP1LC3␤). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3␤ fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.Virus-induced myocarditis is an important cause of morbidity and mortality (1, 2). The enteroviruses have been considered the most common etiologic agents; however, other viruses, such as the adenoviruses, have also been implicated (3). The advent of molecular hybridization and PCR techniques has directly demonstrated infection of the myocardium with these viruses and has provided evidence of a viral etiology for dilated cardiomyopathy (DCM) 5 (1, 3). These findings further support the hypothesis that DCM is in some cases a long term sequela of acute or chronic myocarditis (4, 5), either due to the pathogenic effect of persistent viral replication or to an ongoing autoimmune process secondary to the viral infection.The recognition of viruses or viral particles by the host triggers the activation of an innate immune response that is characterized by the production of mediators such as tumor necrosis factor-␣, interleukins, interferons, and nitric oxide, all of which are toxic to replicating viruses (6). This initial antiviral response by the host is now known to be mediated at least in part by Toll-like receptors (TLRs) (5, 6). It has been shown that double-stranded (ds) RNA is the primary ligand for TLR3 (7). Although most TLR signaling pathways utilize the adaptor molecule MyD88 (myeloid differentiation primary response gene 88), TLR3 signals through the adaptor molecule called Toll-like receptor adaptor molecule 1 (Ticam1) or Toll/Interleukin 1 receptor domain-containing adapter-inducing interferon ␤ (TRIF) (8, 9). Activation of this pathway triggers produ...

Summary The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His → Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

Extended-spectrum β-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M β-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL β-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 β-lactamase. Substitutions of Ser237 and Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. The conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.

Multiple studies have identified changes within the gut microbiome in response to diarrheal-inducing bacterial pathogens. However, examination of the microbiome in response to viral pathogens remains understudied. Compounding this, many studies use fecal samples to assess microbiome composition; which may not accurately mirror changes within the small intestine, the primary site for most enteric virus infections. As a result, the functional significance of small intestinal microbiome shifts during infection is not well defined. To address these gaps, rotavirus-infected neonatal mice were examined for changes in bacterial community dynamics, host gene expression, and tissue recovery during infection. Profiling bacterial communities using 16S rRNA sequencing suggested significant and distinct changes in ileal communities in response to rotavirus infection, with no significant changes for other gastrointestinal (GI) compartments. At 1-d post-infection, we observed a loss in Lactobacillus species from the ileum, but an increase in Bacteroides and Akkermansia, both of which exhibit mucin-digesting capabilities. Concomitant with the bacterial community shifts, we observed a loss of mucin-filled goblet cells in the small intestine at d 1, with recovery occurring by d 3. Rotavirus infection of mucin-producing cell lines and human intestinal enteroids (HIEs) stimulated release of stored mucin granules, similar to in vivo findings. In vitro, incubation of mucins with Bacteroides or Akkermansia members resulted in significant glycan degradation, which altered the binding capacity of rotavirus in silico and in vitro. Taken together, these data suggest that the response to and recovery from rotavirus-diarrhea is unique between subcompartments of the GI tract and may be influenced by mucin-degrading microbes.

Metallo-␤-lactamases, such as IMP-1, are a major global health threat, as they provide for bacterial resistance to a wide range of ␤-lactam antibiotics, including carbapenems. Understanding the molecular details of the enzymatic process and the sequence requirements for function are essential aids in overcoming ␤-lactamase-mediated resistance. An asparagine residue is conserved at position 233 in approximately 67% of all metallo-␤-lactamases. Despite its conservation, the molecular basis of Asn233 function is poorly understood and remains controversial. It has previously been shown that mutations at this site exhibit context-dependent sequence requirements in that the importance of a given amino acid depends on the antibiotic being tested. To provide a more thorough examination as to the function and sequence requirements at this position, a collection of IMP-1 mutants encoding each of the 19 possible amino acid substitutions was generated. The resistance levels toward four ␤-lactam antibiotics were measured for Escherichia coli containing each of these mutants. The sequence requirements at position 233 for wild-type levels of resistance toward two cephalosporins were the most relaxed, while there were more stringent sequence requirements for resistance to ampicillin or imipenem. Enzyme kinetic analysis and determinations of steady-state protein levels indicated that the effects of the substitutions on resistance are due to changes in the kinetic parameters of the enzyme. Taken together, the results indicate that substitutions at position 233 significantly alter the kinetic parameters of the enzyme, but most substituted enzymes are able to provide for a high level of resistance to a broad range of ␤-lactams.The ␤-lactams, which include the penicillins and cephalosporins, are the most widely used class of antibiotics, and resistance to these drugs is a significant clinical problem (7,25). ␤-Lactamases are secreted into the periplasm and hydrolyze the conserved four-membered ␤-lactam ring, rendering the drug inactive. Currently, there are hundreds of ␤-lactamases reported, and they are regarded as the most common mechanism of resistance to this class of antibiotics (43).There are four classes of ␤-lactamases (A to D) based on primary amino acid sequence homology. ␤-Lactamases in classes A, C, and D hydrolyze the drugs by using a catalytic serine as the primary nucleophile (9, 15). Class B ␤-lactamases are metallo-enzymes that require either one or two zinc ions in the active site. The zinc ions in the active site decrease the pK of a coordinated water molecule, resulting in a hydroxide ion that acts as a nucleophile to attack the carbonyl group of the ␤-lactams (14). These enzymes exhibit a broad substrate profile, including carbapenems, which are often held in reserve as antibiotics of last resort (18,26). Mechanism-based inhibitors of the serine-active ␤-lactamases are available for clinical use, but these molecules do not act on class B enzymes (2). The genes encoding metallo-␤-lactamases are often found on mob...

Fluoroquinolones are some of the most prescribed antibiotics in the United States. Previously, we and others showed that the fluoroquinolones exhibit a class effect with regard to the CLSI-established breakpoints for resistance, such that decreased susceptibility (i.e., an increased MIC) to one fluoroquinolone means a simultaneously decreased susceptibility to all. For defined strains, however, clear differences exist in the pharmacodynamic properties of each fluoroquinolone and the extent to which resistance-associated genotypes affect the MICs of each fluoroquinolone. In a pilot study of 920 clinical Escherichia coli isolates, we uncovered tremendous variation in norfloxacin MICs. The MICs for all of the fluoroquinolone-resistant isolates exceeded the resistance breakpoint, reaching 1,000 g/ml. Approximately 25% of the isolates (n ‫؍‬ 214), representing the full range of resistant norfloxacin MICs, were selected for the simultaneous determinations of ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs. We found that (i) great MIC variation existed for all four fluoroquinolones, (ii) the ciprofloxacin and levofloxacin MICs of >90% of the fluoroquinolone-resistant isolates were higher than the resistance breakpoints, (iii) ciprofloxacin and levofloxacin MICs were distributed into two distinct groups, (iv) the MICs of two drug pairs (ciprofloxacin and norfloxacin by Kendall's Tau-b test and gatifloxacin and levofloxacin by paired t test) were similar with statistical significance but were different from each other, and (v) ϳ2% of isolates had unprecedented fluoroquinolone MIC relationships. Thus, although the fluoroquinolones can be considered equivalent with regard to clinical susceptibility or resistance, fluoroquinolone MICs differ dramatically for fluoroquinolone-resistant clinical isolates, likely because of differences in drug structure.Fluoroquinolones, some of the most frequently prescribed antimicrobial agents worldwide, target the bacterial type II topoisomerases gyrase and topoisomerase IV. Type II topoisomerases are essential, ubiquitous enzymes involved in virtually every aspect of DNA metabolism. These enzymes cleave one DNA double helix, pass a second DNA molecule (or a different region of the first DNA molecule) through the break, and religate the broken DNA. Fluoroquinolones increase the longevity of the normally short-lived cleaved DNA-topoisomerase intermediates (reviewed in reference 7). DNA tracking machinery somehow is affected by these intermediates, resulting in multiple subsequent effects, such as chromosome fragmentation, the inhibition of DNA synthesis, and death (reviewed in reference 6).With regard to susceptibility or resistance defined by CLSI breakpoints (Table 1), the fluoroquinolones appear to exemplify a class effect, such that any decrease in susceptibility (i.e., increased MIC) to one drug means a simultaneous decrease for all (2). The fluoroquinolones, however, vary with regard to pharmacokinetic and pharmacodynamic parameters, including potency (reviewed in refer...

Bacterial resistance to β-lactam antibiotics caused by class B metallo-β-lactamases (MBL), especially for certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure activity relationship (SAR) and molecular modeling studies were performed and implications for further inhibitor design are discussed.

e Metallo-␤-lactamases catalyze the hydrolysis of a broad range of ␤-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ␤-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ␤-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ␤-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 M. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.A n increasing prevalence of antibiotic-resistant strains is reducing the available options for treating bacterial infections. ␤-Lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobial agents (31). The major contributors to ␤-lactam antibiotic resistance are ␤-lactamase enzymes, which act by hydrolyzing the four-member ␤-lactam ring (15,62). Mechanistically, this is accomplished either via an active-site serine in the class A, C, and D enzymes or through the use of one or two Zn 2ϩ ions (class B) (4). Class B metallo-␤-lactamases (M␤Ls) are a group of structurally similar enzymes that exhibit a characteristic ␣␤/␤␣ sandwich fold, with the active site located at the interface between domains. This scaffold supports up to 6 residues at the active site that coordinate either one or two zinc ions that are central to the catalytic mechanism (1,6,35). M␤Ls have the capacity to hydrolyze most clinically available ␤-lactam drugs, including extended-spectrum cephalosporins and carbapenems (8,10,32,40,59,61).The IMP-1 metallo-␤-lactamase has been identified in several nosocomial, Gram-negative, pathogenic bacteria, including Pseudomonas aeruginosa and Serratia marcesens (22, 28). The bla IMP-1 gene is carried on an integron element that facilitates genetic transfer and is likely why IMP-1 is found among multiple bacterial species (28, 58). The combination of a broad ␤-lactam antibiotic substrate profile and the genetic ca...