Conformational flexibility in carbapenem hydrolysis drives substrate specificity of the class D carbapenemase OXA-24/40.

The evolution of multidrug resistance in Acinetobacter spp. increases the risk of our best antibiotics losing their efficacy. From a clinical perspective, the carbapenem-hydrolyzing class D ß-lactamase subfamily present in Acinetobacter spp. is particularly concerning because of its ability to confer resistance to carbapenems. The kinetic profiles of class D ß-lactamases exhibit variability in carbapenem hydrolysis, suggesting functional differences. To better understand the structure-function relationship between the carbapenem-hydrolyzing class D ß-lactamase OXA-24/40 found in Acinetobacter baumannii and carbapenem substrates, we analyzed steady-state kinetics with the carbapenem antibiotics meropenem and ertapenem and determined the structures of complexes of OXA-24/40 bound to imipenem, meropenem, doripenem, and ertapenem, as well as the expanded-spectrum cephalosporin cefotaxime, using X-ray crystallography. We show that OXA-24/40 exhibits a preference for ertapenem compared with meropenem, imipenem, and doripenem, with an increase in catalytic efficiency of up to fourfold. We suggest that superposition of the nine OXA-24/40 complexes will better inform future inhibitor design efforts by providing insight into the complicated and varying ways in which carbapenems are selected and bound by class D ß-lactamases.

Cutaneous leukocyte lineages in tolerant large animal and immunosuppressed clinical vascularized composite allograft recipients.

Vascularized composite allografts (VCAs) can restore fully functional anatomic units in patients with limb amputations or severe facial tissue loss. However, acute rejection of the skin is frequently observed and underscores the importance of developing tolerance induction protocols. In this study, we have characterized the skin immune system in VCAs. We demonstrate infiltration of recipient leukocytes, regardless of rejection status, and in tolerant mixed hematopoietic chimeras, the co-existence of these cells with donor leukocytes in the absence of rejection. Here we characterize the dermal T cell and epidermal Langerhans cell components of the skin immune system in our porcine model of VCA tolerance, and the kinetics of cutaneous chimerism in both of these populations in VCAs transplanted to tolerant and nontolerant recipients, as well as in host skin. Furthermore, in biopsies from the first patient to receive a hand transplant in our program, we demonstrate the presence of recipient T cells in the skin of the transplanted limb in the absence of clinical or histological evidence of rejection.

Multiple substitutions lead to increased loop flexibility and expanded specificity in Acinetobacter baumannii carbapenemase OXA-239.

OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced ∼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of ß-lactam drugs.

Expanded Substrate Activity of OXA-24/40 in Carbapenem-Resistant Acinetobacter baumannii Involves Enhanced Binding Loop Flexibility.

Gram-negative bacteria resist ß-lactam antibiotics primarily by deploying ß-lactamase proteins that hydrolytically destroy the antibiotics. In clinical settings, these bacteria are producing variant ß-lactamases with "gain-of-activity" mutations that inactivate a broader range of ß-lactams. Learning how these mutations broaden substrate activity is important for coping with ß-lactam resistance. Here, we investigate a gain of activity mutation in OXA-24/40, a carbapenem-hydrolyzing class D ß-lactamase (CHDL) in Acinetobacter baumannii. OXA-24/40 was originally active against penicillin and carbapenem classes of ß-lactams, but a clinical variant of OXA-24/40, the single-site substitution mutant P227S, has emerged with expanded activity that now includes advanced cephalosporins and the monobactam aztreonam. Using solution-state nuclear magnetic resonance (NMR) spectroscopy, we have compared the site-specific backbone dynamics of wild-type OXA-24/40 and the P227S variant. P227S changes local backbone flexibility in segments that are important for both binding and hydrolysis of carbapenem and cephalosporin substrates. Our results suggest that mutation-induced changes in sequence-specific dynamics can expand substrate activity and thus highlight the role of protein conformational dynamics in antibiotic resistance. To the best of our knowledge, this is the first NMR study of CHDL conformational dynamics and its impact on the expansion of ß-lactam antibiotic resistance.

Clinical Variants of the Native Class D ß-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems.

The threat posed by the chromosomally encoded class D ß-lactamase of Acinetobacter baumannii (OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of ß-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of ß-lactams against this troublesome pathogen.

Structural basis of activity against aztreonam and extended spectrum cephalosporins for two carbapenem-hydrolyzing class D ß-lactamases from Acinetobacter baumannii.

The carbapenem-hydrolyzing class D ß-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for ß-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P → S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower Km values, suggesting that the P → S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the ß5-ß6 loop and the omega loop of the enzymes. The P → S substitution found in OXA-160 and OXA-225 results in a deviation of the ß5-ß6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D ß-lactamases but may also provide a map for ß-lactam improvement.

Structural origins of oxacillinase specificity in class D ß-lactamases.

Since the discovery and use of penicillin, the increase of antibiotic resistance among bacterial pathogens has become a major health concern. The most prevalent resistance mechanism in Gram-negative bacteria is due to ß-lactamase expression. Class D ß-lactamases are of particular importance due to their presence in multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. The class D enzymes were initially characterized by their ability to efficiently hydrolyze isoxazolyl-type ß-lactams like oxacillin. Due to this substrate preference, these enzymes are traditionally referred to as oxacillinases or OXAs. However, this class is comprised of subfamilies characterized by diverse activities that include oxacillinase, carbapenemase, or cephalosporinase substrate specificity. OXA-1 represents one subtype of class D enzyme that efficiently hydrolyzes oxacillin, and OXA-24/40 represents another with weak oxacillinase, but increased carbapenemase, activity. To examine the structural basis for the substrate selectivity differences between OXA-1 and OXA-24/40, the X-ray crystal structures of deacylation-deficient mutants of these enzymes (Lys70Asp for OXA-1; Lys84Asp for OXA-24) in complexes with oxacillin were determined to 1.4 Å and 2.4 Å, respectively. In the OXA-24/40/oxacillin structure, the hydrophobic R1 side chain of oxacillin disrupts the bridge between Tyr112 and Met223 present in the apo OXA-24/40 structure, causing the main chain of the Met223-containing loop to adopt a completely different conformation. In contrast, in the OXA-1/oxacillin structure, a hydrophobic pocket consisting of Trp102, Met99, Phe217, Leu161, and Leu255 nicely complements oxacillin's nonpolar R1 side chain. Comparison of the OXA-1/oxacillin and OXA-24/40/oxacillin complexes provides novel insight on how substrate selectivity is achieved among subtypes of class D ß-lactamases. By elucidating important active site interactions, these findings can also inform the design of novel antibiotics and inhibitors.

Class D ß-lactamases: a reappraisal after five decades.

Despite 70 years of clinical use, ß-lactam antibiotics still remain at the forefront of antimicrobial chemotherapy. The major challenge to these life-saving therapeutics is the presence of bacterial enzymes (i.e., ß-lactamases) that can hydrolyze the ß-lactam bond and inactivate the antibiotic. These enzymes can be grouped into four classes (A-D). Among the most genetically diverse are the class D ß-lactamases. In this class are ß-lactamases that can inactivate the entire spectrum of ß-lactam antibiotics (penicillins, cephalosporins, and carbapenems). Class D ß-lactamases are mostly found in Gram-negative bacteria such as Pseudomonas aeruginosa , Escherichia coli , Proteus mirabilis , and Acinetobacter baumannii . The active-sites of class D ß-lactamases contain an unusual N-carboxylated lysine post-translational modification. A strongly hydrophobic active-site helps create the conditions that allow the lysine to combine with CO2, and the resulting carbamate is stabilized by a number of hydrogen bonds. The carboxy-lysine plays a symmetric role in the reaction, serving as a general base to activate the serine nucleophile in the acylation reaction, and the deacylating water in the second step. There are more than 250 class D ß-lactamases described, and the full set of variants shows remarkable diversity with regard to substrate binding and turnover. Narrow-spectrum variants are most effective against the earliest generation penicillins and cephalosporins such as ampicillin and cephalothin. Extended-spectrum variants (also known as extended-spectrum ß-lactamases, ESBLs) pose a more dangerous clinical threat as they possess a small number of substitutions that allow them to bind and hydrolyze later generation cephalosporins that contain bulkier side-chain constituents (e.g., cefotaxime, ceftazidime, and cefepime). Mutations that permit this versatility seem to cluster in the area surrounding an active-site tryptophan resulting in a widened active-site to accommodate the oxyimino side-chains of these cephalosporins. More concerning are the class D ß-lactamases that hydrolyze clinically important carbapenem ß-lactam drugs (e.g., imipenem). Whereas carbapenems irreversibly acylate and inhibit narrow-spectrum ß-lactamases, class D carbapenemases are able to recruit and activate a deacylating water. The rotational orientation of the C6 hydroxyethyl group found on all carbapenem antibiotics likely plays a role in whether the deacylating water is effective or not. Inhibition of class D ß-lactamases is a current challenge. Commercially available inhibitors that are active against other classes of ß-lactamases are ineffective against class D enzymes. On the horizon are several compounds, consisting of both ß-lactam derivatives and non-ß-lactams, that have the potential of providing novel leads to design new mechanism-based inactivators that are effective against the class D enzymes. Several act synergistically when given in combination with a ß-lactam antibiotic, and others show a unique mechanism of inhibition that is distinct from the traditional ß-lactamase inhibitors. These studies will bolster structure-based inhibitor design efforts to facilitate the optimization and development of these compounds as class D inactivators.

A fluorescent carbapenem for structure function studies of penicillin-binding proteins, ß-lactamases, and ß-lactam sensors.

By reacting fluorescein isothiocyanate with meropenem, we have prepared a carbapenem-based fluorescent ß-lactam. Fluorescein-meropenem binds both penicillin-binding proteins and ß-lactam sensors and undergoes a typical acylation reaction in the active site of these proteins. The probe binds the class D carbapenemase OXA-24/40 with close to the same affinity as meropenem and undergoes a complete catalytic hydrolysis reaction. The visible light excitation and strong emission of fluorescein render this molecule a useful structure-function probe through its application in sodium dodecyl sulfate-polyacrylamide gel electrophoresis assays as well as solution-based kinetic anisotropy assays. Its classification as a carbapenem ß-lactam and the position of its fluorescent modification render it a useful complement to other fluorescent ß-lactams, most notably Bocillin FL. In this study, we show the utility of fluorescein-meropenem by using it to detect mutants of OXA-24/40 that arrest at the acyl-intermediate state with carbapenem substrates but maintain catalytic competency with penicillin substrates.

Lower extremity soft tissue defect reconstruction with the serratus anterior flap.

Reconstruction of limb-threatening lower extremity defects presents unique challenges. The selected method must provide adequate coverage of exposed bone, joints, and tendons while maximizing function of the limb. The traditional workhorse flaps, the free latissimus dorsi and rectus abdominis flaps, have been associated with donor site morbidity and bulkiness that can impair rehabilitation. We report a case series (n = 18) in which the free serratus anterior muscle flap and split thickness skin graft (STSG) was used for lower limb soft tissue coverage. Injuries were due to diabetes (9/18), trauma (7/18), and chronic venous stasis (2/18). A 94% flap survival rate was observed and all but one patient was ambulatory. No donor site morbidity was reported. Our series demonstrates that serratus anterior is an advantageous, reliable free flap with minimal donor site morbidity.

The unique immunobiology of the skin: implications for tolerance of vascularized composite allografts.

PURPOSE OF REVIEW: Vascularized composite allograft (VCA) transplantation restores function and form following major soft tissue and musculoskeletal injury. Lifelong immunosuppression is necessary for graft function and survival but acute skin-targeted rejection episodes remain common. We review recent advances in skin immunobiology, emphasizing findings in clinical and experimental VCAs. We also highlight advances in immunotherapy and tolerance protocols with implications for the prevention of VCA rejection, and ultimately, induction of clinically applicable strategies for VCA tolerance. RECENT FINDINGS: There is now an increasing appreciation for the role of skin-specific mechanisms, including lymphoid neogenesis, in VCA rejection. In contrast, expression of the regulatory master-switch FOXP3 was demonstrated to be significantly upregulated in the skin of tolerant VCAs in large animal models compared with normal skin and rejecting controls. SUMMARY: Most VCA transplant centers continue to utilize antibody-mediated induction therapy and triple agent maintenance immunosuppression. Skin remains the primary target of rejection in VCAs, and current multicenter studies hope to elucidate the mechanisms involved. Proposed standardized procedures for skin biopsies, and diligent reporting of clinical data to the international registry, will be important to maximize the strength of these studies.

The Nuances of Hand Transplantation After Sepsis.

Vascularized composite allotransplantation (VCA) of the upper extremity is an established restorative procedure for selected patients with acquired upper limb loss. The majority of upper limb VCAs performed worldwide have been for victims of various forms of trauma. However, in the developed world, amputation following severe sepsis seems to be an increasingly common indication for referral to hand transplant programs. Unlike trauma patients with isolated limb injuries, patients with amputations as a complication of sepsis have survived through a state of global tissue hypoperfusion and multisystem organ failure with severe, enduring effects on the entire body's physiology. This article reviews the unique considerations for VCA candidacy in postsepsis patients with upper limb amputation. These insights may also be relevant to postsepsis patients undergoing other forms of transplantation or to VCA patients requiring additional future solid organ transplants.

Structural and Dynamic Features of Acinetobacter baumannii OXA-66 ß-Lactamase Explain Its Stability and Evolution of Novel Variants.

OXA-66 is a member of the OXA-51 subfamily of class D ß-lactamases native to the Acinetobacter genus that includes Acinetobacter baumannii, one of the ESKAPE pathogens and a major cause of drug-resistant nosocomial infections. Although both wild type OXA-66 and OXA-51 have low catalytic activity, they are ubiquitous in the Acinetobacter genomes. OXA-51 is also remarkably thermostable. In addition, newly emerging, single and double amino acid variants show increased activity against carbapenems, indicating that the OXA-51 subfamily is growing and gaining clinical significance. In this study, we used molecular dynamics simulations, X-ray crystallography, and thermal denaturation data to examine and compare the dynamics of OXA-66 wt and its gain-of-function variants: I129L (OXA-83), L167V (OXA-82), P130Q (OXA-109), P130A, and W222L (OXA-234). Our data indicate that OXA-66 wt also has a high melting temperature, and its remarkable stability is due to an extensive and rigid hydrophobic bridge formed by a number of residues around the active site and harbored by the three loops, P, Ω, and ß5-ß6. Compared to the WT enzyme, the mutants exhibit higher flexibility only in the loop regions, and are more stable than other robust carbapenemases, such as OXA-23 and OXA-24/40. All the mutants show increased rotational flexibility of residues I129 and W222, which allows carbapenems to bind. Overall, our data support the hypothesis that structural features in OXA-51 and OXA-66 promote evolution of multiple highly stable variants with increased clinical relevance in A. baumannii.

Structures of the class D Carbapenemases OXA-23 and OXA-146: mechanistic basis of activity against carbapenems, extended-spectrum cephalosporins, and aztreonam.

Class D ß-lactamases that hydrolyze carbapenems such as imipenem and doripenem are a recognized danger to the efficacy of these "last-resort" ß-lactam antibiotics. Like all known class D carbapenemases, OXA-23 cannot hydrolyze the expanded-spectrum cephalosporin ceftazidime. OXA-146 is an OXA-23 subfamily clinical variant that differs from the parent enzyme by a single alanine (A220) inserted in the loop connecting ß-strands ß5 and ß6. We discovered that this insertion enables OXA-146 to bind and hydrolyze ceftazidime with an efficiency comparable to those of other extended-spectrum class D ß-lactamases. OXA-146 also binds and hydrolyzes aztreonam, cefotaxime, ceftriaxone, and ampicillin with higher efficiency than OXA-23 and preserves activity against doripenem. In this study, we report the X-ray crystal structures of both the OXA-23 and OXA-146 enzymes at 1.6-Å and 1.2-Å resolution. A comparison of the two structures shows that the extra alanine moves a methionine (M221) out of its normal position, where it forms a bridge over the top of the active site. This single amino acid insertion also lengthens the ß5-ß6 loop, moving the entire backbone of this region further away from the active site. A model of ceftazidime bound in the active site reveals that these two structural alterations are both likely to relieve steric clashes between the bulky R1 side chain of ceftazidime and OXA-23. With activity against all four classes of ß-lactam antibiotics, OXA-146 represents an alarming new threat to the treatment of infections caused by Acinetobacter spp.

The gracilis myocutaneous free flap in swine: an advantageous preclinical model for vascularized composite allograft transplantation research.

Vascularized composite allotransplantation (VCA) has become a clinical reality, prompting research aimed at improving the risk-benefit ratio of such transplants. Here, we report our experience with a gracilis myocutaneous free flap in Massachusetts General Hospital miniature swine as a preclinical VCA model. Fourteen animals underwent free transfer of a gracilis myocutaneous flap comprised of the gracilis muscle and overlying skin, each tissue supplied by independent branches of the femoral vessels. End-to-end anastomoses were performed to the common carotid artery and internal jugular vein, or to the femoral vessels of the recipients. Thirteen of fourteen flaps were successful. A single flap was lost due to compromise of venous outflow. This model allows transplantation of a substantial volume of skin, subcutaneous tissue, and muscle. The anatomy is reliable and easily identified and harvest incurs minimal donor morbidity. We find this gracilis myocutaneous flap an excellent pre-clinical model for the study of vascularized composite allotransplantation.

Vascularized composite allotransplantation: towards tolerance and the importance of skin-specific immunobiology.

PURPOSE OF REVIEW: Vascularized composite allotransplantation (VCA) is increasingly utilized in the restoration of complex injuries and tissue loss. Acute skin-targeted rejection episodes are common and concerns remain regarding the risks of conventional immunosuppression. We review current immunosuppressive regimens for VCA, progress with immunomodulatory and tolerance protocols, and highlight recent advances in cutaneous immunobiology which will have significant implications for future development in the field. RECENT FINDINGS: Advances in induction protocols have demonstrated effective prevention of early graft loss in hand transplantation, although long-term outcomes are still pending. Furthermore, recent findings in leukocyte populations within the skin and their mechanisms of communication reveal that considerable numbers of resident T-effector memory cells, including a T-regulatory subset, exist, and that epidermal Langerhans' cells communicate with these cells, mediating both immunity and tolerance to maintain skin homeostasis. SUMMARY: The majority of VCA centers utilize antibody-mediated induction, followed by double or triple-agent maintenance immunosuppression. A clinical trial of a minimal-immunosuppression protocol based on bone marrow infusion reports encouraging interim results, but long-term follow-up will be required. Skin remains the primary target of rejection in VCA. New data demonstrate extensive T-cell memory resident in skin, and complex interactions between these cells and epidermal Langerhans' cells will have implications for VCA rejection and tolerance, and warrant further investigation in the allogeneic setting.

Hand transplantation: can we balance the risks and benefits?

Asking 'can we balance the risks and benefits?' implies that a quantification of both risk and benefit in hand transplantation (here the terms hand transplant and hand transplantation refer to allotransplantation of the human hand or hand and part or all of the upper limb or limbs) is possible. Despite all we have learned in recent years about hand transplantation, much remains unknown. Even if reliable methods for quantification of risk and benefit were available, fundamental issues relating to effective communication across the gulf of lived experience between the (presumably) handed surgeon and the handless patient remain. Inherent complexities mean some consider hand transplantation an unsolved problem, but we believe the medical and technical considerations fall within the ambit of a competent multidisciplinary team, and that psychosocial and ethical challenges are open to management through robust frameworks for assessment and decision making, underpinned by an extended period of assessment and dialogue, with candid acknowledgement where uncertainty remains. This respects the patient's autonomy while addressing the need for a prolonged period of informing consent.Level of evidence: V.

Sulfonamidoboronic Acids as "Cross-Class" Inhibitors of an Expanded-Spectrum Class C Cephalosporinase, ADC-33, and a Class D Carbapenemase, OXA-24/40: Strategic Compound Design to Combat Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to ß-lactams. One of the most important mechanisms is the production of ß-lactamase enzymes capable of hydrolyzing ß-lactam antibiotics. Co-expression of multiple classes of ß-lactamases is present in CRAB; therefore, the design and synthesis of "cross-class" inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical ß-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C ß-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a Ki = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other ß-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum ß-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

Site-saturation mutagenesis of position V117 in OXA-1 ß-lactamase: effect of side chain polarity on enzyme carboxylation and substrate turnover.

Class D ß-lactamases pose an emerging threat to the efficacy of ß-lactam therapy for bacterial infections. Class D enzymes differ mechanistically from other ß-lactamases by the presence of an active-site N-carboxylated lysine that serves as a general base to activate the serine nucleophile for attack. We have used site-saturation mutagenesis at position V117 in the class D ß-lactamase OXA-1 to investigate how alterations in the environment around N-carboxylated K70 affect the ability of that modified residue to carry out its normal function. Minimum inhibitory concentration analysis of the 20 position 117 variants demonstrates a clear pattern of charge and polarity effects on the level of ampicillin resistance imparted on Escherichia coli (E. coli). Substitutions that introduce a negative charge (D, E) at position 117 reduce resistance to near background levels, while the positively charged K and R residues maintain the highest resistance levels of all mutants. Treatment of the acidic variants with the fluorescent penicillin BOCILLIN FL followed by SDS-PAGE shows that they are active for acylation by substrate but deacylation-deficient. We used a novel fluorescence anisotropy assay to show that the specific charge and hydrogen-bonding potential of the residue at position 117 affect CO(2) binding to K70, which in turn correlates to deacylation activity. These conclusions are discussed in light of the mechanisms proposed for both class D ß-lactamases and BlaR ß-lactam sensor proteins and suggest a reason for the preponderance of asparagine at the V117-homologous position in the sensors.

Tolerance induction strategies in vascularized composite allotransplantation: mixed chimerism and novel developments.

Since the start of the clinical vascularized composite allotransplantation (VCA) era over a decade ago this field has witnessed significant developments in both basic and translational research. Transplant tolerance, defined as rejection-free acceptance of transplanted organs or tissues without long-term immunosuppression, holds the potential to revolutionize the field of VCA by removing the need for life-long immunosuppression. While tolerance of organ and vascularized composite transplants may be induced in small animal models by a variety of protocols, only mixed-chimerism-based protocols have successfully bridged the gap to preclinical study and to clinical trial in solid organ transplantation to date. In this paper we review the mixed-chimerism approach to tolerance induction, with specific reference to the field of VCA transplantation, and provide an overview of some novel cellular therapies as potential adjuvants to mixed chimerism in the development of tolerance induction protocols for clinical vascularized composite allotransplantation.