Exploring the Role of Residue 228 in Substrate and Inhibitor Recognition by VIM Metallo-ß-lactamases.

ß-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete ß-lactams. However, commercially available BLIs are not effective against metallo-ß-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki* values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E*I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.

Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination.

BACKGROUND: Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. METHODS: Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. RESULTS: Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained bla(OXA-23), and four isolates possessed bla(OXA-24/40). Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing bla(KPC-2) or bla(KPC-3) were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). CONCLUSION: In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.

E-Cadherin, NFATC3, and PLP2 Are Differentially Methylated in Multiple Cancers.

It is well documented that cancer cells have abnormal methylation patterns often caused by faulty methylating machinery. Specifically, E-cadherin, NFATC3, and PLP2 are 3 genes known to be aberrantly methylated in cancer cells. These genes are well documented for their role in signaling pathways involved with cell proliferation, adhesion, migration, and other signs of tumor progression. Therefore, changes in gene expression of CDH1, NFATC3, and PLP2 due to aberrant methylation can lead to profound changes in cellular function and tumor formation. In order to ensure that previous in vitro and in vivo methylation studies match what is observed in the clinic, we utilized a bioinformatics approach to complete an extensive analysis of methylation patterns of these 3 genes, analyzing over 5000 patient samples, across all cancers for which both normal and tumor tissues were available. Specifically, we analyzed overall and site-specific methylation patterns, at CpG islands and shores, of all 3 genes across 14 cancer types. Furthermore, we compared these methylation levels in normal and tumor samples of both matched and unmatched patient samples in order to determine any differences between groups. Finally, we examined whether an aberrant DNA methyltransferase, DNMT3B7, known to be expressed in cancer cells and to alter methylation patterns in vitro correlated with altered overall and site-specific methylation of CDH1, NFATC3, and PLP2 in these patient samples. Our results indicate that methylation patterns of CDH1 and NFATC3 were unexpectedly varied across tumors, contrary to previous studies performed in vitro, while PLP2 showed the expected hypomethylation pattern in tumor tissues. We also observed some correlation between DNMT3B7 expression and methylation patterns of these genes, but patterns were inconsistent. Taken together, these results emphasize the necessity for in vivo and patient studies rather than a complete reliance on in vitro data and provide multiple areas of future research.

Elucidating the role of Trp105 in the KPC-2 ß-lactamase.

The molecular basis of resistance to ß-lactams and ß-lactam-ß-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective ß-lactam therapy. Recent crystal structures of KPC-2 and other class A ß-lactamases suggest that Ambler position Trp105 may be of importance in binding ß-lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC-2 by conducting site-saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, -Tyr, -Asn, and -His KPC-2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC-2. Interestingly, most of the variants showed increased MICs to ampicillin-clavulanic acid but not to ampicillin-sulbactam or piperacillin-tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, -Asn, -Leu, and -Val) were studied in detail. Consistent with the MIC data, the Trp105Phe ß-lactamase displayed improved catalytic efficiencies, k(cat)/K(m), toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k(cat)/K(m) toward cefotaxime and imipenem when compared to WT ß-lactamase. The Trp105Asn variant exhibited increased K(m)s for all substrates. In contrast, the Trp105Leu and -Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k(cat)/K(m)) for all substrates. With respect to clavulanic acid, the K(i)s and partition ratios were increased for the Trp105Phe, -Asn, and -Val variants. We conclude that interactions between Trp105 of KPC-2 and the ß-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in ß-lactam and ß-lactamase inhibitor discrimination.