Timothy Palzkill

Metallo-β-lactamase structure and function

β‐Lactam antibiotics are the most commonly used antibacterial agents and growing resistance to these drugs is a concern. Metallo‐β‐lactamases are a diverse set of enzymes that catalyze the hydrolysis of a broad range of β‐lactam drugs including carbapenems. This diversity is reflected in the observation that the enzyme mechanisms differ based on whether one or two zincs are bound in the active site that, in turn, is dependent on the subclass of β‐lactamase. The dissemination of the genes encoding these enzymes among Gram‐negative bacteria has made them an important cause of resistance. In addition, there are currently no clinically available inhibitors to block metallo‐β‐lactamase action. This review summarizes the numerous studies that have yielded insights into the structure, function, and mechanism of action of these enzymes.

Molecular analysis of beta-lactamase structure and function.

The extensive and sometimes irresponsible use of beta-lactam antibiotics in clinical and agricultural settings has contributed to the emergence and widespread dissemination of antibiotic-resistant bacteria. Bacteria have evolved three strategies to escape the activity of beta-lactam antibiotics: 1) alteration of the target site (e.g. penicillin-binding protein (PBPs), 2) reduction of drug permeation across the bacterial membrane (e.g. efflux pumps) and 3) production of beta-lactamase enzymes. The beta-lactamase enzymes inactivate beta-lactam antibiotics by hydrolyzing the peptide bond of the characteristic four-membered beta-lactam ring rendering the antibiotic ineffective. The inactivation of the antibiotic provides resistance to the bacterium. Currently, there are over 300 beta-lactamase enzymes described for which numerous kinetic, structural, computational and mutagenesis studies have been performed. In this review, we discuss the recent work performed on the four different classes (A, B, C, and D) of beta-lactamases. These investigative advances further expand our knowledge about these complex enzymes, and hopefully, will provide us with additional tools to develop new inhibitors and antibiotics based on structural and rational designs.

The protein network of bacterial motility

Motility is achieved in most bacterial species by the flagellar apparatus. It consists of dozens of different proteins with thousands of individual subunits. The published literature about bacterial chemotaxis and flagella documented 51 protein–protein interactions (PPIs) so far. We have screened whole genome two‐hybrid arrays of Treponema pallidum and Campylobacter jejuni for PPIs involving known flagellar proteins and recovered 176 and 140 high‐confidence interactions involving 110 and 133 proteins, respectively. To explore the biological relevance of these interactions, we tested an Escherichia coli gene deletion array for motility defects (using swarming assays) and found 159 gene deletion strains to have reduced or no motility. Comparing our interaction data with motility phenotypes from E. coli, Bacillus subtilis, and Helicobacter pylori, we found 23 hitherto uncharacterized proteins involved in motility. Integration of phylogenetic information with our interaction and phenotyping data reveals a conserved core of motility proteins, which appear to have recruited many additional species‐specific components over time. Our interaction data also predict 18 110 interactions for 64 flagellated bacteria.

The binary protein interactome of Treponema pallidum--the syphilis spirochete.

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this “minimal” bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions (“interologs”) for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed.

β-Lactamases: protein evolution in real time

Abstract The evolution and spread of bacteria resistant to β-lactam antibiotics has progressed at an alarming rate. Bacteria may acquire resistance to a given drug by mutation of pre-existing genes or by the acquisition of new genes from other bacteria. One ongoing example of these mechanisms is the evolution of new variants of the TEM and SHV β-lactamases with altered substrate specificity.

Probing regulon of ArcA in Shewanella oneidensis MR-1 by integrated genomic analyses

BackgroundThe Arc two-component system is a global regulator controlling many genes involved in aerobic/anaerobic respiration and fermentative metabolism in Escherichia coli. Shewanella oneidensis MR-1 contains a gene encoding a putative ArcA homolog with ~81% amino acid sequence identity to the E. coli ArcA protein but not a full-length arcB gene.ResultsTo understand the role of ArcA in S. oneidensis, an arcA deletion strain was constructed and subjected to both physiological characterization and microarray analysis. Compared to the wild-type MR-1, the mutant exhibited impaired aerobic growth and a defect in utilizing DMSO in the absence of O2. Microarray analyses on cells grown aerobically and anaerobically on fumarate revealed that expression of 1009 genes was significantly affected (p < 0.05) by the mutation. In contrast to E. coli ArcA, the protein appears to be dispensable in regulation of the TCA cycle in S. oneidensis. To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map. By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli.ConclusionThese results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

Bufalin Is a Potent Small-Molecule Inhibitor of the Steroid Receptor Coactivators SRC-3 and SRC-1

Virtually all transcription factors partner with coactivators that recruit chromatin remodeling factors and interact with the basal transcription machinery. Coactivators have been implicated in cancer cell proliferation, invasion, and metastasis, including the p160 steroid receptor coactivator (SRC) family composed of SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2), and SRC-3 (AIB1/ACTR/NCOA3). Given their broad involvement in many cancers, they represent candidate molecular targets for new chemotherapeutics. Here, we report on the results of a high-throughput screening effort that identified the cardiac glycoside bufalin as a potent small-molecule inhibitor for SRC-3 and SRC-1. Bufalin strongly promoted SRC-3 protein degradation and was able to block cancer cell growth at nanomolar concentrations. When incorporated into a nanoparticle delivery system, bufalin was able to reduce tumor growth in a mouse xenograft model of breast cancer. Our work identifies bufalin as a potentially broad-spectrum small-molecule inhibitor for cancer.

Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β‐lactamase

In order to understand how TEM‐1 β‐lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161–170 region includes a portion of an omega loop structure, which is involved in the formation of the active‐site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended‐spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild‐type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161‐170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100‐fold increase in ceftazidime resistance were identified. Characterization of altered β‐lactamase enzymes indicated that while their catalytic efficiency (Kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild‐type TEM‐1 β‐lactamase.

A novel Treponema pallidum antigen, TP0136, is an outer membrane protein that binds human fibronectin.

ABSTRACT The antigenicity, structural location, and function of the predicted lipoprotein TP0136 of Treponema pallidum subsp. pallidum were investigated based on previous screening studies indicating that anti-TP0136 antibodies are present in the sera of syphilis patients and experimentally infected rabbits. Recombinant TP0136 (rTP0136) protein was purified and shown to be strongly antigenic during human and experimental rabbit infection. The TP0136 protein was exposed on the surface of the bacterial outer membrane and bound to the host extracellular matrix glycoproteins fibronectin and laminin. In addition, the TP0136 open reading frame was shown to be highly polymorphic among T. pallidum subspecies and strains at the nucleotide and amino acid levels. Finally, the ability of rTP0136 protein to act as a protective antigen to subsequent challenge with infectious T. pallidum in the rabbit model of infection was assessed. Immunization with rTP0136 delayed ulceration but did not prevent infection or the formation of lesions. These results demonstrate that TP0136 is expressed on the outer membrane of the treponeme during infection and may be involved in attachment to host extracellular matrix components.

PBP2a Mutations Causing High-Level Ceftaroline Resistance in Clinical Methicillin-Resistant Staphylococcus aureus Isolates

ABSTRACT Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.